Lipid turnover and GEF recruitment collectively determine Rab5 recruitment and activation during the first step of early endosome formation

Domain: biology
Published: 2026-03-30
Editorial outcome: minor_revision

Abstract
Early endosomes are the pivotal sorting station in eukaryotic cells. A longstanding critical question is how the small GTPase Rab5 is precisely targeted to the correct membrane to initiate early endosome formation. Here, we identify Rabex5 and hRME6 as the two guanine-nucleotide exchange factors (GEFs) that together regulate Rab5 recruitment during early endosome formation. Single-molecule imaging of genome-edited cells reveals that Rabex5 and hRME6 are recruited continuously or transiently to nascent uncoated endocytic carriers, respectively. However, in contrast to uncoated endocytic carriers and other intracellular organelles, directing Rabex5 or its GEF domain to clathrin-coated pits or the plasma membrane fails to trigger Rab5 recruitment and activation. Both in vivo and in vitro experiments show that the plasma membrane-enriched phospholipid PI(4,5)P2 prevents Rab5 association with the plasma membrane. Importantly, we found that impaired hydrolysis of PI(4,5)P2 led to reduced early endosome formation in Lowe syndrome cells. Therefore, the spatiotemporal recruitment and activation of Rab5 during early endosome formation are collectively determined by Rabex5/hRME6 recruitment and PI(4,5)P2 depletion during uncoated endocytic carrier formation.

Introduction
Endocytosis is essential for cells to take up extracellular nutrients, internalize plasma membrane proteins and lipids, and control bacterial and viral infection 1. The internalized cargo is soon delivered to early endosomes, where it can be sorted to recycling endosomes or Golgi for retrieval, or lysosomes for degradation 2, 3. Dysregulation of the biogenesis, distribution, and function of early endosomes is associated with various human diseases including Alzheimer’s disease 4. Despite significant advances in unveiling the structural complexity, signaling functions, and identifying cargo sorting machinery of early endosomes 3, 5, our understanding of the dynamics and mechanisms underlying early endosome biogenesis remains limited.

Rab5, a Rab protein specific to early endosome, plays a particularly critical role in membrane trafficking from the plasma membrane to early endosomes 5. Like other small GTPases, Rab5 functions as a molecular switch that cycles between the GTP-bound active and GDP-bound inactive form. The conversion from the GDP-bound to the GTP-bound form is catalyzed by guanine-nucleotide exchange factors (GEFs), whereas the conversion from the GTP-bound to the GDP-bound form is driven by GTPase activating proteins (GAPs) 6, 7. Rab5 is post-translationally modified at its C-terminus by covalent attachment of two lipophilic geranylgeranyl chains, which allows it to associate with membranes 8. The GDP-bound Rab5 is extracted from membranes and kept soluble in the cytosol in the inactive state by association with the guanine dissociation inhibitor (GDI) 6, 7. The release from GDI and the subsequent GEF-catalyzed GDP-to-GTP exchange enable Rab5 to interact with membranes and recruit effector proteins 6, 7.

Our understanding of GDP/GTP cycling catalyzed by GEFs/GAPs mainly comes from in vitro experiments. Detecting and tracking the dynamic recruitment and activation of Rab5 during early endosome formation in live mammalian cells remains technically challenging. First, Rab5 overexpression leads to early endosome enlargement 9, 10. Second, at least seven putative GEF molecules for Rab5 have been identified, but the precise subcellular localization and relative contribution of each GEF during Rab5 recruitment/activation remain poorly understood. Third, mammalian cells express three Rab5 isoforms, namely Rab5a, Rab5b, and Rab5c. Previous studies suggest that the three isoforms may have distinct subcellular distributions and roles in different tissues or cellular processes 11, 12. The simultaneous knockout of all three isoforms was unsuccessful, and their conditional knockout caused cell death after 48 hours 13, 14. Lastly, early endosome formation begins with the generation of endocytic vesicles from the plasma membrane. The high mobility and small number of molecules recruited to these small, budded vesicles make it challenging to track the recruitment of Rab5 and other regulatory molecules during early endosome biogenesis 15. Together, these technical challenges prevent the mechanistic understanding of when and how Rab5 is recruited to initiate early endosome formation in live cells.

By imaging the endogenous Rab5 in genome-edited cells with single-molecule sensitivity, our previous study showed that Rab5a and Rab5c appeared in newly budded endocytic vesicles, indicating that nascent endocytic carriers recruit Rab5 before their fusion with other carriers or endosomes 15. However, the molecular mechanism that determines the precise spatial and temporal targeting of Rab5 to endocytic vesicles remains unknown. In this study, we developed a live cell system in which endogenous Rab5 is fluorescently tagged, and normally expressed, but cannot get activated. We discovered that the recruitment of Rabex5/hRME6 and the depletion of PI(4,5)P2 from endocytic vesicles together determine the recruitment and activation of Rab5 during the first step of early endosome formation.

Results
Rab5 is recruited specifically to uncoated endocytic carriers in genome-edited cells

Clathrin assembles at the plasma membrane to form clathrin-coated pits, which subsequently pinch off to form clathrin-coated vesicles 16. The disassembly of clathrin coats, initiated by a burst of auxilin, occurs a few seconds after vesicle release and generates uncoated endocytic carriers 17, 18. Our previous work showed that Rab5a and Rab5c are recruited specifically to endocytic carriers soon after their release from the plasma membrane 15. To investigate whether all three Rab5 isoforms exhibit similar recruitment dynamics, we generated an additional genome-edited cell line with endogenous Rab5b tagged with EGFP in SUM159 cells (Supplementary Fig. 1a-c). We then tagged endogenous clathrin light chain A with a near-infrared fluorescent protein miRFP670nano 19 (clathrin-670nano+/+) to create cell lines dual-labeled for clathrin and Rab5a/b/c. To determine the precise timing of Rab5a/b/c recruitment during endocytic carrier generation, we transiently expressed mScarlet-I-auxilin2/GAK in the EGFP-Rab5a/b/c+/+ clathrin-670nano+/+ cells and performed triple-color live-cell imaging by total internal reflection fluorescence (TIRF) microscopy (Supplementary Fig. 1d-f and Supplementary Movie 1). Consistent with previous studies 17, 18, we observed the transient burst-like recruitment of GAK specifically to coated vesicles at the time of clathrin uncoating. Endogenous Rab5a/b/c were recruited after the uncoating but not the assembly stage of clathrin-coated pit formation (Supplementary Fig. 1d-f). Despite the wide range of lifetime distributions of endocytic events, quantitative analysis of the recruitment timing of Rab5a/b/c and GAK to individual endocytic vesicles further confirmed that Rab5a/b/c is recruited after the burst-like signal of GAK (Fig. 1a-c). In our previous work, we have shown that these newly uncoated endocytic carriers contain PI(3,4)P2 and recruit Rab5, yet lack EEA1 and PI3P prior to fusing with endosomes 15. The specific recruitment of endogenous Rab5c after uncoating was also observed in gene-edited HeLa and hTERT RPE-1 (hRPE-1) cells (Supplementary Fig. 1g).

Rab5 switches between the GTP-bound active form and GDP-bound inactive form 6. To investigate how the nucleotide-bound status affects Rab5 recruitment, we transiently expressed mEGFP-tagged wild-type, GTP-bound constitutively active, or GDP-bound constitutively inactive forms of Rab5a/b/c in the clathrin-670nano+/+ cells and performed live-cell TIRF imaging. Like the endogenous Rab5, the wild-type and constitutively GTP-bound forms of Rab5a/b/c were actively recruited to uncoated endocytic carriers (Supplementary Fig. 2a-c). However, compared to wild-type Rab5, the constitutively GTP-bound mutants caused enlargement of intracellular endosomes but showed relatively weaker recruitment to uncoated endocytic carriers. As expected, the constitutively GDP-bound mutants exhibited diffuse cytosolic distribution and were not recruited to uncoated endocytic carriers (Supplementary Fig. 2a-c). These results indicate that GEF-mediated nucleotide exchange facilitates the specific recruitment of Rab5 to uncoated endocytic carriers.

Simultaneous depletion of hRME6 and Rabex5 eliminates Rab5 recruitment to uncoated endocytic carriers and abolishes Rab5-positive early endosome formation

GEFs have been suggested to determine the subcellular targeting of Rab proteins 20. To identify proteins involved in recruiting Rab5 to uncoated endocytic carriers, we transiently expressed low levels of the putative Rab5 GEFs—Rabex5, hRME6, ALS2, ALS2CL, RIN1, RIN2, and RIN3—in clathrin-670nano+/+ cells and analyzed their recruitment dynamics during clathrin-mediated endocytosis. Encouragingly, Rabex5, hRME6, and ALS2CL showed apparent recruitment to uncoated endocytic carriers (Supplementary Fig. 2d). Rabex5 and hRME6 were expressed in SUM159, HeLa, and hRPE-1 cells (Supplementary Fig. 2e). ALS2CL showed high expression in heart and liver tissues 21 but was not detectable in the three human cell lines (Supplementary Fig. 2f). We then focused on the potential role of the two GEFs, Rabex5 and hRME6, in mediating Rab5 recruitment during early endosome formation.

To investigate the functions of Rabex5 and hRME6, we knocked out the expression of either Rabex5, hRME6, or both simultaneously (Rabex5/hRME6-DKO) in gene-edited cells expressing EGFP-Rab5c+/+ clathrin-TagRFP+/+ (Fig. 1d). Using immunoblotting, we confirmed that the knockout of hRME6, Rabex5, or both has a minor effect on the expression of Rab5c and other endosomal proteins, including Rabaptin5 and EEA1 (Fig. 1d). However, Rabex5 knockout resulted in a significant decrease in the number of mEGFP-Rab5c puncta around the bottom surface and perinuclear region (Fig. 1e). Knockout of hRME6 did not affect the number and distribution of mEGFP-Rab5c. Remarkably, Rabex5/hRME6-DKO led to the near complete elimination of EGFP-Rab5c puncta from the cells (Fig. 1e). Instead, we observed some fuzzy structures concentrated around the perinuclear region. Knockdown of hRME6, Rabex5, or both using siRNA had a similar inhibitory effect on the numbers and distribution of EGFP-Rab5c in gene-edited SUM159 and HeLa cells (Supplementary Fig. 3a-d).

By imaging the cells at the basal surfaces using TIRF microscopy, we further showed that simultaneous knockout of hRME6 and Rabex5 was necessary to fully abolish EGFP-Rab5c recruitment to uncoated endocytic carriers (Fig. 1f). To further investigate whether the depletion of hRME6 and/or Rabex5 affected the formation or distribution of early endosomes, we transiently expressed various endolysosomal markers in the cells. We found that hRME6/Rabex5-DKO resulted in the disappearance of EEA1-positive early endosomes (Fig. 1g). Additionally, we observed the disappearance of PI3P- and Rab5a-positive early endosomes in the DKO cells (Fig. 1h and Supplementary Fig. 3e). Rabex5/hRME6-DKO also lead to the significant reduction in the number of the Rab5 subfamily of GTPases Rab21- or Rab22a-positive endosomes (Supplementary Fig. 3f,g). Interestingly, Rabex5/hRME6-DKO did not lead to the disappearance of LAMP1-positive lysosomes (Supplementary Fig. 3h).

As GEF molecules, Rabex5 and hRME6 catalyze the conversion of Rab5 from the GDP-bound state to the GTP-bound form. Consequently, we wondered whether ectopically expressed GTP-bound Rab5 could be targeted to uncoated endocytic carriers and rescue early endosome formation in Rabex5/hRME6-DKO cells. Surprisingly, the constitutively GTP-bound Rab5a/b/c failed to be recruited to uncoated endocytic carriers, and their expression could not rescue the recruitment of endogenous EGFP-Rab5c to uncoated endocytic carriers (Supplementary Fig. 4a-c). The expression of constitutively GTP-bound Rab5a/b/c also failed to restore the formation of EGFP-Rab5c-positive early endosomes (Supplementary Fig. 4d-f). As expected, the wild-type or the constitutively GDP-bound forms of Rab5 were not recruited to uncoated endocytic carriers and were incapable of rescuing early endosome formation in Rabex5/hRME6-DKO cells (Supplementary Fig. 4a-f). These findings suggest that Rabex5- and hRME6-catalyzed GDP-to-GTP conversion is crucial for the recruitment of Rab5 to uncoated endocytic carriers and the formation of Rab5-positive early endosomes.

Distinct recruitment dynamics of endogenous Rabex5 and hRME6 to uncoated endocytic carriers

The finding that Rabex5 and hRME6 collectively control Rab5 recruitment prompted us to investigate how the spatiotemporal recruitment of these two GEFs is coordinated. Earlier studies have shown that ectopically overexpressed Rabex5 induced the enlargement of early endosomes 22-24. The overexpressed Rabex5 colocalized with Rab5 or EEA1 on these enlarged early endosomes around the perinuclear region 22-24. To overcome the limitations arising from overexpression, we fluorescently tagged endogenous Rabex5 with mEGFP by genome editing (Fig. 2a and Supplementary Fig. 5a). The functionality of the Rabex5-mEGFP+/+ cells was confirmed by the uptake and intracellular sorting of fluorescently labeled transferrin and EGF (Supplementary Fig. 5b).

By imaging the Rabex5-mEGFP+/+ cells using TIRF microscopy, we found that endogenous Rabex5 was recruited after clathrin uncoating (Fig. 2b and Supplementary Movie 2). Specifically, Rabex5 was recruited after the burst-like recruitment of GAK and remained associated with uncoated endocytic carriers (Fig. 2c,d). To further characterize the spatiotemporal correlation of endogenous Rabex5 with Rab5, we generated a triple gene-edited cell line expressing Rabex5-mEGFP+/+ mScarlet-I-Rab5c+/+ clathrin-670nano+/+. Triple-color TIRF imaging revealed that endogenous Rabex5 and Rab5c were recruited almost simultaneously and remained associated with uncoated endocytic carriers (Fig. 2e,f). Additionally, endogenous Rabex5 was observed on Rab5c-positive endosomes within the cells (Supplementary Fig. 5c).

RME6 was originally identified as a GEF for Rab5 in Caenorhabditis elegans (C. elegans) 25. Interestingly, instead of colocalizing with Rab5 or EEA1, GFP-tagged RME6 concentrated primarily in clathrin-coated pits at the plasma membrane in C. elegans 25. More recently, by carefully re-examining the subcellular localization of RME6 in C. elegans, it was found that GFP-tagged RME6 localized primarily in the vicinity of the plasma membrane and colocalized with Rab5 26. While the exact subcellular localization of human RME6 (hRME6; also called RAP6 or Gapex-5) has been unclear, it was suggested that hRME6 is involved in the regulation of AP2 uncoating at the plasma membrane of mammalian cells 27. To reveal the genuine subcellular locations and dynamics of hRME6 in mammalian cells, we tagged endogenous hRME6 with mEGFP through genome editing (Fig. 2g and Supplementary Fig. 5d) and verified the functionality of the cells (Supplementary Fig. 5e).

By imaging mEGFP-hRME6+/+ cells using TIRF microscopy, we unexpectedly observed the transient binding of many individual mEGFP-hRME6 spots at the plasma membrane (Fig. 2h and Supplementary Movie 3). Interestingly, mEGFP-hRME6 exhibited a transient burst of recruitment to the nascent endocytic carriers (Fig. 2h and Supplementary Movie 3), occurring immediately after the burst recruitment of GAK (Fig. 2i,j). To elucidate the spatiotemporal relationship between hRME6 and Rab5, we generated a triple gene-edited cell line expressing mEGFP-hRME6+/+ mScarlet-I-Rab5c+/+ clathrin-670nano+/+. TIRF imaging revealed the simultaneous recruitment of hRME6 and Rab5c to the same uncoated endocytic carriers (Fig. 2k and Supplementary Movie 4). However, unlike Rabex5, hRME6 dissociated from uncoated endocytic carriers after its burst-like recruitment (Fig. 2h-l) and was rarely observed on Rab5c-positive endosomes within the cells (Supplementary Fig. 5f). Therefore, despite the distinct recruitment dynamics of Rabex5 and hRME6 to uncoated endocytic carriers, both proteins were recruited alongside Rab5 during the initial formation of uncoated endocytic carriers. Moreover, depletion of either Rabex5 or hRME6 did not affect the recruitment of the other protein (Supplementary Fig. 5g-i), suggesting that these two GEFs are independently recruited to uncoated endocytic carriers.

The Vps9 domain of Rabex5 is necessary for Rabex5 recruitment to uncoated endocytic carriers and Rab5-positive early endosome formation

To reveal the intrinsic connection between the recruitment dynamics and catalytic function of Rabex5, we generated a series of Rabex5 constructs with domain deletions or point mutations as shown in Fig. 3a. Rabex5 is a multidomain protein containing an N-terminal ubiquitin-binding domain (UBD), a long linker, a 4-helix bundle domain (4-HB), a Vps9 domain, a Rabaptin5-binding domain (RpBD), and a C-terminal proline-rich domain (PR) 28, 29. We tagged the mutants with mEGFP and transiently expressed them in cells with Rabex5-KO, and then conducted TIRF imaging to analyze their recruitment dynamics during endocytic vesicle formation. Meanwhile, to evaluate the catalytic function of each mutant, we tagged the mutants with HaloTag and transiently expressed them in EGFP-Rab5c+/+ clathrin-TagRFP+/+ cells with Rabex5/hRME6-DKO. EGFP-Rab5c recruitment to uncoated endocytic carriers and early endosome formation was subsequently analyzed using TIRF microscopy and spinning-disk confocal microscopy, respectively.

Consistent with previous studies 22, we observed that removing the N-terminal UBD or the C-terminal RpBD did not affect Rabex5 recruitment to uncoated endocytic carriers (Supplementary Fig. 5j). Expression of these truncated Rabex5 mutants restored recruitment of Rab5 to uncoated endocytic carriers and formation of Rab5-positive early endosomes in the DKO cells (Fig. 3e and Supplementary Fig. 5k,l). Intriguingly, the removal of the Vps9 domain hindered Rabex5 recruitment to uncoated endocytic carriers, and this mutant failed to restore Rab5 recruitment and early endosome formation (Fig. 3b-e). Rabex5 carrying the D313A mutation in the Vps9 domain, which is known to reduce the GEF activity 30, was still recruited to uncoated endocytic carriers but failed to restore Rab5 recruitment to uncoated endocytic carriers and early endosome formation (Fig. 3b-e and Supplementary Fig. 6j).

Previous studies suggest that Rabex5 is recruited to early endosomes in a complex with Rabaptin5 29, 31. Indeed, like Rabex5, Rabaptin5 was recruited to uncoated endocytic carriers in gene-edited cells expressing mEGFP-Rabaptin5+/+ (Supplementary Fig. 6a-d). Knockdown of Rabex5 impaired the recruitment of Rabaptin5 to uncoated endocytic carriers (Supplementary Fig. 6e,f). However, the depletion of Rabaptin5 did not affect the recruitment of Rabex5 to uncoated endocytic carriers (Supplementary Fig. 6g,h). Nevertheless, Rabaptin5 depletion reduced the number of Rab5c-positive early endosomes (Supplementary Fig. 6i). These results suggest that while Rabaptin5 enhances the GEF activity of Rabex5 to facilitate early endosome formation 24, 28, 29, 31, it is not required for the initial association of Rabex5 with uncoated endocytic carriers.

A burst of hRME6 recruitment triggers Rab5 recruitment to uncoated endocytic carriers and promotes Rab5-positive early endosome formation

hRME6 contains an N-terminal RasGAP domain, a proline-rich region, and a C-terminal Vps9 domain 32 (Fig. 3f). By transiently expressing mEGFP-tagged hRME6 constructs with deletions of individual domains in cells with hRME6-KO, we found that only the full-length hRME6 was recruited in a transient burst to uncoated endocytic carriers (Fig. 3g). Consistent with this observation, only the full-length hRME6 was capable of restoring the recruitment of Rab5 to uncoated endocytic carriers (Fig. 3h) and the formation of Rab5-positive early endosomes (Fig. 3i,j). Mutation of hRME6 at D1429, which corresponds to the conserved D313 residue critical for Rabex5’s GEF activity 30, did not affect hRME6 recruitment to uncoated endocytic carriers (Fig. 3g). However, this mutant failed to restore Rab5 recruitment to uncoated endocytic carriers (Fig. 3h) and early endosome formation (Fig. 3i,j).

The observations that hRME6 is recruited in a transient burst and the re-expression of hRME6 can restore Rab5 recruitment to uncoated endocytic carriers in the Rabex5/hRME6-DKO cells indicate that a burst of hRME6 recruitment is sufficient to induce continuous Rab5 recruitment and activation. Indeed, by transiently expressing hRME6 in the DKO cells, we observed concurrent recruitment of both hRME6 and Rab5 to the same endocytic carrier. However, even after hRME6 dissociation, Rab5c remains associated with the endocytic carrier (Fig. 3k).

Targeting Rabex5 or its Vps9 domain to clathrin-coated pits fails to induce Rab5 recruitment and Rab5-positive early endosome formation

Although Rabex5 and hRME6 exhibit distinct dynamics, both are recruited after uncoating. The next question is whether this specific recruitment timing correlates with the absence of Rab5 recruitment to clathrin-coated pits/vesicles. It has been reported that artificially directing Rabex5 or its Vps9 domain to mitochondria induced the translocation of Rab5a to the mitochondrial membrane 20. This observation led to the hypothesis that the subcellular localization of GEFs determines the specific membrane targeting of Rab5 20, suggesting that Rab5 could potentially be recruited to clathrin-coated pits or vesicles by misdirecting the Vps9 domain to these structures. To explore this possibility, we generated a series of chimeras designed to target the Vps9 domain of Rabex5 to clathrin-coated pits, vesicles, or uncoated endocytic carriers (Fig. 4a).

We chose HIP1R and Epsin1, two clathrin-binding proteins recruited during clathrin-coated pit formation 33, 34, to direct the Vps9 domain to clathrin-coated pits. We used GAK to direct the Vps9 domain to clathrin-coated vesicles (Fig. 4a). The Vps9 domain of Rabex5 was fused to HIP1R, Epsin1, or GAK to create the HIP1R-Halo-Vps9, Halo-Epsin1-Vps9, or Halo-GAK-Vps9 chimeras. In clathrin-TagRFP+/+ cells, HIP1R-Halo-Vps9 and Halo-Epsin1-Vps9 were effectively recruited to the assembling clathrin-coated pits and Halo-GAK-Vps9 was recruited in a transient burst to clathrin-coated vesicles (Supplementary Fig. 7a-c). However, in Rabex5/hRME6-DKO cells, although HIP1R-Halo-Vps9, Halo-Epsin1-Vps9, or Halo-GAK-Vps9 were similarly recruited to clathrin-coated pits or vesicles, they were unable to recruit Rab5 to these structures (Fig. 4b) and failed to promote Rab5c-positive early endosome formation (Fig. 4d,e). Similarly, overexpression of tag-free Rabex5, but not of tag-free Rab5c, Rab5c(Q80L), HIP1R-Vps9, or Epsin1-Vps9, rescued Rab5c-positive early endosome formation in DKO cells (Supplementary Fig. 7d).

We utilized OCRL and Sac2, two proteins recruited to uncoated endocytic carriers and endosomes 35, 36, to direct the Vps9 domain to uncoated endocytic carriers. Similar to OCRL, the OCRL-Vps9 and OCRL-Vps9(D313A) chimeras were recruited to uncoated endocytic carriers (Supplementary Fig. 7e). Remarkably, Halo-tagged OCRL-Vps9, but not OCRL-Vps9(D313A), effectively restored the recruitment of EGFP-Rab5c to uncoated endocytic carriers (Fig. 4c) and the formation of early endosomes in the DKO cells (Fig. 4d,e). The Vps9 domain alone exhibited a cytosolic distribution and could not rescue Rab5c recruitment or early endosome formation (Fig. 3d, 4e). Similarly, Sac2-Vps9 and Sac2-Vps9(D313A) chimeras were recruited to uncoated endocytic carriers (Supplementary Fig. 7f). Expression of Sac2-Vps9, but not Sac2-Vps9(D313A), restored Rab5c recruitment to uncoated endocytic carriers (Supplementary Fig. 7g) and early endosome formation in the DKO cells (Fig. 4d,e). These unexpected results collectively demonstrate that targeting the Vps9 domain to clathrin-coated pits is insufficient to induce Rab5 recruitment.

In cells overexpressing Rabex5, a small amount of ectopically expressed full-length Rabex5 was recruited to clathrin-coated pits. However, endogenous Rab5 remained absent from clathrin-coated pits containing ectopically expressed Rabex5 (Supplementary Fig. 8a,b). Furthermore, targeting more ectopically expressed Rabex5 to clathrin-coated pits using the HIP1R-Halo-Rabex5 chimera still failed to recruit endogenous Rab5c to these sites (Fig. 4f). In contrast, endogenous Rabaptin5 was efficiently recruited to clathrin-coated pits by HIP1R-Halo-Rabex5 (Fig. 4g). In addition to localizing at clathrin-coated pits, HIP1R-Halo-Rabex5 was also found at intracellular carriers, where it restored Rab5-positive early endosome formation in DKO cells (Fig. 4f). These findings indicate that, similar to its Vps9 domain, full-length Rabex5 is not sufficient to induce Rab5 recruitment to clathrin-coated pits.

Targeting Rabex5 or its Vps9 domain to various organelle membranes, but not the plasma membrane, induces Rab5 recruitment

The above results suggest that Rabex5 or its Vps9 domain may not be the sole determinant for the spatial targeting of Rab5. To further investigate this hypothesis, we generated a series of chimeras to redirect the Vps9 domain or full-length Rabex5 to mitochondria, the ER, the Golgi, or the plasma membrane. Consistent with previous observations 20, we found that directing Rabex5 or its Vps9 domain to mitochondria, as well as to the ER and Golgi, effectively recruited endogenous Rab5c to the membrane of these organelles in cells with or without Rabex5/hRME6-DKO (Fig. 5a-c and Supplementary Fig. 8c-e).

Unexpectedly, redirecting the Vps9 domain of Rabex5 to the plasma membrane using the C-terminal membrane targeting sequence of H-Ras (containing two cysteine palmitoylation sites and one CAAX motif, abbreviated as CAAX) 37, did not lead to the recruitment of endogenous Rab5 to the plasma membrane (Fig. 5d). Similarly, targeting full-length Rabex5 to the plasma membrane by CAAX also failed to recruit Rab5 to the plasma membrane (Fig. 5e). Vps9-Halo-CAAX and Rabex5-Halo-CAAX were also localized to intracellular endosomal carriers, where they induced the recruitment of Rab5 in the DKO cells (Fig. 5d,e). Collectively, these results demonstrate that while the Vps9 domain or full-length Rabex5 can effectively target Rab5 to various intracellular organelles, they are insufficient for recruiting Rab5 to the plasma membrane.

The Vps9 domain of Rabex5 interacts with and recruits GDP-bound Rab5

It is established that the GDP-to-GTP switch enables Rab5 to interact with cellular membranes 6, 7. Similar to wild-type Rab5, the constitutively GTP-bound Rab5 was not recruited to the plasma membrane by the Vps9 domain of Rabex5 (Fig. 5f,g). Surprisingly, although the constitutively GDP-bound Rab5 cannot associate with the plasma membrane, it was effectively recruited to the plasma membrane by the mistargeted Vps9 domain (Fig. 5f,g). The Vps9(D313A) mutant could also weakly recruit these GDP-bound Rab5 to the plasma membrane (Fig. 5f,g). Consistent with the observations with the Vps9 domain, mistargeting full-length Rabex5 to the plasma membrane resulted in the recruitment of constitutively GDP-bound but not GTP-bound Rab5 (Supplementary Fig. 8f).

Additionally, although mistargeting the Vps9 domain or Rabex5 to clathrin-coated pits failed to recruit Rab5 to clathrin-coated pits (Fig. 4b,f and Supplementary Fig. 8a), mistargeting the Vps9 domain resulted in the recruitment of constitutively GDP-bound but not GTP-bound Rab5 to clathrin-coated pits (Supplementary Fig. 8g). These results suggest that, compared to the wild-type or constitutively GTP-bound Rab5, the constitutively GDP-bound Rab5 shows a preferential association with the Vps9- or Rabex5-mistargeted plasma membrane. Indeed, immunoprecipitation analysis confirmed that the Vps9 domain interacts with Rab5, exhibiting markedly stronger binding with the constitutively GDP-bound Rab5 (Fig. 5h). The Vps9(D313A) mutant also showed weak binding with the constitutively GDP-bound Rab5 (Fig. 5h).

PI(4,5)P2 inhibits the recruitment of Rab5 to the plasma membrane in cells

The unexpected finding that Rabex5 or its Vps9 domain was unable to recruit endogenous Rab5 or the constitutively GTP-bound Rab5 to the plasma membrane prompted us to investigate additional factors involved in regulating the membrane association of Rab5. Such factor(s) are likely specific to the plasma membrane and may interfere with Rab5 binding or activation. GTPase activating proteins (GAPs) catalyze the conversion of Rab5 from its active GTP-bound form to its inactive GDP-bound form 7. Of the four putative Rab5 GAPs identified in previous studies (RabGAP5, RN-tre, TBC1D17, and TBC1D18), only RN-tre was localized to the plasma membrane, where it was enriched at focal adhesions but absent from clathrin-coated pits 38-41 (Supplementary Fig. 9a,b). Knockdown of RN-tre did not lead to Rab5 recruitment to the Vps9- or Rabex5-mistargeted plasma membrane (Supplementary Fig. 9c). Therefore, the absence of Rab5 recruitment to the Vps9- or Rabex5-mistargeted plasma membrane is less likely related to GAP proteins.

In addition to Rab GTPases, phosphoinositides—a group of signaling phospholipids selectively distributed in various cellular membranes—are another key factor in determining organelle identity and regulating membrane trafficking 42. Among them, PI(4,5)P2 is the most abundant species that is primarily located in the inner leaflet of the plasma membrane 43. We hypothesize that PI(4,5)P2 may inhibit Rab5 recruitment to the plasma membrane, because it is known to be depleted following the budding of endocytic vesicles, a process mediated by the activities of inositol 5-phosphatases 15, 44-46. To test this hypothesis, we first verified that Rab5 recruitment occurred following PI(4,5)P2 depletion during clathrin-mediated endocytosis (Supplementary Fig. 10a). We then assessed whether endogenous Rab5 could be recruited to the Vps9-mistargeted plasma membrane after acute PI(4,5)P2 depletion, which was achieved by rapamycin-induced recruitment of the inositol 5-phosphatase domain of OCRL from the cytosol to the plasma membrane 47. Remarkably, endogenous Rab5c was rapidly recruited to the Vps9-targeted plasma membrane following PI(4,5)P2 depletion (Fig. 6a and Supplementary Movie 5). In contrast, recruitment of the inositol 5-phosphatase domain of the OCRL mutant OCRL(D523G)—which lacks phosphatase activity—had no effect on Rab5 recruitment (Fig. 6b). Cells expressing Vps9(D313A)-Halo-CAAX (Fig. 6c) or Halo-CAAX (Fig. 6d) did not support Rab5 recruitment to the PI(4,5)P2-depleted plasma membrane. Furthermore, we confirmed Rab5 recruitment to the PI(4,5)P2-depleted plasma membrane by the mistargeted full-length Rabex5 (Supplementary Fig. 10b). These results indicate that the recruitment of Rab5c to the plasma membrane depends on both the presence of an active GEF domain and the depletion of PI(4,5)P2.

The dephosphorylation of PI(4,5)P2 by inositol 5-phosphatases generates PI4P. To confirm that it is the depletion PI(4,5)P2, rather than the generation of PI4P, that regulates Rab5 recruitment, we depleted PI4P using the catalytic domain of the S. cerevisiae phosphatase Sac1, and simultaneously depleted both PI(4,5)P2 and PI4P using an OCRL-Sac1 chimera 48. As observed with the depletion of PI(4,5)P2 alone, the simultaneous depletion of PI(4,5)P2 and PI4P resulted in the recruitment of Rab5 to the Vps9-mistargeted plasma membrane (Supplementary Fig. 10c). In contrast, acute depletion of PI4P alone did not affect Rab5 recruitment (Fig. 6e). Additionally, acute depletion of PI(3,4)P2 and PI(3,4,5)P3, two other phosphoinositide species present at the plasma membrane 42, 49, did not result in Rab5 recruitment to the Vps9-mistargeted plasma membrane (Fig. 6f and Supplementary Fig. 10d). These findings suggest that PI(4,5)P2 specifically modulates Rab5 recruitment. Furthermore, we confirmed that PI(4,5)P2 depletion did not lead to the recruitment of Rabex5 to the plasma membrane (Supplementary Fig. 10e), indicating that PI(4,5)P2 does not regulate Rabex5 recruitment.

PI(4,5)P2 inhibits Rab5 recruitment to membranes of PI(4,5)P2-containing liposomes

The inhibitory effect of PI(4,5)P2 on Rab5 recruitment may be direct or through the inhibition of the GEF activity of Rabex5. To distinguish between these possibilities, we devised an in vitro assay. To mimic the cellular conditions under which Rabex5 is targeted to cellular membrane, the purified Vps9 domain of Rabex5 was conjugated to liposomes containing various phosphoinositide compositions. The prenylated Rab5c was purified from membrane fraction of HEK293S GnTI− cells. The GEF activity of Vps9 was examined by incubating it with GDP-loaded Rab5c and GTPγS. After the reaction, the samples were subject to a liposome flotation assay to measure Rab5 binding to liposomes. Our results showed that the liposome-bound Vps9 exhibited GEF activity, efficiently promoting the release of GDP from Rab5c (Fig. 6g). Notably, the presence of PI(4,5)P2, PI(3,4)P2, or PI4P did not affect the GEF activity of the Vps9 domain (Fig. 6g). However, while the incorporation of PI(4,5)P2 into liposomes did not affect the flotation of Vps9-containing liposomes, it markedly reduced the binding of Rab5c to the floated liposome membranes (Fig. 6h and Supplementary Fig. 10f). In contrast, the addition of PI(3,4)P2 or PI4P did not affect Rab5 binding to liposome membranes (Fig. 6h). Therefore, using this in vitro system with minimal components, we demonstrated that PI(4,5)P2 negatively regulates the membrane association of Rab5.

Defective PI(4,5)P2 hydrolysis reduces early endosome formation in Lowe syndrome cells

OCRL is an inositol 5-phosphatase that is recruited to nascent endocytic carriers and early endosomes 35, 36. Mutations in the OCRL gene cause Lowe syndrome, a rare X-linked multisystemic disorder 50. OCRL deficiency impairs PI(4,5)P2 hydrolysis, leading to its abnormal accumulation on early endosomes 51, 52. We therefore hypothesized that the intracellular accumulation of PI(4,5)P2 may hinder Rab5 recruitment, thereby reducing the formation of EEA1-positive early endosomes. Due to the lack of reliable antibodies for Rab5 immunostaining, we stained for endogenous EEA1 in de-identified Lowe syndrome patient fibroblasts and control fibroblasts. We observed a significant reduction in the number of EEA1-positive early endosomes in Lowe syndrome cells (Fig. 7a), while the overall EEA1 protein expression remained unchanged (Fig. 7b). Moreover, the transient overexpression of OCRL increased the number of EEA1-positive early endosomes in Lowe syndrome cells (Supplementary Fig. 10g). These results indicate that timely depletion of PI(4,5)P2 from nascent endocytic carriers is important for early endosome formation.

Methods
Cell culture and chemical treatment

SUM159 cells 60 were cultured at 37°C and 5% CO2 in DMEM/F12 (Gibco), supplemented with 5% FBS (Gibco), 100 U/mL penicillin and streptomycin (Corning), 5 μg/mL insulin (Sigma-Aldrich), and 1 μg/mL hydrocortisone (Sigma-Aldrich), pH 7.4. HeLa cells (CCL-2, ATCC), de-identified Lowe syndrome patient fibroblasts and control fibroblasts 46 were cultured at 37°C and 5% CO2 in DMEM (Corning), supplemented with 10% FBS, and 100 U/mL penicillin and streptomycin. Cells were verified to be mycoplasma-free using the TransDetect PCR Mycoplasma Detection Kit (TransGen Biotech). Rapamycin (Merck Millipore, 553210-100UG, 0.5 or 1 μM) was added to cells during continuous imaging using a syringe pump (Harvard Apparatus).

Plasmids and transfection

The DNA sequences encoding human Rab5a, Rab5b, or Rab5c were amplified by PCR from cDNA clones and inserted into a vector containing mEGFP to generate the plasmids mEGFP-Rab5a, mEGFP-Rab5b, or mEGFP-Rab5c using the Gibson assembly method (pEASY-Uni Seamless Cloning and Assembly Kit, TransGen Biotech). The DNA sequences encoding human Rabex5 (NM_014504.3) or hRME6 (NM_015635.4) were amplified by PCR from the cDNA of HEK293 cells and inserted into a vector containing mEGFP to generate the plasmids Rabex5-mEGFP or mEGFP-hRME6. Rabex5-Halo or Halo-hRME6 were generated by replacing the coding sequence of mEGFP in plasmid Rabex5-mEGFP or mEGFP-hRME6 with the coding sequence of HaloTag. The DNA sequences encoding different domains of Rabex5 or hRME6 were amplified by PCR from Rabex5-mEGFP or mEGFP-hRME6 and then inserted into a vector containing mEGFP to generate the mEGFP-tagged truncated Rabex5 or hRME6. A flexible (GGS)3 or (GGS)5 linker was inserted between mEGFP/HaloTag and the protein-coding cDNA fragments. The DNA sequences encoding HIP1R, OCRL, Epsin1, Sac2, the PTEN and clathrin-binding domains of GAK 17, Sec61β, Golgi84, or the yeast mitochondrial targeting sequence (Mito) 61 were amplified by PCR from the corresponding expression vectors to construct the Halo-tagged constructs using the Gibson assembly method.

The plasmids LYNA11-FRB-ECFP (#38003), mCherry-FKBP-INPP4B (#116864), NES-EGFP-cPHx3 (#116855), and mCherry-FKBP-PTEN (#116866) were obtained from Addgene. mCherry-FKBP-ptaseOCRL, mCherry-FKBP-ptaseOCRL(D523G), mCherry-FKBP-Sac1, and mCherry-FKBP-Sac1(C392S) were generated by replacing PTEN in mCherry-FKBP-PTEN (#116866) with the 5-phosphatase domain of OCRL from mCherry-CRY2-5ptaseOCRL (Addgene, #66836), the D523G mutant of the 5-phosphatase domain of OCRL from mCherry-CRY2-5ptaseOCRL(D523G) (Addgene, #66837), Sac1 from PJ-Sac (Addgene, #38000), or Sac1(C392S) from PJ-DEAD (Addgene, #38002). Plasmid transfection was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.

Generation of knock-in cells using the CRISPR/Cas9 approach

Cells were gene-edited to incorporate EGFP at the N-terminus of Rab5b or Rab5c, mEGFP at the N-terminus of hRME6 or Rabaptin5, and mEGFP at the C-terminus of Rabex5 using the CRISPR/Cas9 approach as described 15, 62. Donor constructs used for homologous recombination were generated by cloning into the pUC19 vector with two ∼600-800-nucleotide fragments of genomic DNA upstream and downstream of the start codon of human RAB5B, RAB5C, GAPVD1, and RABEP1, or the stop codon of RABGEF1, and the open reading frame of EGFP/mEGFP/mScarlet-I using the pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech). A flexible (GGS)3 linker was inserted between the start or stop codon of the gene and the open reading frame of EGFP/mEGFP/mScarlet-I.

Single-guide RNA (sgRNA) targeting human RAB5B (5’-CCTTTCCCATTCTGATAATC-3’), human RAB5C (5’-TGGACGGGCAATGGCGGGTC-3’), human GAPVD1 (encoding hRME6) (5’-AGCCTTTCCCACATTGAAGA-3’), human RABEP1 (encoding Rabaptin5) (5’-CCGCCTGGTCATGGCGCAGC-3’), or human RABGEF1 (encoding Rabex5) (5’-GATCATCCTGCATAAACTTG-3’) was delivered as a PCR amplicon containing a PCR-amplified U6-driven sgRNA expression cassette 62. Cells were transfected with the donor plasmid and the PCR amplicon using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Cells expressing EGFP/mEGFP were enriched by fluorescence-activated cell sorting (FACS) 5-7 days after transfection (FACSAria II or FACSAria Fusion, BD Biosciences). The enriched cells were expanded and then subjected to single-cell sorting into 96-well plates. The genome-edited clonal cells were characterized by PCR, imaging, and western blot analysis.

Generation of knockout cells using the CRISPR/Cas9 approach

Knockout of Rabex5, hRME6, or Rabaptin5 expression was performed using the CRISPR/Cas9 approach as described 62. The sgRNA targeting human RABGEF1 (encoding Rabex5) (5’-AGCCTTAAGTCTGAACGCCG-3’), human GAPVD1 (encoding hRME6) (5’-GAAACTAGATATTCATACTC-3’), or human RABEP1 (encoding Rabaptin5) (5’-TCAGCAACGGGTAGCAGAAT-3‘) was cloned into the lentiviral vector (LentiCRISPR) encoding Cas9 and the sgRNA targeting sequence 63. The cells were infected with the lentivirus and then incubated with puromycin (2 μg/ml) for 2-3 days. The surviving cells were plated into 96-well plates and cultured for another 2-3 weeks. The monoclonal cell populations with mutations in both alleles of Rabex5, hRME6, or Rabaptin5 were identified by sequencing, and the loss of Rabex5, hRME6, or Rabaptin5 protein expression was confirmed by western blot.

To generate the Rabex5 and hRME6 double knockout cell lines, the sgRNA targeting human RABGEF1 or human GAPVD1 was cloned into pSpCas9(BB)-2A-Halo. The Rabex5-KO cells were transfected with the constructed plasmid for 24-36 h, stained with JFX650-HaloTag ligand, and then subjected to single-cell sorting into 96-well plates (FACSAria Fusion, BD Biosciences). The monoclonal cell populations with hRME6 knockout were identified by sequencing and western blot.

Knockdown of Rabex5, hRME6, and Rabaptin5 using siRNA

The siRNA sequences used to knock down the expression of Rabex5, hRME6, and Rabaptin5 were 5’-AAATTAAGCCTCCGAATCA-3’, 5’-TTAGAATAGTGGTGCGTTA-3’, and 5’-GTAGTATGCTGTATGAATA-3’ respectively. Rabex5, hRME6, and Rabaptin5 knockdown was achieved through two sequential transfections using Lipofectamine RNAiMAX (Invitrogen) as described 15. The first transfection, second transfection, and imaging were performed on day one (after overnight plating), day three, and day five, respectively.

Knockdown of RN-tre using shRNA

Lentivirus shRNA containing the target sequence 5’-GTAGATAGTCCCGTGAGATAT-3’ was utilized to knock down the expression of RN-tre. The shRNA was generated by cloning the target sequence into the pLKO.1-TRC cloning vector (Addgene, #10878). The scramble shRNA (Addgene, #1864) was used as a control. shRNA expressing lentivirus was produced in HEK293FT cells. The supernatant containing lentivirus was harvested 48 hours after transfection and concentrated 5-fold (Biogeek, #BG20101L) prior to infection of EGFP-Rab5c+/+ cells. After 24 hours, the cells were replaced with fresh medium containing 2 μg/mL puromycin (InvivoGen). After another 96 hours, the cells were subjected to live-cell imaging and q-PCR analysis.

Live-cell TIRF imaging and imaging analysis

SUM159 cells plated overnight on single-well, 4-well, or 8-well confocal dishes (Cellvis) were imaged in phenol-free DMEM/F12 (Corning) containing 5% FBS and 20 mM HEPES. HeLa cells were imaged in FluoroBrite DMEM (Thermo Fisher Scientific) containing 10% FBS and 20 mM HEPES. The TIRF microscopes used for live-cell imaging were built on a Nikon Ti2-E microscope or a Nikon TiE microscope as described 64. The Nikon Ti2-E microscope was equipped with a manual TIRF Illuminator Unit (Nikon), a CFI Apochromat TIRF 100X objective (1.49 NA, Nikon), a Perfect Focus Unit (Nikon), a UNO Stage Top Incubator (Okolab), OBIS CellX lasers (405, 488, 561 and 637 nm, Coherent), the W-VIEW GEMINI-2C Image Splitting Optics (Hamamatsu), and two EMCCD cameras (Evolve 512 Delta, Photometrics). The 2× tube lens was applied to achieve the final pixel size corresponding to 80 nm of the image. Images were acquired using Micro-Manager 1.4 65. The Nikon TiE microscope was equipped with a motorized TIRF Illuminator Unit (Nikon), a CFI Apochromat TIRF 100X objective (1.49 NA, Nikon), a Perfect Focus Unit (Nikon), a Motorized XY stage (Prior Scientific), a fully enclosed and environmentally controlled cage incubator (Okolab), OBIS 488, 561 and 647 nm lasers (Coherent), W-VIEW GEMINI Image splitting optics (Hamamatsu), and an EMCCD camera (iXon Life 888, Andor Technology). The 1.5× tube lens was applied to achieve the final pixel size corresponding to 86.7 nm of the image. Images were acquired using Micro-Manager 2.0 65. For single-molecular imaging of EGFP/mEGFP-tagged proteins, the 488 nm laser power measured at the objective exit in epi-illumination mode was 1.5 mW. The exposure time was 100 ms per channel, and the EM gain of the EMCCD camera was set to 280 (Evolve 512 Delta) or 300 (iXon Life 888). The imaging interval and duration for each experiment are reported in the respective figure legends. Montages and merged images were generated using Fiji 66. Kymographs were created using the Multi Kymograph plugin in Fiji.

The automated detection and tracking of the recruitment of Rab5 and other proteins during clathrin-mediated endocytosis were performed using the cmeAnalysis software package 67 (https://github.com/DanuserLab/cmeAnalysis) in MATLAB (MathWorks), with clathrin as the “master” and other proteins as the “slave” channel. The minimum and maximum tracking search radii were 1 and 3 pixels, and the maximum gap length in a trajectory was 2 frames. The start buffer and end buffer for the valid tracks were 5 and 10 frames. Valid tracks from single clathrin-coated pits were used for further analysis. Persistent tracks and large clathrin structures (‘plaques’) 68, 69 were excluded from this analysis. The intensity-lifetime cohorts were generated using the cmeAnalysis software package.

To compare the relative timing of GAK and Rab5a/b/c recruitment during clathrin uncoating, the tracks with more than three positive frames in the slave channels were analyzed. The timing of GAK signal appearance was used as the reference point for aligning different tracks. The 10 frames before and after GAK appearance from multiple tracks were averaged and then plotted as heatmaps. To compare the relative appearance time of GAK and Rab5a/b/c in each endocytic event, the appearance times of GAK or Rab5 were divided by the length of the track and then plotted as density scatter plots. The length of the track was determined by the lifetime of clathrin plus the end buffer (10 frames). The timing of GAK or Rab5 appearance was determined by the frame at which the fluorescent intensity of GAK or Rab5 was statistically higher than the local background by the cmeAnalysis software package.

Live-cell spinning-disk confocal imaging and imaging analysis

The spinning-disk confocal microscope was built on the Nikon TiE microscope described above. The microscope was equipped with a CSU-X1 spinning-disk confocal unit (Yokogawa) and an EMCCD camera (iXon Ultra 897, Andor Technology), positioned on the left side port of the microscope.

To quantify the number of Rab5-positive early endosomes in individual cells, the EGFP-Rab5c+/+ clathrin-TagRFP+/+ cells (with or without Rabex5 or/and hRME6 expression) were cultured overnight in confocal dishes (Cellvis), and then imaged from the bottom to the top surfaces of the cells (planes spaced at 0.35 μm) using the spinning-disk confocal microscope. The cell boundaries were manually segmented based on cell fluorescence in Fiji. A maximum-intensity Z projection from four sequential optical sections (starting from the bottom surface) of each cell was generated in Fiji. The projected and binary cell boundary images were loaded into CellProfiler 4 70 (http://www.cellprofiler.org) and the number of spots within the cell boundaries was identified automatically. The results were then exported from Cellprofiler and plotted in GraphPad Prism 9.

To generate the intensity profile of EGFP-Rab5 at and around the plasma membrane, a straight line (31-pixel width) perpendicular to the local plasma membrane was drawn manually. The intensity profiles of both EGFP-Rab5 and Halo-tagged CAAX along this line were measured using Fiji. The position of the cell membrane was defined by the maximum fluorescent signal of CAAX. To determine the background signals for Rab5 and CAAX channels, the average intensity of 21 continuous pixels along this line, positioned 15 pixels outside the cell and away from the cell membrane, was computed. Subsequently, the fluorescence intensity of Rab5 and CAAX for each cell was adjusted by subtracting their corresponding background values and then normalized to their respective maximum intensity. The background-subtracted and normalized intensity profiles from each cell were aligned based on the maximum fluorescent signal of CAAX (set as 0 μm) and then plotted.

To measure the relative enrichment of EGFP-Rab5 at the plasma membrane, the intensity of EGFP-Rab5 at the plasma membrane was determined by averaging the 3 pixels (before, on, after the maximum signal of CAAX) of EGFP-Rab5 (Fmembrane). The intensity of EGFP-Rab5 in the cytosol was determined by averaging 21 continuous pixels inside the cell, positioned 15 pixels away from the cell membrane (Fcytoplasm). Subsequently, the intensity ratio (Fmembrane/Fcytoplasm) was calculated for each cell and then plotted.

Immunofluorescence

Human fibroblasts were cultured overnight on coverslips (NEST, 801010) precoated with poly-D-Lysine (Sigma, P1149-10MG). Subsequently, cells were fixed using 4% paraformaldehyde (Electron Microscopy Sciences, 157-8) and permeabilized with 0.5% Triton X-100 in PBS. After incubation in blocking buffer (1% BSA in PBS) for 1 h at room temperature, the samples were incubated with primary antibodies against EEA1 (BD Biosciences, 610456, 1:400 in 1% BSA) overnight at 4˚C. Following five washes with PBS, the samples were incubated with Alexa Fluor 488-conjugated secondary antibodies or Alexa Fluor 555-conjugated secondary antibodies (Thermo Fisher Scientific, 1:1000 in 1% BSA) for 1 h at room temperature. Then the samples were imaged using the spinning-disk confocal microscope described above. The EEA1-positive endosomes were identified using the TrackMate plugin in Fiji.

Assessment of transferrin and EGF uptake by flow cytometry

Transferrin and EGF uptake were assessed as described 17, 71. In brief, SUM159 cells were cultured overnight in 12-well plates, starved for 1 h in DMEM/F12 (Corning), and then incubated with 5 µg/mL Alexa Fluor 647–conjugated transferrin or 100 ng/mL Alexa Fluor 555–conjugated EGF (Life Technologies) in pre-warmed α-MEM (Corning) at 37°C for 10 min. Then the plates were either washed with ice-chilled PBS and transferred to ice or washed three times with pre-warmed DMEM/F12. Plates washed with DMEM/F12 were replenished with pre-warmed complete culture medium and placed in the incubator for an additional 10, 20, or 30 min. Following the incubation, the cells were washed with ice-chilled PBS. Surface-bound Alexa Fluor 647–conjugated transferrin or Alexa Fluor 555–conjugated EGF was removed through two brief incubations with acid wash medium (150 mM NaCl, 0.125 mM CaCl2, 1 mM MgCl2, and 0.1 M glycine, pH 2.5). The cells were then detached using 0.25% Trypsin and fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were subsequently washed and suspended in PBS. The amount of internalized Alexa Fluor 647–conjugated transferrin or Alexa Fluor 555–conjugated EGF was determined by flow cytometry (FACSAria Fusion, BD Biosciences).

Expression and purification of proteins

To purify prenylated Rab5c, the DNA sequence encoding human Rab5c was cloned into the pEG BacMam vector, with a 10× His tag, EGFP tag, and a PreScission protease recognition site fused to the N-terminus of Rab5c. After transfection, the HEK293S GnTI– cells were cultured at 37°C for 10~12 h, followed by an additional 48 h at 30 °C in the presence of 10 mM sodium butyrate. Cells were harvested by centrifugation at 9,280 g for 20 min and stored at –80°C. The cell pellets were resuspended in Buffer A (50 mM HEPES, 150 mM NaCl, 15% glycerol, 2 μg/mL DNase, 1 mM DTT, 1 mM MgCl2, 1 mM GTP, and a cocktail of protease inhibitors, pH 7.64), sonicated on ice, and centrifuged at 20,000 g for 80 min at 4°C. The pellets were then suspended in Buffer A with the addition of 1% n-dodecyl-β-D-maltoside (DDM) and 0.1% cholesteryl hemisuccinate (CHS) for 2 h at 4°C. Cell debris was removed by centrifugation at 38,900 g for 50 min at 4°C. The supernatants were incubated with anti-GFP nanobody-coupled beads for 2 h at 4°C. The beads were washed with 50 column volumes of Buffer B (25 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM MgCl2, and 0.02% DDM-0.002% CHS, pH 7.64) and then incubated with PreScission protease (4:1 w/w ratio) at 4 °C overnight. The released Rab5c was mixed with GST beads at 4 °C for 1 h to remove the PreScission protease, and then eluted with 4 column volumes of Buffer B. The protein was further purified by size exclusion chromatography using a Superose 6 Increase 10/300 GL column (GE Healthcare, 29-0915-96).

To purify 6x His tagged Vps9, the DNA sequence encoding the Vps9 domain of human Rabex5 was cloned into a pET-28a vector, with an N-terminal 6x His tag. The plasmid was transformed into E. coli strain BL21 (Transgene, #CD601). Protein expression was induced with 0.3 mM isopropyl β-D-1-thiogalactoside when the culture reached an OD600 of 0.6, followed by overnight incubation at 16°C. The bacterial pellet was resuspended in 25 mL of Buffer D (50 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 5 mM β-mercaptoethanol, pH 8.0) supplemented with 10 mM imidazole and 1 mM PMSF, lysed by sonication for 10 min, and centrifuged at 18,000 g for 30 min at 4°C. The supernatant was incubated with 500 µL of nickel-NTA agarose beads (Sigma, #H0537) for 2 h at 4°C. The beads were washed three times with 30 mL of Buffer D containing 30 mM imidazole. His-Vps9 was eluted in 2.5 mL of Buffer D containing 250 mM imidazole and then exchanged into 3.5 mL of storage buffer (20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, pH 7.2) using PD-10 desalting columns (GE healthcare, #17085101).

GEF assay

For GEF assay and liposome flotation assay, POPC (#850457), POPS (#840034), DGS-NTA(Ni) (#790404), PI(4,5)P2 (#850155), PI(3,4)P2 (#850153), and PI4P (#850151) were purchased from Avanti Polar Lipids. POPC and POPS were used to mimic the lipid composition of the plasma membrane 72. All lipids were stored in chloroform at -20°C. The lipids were mixed in a composition of 73% POPC, 15% POPS, 10% DGS-NTA(Ni), and different phosphoinositides. The lipid mixture was evaporated under nitrogen gas and then placed in a SpeedVac for an additional hour. The dried lipids were resuspended in 100 µL of GEF buffer (20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, pH 7.5) to a final concentration of 5 mM and subjected to 11 freeze-thaw cycles. Small unilamellar liposomes were generated by extruding the mixture through 100 nm polycarbonate membranes (Avanti Polar Lipids, #610005) 11 times. The liposomes were aliquoted, snap-frozen in liquid nitrogen, and stored at -80°C.

The GEF activity of Vps9 was assessed using N-Methylanthraniloyl-GDP (MANT-GDP) (Sigma, #69244). To load MANT-GDP onto Rab5c, 70 µL of purified Rab5c (1.8 µg/µL) was exchanged into 70 µL of GEF buffer using spin desalting columns (Thermo Fisher Scientific, #89883). This was then mixed with an equal volume of 2× MANT-GDP loading buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM MgCl2, 10 mM EDTA, 2 mM DTT, and a 20-fold molar excess of MANT-GDP, pH 7.5) and incubated at 20°C for 90 min in the dark. The reaction was terminated by adding 1 M MgCl2 to a final concentration of 10 mM, followed by further incubation for 30 min. Subsequently, 130 µL of MANT-GDP-loaded Rab5c was exchanged into 130 µL of GEF buffer using spin desalting columns.

To prepare Vps9-liposomes, 0.25 µM of His-Vps9 was conjugated to 2.5 mM liposomes via His-NTA interaction in GEF buffer at 4°C for 1 h. A mixture containing MANT-GDP-loaded Rab5c and Vps9-liposomes in GEF buffer was transferred into a 96-well microplate (Thermo Fisher Scientific, #237108). The concentrations in the 50 µL reaction were 5 µM for MANT-GDP-loaded Rab5c, 1 mM for liposomes, and 100 nM for His-Vps9. Fluorescence measurements were performed using a microplate spectrometer (TECAN), with an excitation wavelength of 360 nm and an emission wavelength of 440 nm. The reaction was initiated by adding 3 µL of 8.3 mM GTPγS (final concentration of 500 µM) once the initial fluorescent signal stabilized. Measurements were recorded every 15 s at room temperature. The fluorescence intensity over time was normalized by the mean intensity before GTPγS addition.

Liposome flotation assay

Following the GEF assay, the reaction samples were subjected to the liposome flotation assay. Briefly, 30 µL of the sample was transferred to a polycarbonate tube (Beckman, #343775), mixed with 100 L of 1.9 M sucrose at the bottom of the tube, and overlaid sequentially with 100 L of 1.25 M sucrose and 20 L of GEF buffer. Liposomes floated to the top after ultracentrifugation at 174,000 g for 1 h. Five 50 µL fractions were collected from the top, mixed with SDS loading buffer, and analyzed by Western blot using antibodies against Rab5c (Proteintech, 27219-1-AP, 1:10000).

Western blot analysis

Western blot analysis was performed as described 15. Briefly, cells were lysed at 4˚C for 20 min in RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail (Thermo Fisher Scientific, 78442), and then pelleted at 12,000 g for 15 min at 4˚C. The supernatant was mixed with 5× sample buffer (GenScript, MB01015), heated to 100˚C for 8-10 min, fractionated by SDS-PAGE, and then transferred to nitrocellulose membranes (PALL, 66485). The membranes were incubated in TBST buffer containing 5% skim milk for 1 h at room temperature, followed by overnight incubation at 4˚C with specific primary antibodies. After three washes in TBST (5 min each), the membranes were incubated with the appropriate HRP-conjugated secondary antibody (Beyotime, A0208 or A0216, 1:1,000) for 1 h at room temperature. The membranes were incubated with the SignalFireTM ECL Reagent (Cell Signaling, 6883S) or BeyoECL Moon (Beyotime, P0018FS) and imaged using the Tanon-5200 chemiluminescent imaging system (Tanon) or MiniChemi 610 chemiluminescent imaging system (Sage Creation Science). The primary antibodies used in this study were: GFP (Invitrogen, 14-6674-82, 1:1,000), EEA1 (BD Biosciences, 610456, 1:1,000), GAPDH (Proteintech, 60004-1-Ig, 1:50,000), Rabex5 (Santa Cruz, sc-166611, 1:500), RME6/Gapex-5 (Abcam, ab86033, 1:1,000), Rab5a (Proteintech, 11947-1-AP, 1:1,000), Rab5b (Proteintech, 27403-1-AP, 1:1,000), Rab5c (Proteintech, 27219-1-AP, 1:1,000), Rabaptin5 (Proteintech, 14350-1-AP, 1:1,000), CHC (Abcam, ab21679, 1:1,000), ALS2CL (Santa Cruz, sc-377278, 1:500), OCRL (Proteintech, 17695-1-AP, 1:1,000), and His (MBL Life science, D291-3S, 1:1000).

Co-immunoprecipitation

SUM159 cells were transfected with the specified mEGFP- and Halo-tagged plasmids for 24 h, and then lysed in lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific, 78442) on ice for 30 min. Then the cells were centrifuged at 20,000 g for 15 min at 4℃. The supernatants were collected and incubated with GFP-Nanoab-Agarose beads (Lablead, GNA-25-500) with gentle rotation for 2 h at 4℃. Then the beads were washed five times with lysis buffer, and the bound proteins were eluted using 2× SDS sample buffer, heated to 100˚C for 10 min. The samples were then subjected to immunoblot analysis as described above.

Statistical tests

Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software). The statistical methods used were specified in the figure legends. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001; ns: no significant difference. P < 0.05 was considered statistically significant. No statistical methods were used to predetermine sample sizes.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors address the mechanisms by which the small GTPase Rab5 is targeted to endocytosing membranes resulting in the formation of early endosomes. To this aim, the authors use sets of genetically engineered cell lines expressing tagged variants of Rab5 isoforms, various GTP-exchange factors of Rab5 and additional proteins involved in the formation and uncoating of clathrin-coated vesicles to not only arrive at a precise sequence of recruiting but also to pinpoint the recruiting mechanisms. Using advanced and automated time-lapse imaging, the authors not only show that the two GEFs Rabex and hRM6 cooperate to recruit Rab5 immediately after uncoating of CCVs but also show that the recruitment is impaired if phosphatidylinositol (4,5)bisphosphate, a phosphoinositide highly enriched in plasma membranes, is present on the membrane.\nThis is a high-quality manuscript that, while confirming many previous observations in the mature Rab5 field, clearly goes beyond the state of the art (as far as I can judge), resulting in a coherent and convincing picture of the recruiting mechanisms with many novel elements. The manuscript contains an impressive amount of data, with hundreds of individual experiments in the main and extended figures. While the overall logical flow is perfectly clear, the MS is not always easy to read, and in some parts I needed to go through text and the corresponding figures several times to understand the experiments (particularly the figure legends). Thus, the manuscript may benefit from some editing to improve clarity. For instance, the standard type of analysis as shown the first time in Fig. 1 a-c (and in extended Fig. 1), needs to be better explained – what are lifetime cohorts, and what do the overlaid curves represent? Why is it important to look at these different cohorts – are there any differences between endocytic events that retain clathrin for shorter or longer time periods? Also, it would help readability if some of the non-textbook-standard abbreviations can be avoided (EE, UEC, CCP), and perhaps some of the constructs may be given a more intelligible abbreviation (such as CLTA-670nano, which is essentially a tagged clathrin light chain).\nAside from these minor comments I do not have any serious critique – this is a well-reasoned study of excellent quality. I do not see any room for shortening – the data both in the main figures and the extended figures are all required to support the conclusions of the manuscript.\nMinor technical point:\nExtended Fig. 1g: The legend states that there are fluorescence intensity traces of an endocytic event (as in panels d-f), but I could not detect this in the figure.", "reviewer_2": "The manuscript ‘Lipid turnover and GEF recruitment collectively determine Rab5 recruitment and activation during the first step of early endosome formation’ addresses the central question, which cellular components are required to recruit Rab5 to the nascent uncoated endocytic carrier after loss of the clathrin coat. This work sheds light onto the temporal relationship between different components and their catalytic activity in this process. It identifies Rabex5 and hRME6 as pivotal proteins in this process and, perhaps somewhat unexpected, discover that the lipid composition of the Clathrin coated vesicle additionally prevents early recruitment of Rab5. The hydrolysis of PI(4,5)P2 is prerequisite of Rab5 recruitment and provides an additional regulatory level.\nThe authors provide high-quality live cell imaging in combination with state-of-the art image analysis. They also confirm and extend their imaging results with biochemical and in vitro experiments providing a comprehensive view of the interactions leading to Rab5 recruitment. The sheer number of experiments and their associated controls makes the manuscript challenging yet enjoyable to read. The authors carefully discuss their results and acknowledge that, despite their extensive efforts, some details remain to be characterised. Overall, this work is a crucial step in our undertsanding of the endocytic process and therefore highly relevant. This systematic study will certainly be a valuable resource and provides novel and important information on clathrin mediated endocytosis and endosome formation. I would like to congratulate the authors on their detailed characterisation and their work towards a comprehensive model. I hope that the authors will continue to fill in the remaining gaps in future publications.\nI still would like to see a few points be clarified and / or addressed before publication:\n1) It would be valuable information, if there is a correlation between Rab5 recruitment and onset of movement / increased mobility of the vesicle. I would encourage the authors to revisit their TIRF-imaging data along these lines and include this information in the manuscript.\n2) Variation in ‘Clathrin-density’ on the plasma membrane: Clathrin structures larger than a single CCP are visible for example in Movie 1 and Movie 2. These structures display multiple recruitments of Rab5c and Rabex5, respectively. These could resemble stable clathrin structures as described by Merrifield (for example: Merrifield et al, Cell, 2005; Taylor et al, PLos Biol, 2011; Lampe et al., J Struct Biol, 2016). It would be a valuable information, to briefly describe how these regions are treated during the analysis procedure. As the Rab5 recruitment appears after the scission of the vesicle, I would not assume that there should be a difference between budding sites at regions of different clathrin density, but it would be good if the authors could briefly comment on this.\n3) Imaging parameters: The authors should provide more details about their imaging parameters. At least typical settings (like laser power (in kW/cm²), exposure time and major camera settings) as well as typical values for recorded signal intensities should be mentioned supporting the reproducibility of the data. Alternatively or in addition, the authors might want to consider making at least some of their annotated data publicly available via the BioImage Archive or similar databases.\n4) Schematic overview (Figure 7c), especially lower part on VPS9: In this cartoon, the authors summarize the essential findings of their work. Adding additional details and improve the description of the figure in the legend would certainly help readers to cope with the vast amount of information provided in the manuscript. I would especially motivate the authors to re-visit the lower part of the figure, which lacks any detailed description in the figure legend and cannot be understood without having a careful read of the paper.\nThe authors might want to consider amend the figure such, that it can be used as a graphical summary of their work and help guiding readers as well as emphasizing their major findings, e.g. by adding information about catalytic activity etc..\n5) Minor detail / ambiguity - Extended Data Fig. 9. ‘The failure of Rab5 recruitment to the Vps9- or Rabex5-mistargeted to the plasma membrane is not related to GAP proteins.’ Might be changed to (or similar): ‘The failure of Rab5 recruitment to Vps9- or Rabex5, which were mistargeted to the plasma membrane, is not related to GAP proteins’?", "reviewer_3": "The small GTPase Rab5 is well documented for its function in early endosome fusion and endocytosis. This manuscript investigates the molecular mechanisms underlying the spatiotemporal recruitment of Rab5 during early endosome (EE) formation. Using single-molecule microscopy in genome-edited cells, the authors identify two Rab5 guanine-nucleotide exchange factors (GEFs), Rabex5 and hRME6, as key regulators of Rab5 recruitment and activation on nascent uncoated endocytic carriers (UECs). They show that Rabex5 is continuously recruited to UECs, while hRME6 is recruited transiently. Rabex5 knockout significantly reduces Rab5 recruitment and number of EE while Rabex5/hRME6 double knockout essentially abolish this process. In addition, the study makes an novel observation that the plasma membrane–enriched phosphoinositide PI(4,5)P₂ plays a negative role in Rab5 recruitment and prevents its association with the plasma membrane. This finding is important as it may imply a general role of organelle-specific phosphoinositides in membrane targeting and activation of Rab GTPases during vesicular transport. Finally, impaired PI(4,5)P₂ hydrolysis in Lowe syndrome cells leads to defective Rab5 recruitment and EE formation, linking this mechanism to human disease.\nThe manuscript addresses a longstanding question in endocytic trafficking and presents novel insights into the spatial regulation of Rab5 membrane recruitment and activation. However, several experimental issues and broader implications require clarification or additional evidence before publication.\nSpecific Comments:\n1. The manuscript relies heavily on the concept of UECs, but their precise molecular identity is not well established: do they represent a distinct compartment? How are they biochemically different from EE? More evidence is needed to establish this concept including specific protein and lipid markers.\n2. In addition to Rab5, there are multiple Rabs associated with EE among which Rab21 is also a substrate for Rabex5 and should be included as an important positive control in the Rabex5 knockout experiments. Another EE-associated Rab such as Rab4 or Rab22 that does not respond to Rabex5 may serve as a negative control. Along this line, Rabex5/hRME double knockout cells effectively abolish Rab5-positive EE. Are other EEs, e.g., Rab4 EE or Rab22 EE, affected?\n3. In both the Rabex5/hRME knockout and the Lowe syndrome experiments, a rescue control should be included to strengthen authors’ claim.\n4. Previous studies on Rab5 and Rabex5 should be discussed in more detail to place these findings in context. For example, the first subheading in Results “All three isoforms of Rab5…” is not wrong but imprecise and can be misleading, as the new information is actually for Rab5b while Rab5a and Rab5c results reported previously (ref 15). A more appropriate subheading may be “Rab5b, like Rab5a and Rab5c…”. In addition, the section suggesting “The Vps9 domain is essential for Rabex5 recruitment…) is misleading, as Fig 3 shows that the Vps9 domain alone is insufficient to associate with the membrane, although this Vps9 domain construct is not described in the text. This result and the conclusion that neither the N-terminal UBD nor the C-terminal domain including RpBD is necessary for Rabex5 membrane targeting to EE are consistent with a previous paper addressing Rabex5 membrane targeting domain (ref 22). This and another paper on the same topic (ref 23) are cited but not discussed in the context of new findings."}, "author_response": "Response to the reviewers' comments:\nWe would like to extend our sincere gratitude to the reviewers for dedicating their valuable time\nand providing us with insightful comments and valuable suggestions. During the past three\nmonths, we have devoted extensive efforts to conducting a series of new experiments to\ncomprehensively address all reviewers’ concerns. We do believe that the review process was\nhighly constructive, and the suggested experiments, analyses, and discussions have significantly\nstrengthened our conclusions and improved the work.\nThese new or updated results are shown in the revised manuscript, as well as in this\nresponse letter. We have edited the manuscript text to improve clarity, including the removal of\nnon-textbook-standard abbreviations. These modifications are highlighted in red in the revised\nmanuscript. Detailed point-by-point responses to the reviewers’ comments are enumerated below.\nReviewer #1 (Remarks to the Author):\nIn this study, the authors address the mechanisms by which the small GTPase Rab5 is targeted\nto endocytosing membranes resulting in the formation of early endosomes. To this aim, the\nauthors use sets of genetically engineered cell lines expressing tagged variants of Rab5 isoforms,\nvarious GTP-exchange factors of Rab5 and additional proteins involved in the formation and\nuncoating of clathrin-coated vesicles to not only arrive at a precise sequence of recruiting but also\nto pinpoint the recruiting mechanisms. Using advanced and automated time-lapse imaging, the\nauthors not only show that the two GEFs Rabex and hRM6 cooperate to recruit Rab5 immediately\nafter uncoating of CCVs but also show that the recruitment is impaired if phosphatidylinositol\n(4,5)bisphosphate, a phosphoinositide highly enriched in plasma membranes, is present on the\nmembrane.\nThis is a high-quality manuscript that, while confirming many previous observations in the\nmature Rab5 field, clearly goes beyond the state of the art (as far as I can judge), resulting in a\ncoherent and convincing picture of the recruiting mechanisms with many novel elements. The\nmanuscript contains an impressive amount of data, with hundreds of individual experiments in the\nmain and extended figures. While the overall logical flow is perfectly clear, the MS is not always\neasy to read, and in some parts I needed to go through text and the corresponding figures several\ntimes to understand the experiments (particularly the figure legends). Thus, the manuscript may\nbenefit from some editing to improve clarity. For instance, the standard type of analysis as shown\nthe first time in Fig. 1 a-c (and in extended Fig. 1), needs to be better explained – what are lifetime\ncohorts, and what do the overlaid curves represent? Why is it important to look at these different\ncohorts – are there any differences between endocytic events that retain clathrin for shorter or\nlonger time periods? Also, it would help readability if some of the non-textbook-standard\n1\nabbreviations can be avoided (EE, UEC, CCP), and perhaps some of the constructs may be given\na more intelligible abbreviation (such as CLTA-670nano, which is essentially a tagged clathrin\nlight chain).\nAside from these minor comments I do not have any serious critique – this is a well-reasoned\nstudy of excellent quality. I do not see any room for shortening – the data both in the main figures\nand the extended figures are all required to support the conclusions of the manuscript.\nResponse: We are immensely grateful to the reviewer for the precise summary, positive\ncomments, and very helpful suggestions. We apologize for the lack of clarity in our initial\nsubmission and have now extensively edited the manuscript to address your points and improve\noverall clarity.\nSpecifically, we have made the following revisions:\n(1) Following your suggestion, we have provided a detailed description of the lifetime cohorts in\nthe figure legend for Fig. 1a-c: \"Clathrin-mediated endocytic events with clathrin lifetimes between\n20 and 100 s were identified in 31 (a), 26 (b), or 28 (c) cells. To account for the broad distribution\nof lifetimes, events were grouped into cohorts based on specific clathrin-670nano lifetime ranges.\nFor each cohort, the fluorescence intensity traces for clathrin-670nano (blue), EGFP-Rab5\n(green), and mScarlet-I-GAK (red) were averaged and are shown as mean ± SEM (intensity-\nlifetime cohorts). The number of traces analyzed is shown above each cohort.\"\n(2) We have also provided comments on the endocytic events with shorter or longer lifetime in\nthe text as \"Despite the wide range of lifetime distributions of endocytic events, quantitative\nanalysis of the recruitment timing of Rab5a/b/c and GAK in each endocytic vesicle further\nconfirmed that Rab5a/b/c was recruited after the burst-like signal of GAK (Fig. 1a-c).\"\n(3) In the revised manuscript, we have replaced these non-textbook-standard abbreviations (e.g.\nCCP, CCV, UEC, EE) with their full names (clathrin-coated pit, clathrin-coated vesicle, uncoated\nendocytic carrier, early endosome). We have also defined 'uncoated endocytic carriers' within the\ntext and updated the model in Figure 7c to clearly label these structures, thereby enhancing\noverall clarity.\n(4) We have changed CLTA-670nano to clathrin-670nano, CLTA-TagRFP to clathrin-TagRFP in\nthe revised text and figures.\n(5) Additionally, we have added more information regarding the imaging data collection and\nanalysis, results description and discussion, and other editing in the revised manuscript.\nMinor technical point:\nevent (as in panels d-f), but I could not detect this in the figure.\nResponse: We thank the reviewer for identifying this error. The figure legend has been corrected\nby removing \"the fluorescence intensity traces of an endocytic event\" (new Extended Fig. 1h).\nReviewer #2 (Remarks to the Author):\nThe manuscript ‘Lipid turnover and GEF recruitment collectively determine Rab5 recruitment and\nactivation during the first step of early endosome formation’ addresses the central question, which\ncellular components are required to recruit Rab5 to the nascent uncoated endocytic carrier after\nloss of the clathrin coat. This work sheds light onto the temporal relationship between different\ncomponents and their catalytic activity in this process. It identifies Rabex5 and hRME6 as pivotal\nproteins in this process and, perhaps somewhat unexpected, discover that the lipid composition\nof the Clathrin coated vesicle additionally prevents early recruitment of Rab5. The hydrolysis of\nPI(4,5)P2 is prerequisite of Rab5 recruitment and provides an additional regulatory level.\nThe authors provide high-quality live cell imaging in combination with state-of-the art image\nanalysis. They also confirm and extend their imaging results with biochemical and in vitro\nexperiments providing a comprehensive view of the interactions leading to Rab5 recruitment. The\nsheer number of experiments and their associated controls makes the manuscript challenging yet\nenjoyable to read. The authors carefully discuss their results and acknowledge that, despite their\nextensive efforts, some details remain to be characterised. Overall, this work is a crucial step in\nour undertsanding of the endocytic process and therefore highly relevant. This systematic study\nwill certainly be a valuable resource and provides novel and important information on clathrin\nmediated endocytosis and endosome formation. I would like to congratulate the authors on their\ndetailed characterisation and their work towards a comprehensive model. I hope that the authors\nwill continue to fill in the remaining gaps in future publications.\nResponse: We are deeply grateful to the reviewer for the very precise summary and insightful\ncomments on our work, and we express our sincere gratitude to the reviewer for the highly\nconstructive suggestions. We also thank the reviewer for the recognition of the significance of this\nstudy and supportive comments.\nI still would like to see a few points be clarified and / or addressed before publication:\n3\n1) It would be valuable information, if there is a correlation between Rab5 recruitment and onset\nof movement / increased mobility of the vesicle. I would encourage the authors to revisit their\nTIRF-imaging data along these lines and include this information in the manuscript.\nResponse: We thank the reviewer for the excellent suggestion. As recommended, we have\nrevisited the TIRF-imaging data and analyzed the velocity of endocytic vesicles before and after\nRab5 recruitment. Indeed, the vesicles showed increased movement following Rab5 recruitment.\nThe new results have been provided in Extended Data Fig. S1g in the revised manuscript (also\nshown below for convenience).\nExtended Data Fig. S1g. SUM159 cells gene-edited for EGFP-Rab5a+/+ and clathrin-670nano+/+,\nEGFP-Rab5b+/+ and clathrin-670nano+/+, or EGFP-Rab5c+/+ and clathrin-670nano+/+ were transfected\nwith mScarlet-I-GAK and then imaged at the bottom surface using triple-color TIRF microscopy every\n1 s for 300 s. The velocity distribution of endocytic carriers before and after recruiting EGFP-Rab5a,\nEGFP-Rab5b, or EGFP-Rab5c is shown (median with interquartile range).\n2) Variation in ‘Clathrin-density’ on the plasma membrane: Clathrin structures larger than a single\nCCP are visible for example in Movie 1 and Movie 2. These structures display multiple\nrecruitments of Rab5c and Rabex5, respectively. These could resemble stable clathrin structures\nas described by Merrifield (for example: Merrifield et al, Cell, 2005; Taylor et al, PLos Biol, 2011;\nLampe et al., J Struct Biol, 2016). It would be a valuable information, to briefly describe how these\nregions are treated during the analysis procedure. As the Rab5 recruitment appears after the\nscission of the vesicle, I would not assume that there should be a difference between budding\nsites at regions of different clathrin density, but it would be good if the authors could briefly\ncomment on this.\nResponse: We thank the reviewer for highlighting this intriguing observation and for the excellent\nsuggestion. Indeed, some larger clathrin structures are present on the bottom surface of SUM159\ncells. These structures correspond either to the flat clathrin lattices or ‘plaques’ described by\nMerrifield (Lampe et al., 2016; Merrifield et al., 2005; Taylor et al., 2011), or to aggregates of\nactive coated pits as revealed by live-cell structured illumination microscopy imaging (Li et al.,\n4\n2015). In our previous study, we observed consecutive bursts of GAK recruitment to these long-\nlived, larger clathrin structures in gene-edited SUM159 cells expressing AP2-TagRFP+/+ (He et\nal., 2020). As noted, and similar to GAK, some of these larger structures can also recruit multiple\nrounds of Rab5 and Rabex5. This likely represents the formation of endocytic carriers at the edge\nof a clathrin plaque or from a cluster of active coated pits.\nSince the resolution of our live-cell TIRF imaging was insufficient to resolve individual\nendocytic events within these larger structures, we excluded these long-lived clathrin assemblies\nfrom the analysis. Following the reviewer's suggestion, we have added this clarification to the\nMethods section and cited the relevant work by Merrifield in the revised manuscript (lines 791-\n793 on page 27): \"Valid tracks from single clathrin-coated pits were used for further analysis.\nPersistent tracks and large clathrin structures (‘plaques’) 68, 69 were excluded from this analysis.\"\nFinally, in our imaging experiments, we did not observe apparent difference among budding sites\nlocated in regions with different clathrin densities, which could be an interesting topic for future\nstudy.\n3) Imaging parameters: The authors should provide more details about their imaging parameters.\nAt least typical settings (like laser power (in kW/cm²), exposure time and major camera settings)\nas well as typical values for recorded signal intensities should be mentioned supporting the\nreproducibility of the data. Alternatively or in addition, the authors might want to consider making\nat least some of their annotated data publicly available via the BioImage Archive or similar\ndatabases.\nResponse: We thank the reviewer for the suggestion. The detailed imaging parameters have\nnow been added to the Method section of the revised manuscript (lines 776-783 on page 27):\n\"The 1.5× tube lens was applied to achieve the final pixel size corresponding to 86.7 nm of the\nimage. Images were acquired using Micro-Manager 2.0 65. For single-molecular imaging of\nEGFP/mEGFP-tagged proteins, the 488 nm laser power measured at the objective exit in epi-\nillumination mode was 1.5 mW. The exposure time was 100 ms per channel, and the EM gain of\nthe EMCCD camera was set to 280 (Evolve 512 Delta) or 300 (iXon Life 888). The imaging interval\nand duration for each experiment are reported in the respective figure legends. The peak signal-\nto-noise ratio for purified single mEGFP molecules imaged on coverslips under these single-\nmolecule TIRF conditions was approximately 10.\"\n4) Schematic overview (Figure 7c), especially lower part on VPS9: In this cartoon, the authors\nsummarize the essential findings of their work. Adding additional details and improve the\ndescription of the figure in the legend would certainly help readers to cope with the vast amount\n5\nof information provided in the manuscript. I would especially motivate the authors to re-visit the\nlower part of the figure, which lacks any detailed description in the figure legend and cannot be\nunderstood without having a careful read of the paper. The authors might want to consider amend\nthe figure such, that it can be used as a graphical summary of their work and help guiding readers\nas well as emphasizing their major findings, e.g. by adding information about catalytic activity etc.\nResponse: Thank you for the excellent suggestion. We have now revised the schematic overview\nas well as added a detailed description in the figure legend to improve the clarity (Figure 7c) in\nthe revised manuscript (also shown below for convenience).\nFigure 7c. Schematic summary of the dynamics and regulation of Rab5 during early endosome\nformation. Clathrin-coated pits form at the PI(4,5)P -enriched plasma membrane. After scission,\n2\nendocytic vesicles undergo rapid lipid conversion, including depletion of PI(4,5)P . Auxilin1/GAK is\n2\nthen recruited in a burst-like manner to the budded coated vesicles to generate the uncoated endocytic\ncarriers. The two GEF molecules, Rabex5 and hRME6, are recruited continuously or transiently\nfollowing clathrin uncoating. Unlike uncoated endocytic carriers or endosomes, which lack PI(4,5)P ,\n2\nthe mistargeting of Rabex5 or its Vps9 domain to the plasma membrane or coated vesicles fails to\nrecruit Rab5. Thus, PI(4,5)P depletion and Rabex5/hRME6 recruitment together determine Rab5\n2\nrecruitment and activation during the initial stage of early endosome formation.\n6\n5) Minor detail / ambiguity - Extended Data Fig. 9. ‘The failure of Rab5 recruitment to the Vps9-\nor Rabex5-mistargeted to the plasma membrane is not related to GAP proteins.’ Might be\nchanged to (or similar): ‘The failure of Rab5 recruitment to Vps9- or Rabex5, which were\nmistargeted to the plasma membrane, is not related to GAP proteins’?\nResponse: We thank the reviewer for the excellent suggestion. As advised, we have revised the\nsentence to \"Failure of Rab5 recruitment to plasma membrane-targeted Rabex5 or its Vps9\ndomain is not related to GAP proteins.\".\nReviewer #3 (Remarks to the Author):\nThe small GTPase Rab5 is well documented for its function in early endosome fusion and\nendocytosis. This manuscript investigates the molecular mechanisms underlying the\nspatiotemporal recruitment of Rab5 during early endosome (EE) formation. Using single-molecule\nmicroscopy in genome-edited cells, the authors identify two Rab5 guanine-nucleotide exchange\nfactors (GEFs), Rabex5 and hRME6, as key regulators of Rab5 recruitment and activation on\nnascent uncoated endocytic carriers (UECs). They show that Rabex5 is continuously recruited to\nUECs, while hRME6 is recruited transiently. Rabex5 knockout significantly reduces Rab5\nrecruitment and number of EE while Rabex5/hRME6 double knockout essentially abolish this\nprocess. In addition, the study makes an novel observation that the plasma membrane–enriched\nphosphoinositide PI(4,5)P₂ plays a negative role in Rab5 recruitment and prevents its association\nwith the plasma membrane. This finding is important as it may imply a general role of organelle-\nspecific phosphoinositides in membrane targeting and activation of Rab GTPases during vesicular\ntransport. Finally, impaired PI(4,5)P₂ hydrolysis in Lowe syndrome cells leads to defective Rab5\nrecruitment and EE formation, linking this mechanism to human disease.\nThe manuscript addresses a longstanding question in endocytic trafficking and presents\nnovel insights into the spatial regulation of Rab5 membrane recruitment and activation. However,\nseveral experimental issues and broader implications require clarification or additional evidence\nbefore publication.\nResponse: We sincerely appreciate the reviewer’s insightful comments and excellent suggestions,\nwhich have been instrumental in strengthening our work. In our revised manuscript, we have\nperformed additional experiments, reorganized the data, and revised the text to consolidate our\nfindings and enhance its overall clarity.\n1. The manuscript relies heavily on the concept of UECs, but their precise molecular identity is\nnot well established: do they represent a distinct compartment? How are they biochemically\ndifferent from EE? More evidence is needed to establish this concept including specific protein\nand lipid markers.\nResponse: Thank you for this excellent comment. Indeed, as the reviewer noted, the uncoated\nendocytic carriers (UECs) derived directly from endocytic clathrin-coated vesicles represent an\nintermediate compartment prior to fusion with early endosomes. Canonical early endosomes are\npositive for Rab5 and its effector protein EEA1, as well as the lipid marker PI3P. As demonstrated\nin our previous study (He et al., 2017), uncoated endocytic carriers contain PI(3,4)P , but not PI3P.\nwith pre-existing Rab5/EEA1/PI3P-positive endosomes.\nWe have replicated the findings from our earlier work and further verified that endocytic\ncarriers immediately following clathrin uncoating recruit Rab5 but not EEA1 or a PI3P sensor\n(Reviewer Figure 1). Thus, uncoated endocytic carriers indeed represent the intermediate stage\nbefore fusion with early endosomes. In the revised manuscript, we have cited our prior study and\nincluded the description of the molecular identity of UECs: \"In our previous work, we have shown\nthat these newly uncoated endocytic carriers contain PI(3,4)P₂ and recruit Rab5, yet lack EEA1\nand PI3P prior to fusing with endosomes 15\". Additionally, we have labeled the uncoated endocytic\ncarriers in the updated model and provided a detailed description in the legend for Figure 7c.\n8\n2. In addition to Rab5, there are multiple Rabs associated with EE among which Rab21 is also a\nsubstrate for Rabex5 and should be included as an important positive control in the Rabex5\nknockout experiments. Another EE-associated Rab such as Rab4 or Rab22 that does not respond\nto Rabex5 may serve as a negative control. Along this line, Rabex5/hRME double knockout cells\neffectively abolish Rab5-positive EE. Are other EEs, e.g., Rab4 EE or Rab22 EE, affected?\nResponse: We thank the reviewer for this excellent suggestion. As advised, we have transiently\nexpressed mEGFP-tagged Rab21, Rab22a, and Rab4a in SUM159 cells with Rabex5-KO,\nhRME6-KO, or Rabex5/hRME6-DKO and imaged the live cells using spinning-disk confocal\nmicroscopy. We found that in Rabex5-KO cells, along with Rab5, the number of Rab21-,\nRab22a-, or Rab4a-positive endosomes was decreased. Importantly, and consistent with the loss of\nRab5-positive early endosomes, Rabex5/hRME6-DKO resulted in the near-complete disappearance of\nRab21- (new Extended Data Fig. 3f), Rab22a- (new Extended Data Fig. 3g), Rab4a-positive\nendosomes (Reviewer Figure 2d).\nPreviously, we observed that Rabex5-KO reduces the number of Rab5-positive early\nendosomes, while Rabex5/hRME6-DKO causes Rab5-positive early endosomes to disappear,\nleading to the dispersal of other early endosome-associated proteins such as EEA1 (Fig. 1g),\nAPPL1 (Reviewer Figure 2a), Rabaptin5 (Reviewer Figure 2b), Rabenosyn-5 (Reviewer\nFigure 2c), and 2xFYVE(Hrs) (Fig. 1h). Therefore, it is likely that Rabex5/hRME6-DKO causes\nthe loss of Rab5/EEA1-positive early endosomes, which in turn leads to the dispersal of other\nearly endosome-related proteins, including Rab21, Rab22, and Rab4.\nThe Vps9 domain, together with the HB domain of Rabex5, constitutes the GEF module for\nthe Rab5 subfamily of GTPases, including Rab5 and Rab21. The HB-VPS9 tandem of hRME6 is\nhomologous to the GEF region of Rabex5 (Lodhi et al., 2007). Therefore, whether hRME6 also\nfunctions as a GEF for Rab21 remains a subject for future investigation.\nThese new results and related discussion have been included in the revised manuscript (and\nbelow for convenience).\nExtended Data Fig. S3f,g, SUM159 cells lacking Rabex5 and/or hRME6 expression were transiently\ntransfected with mScarlet-I-Rab5c and mEGFP-Rab21 (f), or mScarlet-I-Rab5c and mEGFP-Rab22a\n(g), and then imaged using spinning-disk confocal microscopy. Representative images show the\ndistribution of mScarlet-I-Rab5c with mEGFP-Rab21 or mEGFP-Rab22a around the middle plane of\nthe cells. Scale bars, 10 μm.\n10\n+/+c5baR-PFGE\n951MUS\n+/+c5baR-PFGE\naLeH\nenalp\nlasaB\nenalp\nelddiM\nenalp\nlasaB\nenalp\nelddiM\na\nControl\nRa\nS\nbe\nU\nx\nM\n5-\nh\nME6-KD\nRabex5/hRME6-DKD b Rabex5/hRME6-DKD\n1 k 8 D 0 CHC 0.4\n0.3\nEEA1\n180\n0.2\n180 hRME6\n130 0.1\n55\nc\nControl Rabe\nH\nx\ne\n5-\nL\nK\na\nD hRME6-KD\nRabex5/hRME6-DKD d Control Rabex5-KD hRME6-KD Rabex5/hRME6-DKD\nkD\n180 hRME6\n130\n55\n35\ne\nh\n2mμ\nrepstopsforebmuN\nns\n✱✱✱✱ ✱ ✱✱✱✱\nContr\nR\no\na\nl bex5-K\nh\nD\nR\nME6-KD DKD\n0.4\n0.3\n0.2\n0.1\nR\no\na\nl bex5-K\nh\nD\nR\nME6-KD DKD\n2mμ\nrepstopsforebmuN\nns\n✱✱✱✱ ✱ ✱✱✱✱\n+/+c5baR-PFGE\na5baR-olaH\negreM\nWT Rabex5-KO hRME6-KO DKO\nWT Rabex5-KO hRME6-KO DKO\n+/+c5baR-PFGE\nolaH-1PMAL\negreM\nf\ng\n12baR-PFGEm\nc5baR-I-telracSm\negreM\nWT Rabex5-KO hRME6-KO DKO\na22baR-PFGEm\nc5baR-I-telracSm\negreM\nWT Rabex5-KO hRME6-KO DKO\n3. In both the Rabex5/hRME knockout and the Lowe syndrome experiments, a rescue control\nshould be included to strengthen authors’ claim.\nexperiments in the Rabex5 and/or hRME knockout SUM159 cells. The re-expression of either full-\nlength Rabex5 (Fig. 3d, 3e) or hRME6 (Fig. 3i, 3j) was able to restore Rab5 recruitment to\nuncoated endocytic carriers and early endosome formation.\nFollowing your advice, we have now performed the rescue experiments in Lowe syndrome\ncells. As expected, transient expression of mEGFP-tagged OCRL restored the formation of EEA1-\npositive early endosomes. These new results are provided in Extended Data Fig. S10g in the\nrevised manuscript (and below for convenience).\nExtended Data Fig. S10g, De-identified Lowe syndrome patient fibroblasts and control fibroblasts\nwere transfected with either mEGFP-OCRL or a vector containing mEGFP alone. The cells were then\nstained for endogenous EEA1 and imaged using spinning-disk confocal microscopy. Numbers of\nidentified EEA1-labeled endosomes are shown on the right (mean ± SEM; 25 cells per group).\nStatistical analysis was performed using the ordinary one-way ANOVA with Tukey’s multiple\ncomparisons test; ***P < 0.001; ****P < 0.0001; ns, no significance. Scale bars, 10 µm.\n4. Previous studies on Rab5 and Rabex5 should be discussed in more detail to place these\nfindings in context. For example, the first subheading in Results “All three isoforms of Rab5…” is\nnot wrong but imprecise and can be misleading, as the new information is actually for Rab5b while\nRab5a and Rab5c results reported previously (ref 15). A more appropriate subheading may be\n“Rab5b, like Rab5a and Rab5c…”.\nResponse: Thank you for the suggestion. In our previous work (ref 15), we generated the gene-\nedited cells expressing EGFP-tagged Rab5a and Rab5c and performed dual-color TIRF imaging.\nWe observed that Rab5a and Rab5c signals appear after uncoating, as indicated by the\ndisappearance of clathrin signal. In the present study, we have generated new cell lines and\nextended this analysis by incorporating a GAK signal to mark the precise timing of uncoating. By\n12\nper performing triple-color TIRF imaging and quantitative imaging analysis—which was absent\nfrom our prior work—we have now verified that Rab5a, Rab5c, and Rab5b are all recruited\nfollowing GAK-mediated uncoating. Accordingly, we have revised the first subheading in the\nResults section to \"Rab5 is recruited specifically to uncoated endocytic carriers in genome-edited\ncells\".\nIn addition, the section suggesting “The Vps9 domain is essential for Rabex5 recruitment…) is\nmisleading, as Fig 3 shows that the Vps9 domain alone is insufficient to associate with the\nmembrane, although this Vps9 domain construct is not described in the text. This result and the\nconclusion that neither the N-terminal UBD nor the C-terminal domain including RpBD is\nnecessary for Rabex5 membrane targeting to EE are consistent with a previous paper addressing\nRabex5 membrane targeting domain (ref 22). This and another paper on the same topic (ref 23)\nare cited but not discussed in the context of new findings.\nResponse: We thank the reviewer for the excellent suggestion. To increase clarity, we have\nrevised the section subheading to \"The Vps9 domain of Rabex5 is necessary for Rabex5\nrecruitment to uncoated endocytic carriers and Rab5-positive early endosome formation\".\nRegarding the results for for UBD and RpBD domains, we have revised the text to \"Consistent\nwith previous studies 22, we observed that removing the N-terminal UBD or the C-terminal RpBD\ndid not affect Rabex5 recruitment to uncoated endocytic carriers\".\nReferences mentioned in this rebuttal document:\nHe, K., Marsland, R., III, Upadhyayula, S., Song, E., Dang, S., Capraro, B.R., Wang, W., Skillern, W.,\nGaudin, R., Ma, M., et al. (2017). Dynamics of phosphoinositide conversion in clathrin-mediated\nendocytic traffic. Nature 552, 410-414.\nHe, K., Song, E., Upadhyayula, S., Dang, S., Gaudin, R., Skillern, W., Bu, K., Capraro, B.R., Rapoport,\nI., Kusters, I., et al. (2020). Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic. J Cell Biol 219.\nLampe, M., Vassilopoulos, S., and Merrifield, C. (2016). Clathrin coated pits, plaques and adhesion. J\nStruct Biol 196, 48-56.\nLi, D., Shao, L., Chen, B.C., Zhang, X., Zhang, M., Moses, B., Milkie, D.E., Beach, J.R., Hammer, J.A.,\n3rd, Pasham, M., et al. (2015). ADVANCED IMAGING. Extended-resolution structured illumination\nimaging of endocytic and cytoskeletal dynamics. Science 349, aab3500.\nA.R. (2007). Gapex-5, a Rab31 guanine nucleotide exchange factor that regulates Glut4 trafficking in\nadipocytes. Cell Metab 5, 59-72.\nMerrifield, C.J., Perrais, D., and Zenisek, D. (2005). Coupling between clathrin-coated-pit invagination,\ncortactin recruitment, and membrane scission observed in live cells. Cell 121, 593-606.\nTaylor, M.J., Perrais, D., and Merrifield, C.J. (2011). A high precision survey of the molecular dynamics\nof mammalian clathrin-mediated endocytosis. PLoS Biol 9, e1000604.\n14"}, {"round": 2, "reviewer_comments": {"reviewer_1": "During revision the authors have carefully addressed all of my (minor) remarks. Thus, I now recommend publication of this excellent manuscript.", "reviewer_2": "All the points I raised in my previous comments have been adequately addressed, except for one minor detail. The author should be able to clarify this by re-analysing the existing data with respect to the onset of vesicle mobility after Rab5 recruitment (see Extended Fig. 1d–g):\nNot all events appear to have an immediate onset of mobility. For example, Extended Fig. 1f shows a rather stationary event, whereas Figs. 1d and 1e seem to show an immediate onset of mobility upon Rab5 recruitment. Is there a time lag between Rab5 recruitment and the onset of mobility? Or is the onset of mobility strictly linked to Rab5 recruitment, with only a small proportion of vesicles remaining stationary? The authors should briefly comment on this and present adequate data in the form of a histogram displaying the time between Rab5 recruitment and the onset of mobility, or another suitable graph, to address this question.", "reviewer_3": "The authors have addressed all my concerns."}, "author_response": "Reviewer #1 (Remarks to the Author):\nDuring revision the authors have carefully addressed all of my (minor) remarks. Thus, I now\nrecommend publication of this excellent manuscript.\nResponse: We are grateful to the reviewer for the recognition of our work.\nReviewer #2 (Remarks to the Author):\nAll the points I raised in my previous comments have been adequately addressed, except for one\nminor detail. The author should be able to clarify this by re-analysing the existing data with respect\nto the onset of vesicle mobility after Rab5 recruitment (see Extended Fig. 1d–g):\nNot all events appear to have an immediate onset of mobility. For example, Extended Fig. 1f\nshows a rather stationary event, whereas Figs. 1d and 1e seem to show an immediate onset of\nmobility upon Rab5 recruitment. Is there a time lag between Rab5 recruitment and the onset of\nmobility? Or is the onset of mobility strictly linked to Rab5 recruitment, with only a small proportion\nof vesicles remaining stationary? The authors should briefly comment on this and present\nadequate data in the form of a histogram displaying the time between Rab5 recruitment and the\nonset of mobility, or another suitable graph, to address this question.\nResponse: We sincerely appreciate the reviewer's recognition of our revised work and for the\ncomments. As suggested by the reviewer during the previous revision, we revisited the TIRF\nimaging data and calculated the average velocity of endocytic vesicles over periods both before\nand after Rab5 recruitment. These analyses indicate that vesicle movement generally increases\nfollowing Rab5 recruitment. However, because the imaging data were acquired at 1-second\nintervals with single-molecule sensitivity, the temporal resolution was insufficient to reliably\ncalculate transient vesicle velocities using only the frames immediately before and after Rab5\nrecruitment. Moreover, the data were collected in two dimensions and therefore do not capture\nthe vertical movement of endocytic vesicles. As a result, we were unable to calculate the time lag\nbetween Rab5 recruitment and the onset of vesicle mobility based on the available data. Given\nthat this point is not directly relevant to our study and to ensure accuracy, we did not include this\nresult in the revised manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed all my concerns.\nResponse: We are grateful to the reviewer for the recognition of our work."}]

Decision letter