MFF budding from mitochondria regulates melanosome size and maturation

Domain: biology
Published: 2026-03-14
Editorial outcome: major_revision

Abstract
Melanosomes are lysosome-related organelles that produce and accumulate melanin. Their maturation is regulated through interactions with mitochondria and involves the export and recycling of proteins via tubular transport and fission events whose mechanisms are unknown. Here, we demonstrate that the mitochondrial fission factor protein (MFF) is involved in melanosome fission. MFF is trafficked between mitochondria and melanosomes and locates at melanosome fission events. Upon downregulation of MFF, but not of dynamin-related protein 1, melanosomes enlarge, intracellular melanin accumulates. melanosomal lumenal catabolism increases, indicating that MFF-dependent melanosomes fission is required for their maturation. We show that MFF interacts with regulators of the ARP2/3 complex, which drives F-actin nucleation. Actin filaments accumulate between melanosomes at MFF-enriched membrane constriction sites, and silencing of ARP2/3 subunits mimicks the increase in melanosome size. MFF regulates actin-dependent fission of melanosomes via the ARP2/3 complex, indicating an extramitochondrial function for MFF in the regulation of melanosome homeostasis.

Introduction
Lysosome-related organelles (LROs) are cell-type-specific organelles of endosomal and/or secretory origin that share adaptive physiological functions, morphological features, and cargoes. Pigmented cells such as epidermal melanocytes and retinal pigment epithelial cells contain melanosomes, a type of LRO that allows compartmentalization of synthesis and storage of melanin pigments 1,2.

Melanosomes share several features with lysosomes. These include acidic intraluminal pH, the presence of lysosomal membrane proteins, luminal proteases, and hydrolases, including cathepsins 3-5. Even so, melanosome identity is determined by the presence of specific melanogenesis-associated machinery, such as the pre-melanosome protein (GP100; also known as PMEL17), Tyrosinase (TYR), and Tyrosinase-related proteins 1 and 2 (TYRP1/TYRP2) 6.

Melanosomes have been proposed to arise from endosomes and multivesicular bodies (MVBs). They are classified into four different stages of maturation based on distinct morphological features. Stage I melanosomes (pre-melanosomes or early melanosomes) are unpigmented endosomal-like compartments lacking melanogenic enzymes and characterized by luminal membranous vesicles 7. Stage II melanosomes exhibit an ellipsoidal shape with striae, due to the presence of GP100 that assembles into amyloidogenic fragments and then into parallel longitudinal fibers within the lumen of the organelle. Stage III melanosomes contain melanogenic enzymes like TYR and TYRP1/2 and visible melanin deposition onto GP100 scaffold/structured sheets. Stage IV melanosomes are fully pigmented and, under physiological conditions, move to the cell periphery where they are secreted and subsequently taken up by keratinocytes8,9 .

The maturation of melanosomes occurs upon delivery/export of melanogenic proteins through the endolysosomal recycling pathway. In line with their endosomal lineage, melanosomes appear to have the plastic and dynamic capacity to undergo homotypic fusion and fission through tubular and/or vesicular membrane remodeling10-16.

This idea is supported by the evidence that myosin 6 (MYO6), along with optineurin (OPTN), Wiskott-Aldrich syndrome protein and SCAR homolog (WASH), and the actin-related protein 2/3 (ARP2/3)-dependent actin nucleation complex participate in melanosome membrane constriction and fission events 15. Specifically, MYO6 mediates the severing of vesicle-associated membrane protein 7 (VAMP7)-positive tubule necks emerging from melanosome membrane subdomains. This process allows the export of non-essential components and fosters proper melanosome maturation 15. Modulators of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) levels, like the kinase PIKfyve, also promote the formation and release of membrane tubules from early melanosomes 17. Therefore, it is likely that different types of machinery regulate melanosome membrane remodeling, depending on the maturation stage.

Mitochondria supply ATP and calcium (Ca2+) to support melanosome maturation and melanin synthesis 18-20. Early-stage melanosomes have been shown to interact more with mitochondria19. Two possible explanations could account for this evidence. First, the proximity between organelles could facilitate the entry of ATP into the melanosomes, thereby fostering melanin synthesis and their maturation; second, it could support the recruitment of specific mitochondrial proteins to the melanosome surface during melanogenesis. Which would be the case still needs to be clarified.

Mitochondria and melanosomes are physically in contact through the outer mitochondrial membrane (OMM) protein mitofusin 2 (MFN2) that regulates mitochondria-melanosome apposition in space and time during active melanogenesis19. Whether the communication between these two organelles extends beyond the established role of MFN2 is unknown, despite mounting evidence for the role of mitochondria contact sites in several cellular processes, like Ca2+ homeostasis, lipid metabolism, and organelle function and dynamics.

While investigating the role of the mitochondrial proteome in melanosome function, we found that the mitochondrial fission factor (MFF) is present on the surface of melanosomes, independently of mitochondria. MFF localization on the melanosome surface enables it to interact with components of the ARP2/3 complex, facilitating early melanosome fission without its mitochondrial pro-fission partner DRP1. The promotion of maturation, and for their function during melanogenesis.

Results
Comparison of pigmented and albino melanocytes identifies MFF involvement in mitochondria-melanosomes crosstalk
To clarify the role of the interaction between mitochondria and melanosomes, we compared wild-type (melan-a) and albino (melan-c2) melanocytes, which lack melanin due to a loss-of-function mutation in the Tyr gene (Fig. S1A, B). These cells lack melanin synthesis, which in turn blocks melanosome maturation and causes an accumulation of early-stage melanosomes (stages I and II).

Given the abnormal accumulation of immature melanosomes in albino cells compared to pigmented cells, we reasoned that mitochondria-early melanosome communication could also be altered. In line with this idea, our analysis confirmed that albino cells accumulate early-stage melanosomes (I/II) and are characterized by increased contact sites with mitochondria (Fig. 1A, B). We observed that, as they mature, melanosomes appear closer to mitochondria, with this proximity being significantly greater in albino cells at stage I than in pigmented cells. (Fig. 1C). In parallel we performed a differential expression analysis on the two cell lines using RNA-seq and found a significant upregulation of mitochondrial genes in albino cells compared to pigmented ones (Fig. 1D, E; Fig. S1FR; Supplementary Data 1).

Then, we isolated mitochondria-melanosome-ER fractions (MMERF) from melan-a and melan-c2 cells and analyzed them by mass spectrometry (MS) (Fig. 1F, G and “Methods” section). To identify membrane-bound proteins involved in mitochondria-organelle interactions, we subjected MMERFs to limited proteolysis (Fig. 1F, G; Fig. S1G). Proteins identified in both treated and untreated fractions were considered luminal, being protected from the added proteases. To confirm which proteins were exposed on the surface of organelles, we exploited the topology of TYRP1. TYRP1 is a melanosome membrane protein with domains facing either the cytosol or the organelle lumen. We used two different antibodies, each of which could target one of the two regions with different exposure. This served as a control to ensure that proteinase K degraded only the exposed, cytosolic parts of proteins (Fig 1L) . Proteins identified only in the untreated MMERF fractions were considered to be exposed to the organelle surfaces due to their susceptibility to proteolysis (Fig. 1F, G, L, and Fig. S1G). A Venn diagram of the proteomic datasets obtained from the two cell lines revealed 109 common proteins, with 136 enriched in melan-a and 242 in melan-c2 (Fig. 1H; Supplementary Data 2). Functional annotation showed that MMERFs from melan-a were enriched in Golgi and proteasome-related proteins, while MMERFs from melan-c2 contained more lysosomal, ER, and outer mitochondrial membrane (OMM) proteins (Fig. 1F). The list of OMM candidates included proteins previously retrieved at mitochondria-derived vesicles (MDVs), membrane contact sites (CTP1A, FUNDC2, MAVS, MTOR, RHOT2, TOM20, TOM34, TOM70) 21 and, unexpectedly, the mitochondrial fission factor MFF.

Although we could not observe morphological differences in the mitochondria network between healthy and pigmented melanocytes (Fig. SI1J, K), we noticed that albino melanocytes were characterized not only by an accumulation of early melanosomes, but also by an increased size of the organelles (Fig. S1J-M). Therefore, we conducted a thorough examination to determine whether there were any changes in melanosome dynamics between the two cell lines (Fig. 1I). Using Lysotracker to monitor melanosome dynamics as done previously22,23, we observed that, despite having larger melanosomes, albino cells showed more fission events (Fig. 1I, J, K). Interestingly, fusion events in albino cells were also more frequent (Fig. S1O, P). These results suggested that the loss of function of TYR in albino cells leads to defective melanosome dynamics: in these cells, melanosome fusion rates would be outpaced by fission due to the inability to progress with melanin synthesis and with maturation of the organelles. Since MFF has been implicated in the division of different organelles, not only mitochondria, we hypothesized that it could participate in melanosome maturation.

MFF can bud from mitochondria to melanosomes and resides at melanosome constriction sites
MFF serves as a membrane adaptor for the dynamin-like GTPase DRP1 in mitochondria and peroxisomal division 24-26. Hence, MFF is also present in non-mitochondrial locations, where it facilitates membrane fission27. We investigated MFF localization using immunogold electron microscopy. MFF was present at the surface of melanosomes at every stage of maturation, independent of the presence of nearby mitochondria (Fig. 2A). To further confirm MFF localization, we used super-resolution microscopy in cells that have been labelled with HMB45, a monoclonal antibody that marks early melanosomes, recognizing an epitope of the protein GP100. Following synthesis, GP100 is subject to several proteolysis steps, resulting in its conversion into the HMB45-reactive low molecular weight form (∼35 kDa)28. This form is known to associate with the fibrillar content of melanosomes: as melanin is produced and deposited on the fibrillar matrix, the HMB45-reactive epitopes gradually become covered, and by the time melanosomes mature into Stage III, reactivity with HMB45 is lost 28. We found MFF-positive puncta at the surface of melanosomes both in melan-a and melan-c2 melanocytes (Fig. 2B, C, D, E). MFF puncta were not positive for TOMM20 or PDH, excluding the possibility of being part of mitochondria or MDVs (Fig. S2B). At steady state, approximately 22% of melanosomes marked by HMB45 were found to have MFF puncta in melan-a cells (Fig. 2F). Some MFF puncta colocalized with the mature melanosome marker TRP1, though to a lesser extent than with HMB45 (Fig. S2A). Quantitative analysis revealed that MFF and mtRFP colocalized less in albino melanosomes compared to pigmented melanocytes, suggesting a larger pool of non-mitochondrial MFF in albino cells (Fig. 2G). Consistent with this, we observed increased colocalization of MFF with HMB45 in albino melanocytes (Fig. S2C). Endogenous MFF was detected not only on the melanosome surface but also at membrane constriction sites and in vesicular/tubular extensions of early melanosomes (Fig. 2H). Quantitative analysis indicated that MFF was associated with approximately 25% of the observed melanosomal fission regions (Fig. 2I), suggesting that there may be distinct fission sites as reported also for other organelles29. Therefore, we sought to understand whether MFF is directly involved in melanosome fission. First, we generated melan-a MELANO-IP cells, and then we verified the presence of MFF in pure melanosomal fractions, free of mitochondrial contamination (Fig. S2D, E). We found that isoform 1 of MFF was enriched in melanosomal fractions, whereas isoform 4 appeared more enriched in the total input (Fig. S2D, E). Overexpression of eGFP-MFF isoform 1 confirmed its localization at HMB45-positive structures and fission sites (Fig. S2F). To verify how MFF could be delivered to melanosomes, we performed time-lapse experiments by marking them with LAMP1-RFP, which had been previously found enriched in these organelles30 and which co-localized with HMB45 in melan-a (Fig. 2B).

With this approach, we observed eGFP-MFF isoform 1 budding from mitochondria and moving toward LAMP1-RFP-positive structures (Fig. 2J). This result was also confirmed by staining stage I/II melanosomes, which have an acidic lumen, with Lysotracker (Fig. S2G). Of note, although we had evidence of MFF being trafficked from mitochondria to melanosomes, we cannot exclude the possibility that cytosolic pools of MFF exist, which may also be independently targeted to these organelles. Super-resolution time-lapse imaging highlighted the presence of MFF puncta at the neck of melanosome tubules, with membrane constriction and scission occurring at or near MFF puncta (Fig. 2K). Figure 2K shows that melanosome membranes can be split into two separate vesicular structures by an MFF punctum, with which they can remain attached (left example) or be released (Fig. 2K, right example). These results demonstrate that MFF associates with melanosomes and that it is enriched at fission sites independently of mitochondrial proximity, likely facilitating melanosome membrane fission.

MFF depletion disrupts melanosome fission and promotes lysosomal protease-mediated degradation
Melanosomes have previously been shown to undergo vesicular and tubular fission, a mechanism critical for regulating their composition and function15. Thus, considering our results, we hypothesized that the recruitment of MFF to the melanosome surface may control their maturation by mediating fission events. Given that MFF is a known receptor for DRP1 (dynamin-related protein 1) - the GTPase responsible for mitochondrial membrane fission—we assessed if Drp1 could be involved in MFF-mediated melanosome fission. We investigated the potential colocalisation of DRP1 and MFF in melan-a cells. Using immunofluorescence, we detected DRP1 at the melanosome surface, similarly to MFF. However, minimal to no colocalization between the two proteins was observed at melanosomes (Fig. 3A). To further explore their eventual function in melanogenesis, we performed siRNA-mediated knockdown (KD) of both Mff and Drp1 in wild-type melanocytes (Fig. 3B; Fig. S3A, B). Interestingly, knocking down either gene led to reduced expression of melanogenic proteins (Fig. S3C-E). However, while intracellular melanin levels were slightly reduced in Drp1-KD cells, they were significantly increased in Mff-KD cells (Fig. S3F). This increase in melanin could be explained by either enhanced melanin synthesis or the accumulation of dysfunctional, immature melanosomes. Given the reduced levels of melanogenic markers, we excluded the former and focused on the possibility of impaired melanosome maturation. Using SR microscopy, we observed that HMB45-positive melanosomes were enlarged only in Mff-silenced melan-a cells (Fig. 3C). The number of HMB45-positive melanosomes remained unchanged in both knockdown conditions (Fig. 3D), but the melanosomes were significantly larger only in Mff-silenced cells (Fig. 3E; Fig. S3H). TEM further confirmed that Mff-KD cells harbored larger melanosomes, often connected by narrow, neck-like structures, or appearing as a fusion of melanosomes at different maturation stages (Fig. 3F; Fig. S3L, M). The presence of these hybrid early–late stage melanosomes suggests the inability to complete fission and that melanogenesis does not progress properly in the absence of MFF. Indeed, we observed a significantly lower frequency of fission events in Mff-KD cells compared to controls (Fig. 3G, H). Additionally, the colocalization of pigmented melanin granules (visible in brightfield) with HMB45-positive melanosomes was significantly higher in Mff-KD cells, further supporting the presence of hybrid melanosomal structures (Fig. 3I, J).

Interestingly, despite reduced fission frequency, melanosomal fission was not entirely abolished in Mff-silenced melanocytes. This is consistent with previous observations that MFF is not present at all visible melanosomal constriction sites (Fig. 2I), and it suggests that alternative fission mechanisms may exist.

To test whether MFF overexpression could influence melanosome morphology, we overexpressed all four MFF isoforms and assessed melanosome size (Fig. S3O). MFF overexpression alone was insufficient to promote melanosome fragmentation, supporting that MFF is not the sole mediator of melanosome division.

Defective melanosome membrane remodeling and fission result in increased melanosome size and accumulation of undegraded or unnecessary proteins, ultimately leading to impaired melanogenesis 15,17. To better understand how MFF and DRP1 depletion differentially impact melanosome homeostasis, we performed RNA sequencing on Mff- and Drp1-silenced melanocytes. Mff silencing led to the upregulation of genes associated with the homeostasis of lysosomes (Capn2, Ctsb, Ctsd, Dpp7, Gabarap, Kxd1, Lipa, Lrrc8a, Mfsd12, Mt1, Myo5a, Plekhm2, Rab7b, Scarb2, Siae, Sidt2, Tpcn1, Tpp1), melanosomes (Capg, Ctsb, Ctsd, Mfsd12, Myh11, Myo5a, Tpp1, Trpv2, Kxd1) or both (Ctsb, Ctsd, Mfsd12) (Supplementary Data 3). These transcriptional changes were not observed in Drp1-silenced cells, which instead showed a broad downregulation of key melanogenesis regulators, including Microphthalmia-associated transcription factor (MITF) (Fig. 4A; Supplementary Data 3). These findings confirm that Mff and Drp1 silencing have distinct functional consequences in pigmented cells. While Mff-KD triggers the upregulation of lysosomal and catabolic genes, including cathepsins (Ctsb, Ctsd) and lysosomal lipase (Lipa), Drp1-KD primarily alters melanocyte differentiation and pigment production by downregulating transcriptional regulators such as MITF. As to the specific upregulation of lysosomal/catabolic genes in Mff-KD, we hypothesized that this response may reflect a compensatory mechanism triggered by impaired melanosome fission. Supporting this, CTSB protein levels were elevated in Mff-silenced melanocytes, which showed an increased colocalization of CTSB and HMB45-positive melanosomes (Fig. 4B–E). CTSB enzymatic activity was also significantly increased in Mff-KD cells (Fig. 4F, G). These data suggest that MFF depletion promotes the trafficking and activity of lysosomal enzymes like CTSB, which are key in the process of melanosomes degradation31,32. Their enhanced melanosome localization could be potentially due to either increased melanosome–lysosome fusion or increased trafficking of lysosomal hydrolases, resulting in their partial conversion to a lysosome-like state. Both scenarios are consistent with a shift toward increased melanosome degradation, which is likely a compensatory response to blocked fission. To confirm this hypothesis, we assessed melanosomal acidification, a hallmark of lysosomal activity and of early melanosome, whose pH undergoes neutralization during maturation. Acidification was significantly increased in Mff-KD cells, but it was rescued to control levels by co-silencing CTSB (Fig. 4H, I). Notably, combined silencing of Mff and Ctsb restored the expression of melanogenesis markers (downregulated in Mff-KD alone) and further increased both melanin content and melanosome size, compared to Mff silencing alone (Fig. 4J, K, and Fig. S4C-I). Consequently, when CTSB-mediated melanosomal degradation was suppressed in Mff-KD cells, a marked accumulation of early-stage melanosomes was observed per total cell area (Fig. 4L), which was not detected in Mff silencing alone (Fig. 3D). These findings indicate that the degradation of key melanogenic proteins (such as TYR, TRP1, and GP100) is enhanced in Mff-KD cells due to increased recruitment and activity of lysosomal enzymes within the melanosome lumen. This could represent a negative feedback mechanism aimed at reducing melanosome buildup when fission is impaired.

In summary, MFF depletion impairs early-stage melanosome maturation and triggers a compensatory upregulation of lysosomal catabolism to prevent the accumulation of dysfunctional or oversized melanosomes in response to decreased DRP1-independent melanosome fission.

MFF-dependent melanosome fission requires ARP2/3-mediated F-actin assembly
Our data suggest that MFF can modulate the maturation of melanosomes by controlling their membrane fission independently of DRP1. To shed light on the molecular mechanisms of MFF-mediated melanosome fission, we determined the MFF interactome by immunoprecipitating it from melan-a whole cell lysate and identified the co-purified proteins by MS (Supplementary Data 3). The list of interacting proteins was enriched in actin cytoskeleton organization regulators, including subunits of the WASH core complex (Fig. 5A). This complex functions as an actin nucleation-promoting factor at the surface of endosomes and melanosomes, recruiting the ARP2/3 complex and allowing actin polymerization for vesicle/tubule fission 15,33. FAM21C and Strumpellin (Kiaa0196) are key members of the WASH complex that appeared as hits from our IP (Fig. 5A). After confirming their interaction with MFF (Fig. 5B) and close apposition with early and late-stage melanosomes (Fig. S5G), we verified if their downregulation could affect early melanosome morphology (Fig. S5A, B, C). In melan-a cells, depletion of either of the two leads to no significant changes (Fig.S5D, E). These results suggest that the different components of the complex may have redundant functions and may compensate for each other. A gene ontology (GO) analysis of the MFF interactome also highlighted that the actin nucleation complex may be involved in MFF function (Fig. 5C). Therefore, to clarify the contribution of actin filaments to MFF-mediated melanosome fission, we co-stained melan-a expressing eGFP-MFF with phalloidin and HMB45 (Fig. 5D). We observed F-actin at and in between MFF-positive melanosome constriction sites (Fig. 5D,E). Because ARP2/3 complex-mediated actin nucleation was among the enriched GO terms (Fig. 5C), we sought to understand whether the effect of MFF depends on the ARP2/3 complex by silencing two different subunits: ARPC1A and ARPC3 (Fig. S5H-J). Their silencing led to increased early melanosome size, which was more noticeable when ARPC1A was silenced (Fig. 5F). ARPC1A silencing also led to a change in intracellular melanin levels (Fig. S5K. Besides, we confirmed ARP2/3 complex subunit localization at MFF-HMB45-positive melanosomes (Fig. 5G), and we found that MFF puncta locate in proximity to the enlarged melanosomes in ARPC1A/ARPC3-silenced cells (Fig. S5L). This evidence suggests that the recruitment of MFF to melanosomes is independent of the ARP2/3 complex. Silencing of MFF led to increased melanosome size, consistent with a role in promoting organelle fission. However, co-treatment with the Arp2/3 inhibitor CK-666 (a small molecule that stabilizes the ARP2/3 complex in its inactive state and thereby blocks actin nucleation) abrogated this phenotype, suggesting that melanosome enlargement in the absence of MFF requires active actin polymerization (Fig. 5H).

Methods
Cell culture: melan-a and melan-c2
Melan-a melanocytes are normal, immortalized murine melanocyte cells derived from C57BL/6J mice. Melan-a melanocytes were obtained from the Functional Genomics Cell Bank at St. George’s University of London, UK42. Melan-a melanocytes were maintained in a RPMI-1640 medium (Euro Clone, Cat. No. ECB2000L; Supplementary Table 1) supplemented with 10% fetal bovine serum (FBS) (Euro Clone, Cat. No. ECS0165L), 100 U/mL penicillin, 100 µg/mL streptomycin, and 200 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Cat. No. P8139). PMA was diluted in phosphate-buffered saline (PBS) containing 1% FBS to obtain a 40 μM stock solution. Melan-c2 melanocytes are immortalized tyrosinase mutant murine melanocytes (Tyrc-2J/Tyrc-2J, a functionally null allele) derived from C57BL/6J. Cells were maintained in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 200 nM PMA and 2 nM cholera toxin diluted in water (Sigma-Aldrich, St. Louis, MO, Cat. No. C8052). Both cell lines were kept at 70% confluency between experiments at 37°C in a 10% CO2 incubator.

Virus production and generation of melan-a stably expressing HA-MelanoTag and Myc–MelanoTag
Lentiviruses were produced by co-transfection of HEK 293T cells with a target lentiviral transfer vector (2,5 μg HA–MelanoTag (Addgene #160369) and Myc–MelanoTag (Addgene #160368)), pMD2.G (0,5 μg, Addgene #12259), and psPAX2 (2 μg, Addgene #12260) plasmids. Virus-containing supernatants were collected 48 h post-transfection, and transduction was performed. About 2,0x106 melan-a cells were incubated for infection with 5 mL of viral supernatant, 8 μg/ml polybrene, and 5 mL culture medium in a T75 flask. Infected cells were passaged into medium containing puromycin (3 μg/ml) 36–48 h post-transduction. Selection was maintained for 7 days before use of the cells.

Intracellular melanin content assay
1 x 106 cells were plated on a 100 mm cell culture Petri dish. After 66 h, cells were scraped in PBS and lysed using RIPA buffer (50 mM TRIS pH 8, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA) supplemented with protease (Thermo Fisher Scientific, Cat. No. A32955) and phosphatase (Thermo Fisher Scientific, Cat. No. A32957) inhibitors for 30 min, on ice. After centrifugation at 16,500 x g for 15 min at 4ºC, the supernatant was collected for protein assay, and the melanin pellet was solubilized in 1 N NaOH with 10% DMSO for 90 min at 80ºC. Then, sample absorbance was acquired at 450 nm and the values were compared with a standard curve of synthetic melanin (Sigma-Aldrich, #M8631). Samples were normalized for total protein content (Bradford method, Bio-Rad, Cat. No. 5000006).

Mitochondria-Melanosome-ER- fraction (MMERF) isolation
Cells were plated at 80% confluency in a Corning® square dish (245x245 mm; CLS431110-16EA Corning). After 24 h, they were harvested and washed once with cold PBS. The cell pellet was then resuspended in 700 l of mitochondrial isolation buffer (containing 200 mM mannitol, 70 mM sucrose, 1 mM EGTA, 10 mM HEPES; 1X protease inhibitor cocktail (Thermo Scientific, Cat. No. A32955)). Homogenization was performed on ice using a 1 ml insulin syringe. Cells were centrifuged at 1,000 x g for 10 min at 4ºC, and the supernatant was collected while the pellet, corresponding to the PNS fraction, was retained for further processing. The supernatant was spun at 12,500 x g for 15 min at 4ºC. After centrifugation, the new supernatant (enriched with cytosolic and microsome components) and a part of the pellet (corresponding to crude mitochondria) were stored for Western Blot analyses. Crude mitochondria were washed with 1 ml isolation buffer and centrifuged again at 12,500 x g for 10 min, thereby obtaining MMERF. The pellet was resuspended in 60 µl of mitochondrial isolation buffer and protein concentration was determined using Bio-Rad Protein Assay Reagent (Bio-Rad, Cat. No. 500000). MMERF were treated on ice for 15 min with proteinase K (Invitrogen, Cat. No. AM2548) in a final concentration of 15 µg/ml diluted in mitochondrial isolation buffer. Proteinase K was subsequently blocked with 5 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich Cat. No. 78830) for 5 min on ice. 20 µg of each sample (either proteinase K-treated or not) was loaded for western blotting; 70 µg were used for the MS analysis, and 15 µg of MMERF were fixed for TEM.

Mass spectrometry sample preparation
Proteins were precipitated and concentrated by adding 4 volumes of ice-cold MS-grade Acetone (over-night incubation) and pelleted at 20,000 xg for 20 min, at 4°C. The protein pellet was additionally washed with ice-cold 70% ethanol. Then a standardized in solution digestion protocol was applied. Briefly, dried proteins were dissolved in 6M guanidine hydrochloride (GnHCl), 50 mM Tris pH=8.5, reduced by incubation with 10 mM Tris-(2-carboxyethyl)phosphine-hydrochloride (TCEP) and alkylated with 40 mM chloroacetamide (CAA). Then, they were digested overnight (37°C) adding a mix of Lys-C/Trypsin hydrolases in a 1:50 enzyme to protein ratio. The hydrolysis reaction was quench by adding 1% trifluoracetic acid (TFA). The resulting peptide mixtures were desalted using the Stop and Go Extraction (STAGE) procedure and then lyophilized and stored at -20°C until the MS analysis (PMID: 12585499). All the experiment were performed in triplicate starting from three different immuno-precipitations. As controls, we performed, in triplicate, immuno-precipitations without the specific antibody against our target.

Mass Spectrometry Analysis
Instruments for LC-MS/MS analysis consisted of a NanoLC 1200 (Thermo Fisher Scientific) coupled via a nano-electrospray ionization source to the quadrupole-based Q Exactive HF (ThermoFisher Scientific) benchtop mass spectrometer43. Peptide separation was carried out according to their hydrophobicity on a home-made column, 75 µm ID, 8 µm tip, 250 mm bed packed with Reprosil-PUR, C18-AQ, 1.9 µm particle size, 120 Å pore size (New Objective, Inc., Cat. No. PF7508-250H363), using a binary buffer system consisting of solution A: 0.1% formic acid and B: 80% acetonitrile, 0.1% formic acid. Total flow rate: 300 nl/min; LC linear gradient: after sample loading, the run starts at 5% buffer B for 5 min, followed by a series of linear gradients, from 5% to 30% B in 90 min, then a 10 min step to reach 50% and a 5 min step to reach 95%. This last step was maintained for 10 min.MS spectra were acquired using 3E6 as an AGC target, a maximal injection time of 20 ms, and a 120,000 resolution at 200 m/z. The mass spectrometer operated in a data-dependent Top20 mode with subsequent acquisition of higher-energy collisional dissociation (HCD) fragmentation MS/MS spectra of the top 20 most intense peaks. Resolution for MS/MS spectra was set to 15,000 at 200 m/z, AGC target to 1E5, max injection time to 20 ms, and the isolation window to 1.6 Th. The intensity threshold was set at 2.0E4 and Dynamic exclusion at 30 s.

Mass spectrometry Data Processing
MaxQuant software (1.6.2.10) was used for protein identification and label-free quantification. Trypsin (Thermo Fisher Scientific) was chose as the enzyme and the number of missed cleavages was set to 1. Cysteine carbamido-methylation was set as a fixed modification, whilst methionine oxidation and N-term acetylation were used as variable modifications. Uniprot Mus Musculus (UP000000589) FASTA file was used as database (accessed in 2023). The maximum false discovery rate (FDR) for peptide spectral match was set to 1%, using a minimum of one sequence-unique peptides was required for identification. Statistical analyses for quantitative evaluation were performed using the open-source Perseus software (1.6.2.3). Two-sample t-test analysis was applied, setting the level of significance equal to 0.05. Proteins with at least one-fold changes were considered differentially regulated. Proteins of interest were further investigated for GO enrichment terms with ShinyGo platform. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository44 with the dataset identifier PXD051372.

Library preparation and mRNA sequencing
Melan-a and melan-c2 cells underwent lysis and RNA extraction using Trizol® (Thermo Scientific Cat. No. 15596018) according to the manufacturer’s protocol. The purity and concentration of samples were checked with a NanoDrop spectrophotometer (ND 1000, Thermo Fisher Scientific). The libraries were generated with the QuantSeq 3' mRNA-seq Library Prep kit (Lexogen) and oligodT priming. After the first strand synthesis, the RNA was removed. A second strand synthesis was initiated using random priming followed by magnetic bead-based purification. The libraries were then amplified, introducing the sequences required for cluster generation. External barcodes were introduced during the PCR amplification step. Library quantification and quality control were performed using a fluorometer (Qubit, Thermo Fisher scientific) and Bioanalyzer 2100 (Agilent). NGS reads were generated towards the poly(A) tail. Sequencing was performed on NextSeq500 Illumina instrument to produce at least 5 x 106 reads (75 bp SE) for each sample.

Analysis of RNA-Seq data
Quality of sequencing reads was evaluated with fastqc v0.11.9, plus multiqc v1.6 45, then the reads were trimmed with fastp v0.20.1 46 by removing 15 nt at 5’, 2 nt at 3’, discarding all those reads containing “N” characters, removing heading and trailing homopolymers, removing heading and trailing low-quality portions and retaining only trimmed reads with length of at least 35 nt. All the other settings for the trimming were left as default. Trimmed reads were mapped on the cDNA sequences from the reference mouse genome (GRCm39) with salmon v1.8 47 with the validate Mappings flag and by providing the transcript to gene map in order to obtain gene-level expression measurements in terms of counts. A first check of coherence between samples was performed by visual inspection of Principal Component Analysis (PCA) and Multi-Dimensional Scaling (MDS) plots. Differential gene expression analysis was carried out with the edgeR package48: briefly genes with low counts were discarded (filterByExpr function), then expression values were normalized, and a Generalized Linear Model (GLM) was fitted on the data. Differential expression between all possible pairs of samples was assessed using a quasi likelihood F-test for generalized linear models as implemented in edgeR, and p-values were adjusted for multiple testing using the Benjamini–Hochberg false discovery rate (FDR) correction. Genes with an FDR-adjusted p-value < 0.05 (two-sided test) were considered differentially expressed. Gene Set Enrichment Analysis (GSEA) was performed on the Gene Ontology (GO) and annotations49. Specifically, a hypergeometric test was applied to the DEGs sets (also splitting up- and down-regulated DEGs)50 using only expressed genes as “universe” (i. e. those obtained from the FilterByExpr function) to avoid biases51. Only GO terms supported by an FDR-corrected p-value < 0.05 were considered as significantly enriched. Data visualizations were produced with seaborn and plotly.

Immunoblotting
Melanocytes were lysed with ice-cold RIPA buffer supplemented with protease inhibitor cocktails (Thermo Fisher Scientific, Cat. No. A32955). Cell lysates were then centrifuged at 16,500 x g for 15 min at 4°C and the supernatant protein content was determined using the Bradford reagent (Bio-Rad, Cat. No. 5000006). After heating at 70°C for 10 min, equal amounts of protein (30 μg) were loaded onto precast gels (ExpressPlus™ PAGE Gel, Genscript 4-12% or 4-20%) and separated electrophoretically in Tris-MOPS running buffer (Genscript, Cat. No. M00138). After electrophoresis, the proteins were blotted onto a polyvinylidene fluoride membrane (PVDF) (EMD Millipore, Merck, Cat. No. IPVH85R). The blocking was performed for 1 h at RT using 5% milk (Sigma Aldrich, Cat. No. 70166) in Tris-buffered saline with 0.1% Tween 20 (TBS-T), and the membrane was incubated with the selected primary antibodies O/N at 4°C (listed in Supplementary Table 2). After washing, the membranes were incubated with HRP-conjugated anti-Rabbit secondary antibody (1:4000; Cell Signalling, Cat. No. 7074-P2) and HRP-conjugated anti-Mouse secondary antibody (1:2000/1:4000, Cell Signalling, Cat. No. 7076-P2) for 1 h at RT. Secondary antibodies details are provided in Supplementary Table 3. The signal was visualized using an enhanced chemiluminescence kit (Luminata Forte Western HRP (Millipore Sigma WBLUF0500) according to the manufacturer's instructions and acquired using iBright™ CL1500 (Invitrogen), ImageQuant LAS 4000 Mini (GE Healthcare), or the ChemiDoc MP Imaging System (BioRad). For two-color fluorescence imaging of membranes Odyssey CLx Imager from LI-COR Biosciences was used.

Melanosome immunoprecipitation (Melano-IP)
Melan-a cells (3 × 106) expressing MelanoTag were plated into a 15-cm plate. Two days later, cells were collected for Melano-IP. Cells were washed and collected by scraping in ice-cold KPBS (136 mM KCl, 10 mM KH2PO4, pH 7.25 adjusted with KOH)30 before pelleting by centrifugation at 1000g for 2 min at 4 °C. Pellets were resuspended in 950 μl ice-cold KPBS, and 50 μl of the cell suspension was taken as a whole-cell fraction (Input). Homogenization was performed on ice using a 1 ml insulin syringe, and then samples were centrifugated at 1000g for 2 min at 4 °C. Supernatants were transferred into new 1.5-ml tubes containing 100 μl of KBPS-washed Pierce™ Anti-HA Magnetic Beads (Thermo Fisher) and incubated rocking for 15 min at 4 °C. Beads were washed 3 times with 1 mL ice-cold KPBS, transferring to a new 1.5-ml tube each time. Whole-cell and immunoprecipitated samples were then lysed in 50 μl ice-cold RIPA buffer supplemented with protease inhibitor cocktails (Thermo Fisher Scientific, Cat. No. A32955).

MFF Immunoprecipitation
Protein A beads (protein A - Agarose, Roche, Cat. No. 11719408001) were prepared in PBS with 0.1% BSA, and left to rotate at 4ºC for 30 min. They were washed three times with 400 µl of PBS and centrifuged at 200 x g for 1 min. Then, beads were resuspended in lysis buffer (50 mM Tris-HCl pH 7,5, 150 mM NaCl, 1 mM EGTA pH 7,5, 1 mM EDTA pH 7,5, and 1% Triton X-100) supplemented with protease inhibitor, and incubated with 3 µg of each antibody on a rotator at 4ºC O/N. Protein A beads were washed with lysis buffer and centrifuged three times at 200 x g for 1 min. Whole cell lysates were prepared by collecting cells in cold PBS and resuspension in lysis buffer. The cell lysate was kept on ice for 10 min, sonicated (four times 5 pulses of 0.5 s), and then centrifuged at 14,000 x g for 15 min. The resulting supernatant was quantified with Bradford protein content. 2 mg of proteins were incubated with the bead-antibody conjugate O/N at 4ºC. The next day, the beads-antibody-whole lysate complexes were spun five times at 200 x g for 30 s to remove the supernatant and add clean, fresh lysis buffer (300 µl each time). IP elution was performed by resuspension of the last in 50 µl of 2x Laemmli buffer containing 200 mM β-mercaptoethanol (Merck, Cat. No. M3148) with further denaturation for 5 min at 95ºC.

Cloning and plasmids
We generated eGFP-MFF plasmids (pEGFP-FlagMff-iso1, 2, 3, 4) starting from melan-a cells. RNA from melan-a cells was extracted using RNeasy Mini Kit (Qiagen, Cat. No. 74104) following the manufacturer’s instructions. RNA was reverse transcribed in cDNA using GoScript Reverse Transcriptase (Promega, Cat. No. A5003). For Mff amplification, PCR was performed using melan-a cDNA, PrimeSTAR GXL DNA Polymerase (Takara technologies, Cat. No. R050A), and the primers specified in Supplementary Table 4. The amplicons were run in a 2% agarose gel to distinguish the different Mff isoforms according to their nucleotide sizes. The band corresponding to Mff isoform 2 appearing at ~720 bp was gel purified using Qiagen Gel Extraction Kit (Qiagen, Cat. No. 28704). The amplicon of Mff isoform2 and the vector pEGFP-C1 (Addgene, Cat. No. 6084-1) were digested with HindIII-HF (New England Biolabs NEB Cat. No. R3104S) and BamHI-HF (New England Biolabs NEB Cat. No. R3136S) restriction enzymes, run in a 2% agarose gel and gel purified. The insert was ligated to the vector using T4 DNA Ligase (New England Biolabs NEB Cat. No. M0202S) following the manufacturer’s instructions using a 3:1 ratio. The insertion was verified by Sanger sequencing.

Electroporation into melan-a and melan-c2 of siRNA and plasmids
Microporation was done using either Neon™ Transfection System (Invitrogen, Cat. No. MPK5000) or Lonza® Nucleofector® electroporation system. Melan-a and melan-c2 at 80% confluency were detached with trypsin, counted, and centrifuged in serum-containing medium at 500 x g for 5 min. The cell pellet was resuspended in warm (37°C) PBS. Then, 1,2 × 105 cells were electroporated with siRNA and/or plasmid DNA according to the manufacturer's instructions (10 µl volume, 40 nM final concentration of a pool of 4 siRNAs; sigenome SMART pool, Horizon Discovery 1 µg/ul for plasmids; Supplementary Data 4). After microporation (1,200 V, 20 ms, 3 pulses), cells were immediately resuspended with media (RPMI-1640 medium supplemented with 10% FBS) and 200 nM PMA, without antibiotics) and plated. 66 h after electroporation, cells were either fixed or collected for further analysis.

Melan-a cell lentiviral transduction
For viral transduction, 10000 melan-a cells per well were seeded in 96-well plates, and cultured for 24 h at 37 °C in a humidified incubator with 10% CO₂. Cells were then transduced with lentiviral particles encoding either shRNA targeting Mff or the non-targeting control (MISSION® pLKO.1-puro Non-Target shRNA Control Transduction Particles; Sigma-Aldrich, SHC016V). For Mff silencing, individual shRNA constructs were employed: TRCN0000174665 (target sequence: GATCGTGGTTACAGGAAATAA) and TRCN0000174539 (target sequence: CTTCATTAAGACGTCAGATA; Sigma-Aldrich, MISSION® shRNA library). Transduction was performed in complete growth medium supplemented with 10 µg/mL polybrene (Sigma-Aldrich, #TR-1003) for 24 h at 37 °C in a humidified incubator with 5% CO₂. Following transduction, cells were cultured in fresh complete growth medium for additional 24 h prior to downstream analyses.

HTS acquisition and analysis of mitochondria and melanosome morphology
Melan-a/c2 cells were seeded on a 96-well TC-Treated Black µCLEAR microplate (Greiner Bio-One, Cat. No. 655090) and incubated in growth media for 48 h. Melanocytes were fixed with PBS containing 3.7% formaldehyde (Sigma-Aldrich, Cat. No. 252549) for 15 min, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Cat. No. T8787 ) for 15 min and blocked with 5% BSA (Carlo Erba Reagents, Cat. No. A268131000) diluted in PBS for 1 h at RT. Cells were stained with the primary antibodies anti‐TOM20 (diluted 1:150, Sigma-Aldrich, Cat. No. HPA011562) and HMB45, which is already diluted and ready-to-use (Agilent Technologies, Cat. No. GA052) O/N at 4ºC. The cells were then washed three times with 5% BSA in PBS and incubated with the following secondary antibodies: Goat anti-Rabbit IgG (H + L) Alexa fluor 488 Secondary Antibody diluted 1:250 (Invitrogen Thermo Fisher Scientific, Cat. No. A-11070), Goat anti-Mouse IgG (H+L) Alexa Fluor 568 diluted 1:250 (Thermo Fisher Scientific, Cat. No. A-11004). Nuclei were stained with Hoechst 33342 200 μg/mL in PBS (Thermo Fisher Scientific, Cat. No. H1399) for 10 min at RT. Cells were later incubated with phalloidin 680 (Abcam, Cat. No. ab176760, diluted 1:500 in 1% BSA in PBS) for 90 min at RT for total cell area detection. After incubation, melanocytes were washed 3 times with PBS. Images were acquired using the Operetta CLS™ High‐Content Analysis System (PerkinElmer), with a 63× Water/1.15 NA objective (High Content Imaging was performed at the HiTS@uniPD Facility, Department of Biology, Padua). Alexa Fluor 488/568, Hoechst, and phalloidin images were captured using 8 Z-stack planes at 1-micron step size using 460–490 and 355–385 nm excitation LEDs and emission filters 500-550 and 570-650, respectively.

Automated image analysis was performed with the Harmony® Analysis Software (PerkinElmer); a proprietary supervised machine learning algorithm (PhenoLOGIC™, Harmony® Analysis Software, Perkin Elmer) was used to quantify single-cell mitochondria and melanosome number and intensity, in addition to their mean area (px²), perimeter (px), and length (px). Z-stacks were processed to obtain maximum projections; nuclei segmentation based on Hoechst was followed by identification of the cytoplasm and HMB45/Tom20 positive particles.

Immunofluorescence and imaging
Immunofluorescence was performed as previously described52. Cells were fixed with PBS containing 3.7% formaldehyde (Sigma-Aldrich, Cat. No. 252549) for 15 min, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Cat. No. T8787) for 15 min and blocked with 5% BSA in PBS for 1 h, and then incubated O/N with primary antibodies diluted in PBS with 5% BSA at 4ºC. After three washes with 5% BSA in PBS, cells were incubated with appropriate secondary antibodies prepared in 5% BSA in PBS solution for 1h at RT. Samples were rinsed three times with PBS and incubated with Hoechst 33342 (200 μg/mL for 10 min, diluted in PBS). After three additional washes with PBS, coverslips were briefly rinsed in MilliQ water and mounted onto glass slides using Mowiol. Cells were imaged using either an Axio Imager M2 laser scanning microscopy (LSM) 900 microscope (Carl Zeiss), equipped with an Airyscan module and a Plan-Apochromat 63x/1.40 Oil DIC M27 objective, or for super-resolution imaging, an Elyra 7 SIM2 (Carl Zeiss Microscopy) equipped with a Plan-APOCHROMAT 63x/1.4 Oil DIC M27 objective. Images were acquired depending on the needs with the lasers (405, 488, 561, and 640 nm) and dichroic filters QBS 405, 493,575, 653 and TBS 405, 493, 610 (16 bit) either in confocal or Airyscan mode (these were deconvoluted with standard Airyscan processing). SIM acquisition utilized all available fluorophores with appropriate laser lines and filters (filters: BP 495-550 + LP 655, BP 420-480 +BP 570-640 + LP 740, BP 495-550 + LP 655; camera exposure time of 50 ms; 13 phases, 16-bit). Z-stacks were acquired to ensure full volumetric coverage of the cellular structures, with optimal slice intervals for processing. Post-acquisition, raw SIM datasets were processed using the integrated ZEISS ZEN Black software with the SIM² algorithm to reconstruct super-resolved images (3D, leap). Quantification of MFF puncta and their colocalization with melanosome constriction points was performed on fixed melan-a cells stained for MFF and HMB45. MFF puncta and their overlap with fission zones were manually counted within two to three regions of interest (ROIs) per cell, each measuring 40 µm².

Live imaging and quantification of melanosome fission and fusion
Live imaging was performed on melan-a cells previously transfected with the eGFP-MFF (isoform 1) plasmid and LAMP1-RFP, cultured on an ibidi µ-Slide 8 Well chambers (Cat. No. 80826). For LysoTracker-based imaging, cells were transfected with eGFP-MFF only when required. On the day of imaging, cells were incubated with 100 nM LysoTracker Deep Red (Molecular Probes, Thermo Fisher Scientific, Cat. No. L12492) and with 50 nM of MitoTracker™ Green FM (Molecular Probes, Thermo Fisher Scientific, Cat. No. M7514) in complete medium for 45 minutes. After incubation, the medium was replaced with fresh complete medium for microscopy.

Live-cell super-resolution imaging was performed using a Zeiss Elyra 7 system equipped with Lattice SIM² (Carl Zeiss Microscopy) using a 13-phase acquisition protocol across all channels. Time-lapse videos were acquired over 30 cycles under physiological conditions (37 °C, 5% CO₂). Image reconstruction was carried out in ZEN Black (Carl Zeiss Microscopy) using the SIM² algorithm in two-step burst mode (2D Leap), yielding an approximate lateral (x, y) resolution of ~31 nm after deconvolution. Melanosome fission and fusion events were tracked using the TrackMate plugin in Fiji. Analyses were conducted either on defined regions of interest (ROIs) comparing melan-a and melan-c2 cells, or across full-cell areas following HUANG thresholding, using cells labeled with LysoTracker Deep Red or electroporated with LAMP1-RFP. To minimize artifacts from focal plane shifts, the number of fission (splitting) events was subtracted from the number of fusion (merging) events per spot detection. Spot area and perimeter measurements were extracted from the same plugin, considering only LysoTracker-labeled structures with a detected area exceeding 0.5 µm².

Colocalization analysis
For colocalization analysis of MFF, HMB45, and mito-RFP in melan-a and melan-c2 cells, Z-stacks were acquired with a step size of 0.15 µm. Mito-RFP positive cells were processed using standard Airyscan super-resolution settings, and standard size ROIs (11.5 µm x 11 µm = 126.5 µm2) were drawn in the cell cytosol. At least three per cell were analyzed. Maximum z-projections of all acquired stacks were generated for the three channels in the analyzed ROIs. On these ROIs, the Manders overlap coefficient was quantified using the JACoP plugin in the Fiji program53,54. The same analysis and ROI size were used for colocalization analysis of MFF, HMB45, and mito-RFP in CTSB silencing conditions (Table 3). The same procedure was used for colocalization analysis of HMB45 and CTSB in siRNA control/MFF conditions, but in total cell area ROIs. HMB45 colocalization with melanin was quantified from single-plane confocal images of the respective conditions, considering five cells per condition (two ROIs per cell, 10 µm x 10 µm = 200 µm2). Single plane images were selected for analysis based on the focal plane that allowed the maximum number of melanin BF spots to be identified.

Magic Red® (Cathepsin B assay) and acridine orange (AO)
Melan-a cells were seeded on a 96-well Ultra microplate (PerkinElmer) after electroporation with the appropriate siRNA conditions and incubated in growth media for 66 h. Melanocytes were incubated for 1 h at 37ºC and 10% CO2 with Magic Red reagent (Cathepsin B Assay Kit - Magic Red® Abcam, Cat. No. AB270772) diluted in DMSO according to the manufacturer's instructions. For AO staining (Sigma Aldrich, Cat. No. 158550), cells were incubated at a final concentration of 2.5 μM for 30 min at 37ºC and 10% CO2. Then, live cells were stained with Hoechst 33342 (200 μg/mL for 10 min diluted in RPMI 1640 without phenol red supplemented with 1% FBS). Melanocytes were washed once with RPMI 1640 without phenol red, supplemented with 1% FBS, and imaged in the same medium. Images were acquired using the Operetta CLS™ High‐Content Analysis System (PerkinElmer), with 63× Water/1.15 NA. Magic Red (excitation 530-560 nm; emission 600-640 nm), AO (excitation 450-490 nm; emission 590-650 nm).

Transmission electron microscopy (TEM)
8 x 104 melan-a/c2 cells per well were plated into 24-well plates. After 48h, cells were fixed, and sample preparation was performed as previously described. Samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, O/N at 4°C. The samples were postfixed with 1% osmium tetroxide plus potassium ferrocyanide 1% in 0.1 M sodium cacodylate buffer for 1 h at 4°C. After three water washes, samples were dehydrated in a graded ethanol series and embedded in an epoxy resin (Sigma-Aldrich). Ultrathin sections (60-70 nm) were obtained with Leica Ultracut EM UC7 ultramicrotome, counterstained with uranyl acetate and lead citrate, and viewed with Tecnai G2 (FEI) transmission electron microscope operating at 100 kV. Images were captured with a Veleta (Olympus Soft Imaging System) digital camera. TEM images and experiments were performed at the Electron Microscopy Facility of the University of Padua. The TEM images and experiments were performed by the University of Padua electron microscopy facility. Mitochondria-melanosome distances and perimeters were quantified with the ImageJ (National Institutes of Health). At least four biological replicates were performed per cell line, with a minimum of five cells collected from each sample. The surface of every cell was completely covered by different images. The melanosome-mitochondria contact coefficient (MELAMICC) was used to represent the extent of physical interaction between the melanosome and mitochondria. This index considered the melanosome and mitochondria perimeter and the respective distance between the two organelles. Contacts with distances between organelle surfaces bigger than 120 nm were excluded. The MMERF was pre-embedded in 3% LMP agarose in 0.1 M cacodylate buffer and processed following the standard EM cell protocol.

Statistical Analysis
Unless otherwise stated, data are presented as mean ± SEM. All independent experiments represent biological replicates. Unless otherwise indicated, all experiments were performed at least three times. The statistical tests used are specified in the corresponding figure legends. Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript was designed to investigate the role of mitochondria in melanosome biogenesis. The authors unexpectedly discovered the role of mitochondrial fission factor (MFF) in mediating melanosome fission. Whereas MFF was known to mediate the fission of mitochondria and peroxisomes, the discovery that it mediates fission in melanosomes independent of DRP1 is novel. MFF-deficient cells display enlarged early melanosomes, melanosome hybrids, intracellular accumulation of melanin, and increased catabolism. Results are relevant in advancing our understanding of related human diseases. While the results are interesting, MFF or DRP1 inhibition is based on results in one cell line using one shRNA-mediated knockdown. The knockdown's potential off-target effect is not addressed; as a result, the conclusions are not sold. There are also problems with over- or misinterpretations of the data.\n1. The method to isolate mitochondria-melanosome-ER fractions appears to be based on the method for mitochondria isolation but not particular for the proteins at the contact sites. Proteins from other organelles are identified as a result. Therefore explicitly referring to the results as for the melanosome-mitochondria interface is problematic.—line 133\n2. Line 137- the conclusion that MFF buds from mitochondria to melanosomes is not supported by the data in Fig 2. Whereas mitochondria release of MFF spots is visible, the evidence that melanosomes acquire these spots is lacking. One would need to label MFF on mitochondria, such as photoconversion, and then track their appearance on melanosomes to be sure.\n3. Line 181, the description of Fig. 3D results does not match the figure.\n4. Fig. 3 primarily focuses on early melanosomes. However, MFF is localized to melanosomes of all maturation stages. Is there any enlargement of more mature melanosomes? A related question is: The mutant cells have higher MFF levels and larger melanosomes. Will over-expression of MFF alone cause enlargement of melanosomes?\n5. The role of CTSB is not clearly defined and discussed. Why would CTSB deficiency lead to reduced intracellular melanin levels and enlarged melanosomes?\n6. Line 214, the conclusion that removing unneeded material required MFF activity is not supported. Which unneeded material accumulated as a result of MFF KD? So are similar discussions in lines 250-251.\n7. Conclusions in lines 234-237 are exactly opposite of what the data suggested. CK-666 reversed the MFF KD phenotype, suggesting ARP2/3 is downregulated by MFF to support fission. Again, making a conclusion based on one inhibitor data is problematic. Knocking down or out ARP2/3 can be attempted.\n8. lines 267, it would be better to discuss the genetic causes of the diseases and see if any is linked to fission or MFF deficiency.\n9. Fig 1: What is the difference between cytosolic and lumen TYRP1? Why are both present on the MMERF fraction and both susceptible to PK? It would be helpful to include the sample of whole cells to determine if the two cells have different materials to begin with.\n10. Fig 2: It would be necessary to add the lines to B to show the positions of the line scan. F is hard to see the GFP or magenta signal. The arrows are not helpful.\n11. Fig 3. The image quality of A is poor. At the current magnification, it is hard to appreciate if the arrows indeed point to melanosomes with different morphology in D. It is hard to appreciate the lumen accumulation of cargo in G. It is not clear what the difference is between the two BF panels in H.\n12. Scales in Fig S4 are missing or too small to see.\n13. Typos in lines 79, 215, 293, and figure 1 legend.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This study describes a novel role for MFF, or mitochondrial fission factor, in the regulation of melanosome fisson and maturation. The authors begin with a discovery-based platform involving transcriptomics and proteomics of two cell lines – melan-a cells, which represent a wild-type melanocyte maturation model, and melan-c2 cells, which harbor a loss-of-function mutation in a key protein involved in melanogenesis and thus are disrupted in their maturation. The authors preliminarily find that MFF is enriched in melan-c2 cells, and propose that it localizes to endogenous melanocyte membranes using immunogold-labeling coupled with EM, as well as fluorescence microscopy. The authors then find that genetic ablation of MFF, but not the mitochondrial fission factor DRP1, causes increased early melanosome size and increased intracellular melanin level, which is proposed to reflect impaired melanocyte maturation. The authors perform RNA-seq on melanocytes harboring MFF knockdown and find differential expression of ‘catabolism’ proteins, leading the authors to suggest that MFF activity is required for the removal of unwanted material during maturation. Finally, the authors perform IP-MS to identify interacting proteins with MFF and identify the ARP2/3 complex, which they propose is required for MFF-dependent melanosome fission.\nWhile the study has interesting observations, I found many of the conclusions to be overstated and further find the model for MFF-mediated function at melanosomes unclear. I elaborate on these points further below.\nMajor points:\n1. The authors begin their study by “devis[ing] a proteomic pipeline aimed at identifying the proteins enriched on the surface of the co-sedimenting organelles” – meaning the mitochondria-melanosome interface. This is a very exciting and interesting proposal, and I was eager to read more about the technical details about how the authors were able to enrich for mitochondrial-melanosome interfaces. However, I found that the methods of the “MMERF” isolation as underwhelming, as the method essentially describes a crude mitochondrial prep. While such a prep would likely contain “mitochondria-melanosome-ER fractions” or MMERF, as the authors state, it does not provide a method to “provide a compendium of the proteome of the melanosome-mitochondria interface” as claimed in line 133. This is simply a heavy membrane fraction in cells in which melanosome maturation is enabled or impaired. This is not to say that the dataset generated by the authors is not of value; it is indeed an appropriate experimental approach to identify how mitochondria and mitochondria-associated proteins differentially accumulate in a model of disrupted melanocyte maturation. The authors should amend their claim on line 133 mentioned above and render it more reflective of the dataset collected.\n2. The authors choose to focus on MFF, which is shown to be enriched in melan-c2 ‘MMERF’ fractions and is digestable by proteinase K, suggesting it is membrane localized and could be present at an intraorganellar contact. However, the authors do not provide sufficient rationale for studying MFF versus other targets. While the claim that MFF is increase in melan-c2 cells is true, Figures 1A and 1B suggest that most mitochondrial genes are increased in melan-c2 cells. Figure 1D suggests that mitochondria are larger in melan-c2 cells than melan-a cells, and all mitochondrial targets examined in Figure 1E are elevated. Why do the authors choose to study MFF in the context of such large organellar changes across mitochondria?\n3. In Figure 2, the authors use immunogold staining and fluorescence microscopy to show MFF proximal to the melanosome membrane, and suggest that MFF co-localizes to sites of melanosome fission, leading to the claim that “MFF is found on the surface of melanosomes and is present at melanosome constriction and fission events.” I have questions regarding this claim.\na. The data are not quantified, making them difficult to evaluate. How frequent is MFF presence on the melanosome membrane? Does this change during differentiation states? How often is MFF found at melanosome constriction sites? Are there constriction sites that occur that are absent for MFF? These are important details to understand the necessity of MFF for membrane constriction in this model system.\nb. The authors make a statement in their abstract that their data “unveil an extramitochondrial role for MFF in the regulation of melanosome morphology and maturation.” However, the authors have not demonstrated that MFF is exclusively at the melanosome membrane, rather than a model in which MFF resides on the mitochondrial membrane but enriches at a melanosome contact site. If the authors truly believe that MFF is acting in a melanosome-autonomous manner (that is, completely independent of mitochondrial targeting), more data is required to substantiate this claim.\nc. To follow up on point 2b, it is unclear what the authors mean by an “extramitochondrial role” for MFF. Do the authors propose that MFF independently targets to melanosomes, or that it shuttles there by way of mitochondria? Or is actually localized on mitochondria but at melanosome contact sites? The authors should be more clear on their model.\nd. The authors repeatedly make claims about the role of MFF in membrane fission, but this is never shown. Rather, it is inferred that without MFF, melanosomes become enlarged and this could be due to inefficient organellar fission. The authors should show that MFF directly enables membrane fusion or soften their language to suggest that this model is consistent with membrane fission, although the mechanisms by which this occur are unclear.\n4. In Figure 3, the authors make the claim that MFF, but not DRP1, is required for melanosome maturation and function. This is surprising, as the current literature postulates that MFF is solely an adaptor for DRP1 to mediate mitochondrial membrane fission, but does not have intrinsic membrane fission capabilities itself. The authors only assay DRP1 knockdown in HMB45 spot perimeter and number of HMB45 spots per cells in this figure. Is it possible that DRP1 is not sufficiently knocked down and MFF could be utilizing small amounts of DRP1 to enable differentiation? Does DRP1 ever colocalize with HMB45 during melanocyte maturation, similar to MFF in Figure 2? Does it ever occur proximal to HMB45 positive structures via EM, similar to MFF in Figure 2? Further studies should be carried out with DRP1, as negative data is meaningful in support of the authors’ claims. These experiments also highlight the importance of quantifying these events for both MFF and DRP1.\n5. The authors end the paper by performing IP-MS to identify interaction partners with MFF. However, the authors do not comment on whether this pulled down MFF associated with melanosomes or mitochondria. It is likely that the bulk of MFF associates with mitochondria rather than melanosomes, rendering the stoichiometry and meaning of global MFF interactions presented in this figure unclear.\na. To follow up on point 4 – the authors suggest that “MFF-dependent melanosome fission relies on ARP2/3 mediated actin assembly”. The authors somewhat suggest that this may be due to different subunits of the WASH complex, but suggest that phenotypes were not seen due to potential redundancies and compensation. The authors should use combinatorial knockdowns to explore this further, as the proposal that DRP1 is not required for membrane fission begs the question of which molecular components are involved in membrane fission.\nb. Is it possible to further enrich melanosomes specifically and perform the MFF pulldown with this specific fraction? If many of these hits persist in this experiment, it would suggest that the MFF-ARP2/3 interaction is important specifically at melanosomes, instead of at mitochondria which could indirectly affect melanosome function.\n6. MFF runs as a doublet throughout the western blotting in the manuscript. Is it possible that these bands represent different MFF isoforms that localize to different organelles? Does this doublet change in abundance throughout melanocyte maturation? Could the authors purify melanosomes away from mitochondria and see one versus another isoform of MFF on melanosomes? This could be a very compelling piece of data that would also address some of the comments raised in point 2 above.", "reviewer_4": "The manuscript by Rebelo et al. describes a new role for MFF, a mitochondrial fission factor, in melanosome size and maturation. This is an interesting finding that contributes to the growing body of evidence on the importance of organelle contacts for melanosome biogenesis, maturation and function. However, this finding is not entirely novel as mitochondrial-melanosome contacts were reported 10 years ago by the group of Maria Vittoria Schiaffino. Moreover, this group found that the mitochondrial protein MFN2 regulates melanosome biogenesis. In terms of mechanism, it is suggested that MFF-dependent melanosome fission depends on Arp2/3-mediated actin assembly. While this is interesting, it does not provide enough mechanistic detail for the journal's standard (see point 2).\n1 - One major issue is the lack of quantification of melanosome maturation in a manuscript on this subject. Indeed, authors did not quantify melanosome stages by EM, which is essential to support the claims made. This applies to several figures in the manuscript, including Fig. S1A/B, 2A - to know if MFF is present in all melanosome stages in the same proportion - and 3G.\n2- Another major issue is the use of an inhibitor only to interfere with Arp2/3 and draw conclusions about the involvement of the complex and actin nucleation. Indeed, pharmacological approaches, as any other, have pitfalls. Therefore, the findings should be validated with a genetic approach, by depleting Arp2/3.\n3 - The evidence that MFF also localizes to stage III and IV melanosomes seems to be largely ignored by the authors and not considered in the interpretation of the findings.\n4 - Regarding the explanation why Mff-KD cells are more pigmented, it is not likely that immature melanosomes accumulate melanin (stage II melanosomes should have little pigment). Moreover, a negative feedback loop is expected to occur, downregulating melanogenesis, and this is likely why the authors detected lower levels of melanogenic markers. However, this should not lead to increased pigmentation. Therefore, other mechanisms should be considered.\nMinor issues:\n- Fig. 2G, H does not exist.\n- Reference 8 is old and there are several more recent ones."}, "author_response": "We would like to thank the reviewers for evaluating our work and for their constructive suggestions.\nOverall, the reviewers recognised the novelty of our findings, particularly with regard to the role of\nMFF in melanosome fission and function, independently of DRP1. By addressing their comments, we\nhave strengthened the manuscript's conclusions and main message. The new data presented provide\nrobust evidence that MFF plays a role in melanosome fission that is independent of DRP1. This is\nsupported by a new dataset highlighting the association of MFF with the ARP2/3 complex and F-actin\nnucleation. Below, we provide a point-by-point response to each comment from the reviewers. Their\ncomments are in italic black and our responses are in red.\nReviewer #1\nThe manuscript was designed to investigate the role of mitochondria in melanosome biogenesis. The\nauthors unexpectedly discovered the role of mitochondrial fission factor (MFF) in mediating\nmelanosome fission. Whereas MFF was known to mediate the fission of mitochondria and\nperoxisomes, the discovery that it mediates fission in melanosomes independent of DRP1 is novel. MFF-\ndeficient cells display enlarged early melanosomes, melanosome hybrids, intracellular accumulation\nof melanin, and increased catabolism. Results are relevant in advancing our understanding of related\nhuman diseases. While the results are interesting, MFF or DRP1 inhibition is based on results in one\ncell line using one shRNA-mediated knockdown. The knockdown's potential off-target effect is not\naddressed; as a result, the conclusions are not sold. There are also problems with over- or\nmisinterpretations of the data.\nResponse: Thank you for your valuable feedback on our manuscript. For clarification, we employed a\npool of four distinct siRNAs (siGENOME SMARTpool, Horizon Discovery) for all the knockdowns\ndescribed in the manuscript. This approach was chosen to minimize potential off-target effects in each\ntransfection. We have specified these details in the Methods section of the manuscript and Table 3.\nComment 1: The method to isolate mitochondria-melanosome-ER fractions appears to be based on\nthe method for mitochondria isolation but not particular for the proteins at the contact sites. Proteins\nfrom other organelles are identified as a result. Therefore explicitly referring to the results as for the\nmelanosome-mitochondria interface is problematic.—line 133\nResponse: We agree with this observation and have accordingly revised the method description in\nthe manuscript (line 117-123 and 394-409). However, we would like to emphasize that the novelty of\nour approach lies not in the isolation method itself, but in the subsequent digestion step we\nintroduced. By applying proteinase K digestion to the isolated fractions, we effectively removed\nlumenal organellar proteins from consideration, thereby significantly narrowing the pool of potential\ncandidates involved in mitochondria communication with other organelles in the two models of\nmelanocytes, focusing only on the proteins that are exposed to the cytosol. We also explored\nalternative approaches to isolate mitochondria–melanosome contact sites directly. However, these\nattempts were consistently limited by technical challenges, including high levels of contamination with\nmitochondria-ER contact sites. To further support our findings, we have now included an additional\ndataset based on melanosome immunoprecipitation, confirming the presence of MFF at\nmelanosomes regardless of mitochondrial association (Fig. S2D).\nComment 2: Line 137- the conclusion that MFF buds from mitochondria to melanosomes is not supported by the data in Fig 2. Whereas mitochondria release of MFF spots is visible, the evidence that\nmelanosomes acquire these spots is lacking. One would need to label MFF on mitochondria, such as\nphotoconversion, and then track their appearance on melanosomes to be sure.\nResponse: Thank you for your thoughtful suggestion. While exploring this aspect would indeed be\ninteresting, we would like to clarify that we do not propose that MFF is internalized into melanosomes\nor localized within the melanosomal lumen. Our data do not show complete colocalization between\nMFF and HMB45, which is a well-established lumenal melanosome marker. Instead, we observe that\nMFF is consistently positioned adjacent to HMB45 signals, suggesting its localization at the\nmelanosome membrane, specifically on the cytosolic face. This possibility is further supported by our\nimmunogold labeling experiments, which show that MFF localizes to the melanosome surface. To\nfurther clarify this, we have revised Fig. 2 and Fig. S2 by adding other images, obtained by resolution\nimaging approaches. We believe the improved figure quality now more clearly demonstrates the\nmembrane localization of MFF, which may not have been as evident in the initial version of the\nmanuscript. Additionally, we have included a new dataset employing MELANO-IP, a system that\nenables the isolation of pure melanosomes, free from mitochondrial contamination. Using this\napproach, we were able to pull down MFF from purified melanosome fractions, providing\ncomplementary biochemical evidence that MFF is associated with melanosomes independently of\nmitochondria.\n3. Line 181, the description of Fig. 3D results does not match the figure.\nResponse: We apologise for this oversight. We have thoroughly reviewed our image dataset and\nreplaced the previous panels with more representative examples. Additionally, we have revised the\ncorresponding text in the manuscript to better reflect these changes.\n4. Fig. 3 primarily focuses on early melanosomes. However, MFF is localized to melanosomes of all\nmaturation stages. Is there any enlargement of more mature melanosomes? A related question is:\nThe mutant cells have higher MFF levels and larger melanosomes. Will over-expression of MFF alone\ncause enlargement of melanosomes?\nResponse: We appreciate the reviewer’s observation and agree that the presence of MFF at later-\nstage melanosomes (stages III and IV) deserves further discussion. Although our super-resolution\nmicroscopy occasionally detected MFF signal at these stages (Fig. 2 and Fig. S2), its localization at\nstages III and IV was significantly reduced compared to early-stage melanosomes. Importantly, we did\nnot observe MFF enrichment at constriction or fission sites in TYRP1-positive melanosomes,\nsuggesting that MFF is unlikely to play an active role in fission events at these later stages of\nmaturation. One possible explanation for the partial colocalization observed could be that MFF\nlocalization to Stage III/IV melanosomes could reflect hybrid or transitional stages that cannot be\ndistinguished with the currently available markers. This may account for the partial colocalization\nobserved. Alternatively, MFF may have a distinct, yet unexplored, function in the later stages of\nmelanosome maturation.\nRegarding the second point, we have now included the quantification of the melanosome perimeter\nin the TEM dataset shown in Fig. 3G (now Fig. 3F), with the corresponding graph provided in Fig. S3M.\nThese data indicate a tendency towards increased perimeter also in the case of late-stage\nmelanosomes.\nFurthermore, our investigation was initiated using an albino melanocyte model, which predominantly\ncontains early-stage melanosomes. In this context, we observed the specific enrichment and altered\nexpression of MFF. To further support our focus on early-stage melanosomes and the functional\ninvolvement of MFF, we have included new data that quantify melanosome dynamics in both melan-\na and melan-c2 cells (Fig. 1I–K). These analyses revealed an increased number of fission events in\nearly-stage albino melanosomes, which further demonstrates the occurrence of altered melanosome\ndynamics and the eventual functional role for MFF in this process. Of note, melanosome fission events\nindependent of MFF also exist, as our quantification showed that MFF localizes to ~20% of constriction\nsites (Fig. 2I).\nFinally, as requested by the reviewer, we assessed the effect of MFF overexpression by expressing all\nfour isoforms and measuring the size of early melanosomes via HMB45 staining (Fig. S3O). We did not\nobserve significant changes, likely due to the fact that MFF overexpression alone may be insufficient\nto drive a phenotype.\n5. The role of CTSB is not clearly defined and discussed. Why would CTSB deficiency lead to reduced\nintracellular melanin levels and enlarged melanosomes?\nResponse: We thank the reviewer for raising this important point. Previous studies have shown that\nCTSB is present in melanosomes and contributes to melanosome maturation and degradation.\nAdditional data, summarised in lines 240–225 and discussed in lines 333–341, supports and expands\nupon this knowledge. In particular, we found significant differences in the transcriptional response to\nMff and Drp1 silencing, particularly regarding the regulation of lysosomal and catabolic gene\nexpression (Fig. 4A). Specifically, Mff silencing leads to a marked upregulation of lysosomal enzymes—\nincluding Ctsb, Ctsd, and Lipa—while Drp1 silencing does not induce this response and instead results\nin the downregulation of genes involved in melanosome biogenesis, such as the melanogenesis master\nregulator Mitf (as shown in our RNA-seq data). These differences suggest that MFF and DRP1 regulate\nmelanosome homeostasis via distinct mechanisms. We hypothesize that the upregulation of Ctsb in\nMff-KD cells is a compensatory response to defective melanosome fission, promoting intraluminal\ndegradation to prevent excessive melanosome buildup. Indeed, we show that co-silencing Ctsb and\nMff results in a significant exacerbation of melanosome enlargement compared to Mff silencing alone.\nAdditionally, this condition restores the levels of melanogenic markers such as TYR and TRP1, which\nare otherwise reduced in Mff-KD cells, and rescues the increased acidification observed in\nmelanosomes upon Mff silencing (Fig. 4H–K, S4C–I). These findings suggest that Ctsb upregulation in\nMff-KD cells facilitates a degradation pathway aimed at reducing the accumulation of melanogenic\nproteins and dysfunctional organelles caused by impaired fission. This idea is further supported by the\nincreased colocalization of CTSB with HMB45-positive melanosomes in MFF-KD cells (Fig. 4B–E).\nFinally, TEM images from Ctsb-KD melan-a revealed the presence of melanosomes with altered\nmorphology (Fig. S4). Together, these results suggest that Ctsb upregulation mitigates the\naccumulation of immature melanosomes via a lysosomal/autophagic degradation pathway that is\nactivated in response to the disruption of melanosomal dynamics by Mff silencing.\n6. Line 214, the conclusion that removing unneeded material required MFF activity is not supported.\nWhich unneeded material accumulated as a result of MFF KD? So are similar discussions in lines 250-\n251.\nResponse: Thanks for this comment: we have revised the conclusions. We do agree that we cannot\nassume which material is accumulated, however our additional data on Mff-KD, Ctsb-KD, and\nMff/Ctsb-KD cells confirm the increased degradative activity of early melanosomes in Mff-KD cells\n(Fig.4) Indeed, Mff silencing leads to increased acidification of the melanosome lumen and increased\nlevels and activity of cathepsin. Furthermore, as described before, co-silencing of Mff and Ctsb could\nrestore the expression levels of melanogenesis proteins, which are likely to be one example of the\ntype of material degraded.\n7. Conclusions in lines 234-237 are exactly opposite of what the data suggested. CK-666 reversed the\nMFF KD phenotype, suggesting ARP2/3 is downregulated by MFF to support fission. Again, making a\nconclusion based on one inhibitor data is problematic. Knocking down or out ARP2/3 can be attempted.\nResponse: To further clarify the mechanism by which MFF may promote early melanosome fission,\nwe followed the reviewer’s suggestion and investigated the role of the ARP2/3 complex in\nmelanosome morphology by siRNA. We performed knockdown experiments targeting two ARP2/3\nsubunits, ARPC1A and ARPC3, and observed an increase in melanosome size in both cases,\nphenocopying the effects of Mff knockdown (Fig. 5G). Importantly, MFF puncta remained detectable\nat early-stage melanosomes under these conditions, indicating that the recruitment of MFF is\nindependent of the ARP2/3 complex. This supports our model in which MFF may interact with ARP2/3\nregulators to specifically drive fission (Fig. 5H). To further support the idea that MFF and actin could\ncooperate to control melanosome membrane dynamics, we performed additional microscopy\nexperiments. We found that F-actin is enriched at melanosome fission sites marked by MFF (Fig. 5C,\nD), strengthening our hypothesis that MFF-dependent melanosome fission is mediated by actin\nnucleation factors.\n8. lines 267, it would be better to discuss the genetic causes of the diseases and see if any is linked to\nfission or MFF deficiency.\nResponse: We appreciate this suggestion, however, the involvement of MFF in skin physiology has\nnever been explored before, and we could not find any relevant data or literature. Moreover, the MFF\nKO mouse is not viable. We believe that due to the impact of MFF deletion in other tissues, the effects\nin skin may have been overlooked.\n9. Fig 1: What is the difference between cytosolic and lumen TYRP1? Why are both present on the\nMMERF fraction and both susceptible to PK? It would be helpful to include the sample of whole cells\nto determine if the two cells have different materials to begin with.\nResponse: There are no cytosolic or lumenal forms of the protein TYRP1. TYRP1 is a melanosome\nmembrane protein with domains exposed both to the cytosol and lumen. We took advantage of this\ntopology and used two different antibodies, each of which could target one of the two differently\nexposed regions. This served as a control to ensure that Proteinase K degraded only the cytosolic,\nexposed parts of proteins. We have added this explanation to the result section, lines 121-124.\n10. Fig 2: It would be necessary to add the lines to B to show the positions of the line scan. F is hard\nto see the GFP or magenta signal. The arrows are not helpful.\nResponse: We revised all the microscopy figures to improve their resolution and visualization.\n11. Fig 3. The image quality of A is poor. At the current magnification, it is hard to appreciate if the\narrows indeed point to melanosomes with different morphology in D. It is hard to appreciate the\nlumen accumulation of cargo in G. It is not clear what the difference is between the two BF panels in\nH.\nResponse: We have changed all the figures with better resolution and image quality.\n12. Scales in Fig S4 are missing or too small to see.\nResponse: The scale was increased in size; some of these panels are now on Fig.S3 due to changes in\nthe order, or addition of new panels, in the figures of the manuscript.\n13. Typos in lines 79, 215, 293, and figure 1 legend.\nResponse: These typos were corrected.\nReviewer #2\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nReviewer #3\nThis study describes a novel role for MFF, or mitochondrial fission factor, in the regulation of\nmelanosome fission and maturation. The authors begin with a discovery-based platform involving\ntranscriptomics and proteomics of two cell lines – melan-a cells, which represent a wild-type\nmelanocyte maturation model, and melan-c2 cells, which harbor a loss-of-function mutation in a key\nprotein involved in melanogenesis and thus are disrupted in their maturation. The authors preliminarily\nfind that MFF is enriched in melan-c2 cells, and propose that it localizes to endogenous melanocyte\nmembranes using immunogold-labeling coupled with EM, as well as fluorescence microscopy. The\nauthors then find that genetic ablation of MFF, but not the mitochondrial fission factor DRP1, causes\nincreased early melanosome size and increased intracellular melanin level, which is proposed to reflect\nimpaired melanocyte maturation. The authors perform RNA-seq on melanocytes harboring MFF\nknockdown and find differential expression of ‘catabolism’ proteins, leading the authors to suggest\nthat MFF activity is required for the removal of unwanted material during maturation. Finally, the\nauthors perform IP-MS to identify interacting proteins with MFF and identify the ARP2/3 complex,\nwhich they propose is required for MFF-dependent melanosome fission.\nWhile the study has interesting observations, I found many of the conclusions to be overstated and\nfurther find the model for MFF-mediated function at melanosomes unclear. I elaborate on these points\nfurther below.\nMajor points:\nComment 1: The authors begin their study by “devis[ing] a proteomic pipeline aimed at identifying the\nproteins enriched on the surface of the co-sedimenting organelles” – meaning the mitochondria-\nmelanosome interface. This is a very exciting and interesting proposal, and I was eager to read more\nabout the technical details about how the authors were able to enrich for mitochondrial-melanosome\ninterfaces. However, I found that the methods of the “MMERF” isolation as underwhelming, as the\nmethod essentially describes a crude mitochondrial prep. While such a prep would likely contain\n“mitochondria-melanosome-ER fractions” or MMERF, as the authors state, it does not provide a\nmethod to “provide a compendium of the proteome of the melanosome-mitochondria interface” as\nclaimed in line 133. This is simply a heavy membrane fraction in cells in which melanosome maturation\nis enabled or impaired. This is not to say that the dataset generated by the authors is not of value; it\nis indeed an appropriate experimental approach to identify how mitochondria and mitochondria-\nassociated proteins differentially accumulate in a model of disrupted melanocyte maturation. The\nauthors should amend their claim on line 133 mentioned above and render it more reflective of the\ndataset collected.\nResponse: We agree with your observation and have accordingly revised the method description in\nthe manuscript (line 117-123 and 394-409). However, we would like to emphasize that the novelty of\nour approach lies not in the isolation method itself, but in the subsequent digestion step we\nintroduced. By applying proteinase K digestion to the isolated fractions, we effectively removed\nlumenal organellar proteins from consideration, thereby significantly narrowing the pool of potential\ncandidates involved in mitochondria communication with other organelles in the two models of\nmelanocytes, focusing only on the proteins that are exposed to the cytosol. We also explored\nalternative approaches to isolate mitochondria–melanosome contact sites directly. However, these\nattempts were consistently limited by technical challenges, including high levels of contamination with\nmitochondria-ER contact sites. To further support our findings, we have now included an additional\ndataset based on melanosome immunoprecipitation, which confirms the presence of MFF at\nmelanosomes regardless of mitochondrial association (Fig. S2D).\n2. The authors choose to focus on MFF, which is shown to be enriched in melan-c2 ‘MMERF’ fractions\nand is digestable by proteinase K, suggesting it is membrane localized and could be present at an\nintraorganellar contact. However, the authors do not provide sufficient rationale for studying MFF\nversus other targets. While the claim that MFF is increase in melan-c2 cells is true, Figures 1A and 1B\nsuggest that most mitochondrial genes are increased in melan-c2 cells. Figure 1D suggests that\nmitochondria are larger in melan-c2 cells than melan-a cells, and all mitochondrial targets examined\nin Figure 1E are elevated. Why do the authors choose to study MFF in the context of such large\norganellar changes across mitochondria?\nResponse: We are grateful for this feedback, and we acknowledge the need to better support the\nrationale. To this aim, we have provided additional quantitative analyses and datasets. First, we\nexpanded the data originally presented in Fig. S1A/B (now Fig. 1A) and provided new results in Fig. 1B,\n1C, and Fig. S1C–E to better highlight the differences in melan-a and melan-c2 related to melanosomes\nand mitochondria dynamics. Specifically, we quantified the percentage of melanosomes at various\nmaturation stages that are in contact with mitochondria in both melan-a and melan-c2 cell lines.\nAdditionally, we measured the distances between mitochondria and melanosomes at all stages of\nmaturation, along with the perimeters of each organelle when in contact. To provide a thorough view\nof these interactions, we developed a new index—the\nMelanosome-Mitochondria Contact Coefficient (MELAMICC)—calculated as follows: MELAMICC = melanosome perimeter / (mitochondria perimeter\n× mitochondria–melanosome distance), similar to the ERMICC index, which was previously suggested\nfor mitochondria-ER contact sites (Naon et al, PNAS 2016). This coefficient provides a normalized\nmeasure of contact, allowing comparison of contact sites across different melanosome stages. Our\nfindings revealed a significant increase in mitochondria–melanosome contacts in albino melanocytes,\nwhich are also characterized by an accumulation of early-stage melanosomes (Stages I and II). These\nobservations led us to hypothesize that mitochondria-associated proteins, particularly those exposed\nto the cytosol, could play a role in hetero-organelle communication. From our analysis of outer\nmitochondrial membrane (OMM) proteins (listed in Fig. 1H), fewer than ten proteins met these\ncriteria. Among this subset, only three were transcriptionally upregulated in albino melanocytes.\nNotably, MFF (Mitochondrial Fission Factor) stood out as particularly intriguing. We further observed\naltered expression and specific enrichment of MFF in albino melanocytes, along with increased\nmelanosome size, suggesting a potential dysfunction in the regulation of their morphology in albino\ncells. To investigate this further, we performed additional analyses of melanosome dynamics in melan-\na and melan-c2 (Fig. 1I–K). These data showed a higher number of fission events in early-stage\nmelanosomes in albino cells than in pigmented controls. We believe this is due to blocked\nmelanosome maturation and subsequent outgrowth of melanosomes in albino cells, as a\ncompensatory response to the lack of TYR activity. Given that MFF is a regulator of mitochondria and\nperoxisome dynamics, and being the only organelle dynamic protein highlighted by our analyses, we\nhypothesized that it could play a role in melanosomal membrane dynamics.\n3. In Figure 2, the authors use immunogold staining and fluorescence microscopy to show MFF\nproximal to the melanosome membrane, and suggest that MFF co-localizes to sites of melanosome\nfission, leading to the claim that “MFF is found on the surface of melanosomes and is present at\nmelanosome constriction and fission events.” I have questions regarding this claim.\na. The data are not quantified, making them difficult to evaluate. How frequent is MFF presence on\nthe melanosome membrane? Does this change during differentiation states? How often is MFF found\nat melanosome constriction sites? Are there constriction sites that occur that are absent for MFF?\nThese are important details to understand the necessity of MFF for membrane constriction in this\nmodel system.\nResponse: To address this relevant point, besides adding the requested quantifications, we have\nperformed other experiments, now included in Fig. 2 and Fig. S2. Specifically, we quantified how\nfrequently MFF is present in melanosomes; we found MFF at approximately 20% of HMB45-positive\nmelanosomes (Fig. 2F). In contrast, the presence of MFF on TYRP1-positive melanosomes (stage III/IV)\nwas barely detectable (Fig.S2A). Importantly, we did not observe MFF localization at membrane\nconstriction sites in TYRP1-positive melanosomes, unlike what was observed in those marked by\nHMB45. Therefore, we restricted our quantification of MFF at melanosomal constriction sites to the\nHMB45-positive population. From this analysis, we concluded that MFF is not universally present at\nall melanosome membrane fission sites and is enriched in 22% of observed melanosome membrane\nconstrictions (Fig. 2I).\nb. The authors make a statement in their abstract that their data “unveil an extramitochondrial role\n)for MFF in the regulation of melanosome morphology and maturation.” However, the authors have\nnot demonstrated that MFF is exclusively at the melanosome membrane, rather than a model in which\nMFF resides on the mitochondrial membrane but enriches at a melanosome contact site. If the authors\ntruly believe that MFF is acting in a melanosome-autonomous manner (that is, completely\nindependent of mitochondrial targeting), more data is required to substantiate this claim.\nResponse: We understand the concerns raised by the reviewers, and as previously mentioned, we\nhave included several additional datasets to show that MFF localises to melanosome membranes\nregardless of the presence of mitochondria. We have revised Fig. 2 and Fig. S2 in the updated\nmanuscript. In the newly presented data, we did not observe colocalization of MFF puncta at\nmelanosomes with either PDH or TOMM20—two well-established mitochondrial markers—thereby\nexcluding the possibility that these structures are mitochondria or mitochondrial-derived vesicles\n(MDVs) (Fig. S2B). Additionally, we generated MELANO-IP melan-a cells to isolate highly purified\nmelanosome fractions, which were confirmed to be free of mitochondrial contamination (Fig. S2D).\nWe detected MFF in these purified fractions, while mitochondrial markers were absent, further\nsupporting that MFF is associated with melanosomes independently of mitochondrial contact.\nc. To follow up on point 2b, it is unclear what the authors mean by an “extramitochondrial role” for\nMFF. Do the authors propose that MFF independently targets to melanosomes, or that it shuttles\nthere by way of mitochondria? Or is actually localized on mitochondria but at melanosome contact\nsites? The authors should be clearer on their model.\nResponse: We believe that both scenarios are possible. We observed instances where MFF appears\nto bud from mitochondria toward melanosomes (Fig. 2J and Fig. S2G), suggesting a potential direct\ntransfer. However, we cannot exclude the possibility that cytosolic pools of MFF exist and may also be\ntrafficked independently to melanosomes. That said, considering that MFF was identified in our\nenriched fractions from albino cells, which exhibit increased contact between mitochondria and early-\nstage melanosomes, we propose that this organelle proximity may facilitate enhanced MFF trafficking\nfrom mitochondria to melanosomes. To reflect this, we have revised the manuscript text to\nacknowledge that both mechanisms should be considered as potential contributors to MFF\nlocalization at melanosomes (line 323-326).\nd. The authors repeatedly make claims about the role of MFF in membrane fission, but this is never\nshown. Rather, it is inferred that without MFF, melanosomes become enlarged and this could be due\nto inefficient organellar fission. The authors should show that MFF directly enables membrane fusion\nor soften their language to suggest that this model is consistent with membrane fission, although the\nmechanisms by which this occurs are unclear.\nResponse: We appreciate the reviewers' concerns and have added several new datasets to\ndemonstrate more clearly the extent of MFF involvement in early melanosome fission. First, using\nsuper-resolution, we observed MFF puncta localizing at the necks of melanosomal tubules, where\nmembrane constriction and subsequent scission usually occur (as reported for example by Ripoll et al,\n2014, Fig. 2H). Additionally, live-cell imaging (Fig. 2K, left panels) shows a single MFF punctum\nmediating membrane splitting of a melanosome, with one of the resulting vesicular structures\nremaining associated with MFF. A second example (Fig. 2K, right panels) further illustrates the division\nof a melanosomal structure into two distinct compartments beneath an MFF punctum. Besides, MFF-\nsilenced melan-a cells showed a significant reduction in the frequency of melanosome fission events\n(Fig. 3H). Considering that MFF is enriched at only ~22% of melanosome membrane constriction sites\n(Fig. 2I), these findings suggest that while MFF is not required for all melanosome fission events,\nit plays a significant role in facilitating a subset of them. In MFF-depleted cells, fission can still occur,\nalthough at reduced levels. To further clarify the mechanism by which MFF may promote early\nmelanosome fission, we followed the reviewer’s suggestion and investigated the role of the ARP2/3\ncomplex in melanosome morphology. We performed knockdown experiments targeting two ARP2/3\nsubunits, ARPC1A and ARPC3, and observed an increase in melanosome size in both cases,\nphenocopying the effects of MFF knockdown (Fig. 5G). Importantly, MFF puncta remained detectable\nat early-stage melanosomes under these conditions, indicating that MFF recruitment is independent\nof the ARP2/3 complex. This supports our model in which MFF may interact with ARP2/3 regulators\nto drive fission (Fig. 5H). Finally, to further support the connection between MFF and actin in membrane\ndynamics, we show that F-actin is enriched at melanosome fission sites marked by MFF (Fig. 5C,\nD), strengthening our hypothesis that MFF-dependent melanosome fission is mediated through localized\nactin nucleation.\n4. In Figure 3, the authors make the claim that MFF, but not DRP1, is required for melanosome\nmaturation and function. This is surprising, as the current literature postulates that MFF is solely an\nadaptor for DRP1 to mediate mitochondrial membrane fission, but does not have intrinsic membrane\nfission capabilities itself. The authors only assay DRP1 knockdown in HMB45 spot perimeter and\nnumber of HMB45 spots per cells in this figure. Is it possible that DRP1 is not sufficiently knocked down\nand MFF could be utilizing small amounts of DRP1 to enable differentiation? Does DRP1 ever colocalize\nwith HMB45 during melanocyte maturation, similar to MFF in Figure 2? Does it ever occur proximal to\nHMB45 positive structures via EM, similar to MFF in Figure 2? Further studies should be carried out\nwith DRP1, as negative data is meaningful in support of the authors’ claims. These experiments also\nhighlight the importance of quantifying these events for both MFF and DRP1.\nResponse: Thank you for your thoughtful comments. Regarding the efficiency of DRP1 silencing, we\nconfirm that knockdown was highly effective, with more than 98% reduction observed consistently\nacross all replicates. This is clearly demonstrated in Fig. 3B and the source data of this figure. Given\nthis level of knockdown, we believe the residual DRP1 is unlikely to be sufficient to maintain MFF-\ndependent melanosome function. As requested, we examined the subcellular localization of both\nDRP1 and MFF at melanosomes (Fig. 3A). While we do observe DRP1 on melanosome membranes, we\ndid not detect any clear colocalization with MFF at these sites, unlike in the case of mitochondria,\nwhere DRP1 and MFF colocalize, as indicated by the orange arrows in Fig. 3A. In contrast, MFF and\nDRP1 were detected at distinct regions of the melanosome membrane, as shown by the white and\nyellow arrows in Fig. 3A.\nTo further explore the functional differences between DRP1 and MFF in melanosome biology, we also\nanalyzed transcriptomic changes following their individual knockdowns. We have now added the RNA-\nseq data for DRP1-silenced cells (Fig. 4A). Interestingly, the transcriptional profiles resulting from MFF\nand DRP1 knockdowns were markedly distinct. MFF silencing led to the upregulation of pathways\nassociated with melanosome and lysosome catabolism, consistent with a compensatory response to\ndisrupted early-stage melanosome dynamics. In contrast, DRP1 silencing resulted in the\ndownregulation of several key melanosome-specific genes, including MITF, a master regulator of\nmelanocyte function and melanosome biogenesis. These findings suggest that while both DRP1 and\nMFF can localize to melanosomes, their functional roles in this context differ substantially. DRP1 may\ninfluence melanosome gene regulation more broadly (which was previously demonstrated), whereas\nMFF appears to play a more specific role in melanosome maturation/ segregation.\n5. The authors end the paper by performing IP-MS to identify interaction partners with MFF. However,\nthe authors do not comment on whether this pulled down MFF associated with melanosomes or\nmitochondria. It is likely that the bulk of MFF associates with mitochondria rather than melanosomes,\nrendering the stoichiometry and meaning of global MFF interactions presented in this figure unclear.\nResponse: We agree with the reviewer that the source of the material used for MFF\nimmunoprecipitation (MFF-IP) should be clearly stated. We have rephrased the relevant section in line\n260 and in the methods section for clarity. The MFF-IP was performed using whole-cell lysates from\nmelan-a cells, not from isolated organelle fractions. We acknowledge that many of the MFF interactors\nidentified in this experiment may reflect MFF’s known association with mitochondria, rather than\ndirect interactions at melanosomes. However, when we analysed the enrichment of biological\nprocesses among the identified interactors, we found that the most significantly enriched pathway\nwas “regulation of ARP2/3 complex-mediated actin nucleation,” as shown in Fig. 5E. Given that\nARP2/3-mediated actin dynamics have previously been implicated in melanosome fission (Ripoll et\nal,2018), we consider this the most likely mechanism through which MFF may modulate melanosome\nmembrane fission. To support this hypothesis, we have performed new experiments and updated Fig.\n5 by including the respective results. First, we investigated whether branched F-actin and the ARP2/3\ncomplex are spatially associated with melanosomes using super-resolution immunofluorescence\nmicroscopy. We labelled cells with phalloidin (to visualize F-actin) or an antibody targeting ARPC3 (a\ncore subunit of the ARP2/3 complex). Both were found to localize at sites of membrane constriction\non melanosomes enriched with MFF (Fig. 5C and Fig.5F). In addition, we performed knockdown\nexperiments targeting two ARP2/3 subunits, ARPC1A and ARPC3. In both conditions, we observed an\nincrease in melanosome size, phenocopying the effect seen upon MFF depletion (Fig. 5G). Notably,\nMFF puncta remained present at early-stage melanosomes following ARP2/3 complex silencing,\nindicating that MFF localization is independent of ARP2/3 complex presence. This finding supports our\ninterpretation that MFF likely functions in coordination with ARP2/3 regulators, rather than being\nrecruited by the complex itself, to promote melanosome fission (Fig. 5H).\na. To follow up on point 4 – the authors suggest that “MFF-dependent melanosome fission relies on\nARP2/3 mediated actin assembly”. The authors somewhat suggest that this may be due to different\nsubunits of the WASH complex, but suggest that phenotypes were not seen due to potential\nredundancies and compensation. The authors should use combinatorial knockdowns to explore this\nfurther, as the proposal that DRP1 is not required for membrane fission begs the question of which\nmolecular components are involved in membrane fission.\nResponse: We agree with the reviewer, and we have addressed these points with other experiments.\nAs silencing of Fam21 and Washc5 did not yield significant changes, we decided to target directly the\nactin nucleator complex ARP2/3: we found that downregulation of ARPC1A and ARPC3 could mimic\nthe effect of Mff silencing, supporting their role in melanosome fission (Fig. 5G).\nb. Is it possible to further enrich melanosomes specifically and perform the MFF pulldown with this\nspecific fraction? If many of these hits persist in this experiment, it would suggest that the MFF-ARP2/3\ninteraction is important specifically at melanosomes, instead of at mitochondria which could indirectly\naffect melanosome function.\nResponse: Thanks for raising this point. Besides isolating pure melanosome fractions (see response to\npoint 6), we performed co-localization experiments and showed that ARPC3 and F-actin co-localize\nwith melanosome fission sites marked by MFF (Fig. 5C, D). As pointed out by the reviewer, this data\ntogether and Arpc1a and Arp3 silencing results, suggest that MFF-ARP2/3 interaction is important\nspecifically at melanosomes.\n6. MFF runs as a doublet throughout the western blotting in the manuscript. Is it possible that these\nbands represent different MFF isoforms that localize to different organelles? Does this doublet change\nin abundance throughout melanocyte maturation? Could the authors purify melanosomes away from\nmitochondria and see one versus another isoform of MFF on melanosomes? This could be a very\ncompelling piece of data that would also address some of the comments raised in point 2 above.\nResponse: Thank you for your thoughtful comments. Indeed, the different bands represent MFF\nisoforms. We generate MELANO-IP melan-a cells to isolate highly purified melanosome fractions,\nwhich were confirmed to be free of mitochondrial contamination (Fig. S2D). In these purified fractions,\nwe detected MFF isoforms curiously differently enriched in melanosome pure fractions (Fig.S2D, E).\nWe quantified the enrichment level and we observed that isoform 1 was more abundant in the\nmelanosome pull-down than in the input (whole lysate). In the latter, however, isoform 4 was found\nto be more prevalent. We overexpressed all isoforms and quantified HMB45-positive melanosome\nsize (Fig.S3O). We did not observe any significant changes, but this is perhaps not surprising, as\noverexpression alone may be insufficient to drive the phenotype.\nReviewer #4\nThe manuscript by Rebelo et al. describes a new role for MFF, a mitochondrial fission factor, in\nmelanosome size and maturation. This is an interesting finding that contributes to the growing body\nof evidence on the importance of organelle contacts for melanosome biogenesis, maturation and\nfunction. However, this finding is not entirely novel as mitochondrial-melanosome contacts were\nreported 10 years ago by the group of Maria Vittoria Schiaffino. Moreover, this group found that the\nmitochondrial protein MFN2 regulates melanosome biogenesis. In terms of mechanism, it is suggested\nthat MFF-dependent melanosome fission depends on Arp2/3-mediated actin assembly. While this is\ninteresting, it does not provide enough mechanistic detail for the journal's standard (see point 2).\n1 - One major issue is the lack of quantification of melanosome maturation in a manuscript on this\nsubject. Indeed, authors did not quantify melanosome stages by EM, which is essential to support the\nclaims made. This applies to several figures in the manuscript, including Fig. S1A/B, 2A - to know if\nMFF is present in all melanosome stages in the same proportion - and 3G.\nResponse: We appreciate the reviewer’s insightful comments and agree that the previously missing\nquantifications were essential to strengthen our conclusions. We added all the requested\nquantifications to the revised manuscript. Specifically, we have included quantifications for the\nprevious Fig. S1A/B (now Fig. 1A), as shown in Fig. 1B, 1C, and Fig. S1C–E. We quantified the\npercentage of melanosomes at different maturation stages that were in contact with mitochondria in\nboth melan-a and melan-c2 cell lines. We also measured the distances between mitochondria and\nmelanosomes at all maturation stages, as well mitochondria and melanosome perimeters when in\ncontact. To provide a thorough view of these interactions, we developed a new index—the\nMelanosome-Mitochondria Contact Coefficient (MELAMICC)—calculated as follows: MELAMICC =\nmelanosome perimeter / (mitochondria perimeter × mitochondria–melanosome distance), similar to\nthe ERMICC index that was previously suggested for mitochondria-ER contact sites (Naon et al, PNAS\n2016). This coefficient provides a normalized measure of contact, allowing comparison of contact sites\nacross different melanosome stages. Our findings revealed a significant increase in mitochondria–\nmelanosome contacts in albino melanocytes, which are also characterized by an accumulation of\nearly-stage melanosomes (Stages I and II). Notably, no mature melanosomes were observed in these\ncells, precluding quantification of later stages. To precisely quantify the number of melanosomes\ncarrying MFF, we then decided to perform super-resolution microscopy experiments (Fig. 2). We\nobserved that approximately 20% of early-stage melanosomes are positive for MFF (Fig. 2F). Given the\nabsence of MFF at late-stage melanosomes (TYRP1-positive) and in their constriction sites, these\nstages, we focused on early stages. We quantified the number of times that MFF is observed at early\nmelanosome fission regions (approximately 20% of early-stage melanosomes are positive for MFF)\n(Fig. 2I). Lastly, we have now included the quantification of the TEM images shown in the original Fig.\n3G (now Fig. 3F), and provided the corresponding graph in Fig. S3M.\n2- Another major issue is the use of an inhibitor only to interfere with Arp2/3 and draw conclusions\nabout the involvement of the complex and actin nucleation. Indeed, pharmacological approaches, as\nany other, have pitfalls. Therefore, the findings should be validated with a genetic approach, by\ndepleting Arp2/3.\nResponse: We thank the reviewer for this valuable suggestion and have now included additional data\naddressing the role of the ARP2/3 complex in melanosome morphology. Specifically, we performed\nknockdown experiments targeting two different subunits of the ARP2/3 complex, ARPC1A and ARPC3.\nIn both cases, we observed an increase in melanosome size, consistent with the phenotype seen upon\nMFF knockdown (Fig. 5). Notably, MFF puncta remained detectable at early-stage melanosomes under\nthese silencing conditions, indicating that MFF localizes to melanosomes independently of the ARP2/3\ncomplex itself. This supports our interpretation that MFF may function via interaction with ARP2/3\nregulators to facilitate melanosome fission. Furthermore, we immunostained for one ARP2/3 complex\nsubunit and confirmed its localization to early melanosomes (Fig. S5F, G). To further support the link\nbetween MFF and actin dynamics, we demonstrated the enrichment of F-actin at melanosome fission\nsites marked by MFF localisation (see Figures 5C and 5D). This finding reinforces our model that MFF-\ndependent melanosome fission is mediated through the localised nucleation of actin.\n3 - The evidence that MFF also localizes to stage III and IV melanosomes seems to be largely ignored\nby the authors and not considered in the interpretation of the findings.\nResponse: We appreciate the reviewer’s observation and agree that MFF localization to later-stage\nmelanosomes (Stage III and IV) warrants further discussion. Although our super-resolution\nmicroscopy images occasionally revealed MFF signal at these later stages (Fig. 2 and Fig. S2), its\npresence was visibly lower compared to early-stage melanosomes. Importantly, we did not observe MFF\nenrichment at constriction or fission sites in TYRP1-positive melanosomes, suggesting that MFF may not\nbe actively involved in fission events at these later maturation stages. To clarify this, we have expanded\nthe discussion in the revised manuscript. We now consider the possibility that MFF localization to stage\nIII/IV melanosomes may reflect hybrid or transitional maturation stages that cannot be fully\ndistinguished using current markers. This could explain partial colocalization. Alternatively, MFF may\nserve a different, yet unexplored, role at these stages. However, we did not pursue this possibility as\nthe focus of our current study was on early melanosomes. Indeed, we initiated our investigation using\nan albino melanocyte model that exclusively contains early-stage melanosomes, as it harbours a\nTyrosinase mutation that impedes melanin synthesis. In this model, we observed specific enrichment\nand altered expression of MFF. To further support our focus on early-stage melanosomes and MFF,\nwe included new data quantifying melanosome dynamics in melan-a and melan-c2 cells (Fig. 1I–K).\nThese analyses revealed an increased number of fission and fusion events in early-stage melanosomes\nin albino cells, confirming the presence of altered melanosomal dynamics and supporting a functional\nrole for MFF in this process.\n4 - Regarding the explanation of why Mff-KD cells are more pigmented, it is not likely that immature\nmelanosomes accumulate melanin (stage II melanosomes should have little pigment). Moreover, a\nnegative feedback loop is expected to occur, downregulating melanogenesis, and this is likely why the\nauthors detected lower levels of melanogenic markers. However, this should not lead to increased\npigmentation. Therefore, other mechanisms should be considered.\nResponse: We appreciate the reviewer’s comment regarding the increased pigmentation observed in\nMff-KD cells. While we agree that immature melanosomes (e.g., stage II) typically contain little\npigment, we hypothesize that their pigmentation could increase in Mff-silenced cells due to impaired\nprogression of maturation into later stages. Specifically, we hypothesize that MFF-mediated fission\nmay participate in the removal of unnecessary material: indeed, similarly to other organelles,\nmelanosomes must control their size to maintain their homeostasis. Disruption of cargo recycling\ncould lead to the accumulation of enlarged melanosomal structures, characterized by abnormal\nmaturation and eventual pigmentation. In line with this idea, we observed: enlarged or doublet\nmelanosome-like structures in Mff-KD cells (Fig. S3N); a trend toward increased size not only of early\nbut also of late-stage melanosomes by TEM (Fig. S3M); and a higher colocalization of the early\nmelanosome marker HMB45 with melanin (Fig. 3I).\nBesides, we found that Mff-silenced melanocytes are characterized by a significant upregulation of\nlysosomal and catabolic genes, including Ctsb, Ctsd, and Lipa, a transcriptional response not observed\nin Drp1-KD cells. Interestingly, unlike Mff, knockdown of Drp1 led to downregulation of the key\nmelanogenesis regulator Mitf (Fig. 4A), indicating divergent cellular responses to the loss of each\nprotein.\nThe specific upregulation of lysosomal enzymes in Mff-KD cells may represent a compensatory\nmechanism triggered by impaired melanosome fission, aimed at increasing melanosome degradation\nand at limiting the accumulation of enlarged, dysfunctional organelles. However, if this catabolic\nresponse was not sufficient to compensate for the lack of MFF-mediated fission, melanosomes that\nare enlarged and only partially mature may persist. Supporting this idea, co-silencing Ctsb with Mff\nrescued the elevated melanosomal acidification (Fig. 4H, I), restored the expression of melanogenesis\nmarkers, and further increased both melanin content and melanosome size compared to Mff silencing\nalone (Fig. 4J, K; Fig. S4C–J). Moreover, this condition led to a significant accumulation of early-stage\nmelanosomes per total cell area (Fig. 4L), a phenotype absent in Mff-KD alone (Fig. 3D). These findings\nsuggest that increased lysosomal enzyme activity, particularly CTSB, contributes to the degradation of\nkey melanogenic proteins (e.g., TYR, TRP1, GP100), forming part of a negative feedback loop that\nattempts to limit melanosome buildup.\nAnd Minor issues:\n- Fig. 2G, H does not exist.\nResponse: We correct this misinformation in the manuscript.\n- Reference 8 is old and there are several more recent ones.\nResponse: We added to the manuscript a more recent reference in addition to reference 8."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my concerns.", "reviewer_3": "This is a revision for a manuscript outlining a role for mitochondrial fission factor (MFF) in the regulation of melanosome fission. The authors have presented a thoughtful response to reviewers, and have satisfactorily addressed my concerns in the first presentation of this manuscript.", "reviewer_4": "The authors thoroughly reviewed the manuscript and considered all the reviewers' comments and suggestions. As a result, the manuscript was vastly improved. Nevertheless, the question of the potential off-target effects was not addressed. Contrary to what the authors state, the use of siRNA pools does not minimize these efects and can even exacerbate them, as four siRNA oligos are present in the pool. Therefore, a rescue experiment is warranted."}, "author_response": "Response to Reviewers\nReference number: NCOMMS-24-37754A\nWe would like to thank the reviewers for their thoughtful evaluations and for acknowledging that the\nrevised manuscript satisfactorily addresses all points raised during the initial submission. We are\npleased that the reviewers found that the revised work has improved the overall quality and clarity of\nthe manuscript.\nRegarding the remaining concern about potential off-target effects and the suggestion to perform a\nrescue experiment, we fully agree that addressing off-target contributions is important. However,\nseveral technical constraints prevented us from accomplishing a definitive rescue.\nFirst, rescue with murine MFF was not feasible because the siRNA pool used in our experiments targets\nthe mouse MFF coding sequence, as shown in the table provided below (Table 1). This results in rapid\nsilencing of not only the endogenous, but also the exogenous mouse construct.\nSecond, although we attempted rescue with two different constructs encoding for human MFF\n(tagged either with V5 or with GFP), this strategy was also unworkable: the expression kinetics of the\nhuman construct did not match the siRNA knockdown window, and, critically, one of the siRNA oligos\nin the pool also targets human MFF, thereby preventing stable and interpretable rescue (Fig.1).\nThird, we tried to generate a stable MFF knockdown cell line, but this approach proved unsuitable for\nour model. Indeed, likely due to the primary-like nature of melan-a, after antibiotic selection and cell\nexpansion the cells showed substantial alterations in cell morphology and pigmentation levels.\nTherefore, to address the reviewer’s concerns and independently validate the phenotype while\nminimizing off-target effects, we used an alternative knockdown approach. Instead of using a pool of\nsiRNA, we took advantage of Lentiviral particles carrying an individual shRNA targeting murine MFF.\nSpecifically, we tested two individual shRNAs. Our Western blot analysis showed that each of these\nconstructs was able to decrease MFF levels, albeit to a lesser extent than the siRNA pool (Fig.2).\nThe shRNA that most effectively reduced MFF levels produced a phenotype similar to that observed with\nthe siRNA pool, although the effect size was smaller (Fig.3). This difference corresponds precisely to\nthe distinct levels of MFF depletion achieved: our quantifications suggest that the siRNA pool reduces\nMFF by ~90–95%, whereas the single shRNA reduces MFF by ~50%. The proportionality between MFF\ndepletion and phenotypic strength strongly supports the specificity of the effect and argues against\noff-target artifacts. Taken together, these results provide evidence that the observed phenotype is\ncaused by the specific depletion of MFF, rather than by off-target effects.\nOverall, we believe that the additional experiments and clarifications provided in the revised\nmanuscript substantially strengthen the rigour and robustness of our conclusions on the central role\nof MFF in regulating melanosome fission by an actin-dependent mechanism. We therefore hope that\nyou will now consider the manuscript suitable for publication.\nTable 1. siGENOME Mouse Mff siRNA – SMARTpool (Dharmacon, Cat. No. M-041534-00-0020)\nsiRNA sequence Target\nsiRNA1 UGAGAGACCACUAGAUUUC Mouse\n TGAGAGACCACTAGATTTC\nsiRNA2 CGUCAGGUAUGGCAUUUCA Mouse - Human\n CGTCAGGTATGGCATTTCA\nsiRNA3 GGAUCGUGGUUACAGGAAA Mouse\n GGATCGTGGTTACAGGAAA\nsiRNA4 UUAGUCGAAUUCAGUAUGA Mouse\n TTAGTCGAATTCAGTATGA\nA\nB\nFigure 1. Expression of human MFF in melan-a. Melan-a cells were electroporated with an siRNA pool\ntogether with a plasmid encoding either V5-tagged or GFP-tagged human MFF. A) Cells were fixed and\nstained as described in the Materials and Methods section of the manuscript. A rabbit monoclonal\nanti-V5 tag antibody (Cell Signaling Technology, #13202) was used at a dilution of 1:50 in 5% BSA,\nfollowed by incubation with a fluorophore-conjugated secondary antibody (as described in the\nMaterials and Methods section). Images were acquired using an Operetta high-content imaging\nsystem in confocal mode with a 63× objective. Representative maximum-intensity projections are\nshown. Channels are displayed as follows: phalloidin (red), Hoechst (blue), and V5 (green). B) Overview\nof GFP-tagged MFF expression in melan-a cells. Melan-a cells were electroporated with a plasmid\nencoding GFP-tagged MFF. Images were acquired using the Operetta high-content imaging system in\nepifluorescence mode with a 63× objective. The panel shows a stitched overview of multiple fields\nfrom a single well. Red arrows indicate GFP-positive cells.\nFigure 2. Knockdown of MFF in melan-a cells via shRNA. (A) Western blot analysis showing\ndownregulation of MFF expression in cells transduced with Lentiviral particles carrying shRNA\ntargeting MFF (MERCK ready to use lentiviral particles). In particular we used, as a control, MISSION®\npLKO.1-puro Non-Target shRNA Control Transduction Particles (SHC016V) and, for MFF knockdown,\nTRCN0000174665 Transduction Particles (targeting sequence GATCGTGGTTACAGGAAATAA) and\nTRCN0000174539 Transduction Particles (Targeting sequence CTTCATTAAGACGTCAGATAA). Vinculin\n(VCL) was used as a loading control. (B) Densitometric quantification of MFF expression in non-\ntargeting (NT) and shRNA-539 samples. Data are presented as mean ± SEM; N = 3 independent\nexperiments.\nFigure 3. MFF knockdown with shRNA increases melanosome size in melan-a cells. Melan-a cells\nwere seeded in 96-well plates and treated the next day with shRNA-539 targeting MFF or the non-\ntargeting control. After 48 hours, cells were fixed and stained with HMB45 and Tom20 antibodies as\ndescribed in the Materials and Methods section of the manuscript. Images were acquired using the\nOperetta high-content imaging system. Quantification of melanosome size is shown (N = 4\nindependent experiments). Data are presented as mean ± SEM; statistical significance was assessed\nusing an unpaired t-test (p < 0.05)."}, {"round": 3, "reviewer_comments": {"reviewer_4": "Although the use of another method to silence MFF (in this case, shRNA) does not fully exclude potential off-target effects, it strengthens significantly the conclusions. Therefore, the manuscript can now be accepted for publication, with the inclusion of the data supplied in the rebuttal letter as supplementary data."}, "author_response": "Response to Reviewers\nReference number: NCOMMS-24-37754\nWe would like to thank the reviewers for evaluating our work, and for providing us with constructive\nsuggestions. Overall, the reviewers recognised the novelty of our findings, particularly with regard to the role\nof MFF in melanosome fission and function, independently of DRP1. By addressing their comments related\nto the use of siRNA pools, we have strengthened the manuscript's conclusions and main message. The new\ndata presented further confirm the role of MFF in melanosome fission. Below, we provide a point-by-point\nresponse to each comment from the reviewers. Their comments are in italic and our responses are in bold.\nReviewer #4\nAlthough the use of another method to silence MFF (in this case, shRNA) does not fully exclude potential off-\ntarget effects, it strengthens significantly the conclusions. Therefore, the manuscript can now be accepted for\npublication, with the inclusion of the data supplied in the rebuttal letter as supplementary data.\nWe thank the reviewer for this comment. We have included the data supplied in the rebuttal letter as\nsupplementary data, as requested. Specifically, they have been included as panels P,Q and R in\nSupplementary Figure 3. Material and methods used to perform these experiments have been included in\nthe manuscript text (rows 573-582)."}]

Decision letter