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# Microtubules coordinate mitochondria transport with myofibril morphogenesis during muscle development

Jerome Avellaneda, Duarte Candeias, Ana da Rosa Soares, Edgar R Gomes, Nuno Miguel Luis, Frank Schnorrer

► To cite this version:

Jerome Avellaneda, Duarte Candeias, Ana da Rosa Soares, Edgar R Gomes, Nuno Miguel Luis, et al. Microtubules coordinate mitochondria transport with myofibril morphogenesis during muscle development. Developmental Cell, In press, 10.1016/j.devcel.2025.06.033. hal-05228474

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Submitted on 28 Aug 2025

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# Developmental Cell

## Microtubules coordinate mitochondria transport with myofibril morphogenesis during muscle development

### Graphical abstract

<figure><img src="image_3.png" /></figure>

### Authors

Jerome Avellaneda, Duarte Candeias, Ana da Rosa Soares, Edgar R. Gomes, Nuno Miguel Luis, Frank Schnorrer

### Correspondence

nuno.luis@univ-amu.fr (N.M.L.), frank.schnorrer@univ-amu.fr (F.S.)

### In brief

Avellaneda et al. identify that kinesin-dependent transport is required to relocate mitochondria into close proximity to the assembling myofibrils in *Drosophila* and mouse muscles. Microtubules are in intimate contact with the assembling myofibrils and thus coordinate mitochondria and myofibril morphogenesis.

### Highlights

* Mitochondria, microtubules, and myofibrils are linked during muscle development

* Microtubules transport mitochondria in between assembling myofibrils

* Kinesin-dependent mitochondria transport is conserved from fly to mouse muscle

* Severing microtubules has acute effects on mitochondria and myofibrillogenesis
<figure><img src="image_4.png" /></figure>

## Article

# Microtubules coordinate mitochondria transport with myofibril morphogenesis during muscle development

Jerome Avellaneda,<fnref n="1" /> Duarte Candeias,<fnref n="2" /> Ana da Rosa Soares,<fnref n="2" /> Edgar R. Gomes,<fnref n="2" />,<fnref n="3" /> Nuno Miguel Luis,<fnref n="1" />,*
and Frank Schnorrer<fnref n="1" />,<fnref n="4" />,*

## SUMMARY

Muscle cells contain numerous energy-producing mitochondria and contractile myofibrils, whose myosin motors need ATP to generate force. Thus, myofibrils and mitochondria are in intimate contact in mature muscles. However, how their morphogenesis is coordinated during development remains largely unknown. Here, we used *in vivo* imaging to investigate myofibril and mitochondria network dynamics in developing *Drosophila* flight muscles. We found that mitochondria intercalate from the surface of actin bundles to their interior, and concomitantly, actin filaments condense to individual myofibrils. This ensures that mitochondria locate in proximity to every myofibril. Notably, antiparallel microtubules bundle with the assembling myofibrils, suggesting a key role in myofibril orientation. Indeed, microtubule severing affects myofibril orientation, whereas kinesin knockdown specifically blocks mitochondria intercalation. Importantly, mitochondria intercalation and their kinesin-dependent microtubule-based transport are conserved in mammalian muscle. Together, these data identify a key role for microtubules in coordinating mitochondria and myofibril morphogenesis to build functional muscles.

## INTRODUCTION

Complex animals possess several muscle fiber types to support different contraction patterns mediating locomotion and pumping of body fluids. The different fiber types contain particular architectures of their contractile myofibrils and of their energy-producing mitochondria. The most notable difference in mammals is the extremely dense mitochondria network in cardiac muscle, compared with fewer mitochondria in fast glycolytic skeletal muscles, with oxidative skeletal muscles showing an intermediate phenotype.<fnref n="1" />,<fnref n="2" /> This is accompanied by striking biomechanical differences of the sarcomeres, with the rigid cardiac sarcomeres oscillating continuously throughout life, while the more compliant skeletal muscles undergo deliberate boosts of activity that are particularly intense in fast glycolytic fibers and more moderate but long lasting in slow oxidative fibers.<fnref n="3" /> These biomechanical differences are largely regulated by differences in the sarcomere protein isoforms,<fnref n="4" /> while the metabolism is controlled by their mitochondria content.<fnref n="2" /> Thus, muscle physiology depends on the proper coordination of mitochondria morphogenesis with myofibril morphogenesis.

The variability of mitochondria morphologies and their impact on the physiology of different cell types in a complex organism

are still underappreciated. Not only does the mitochondria density vary in the different muscle types but also the position and relative orientation of mitochondria with respect to the contractile myofibrils. In oxidative skeletal muscles and particularly in the heart, the mitochondria are located in close proximity to myofibrils.<fnref n="1" />,⁵,⁶ Hence, mitochondria are maximally close to the ATP-consuming myosin motors.

A similar phenomenon is observed in *Drosophila* adult muscle types. The high-energy-consuming, oscillating flight muscles contain a particularly high mitochondria density, with mitochondria surrounding each of the individual fibrillar myofibrils, thus supporting them maximally with ATP.⁷,⁸ By contrast, the tubular leg muscles concentrate most mitochondria around their centrally localized nuclei, away from the cross-striated, aligned myofibrils.<fnref n="2" />,⁷ These different architectures are regulated by the master regulator of flight muscle development, the transcriptional regulator Spalt (Salm).⁷,⁹,¹⁰ However, to date we do not understand how the intimate, fiber-type-specific interactions between myofibrils and mitochondria are achieved during development.

*Drosophila* flight muscles are an excellent model to study muscle development.¹¹ Each large flight muscle fiber assembles all of its 2,000 myofibrils simultaneously at 30 h after puparium

<footnote n="1">¹Aix Marseille University, CNRS, IBDM, Turing Centre for Living Systems, 13288 Marseille, France</footnote>

<footnote n="2">²Gulbenkian Institute for Molecular Medicine (GIMM), 1649-035 Lisboa, Portugal</footnote>

<footnote n="3">³Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal</footnote>

<footnote n="4">⁴Lead contact</footnote>

<footnote>*Correspondence: nuno.luis@univ-amu.fr (N.M.L.), frank.schnorrer@univ-amu.fr (F.S.)</footnote>

<footnote>https://doi.org/10.1016/j.devcel.2025.06.033</footnote>
A

In vivo acquisition of mitochondria and actin morphogenesis

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B

High resolution of mitochondria intercalation

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C

Developing flight muscles cross-section

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D

Mitochondria and actin morphogenesis cross-section scheme in flight muscle

<figure><img src="image_8.png" /><figcaption>**Figure 1. Mitochondria intercalation into actin bundles**</figcaption></figure>

(A) Stills from a developing flight muscle, labeled for actin (LAD9-GFP) and mitochondria (UAS-mit-Cherry; Mef2-GAL4) before (24 and 26 h APF) and after mitochondria intercalation (28 and 30 h APF); yz cross-sections are below (Video S1).

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**Developmental Cell**

Article

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formation (APF) from less well-organized actomyosin bundles.12,13 The assembled myofibrils segregate into large bundles,14 with mitochondria having intercalated between each of the myofibrils at 32 h APF.7 Subsequently, myofibrils mature and grow in diameter, while also the neighboring mitochondria expand in size to fully surround and mechanically insulate the myofibrils at the adult stage.7,15 How mitochondria-myofibril intercalation and their homogenous distribution along all myofibrils are regulated is not understood.

Skeletal muscle fibers are large syncytial cells that have lost the classical centriole-based microtubule organizing center early during development and instead organize their microtubules around their perinuclear space.<sup>16-18</sup> Microtubules are known to be required for nuclear transport in developing muscles in flies and mammals.<sup>19</sup> They might directly or indirectly also be needed for myofibrillogenesis,<sup>20</sup> though this hypothesis has been challenging to test *in vivo* due to the difficulty of acute microtubule manipulations in such settings. It is well established that mitochondria require microtubules for long-range transport along the axon and dendrites of neurons.<sup>21</sup> However, how mitochondria are transported in developing muscles has been little investigated.

Here we found an essential role for microtubules in transporting mitochondria at long-range during the key developmental stage of myofibril assembly in *Drosophila* flight muscles. This microtubule-based transport mechanism is also conserved during mammalian muscle differentiation, ensuring that mitochondria are positioned optimally to fuel the high energy requirements of the myosin motors in mature muscles.

RESULTS

Mitochondria intercalate rapidly into actin filament bundles

To study the coordination of myofibril with mitochondria morphogenesis, we used the indirect flight muscles of *Drosophila*. We recently found that mitochondria intercalate between the myofibril bundles between 24 and 32 h APF.⁷ To characterize the dynamics of the intercalation process in detail, we acquired two-color *in vivo* time-lapse movies, labeling actin and mitochondria, to follow flight muscle morphogenesis in fully intact pupae from 24 h to 30 h APF (Figure 1A; Video S1). These movies revealed interesting dynamics: at 24 h APF, actin filaments are largely located at the periphery of the myotube, while mitochondria are present at distinct central regions (in proximity to the nuclei²²). At 26 h, actin filaments consolidate into large, distinct bundles throughout the muscle fiber, while mitochondria are located at their surface. Surprisingly, this changes dramatically after 26 h, when most mitochondria intercalate into the actin filament bundles until 28 h APF, while the actin bundles consolidate and form distinct myofibrils at

30 h APF (Figure 1A; Video S1). Thus, mitochondrial intercalation occurs within 2 h and appears to be coordinated with myofibril assembly.

To characterize the superposition of mitochondria with actin bundles at high resolution, we performed super-resolution Airy-scan confocal imaging of flight muscles fixed at 26 and 28 h APF. As seen in the live movies, at 26 h APF, distinct actin filament bundles have formed in each muscle fiber. They contain dense actin filaments, which are not yet assembled into individual myofibrils. The mitochondria are excluded from these bundles while being in close contact at their surface (Figures 1B and 1B'). At 28 h APF, the actin filaments in each bundle have condensed further, generating space in between them, which is now occupied by the intercalated mitochondria (Figures 1B and 1B'). This demonstrates that mitochondria intercalate into actin bundles concomitantly with the start of myofibrillogenesis.

To observe the relative arrangement of the mitochondria with the assembling myofibrils at even higher resolution, we generated cryo-cross-sections of developing flight muscles. These cross-sections confirmed that mitochondria are in contact with, but largely excluded from, the actin filament bundles at 26 h APF (Figure 1C). Whereas at 28 h APF, actin filaments have condensed at the periphery of each bundle, and mitochondria have intercalated into the center of each bundle. This arrangement becomes more refined at 30 h APF, when all distinct myofibrils have assembled and the mitochondria have redistributed in between them, insulating each myofibril from its neighbors (Figure 1C). Taken together, our results show that mitochondrial reorganization coincides with myofibrillogenesis. From these observations, we define three main steps: first, dense actin bundles exclude mitochondria at their surface; second, the condensation of actin filaments to myofibrils coincides with mitochondrial intercalation into the bundles; and third, when myofibrils have fully assembled, the mitochondria have distributed homogenously between them (Figure 1D).

Mitochondria dynamics change upon intercalation

Having shown that the mitochondria intercalation process is accomplished after only 2 h, we aimed to determine the dynamics of mitochondria transport. Expression of GFP fused to a mitochondrial matrix targeting signal (mit-GFP) together with fast frame acquisition (sub-second) determined the individual mitochondria transport speed to be 0.7 µm/s at the pre-intercalation stage at 25 h APF (Figure S1; Video S2). Thus, mitochondria can move locally in the muscle fibers at rather high speed.

In order to quantify the net translocation of mitochondria throughout the myofiber, we employed the photoconvertible marker Dendra2 localized to the mitochondrial matrix (mit-Dendra2), which allowed us to convert mitochondria from green to

(B) Airyscan images of dissected flight muscles, co-labeled for actin (phalloidin-rhodamine) and mitochondria (mit-GFP, enhanced by a GFP nanobody). (B') Intensity profiles of actin (magenta) and mitochondria (green) are plotted along the white line in (B). Note the overlap of mitochondria and actin at 28 h but not at 26 h APF.

(C) Cryo-cross-sections of flight muscles expressing *Mef2*-GAL4, UAS-mit-GFP at 26, 28, and 30 h APF were stained with phalloidin-rhodamine (actin, red), GFP nanobody (mitochondria, green), and DAPI (nuclei, blue). Left column shows overview cross-sections of 5–6 flight muscle cells (white dashed lines), and the 3 right-most panels are Airyscan acquisitions of myofibril bundles (one bundle is highlighted by the yellow line). Note that mitochondria have intercalated at 28 h APF and completely ensheath individual myofibrils at 30 h APF.

(D) Schemes of mitochondria and actin morphogenesis in flight muscles representing cross-section views of one fiber (top) and a zoom into two actin bundles/myofibril bundles (bottom). The major morphogenesis steps are indicated. Scale bars represent 10 µm in all panels, except 50 µm in the left column C. See also Video S1.
<figure><img src="image_10.png" /><figcaption>B 25 h APF: pre-intercalation</figcaption></figure>

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Mitochondria movement

<figure><img src="image_12.png" /><figcaption>C 32 h APF: post-intercalation</figcaption></figure>

<figure><img src="image_13.png" /></figure>

Mitochondria movement

<figure><img src="image_14.png" /><figcaption>D 25 h APF: pre-intercalation</figcaption></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>E 32h APF: post-intercalation</figcaption></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>F Net movement in 20 min</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>H Distance travelled nonstop</figcaption></figure>

<figure><img src="image_20.png" /></figure>

Mitochondria speed

<figure><img src="image_21.png" /></figure>

Transport direction

<figure><img src="image_22.png" /><figcaption>G Tracking of individual mitochondria during or after intercalation at 27 h APF</figcaption></figure>

mitochondria inside bundle after intercalation:

<figure><img src="image_23.png" /></figure>

mitochondria outside bundles during intercalation:

<figure><img src="image_24.png" /></figure>

(legend on next page)
red fluorescence in a defined region of the muscle fiber and then monitor their transport dynamics (Figure 2A). Surprisingly, by quantifying the speed of translocation along the long axis of the muscle at 25 h APF, we found that mitochondria move in bulk only over relatively short distances within 30 min (Figures 2B and 2F; Video S3). Conversely, after mitochondrial intercalation at 32 h APF, mitochondria translocate rapidly across large distances along the long axis of the flight muscle fiber (Figure 2C and 2F; Video S3). This demonstrates that mitochondria transport dynamics change dramatically in the muscle cells, promoting mitochondria long-range transport after intercalation occurred.

Having identified this fast transport dynamic, we wanted to clarify how directed the movement is. To this end, we photo-converted a smaller region per muscle fiber and followed the translocation of mitochondria along the long axis as well as the short axis of the muscle. At 25 h APF, we found almost no translocation along either axis (Figures 2D and 2F; Video S4). At 32 h APF, we confirmed the above-identified fast translocation of the mitochondria along the long axis of the muscle. However, along the short axis, we found no translocation (Figures 2E and 2F; Video S4). These results show that the long-range mitochondria translocation is specifically oriented along the long axis, strongly suggesting an active transport mechanism.

## During intercalation, mitochondria move along the short axis into actin bundles

Having shown that mitochondria gain long-range movement after intercalation, we wanted to follow them directly during the intercalation process. We selected the 27 h APF time point, while mitochondria intercalation was ongoing, and followed individual mitochondria at high time resolution. Dependent on the location of mitochondria, we identified distinct transport behaviors: mitochondria already located within actin filament bundles (post-intercalation) move with slightly higher speed, covering a longer distance, and are transported exclusively along the axis of the filament bundles (0° deviation) (Figures 2G and 2H; Video S5). By contrast, mitochondria still located outside actin filament bundles (pre-intercalation) move with slightly slower speed, cover less distance, and, importantly, deviate strongly from the filament bundle axis (about 45° deviation) (Figures 2G and 2H; Video S5). Thus, mitochondria outside the bundles are trans-

ported along the short axis, resulting in the intercalation into the neighboring actin bundle. Once intercalated, mitochondria accelerate and move long distances, strictly following the actin bundle direction.

## Actin and microtubule bundles are closely associated

How are mitochondria transported during flight muscle development? The microtubule cytoskeleton is the obvious candidate, as it is the main transport route for mitochondria in various cell types²³; however, the function of microtubules is not well explored during flight muscle development.²⁰ We first analyzed the relation of microtubules to the actin filaments during flight muscle development. By imaging flight muscles expressing Lifeact-Ruby and Tau-GFP to mark actin and microtubules, respectively, we found that both co-localize extensively (Figures 3A and S2A; Video S6). At 24 h APF, actin filaments and microtubules largely accumulate at the periphery of the myotube, while a microtubule mesh is visible in its center, around the centrally located nuclei (dark circles in Figure S2A²²). At 26 h APF, actin filaments and microtubules start to bundle together simultaneously, and as myofibrils assemble at 29 h APF, microtubules and actin remain closely associated (Figures 3A and S2A; Video S6).

To increase the resolution, we generated cross-sections of 26, 28, and 30 h APF flight muscles expressing β-Tub60D-GFP to label the microtubules, co-stained them with phalloidin, and imaged them with Airyscan super-resolution microscopy. This confirmed the high concentration of the microtubules in the assembled actin filament bundles at 26 h APF, with some microtubules still localized between actin bundles (Figure 3B). This is consistent with mitochondria being transported along microtubules into the actin filament bundles after 26 h APF. This inter-bundle microtubule mesh is still observed at 28 h APF, while it disappears at 30 h APF (Figure 3B). At 30 h APF, we found the microtubules organized around each individual myofibril (Figures 3B and B'), which is consistent with electron-microscopy data of the same stage.²⁴,²⁵ The space between the microtubules is probably occupied by the intercalated mitochondria at 30 h APF (Figures 3B and B'). These inter-bundle microtubules are highly dynamic and organized in an anti-parallel fashion, as judged by their rapid bidirectional sliding behavior, revealed by the signal spread upon photoconversion of td-EOS-α-Tubulin (Figure S2B).²⁶ Microtubules remain in proximity to the myofibrils until 48 h, while

**Figure 2.** Mitochondria transport dynamics switches after intercalation

(A) Schematic overview of a photo-conversion experiment using mit-Dendra2 and Mef2-GAL4 to label mitochondria sub-populations. (B and C) Live imaging of developing flight muscles expressing mit-Dendra2 (by Mef2-GAL4) at 25 h APF (B) and 32 h APF (C), after photo-conversion. The magenta rectangles label the photo-converted areas. Top images show a composite of non-converted mit-Dendra2 in green and photo-converted in magenta. The photo-converted mit-Dendra2 is shown in gray below at the indicated time after conversion. Plot profiles quantify the photo-converted mit-Dendra2 intensities. Note the slow spread at 25 h APF compared with the fast spread at 32 h APF (Video S3).

(D and E) Photo-conversion of mitochondria in a thin rectangular area to compare the mit-Dendra2 spread along the short and long axes before (D) or after intercalation (E). Images were acquired immediately (1 min) or 20 min after photoconversion (Video S4).

(F) Quantification of the fluorescence spread after 20 min along the short or long axis at 25 and 32 h APF from data in (D) and (E). Note the absent spread along the short axis. *n* = 6, 4, 5, 10 animals, respectively; see Data S1; Tukey's multiple comparisons test: \*\*\*p < 0.0001, n.s. *p* > 0.05.

(G) Stills of a movie from a 27 h APF flight muscle expressing mit-GFP (Mef2-GAL4, Video S5) highlighting the transport of one mitochondrion inside a bundle (yellow to orange arrowhead) and a second one located outside a bundle (light blue to dark blue arrowhead). Note the different directions of travel. The distance of travel in one direction is indicated.

(H–J) Quantification of individual mitochondrial transport in flight muscles at 26–27 h APF as distance travelled continuously in the same direction (H), speed (l), and orientation relative to long muscle axis (J). Note that mitochondria outside the bundles move a shorter distance along the short axis (n = 22 individual mitochondria tracked from 5 animals), whereas intercalated mitochondria move a longer distance along the long axis (n = 40 individual mitochondria tracked from 5 animals). See Data S1, Mann-Whitney tests \*\*p < 0.01, \*\*\*p < 0.0001. Scale bars represent 10 µm in (B–E) and 5 µm in (G). See also Figure S1, Videos S2–S5, and Data S1.
A

<figure><img src="image_25.png" /></figure>

B

<figure><img src="image_26.png" /></figure>

B'

<figure><img src="image_27.png" /><figcaption>Figure 3. Myofibrils form in close association with microtubules</figcaption></figure>

(A) Longitudinal confocal images of developing flight muscle at the indicated time points labeled for actin (Lifeact-Ruby, Mef2-GAL4) and microtubules (Mhc-Tau-GFP). Note how actin and microtubules overlap at this resolution (Video S6).

(B) Airyscan acquisitions of flight muscle cross-sections at 26, 28, and 30 h APF labeled for actin (phalloidin) and microtubules (β-Tub60D-GFP).

(B') Zoom-in of the 30 h APF stage at the indicated yellow box in (B). Note that microtubules largely surround each assembled myofibril at this stage. Scale bars represent 10 µm in (A) and (B) and 1 µm in (B'). See also Video S6.

(Figure 4A). This is consistent with our above findings that actin filament bundles intimately associate with microtubules (Figures 3A and 3B) and that mitochondria intercalate into the actin filament bundles until 28 h APF (Figure 1B). Importantly, the few non-intercalated mitochondria that are still present outside the microtubule bundles are often associated along transversely oriented microtubules that can mediate transport along the short muscle axis (Figure 4B), which occurs on average at about 45° from the long filament bundle axis (Figures 2G and 2H). This microtubule population probably corresponds to the mesh of microtubules best observed in cross-sections (Figure 3B). Overall, these results support a mechanism by which mitochondria intercalate into the actin filament bundles through microtubule-dependent transport.

Microtubules are required for actin
bundling and mitochondria
positioning

To functionally test the microtubule-
dependent mitochondrial intercalation
hypothesis, we aimed to deplete microtu-

bules by expression of the microtubule-severing protein spasin.<sup>27-29</sup> However, since microtubules are important for many cellular functions, the expression of spasin must be precisely controlled in time. To do so, we employed the recently developed photoactivatable *shine*-GAL4, which enables light-controlled expression within 1–2 h.<sup>30</sup> *shine*-GAL4 UAS-*spasin* flies raised in the dark are wild type, while exposing them to light starting at 23 h APF is pupal lethal. This demonstrates the absence of leakage of *shine*-GAL4 activity in the dark.

To test the effect of spasin expression on myofibrillogenesis, we raised *shine*-GAL4 UAS-*spasin* pupae in the dark and exposed them to light at 23 h APF and then analyzed the flight muscles at 32 h APF (Figures 5A and 5B). We found that these flight muscles show severe myofibril defects: myofibrils do not

the β-Tubulin60D signal has strongly reduced when myofibrils
have grown in diameter at 72 and 90 h APF (Figure S2C). Taken
together, our results show a close association between the actin
and the microtubule cytoskeleton before and during myofibrillo-
genesis, which supports a possible role of microtubules during
mitochondria intercalation.

Mitochondria follow microtubules into the actin bundles

To visualize microtubules and mitochondria simultaneously, we co-labeled them in developing flight muscles by expressing β-Tub60D-GFP and mit-Kate2 and imaged them with Airyscan super-resolution microscopy. We found that mitochondria are excluded from the microtubule bundles at 26 h APF, whereas they have intercalated inside the bundles at 28 h APF
A

<figure><img src="image_28.png" /></figure>

B

<figure><img src="image_29.png" /></figure>

form distinct bundles and run in variable directions in the same muscle fiber, compared with their regular arrangement in bundles in control pupae raised in constant darkness (Figure 5B). Furthermore, the nuclei are not restricted to the inter-bundle space as they are in wild-type fibers but distributed more homogeneously (Figure 5B). This demonstrates that microtubules are required after 23 h APF for myofibrillogenesis and correct myofibril orientation.

To directly investigate a role of microtubules for mitochondria transport and actin filament bundling, we exposed *shine-GAL4* UAS-*spastin* pupae to light at 23 h APF and analyzed their flight muscles only 2 h later at 25 h APF (Figures 5A and 5C). We found that microtubule severing during this brief developmental period is sufficient to completely block actin filament bundle formation. Instead, actin filaments form a homogenous mesh with interspersed nuclei, and consequently the mitochondria are not reorganizing into their bundle-associated pattern as they do in controls (Figure 5C). This demonstrates that microtubules are critical for actin filament bundling and mitochondria localization.

Inducing the expression of *spastin* only after 30 h APF does not strongly perturb mitochondria positioning relative to the myofibrils at 48 h, while mitochondria appear slightly smaller and sarcomeres slightly shorter at 90 h APF (Figure S3). This suggests that microtubules have no major role in myofibril maturation, consistent with their disassembly after 48 h APF (Figure S2C), whereas they are needed during the preceding steps of actin bundling, myofibril assembly, and mitochondria intercalation.

<caption>**Figure 4.** Mitochondria are associated with microtubules</caption>

(A) Flight muscles labeled for mitochondria (mit-Kate2, Mef2-GAL4) and microtubules (β-Tub60D-GFP) at 26, 28, and 30 h APF were imaged with Airyscan microscopy. Note that mitochondria are located on the surface of microtubule bundles at 26 h APF but have intercalated at 28 h APF. yz optical cross-sections are shown on the right.

(B) Close ups of similar images shown in (A) focusing on two different single mitochondria that are not yet intercalated (labeled by orange arrows; top and bottom). Note their close association with microtubules at 28 h APF. Scale bars represent 10 µm (A) and 5 µm (B).

**Kinesin is necessary for
mitochondria intercalation**

As the complete disassembly of microtubules affects the bundling of the actin filaments, it is trivial that the mitochondria are also mis-localized. To investigate more specifically the role of microtubule transport in mitochondria intercalation, we have performed a candidate RNAi screen and identified an important role for the microtubule motor *kinesin heavy chain* (*khc*). Knocking-down *khc* in muscle by RNAi (*khc-IR*) using *Mef2-GAL4* results in late pupal lethality, demonstrating an important role for *khc* for muscle development or muscle function.³¹

Investigating the flight muscle morphology of these late pupae showed a dramatic agglomeration of mitochondria outside of the myofibril bundles in mature muscles at 90 h APF, demonstrating that *khc* is indeed important for normal mitochondrial localization (Figures S4A and S4B). As a consequence, the diameter of the *khc-IR* myofibrils is enlarged and more variable compared with wild type (Figures S4C and S4D). To test if this sarcomere phenotype could be caused by changes in sarcomere protein isoform expression, which differs between fibrillar flight and cross-striated leg muscle types,¹⁰ we quantified the expression levels of the flight muscle sarcomere-specific proteins Actin88F and Flightin (Figures S4E and S4F). Both proteins are expressed at normal levels, demonstrating that the *khc-IR* flight muscles retain their fibrillar fate identity.

This suggests that kinesin is the motor protein transporting mitochondria during mitochondria intercalation. To test this, we analyzed *khc-IR* flight muscles at 32 h APF and found that actin filaments bundle normally and form myofibrils. This suggests that the microtubule network remains intact upon *khc* knock-down. Strikingly, most mitochondria, however, remain in the space outside of the myofibril bundles in large aggregates upon *khc* knockdown (Figures 6A and 6B). Thus, kinesin is required for mitochondria intercalation into the myofibril bundles before 30 h APF.

To quantify transport speed directly, we tested the mobility of the mitochondria in *khc-IR* muscles using our established photo-conversion assay of labeled mitochondria. We found that, in
<figure><img src="image_30.png" /><figcaption>**Figure 5.** Microtubules are required for myofibrilogenesis</figcaption></figure>

(A) Scheme of temporal spastin expression via light activation by *shine*-GAL4 and the sample collection time points.

(B and C) Confocal images of developing flight muscles from *shine*-GAL4; UAS-*spastin* pupae raised in the dark (control, top rows in B and C) or exposed to light after 23 h APF to induce spastin (bottom rows in B and C) and labeled for actin (phalloidin) and nuclei (DAPI) at 32 h APF (B) or labeled for mitochondria (anti-ATP5α) and actin (phalloidin) at 25 h APF (C). Note the severe myofibril orientation defects upon spastin expression in (B) and the defects in actin bundling in (C). Scale bars represent 5 µm. See also Figure S3.

contrast to wild type, mitochondria in *khc* knockdown flight muscles remain immobile at 32 h APF (Figures 6C–6E). This demonstrates that kinesin is required to transport mitochondria at long range in developing flight muscles and hence very likely also mediates the lateral transport from outside to inside the bundles that we have observed at 27 h APF (see Figure 2G).

Mitochondria hyperfusion can also block their intercalation.⁷ To rule out that a gain of mitochondria size is responsible for the inter-

calation block in *khc* knockdown muscles, we overexpressed the fission-promoting protein Drp1 in *khc-IR* muscle. We found that mitochondrial hyper-fission does not rescue the *khc-IR* mitochondria clustering phenotype in flight muscles of 90 h APF pupae (Figure S4G), demonstrating that the *khc-IR*-induced intercalation block is not caused by an increase in mitochondria size.

How are kinesin motors interacting with mitochondria during the transport process? The outer mitochondrial membrane
A

<figure><img src="image_31.png" /></figure>

C

Mitochondria dynamics in *Mef2-GAL4; khc-IR* 32 h APF

<figure><img src="image_32.png" /></figure>

D

<figure><img src="image_33.png" /></figure>

E

Mitochondria displacement

<figure><img src="image_34.png" /></figure>

protein Miro links kinesin motors to mitochondria during axonal transport,²³,³² and Miro function in muscles is required for normal locomotion.⁸ Hence, we investigated *Miro* knockdown muscle phenotypes. While we did not find obvious defects in mitochondria localization in flight muscles, we identified a striking phenotype in leg muscles: all mitochondria cluster in the center of the tubular *Miro* knockdown leg muscles, in the proximity of the nuclei, while in the wild-type leg muscles, mitochondria extend in between the myofibrils to the fiber surface (Figure S4H). We found the same defect in *khc* know-down leg muscles. Hence, kinesin-dependent mitochondria transport is not restricted to flight muscles and uses the known linker protein Miro in leg muscles.

## Mitochondria intercalation and microtubule-dependent transport are conserved in mammalian muscles

To test if the microtubule-based mitochondria reorganization mechanism is conserved in developing mammalian muscles, we investigated mitochondria localization in mouse primary myofibers differentiated *in vitro* that recapitulate early muscle

<caption>**Figure 6.** Kinesin-dependent mitochondria transport</caption>

(A and B) Flight muscles from control (*Mef2*-GAL4) (A) or kinesin knockdown (*Mef2*-GAL4, UAS-*khc-IR*) (B) were labeled at 32 h APF for actin (phalloidin) and mitochondria (mit-GFP). x-y views are shown to the left and y-z sections at the indicated white lines to the right. Note that kinesin knockdown results in large mitochondria clusters and blocks intercalation.

(C and D) Live imaging of mit-Dendra2 in flight muscles at 32 h APF in control (*Mef2*-GAL4, UAS-mit-Dendra2) and after kinesin knockdown (*Mef2*-GAL4, UAS-*khc-IR*, and UAS-mit-Dendra2). The magenta rectangles label the photo-converted areas. Top row shows non-converted mit-Dendra2 in green and photo-converted mit-Dendra2 in magenta. The photo-converted mit-Dendra2 is shown in gray at the indicated time after conversion.

(E) Quantification of the photo-converted mit-Dendra2 spread in the long and the short perpendicular cell axis, 20 min after photo-conversion (respectively *n* = 6, 5, 6, 6 animals; see Data S1). Note the nearly complete absence of spread upon *khc-IR* compared with the fast spread at 32 h APF in control. Tukey's multiple comparisons test: \*\*\*p < 0.0001. All scale bars represent 10 µm. See also Figure S4, Video S7, and Data S1.

morphogenesis.³³ According to actin filament morphologies and nuclear positioning, we classified developing myofibers into one of four types (Figures 7A and S5): in type 1, mitochondria are largely excluded from a dense actin filament mesh at the cell cortex and rather cluster around the nuclei. In types 2 and 3, nuclei have distributed and actin filaments have oriented along the myofiber axis, with the

first periodic myofibrils appearing, while mitochondria progressively locate in between the assembling myofibrils. Finally, in type 4, the nuclei have moved to the periphery, the sarcomeres have matured into periodic repeats, and neighboring myofibrils have aligned into their typical cross-striated pattern. Interestingly, the mitochondria are located in between bundles of myofibrils and appear elongated along the long axis of the type 4 fibers, while some mitochondria are even present at the periphery of the type 4 fibers (Figures 7A and S5). The transition from type 1 and 2 myotubes to type 3 and 4 myofibers occurs between days 2 and 4 in culture (Figures 7A and 7B). This shows that within 4 days of culture, mouse mitochondria dynamically change their location, concomitantly with myofibrillogenesis, very reminiscent of the intercalation observed in the developing insect flight muscle.

To test how mitochondria dynamics changes during myofiber morphogenesis, we employed our above-described mitochondria photoconversion experiment by transfecting myotubes with mit-Dendra2. We photoconverted mitochondria in muscle cells of type 2 and type 4 (before and after the mitochondria re-locate, respectively) and followed the spread of the signal to
A

<figure><img src="image_35.png" /></figure>

B

<figure><img src="image_36.png" /></figure>

C

control

<figure><img src="image_37.png" /></figure>

D

nocodazole treatment

<figure><img src="image_38.png" /></figure>

E

<figure><img src="image_39.png" /></figure>

F

control RNAi

<figure><img src="image_40.png" /></figure>

kif5b siRNA-1

<figure><img src="image_41.png" /></figure>

kif5b siRNA-2

<figure><img src="image_42.png" /></figure>

G

Mitochondria
clustering index

<figure><img src="image_43.png" /><figcaption>**Figure 7.** Microtubule-dependent transport of mitochondria is conserved in mammalian muscle</figcaption></figure>

(A) *In vitro* differentiation of mouse myoblasts into mature myofibers classified into 4 differentiation types. Cells were transfected with 4xmts-mNeonGreen to label mitochondria, fixed, and labeled with phalloidin (actin), anti-tyrosinated α-tubulin (microtubules, YL1/2), and DAPI (nuclei). Criteria for classification are shown in Figure S5.

(B) Quantification of the myofiber type distribution during 4 days of culture shows a progressive increase in differentiated myofibers (types 3 and 4).

(C and D) Cultured myofibers were transfected with mit-Dendra2 and mitochondria and photoconverted within the indicated magenta boxes. Type 2 and type 4 fibers were imaged immediately after photoconversion and then every 2 min. Top images show non-converted mit-Dendra2 in green and photo-converted mit-Dendra2 in magenta. The photo-converted mit-Dendra2 is shown in gray below at the indicated time after conversion. In (D), the cultured myofibers were treated with 10 µg/mL nocodazole, 5 min prior to photo-conversion. Plot profiles below quantify the photo-converted mit-Dendra2 over time. Note the slow spread of type 2 compared with the faster spread of type 4 myofibers and the absence of spread upon nocodazole treatment. Scale bars are 10 µm in (A) and 5 µm in (C) and (D).

(E) Quantification of the fluorescence signal spread after 30 min from data in (C) and (D) (N = 5, 5, 8, 4, respectively; see Data S1). Tukey's multiple comparisons test: \*\*\*p < 0.001.

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quantify mitochondria long-range movement. Similar to the pre-
intercalation stage in flight muscles, we found little mitochondria
long-range translocation at stage 2, while at stage 4 mitochon-
dria move over longer distances (Figures 7C and 7E). Thus, mito-
chondria mobility in mouse muscles strongly increases after
mitochondria relocation, similarly to the post-intercalation stage
in the flight muscles, which again suggests a microtubule-based
transport mechanism.

To directly test for a function of the microtubules, we acutely
depolymerized the microtubules with nocodazole for 5 min and
then imaged mitochondria after photoconversion. We found
mitochondria long-range transport stops almost completely af-
ter nocodazole treatment (Figures 7D and 7E), consistent with
microtubules being essential for the mitochondria transport in
type 4 mouse muscle fibers.

As nocodazole is affecting most microtubule-based functions
in muscle cells, we aimed at a complementary and more specific
functional test. Since the kinesin motor KIF5B is required for cor-
rect nuclear distribution in mouse muscle fibers,¹⁹,³⁴ we tested if
KIF5B would also be involved in mitochondria transport. We
knocked down KIF5B using two independent small interfering
RNA (siRNA) constructs and analyzed the mitochondria distribu-
tion in mature type 4 mouse muscle fibers that show clear sarco-
mere striations. Interestingly, we found that KIF5B knockdown
blocked the normal relocation of the mitochondria between the
assembled myofibrils and resulted in prominent mitochondrial
clusters in the vicinity of the clustered nuclei (Figures 7F and 7G).

Taken together, these data demonstrate that mitochondria are
transported along the microtubule cytoskeleton by kinesin motor
proteins in order to coordinate with the assembling and maturing
myofibrils during mouse myofiber differentiation. Hence,
microtubules coordinate mitochondria transport with myofibril
morphogenesis in developing *Drosophila* and mouse myofibers.

DISCUSSION

The two key components of muscles are the contractile myofibrils and the energy-producing mitochondria. Depending on the biomechanical and physiological properties of the muscle type, the interplay between these components results in fiber-type-specific arrangements,<sup>1,5,12</sup> which are important for muscle function. In the *Drosophila* flight muscle, mitochondria intercalate between the assembled myofibrils during development, and this intercalation then feeds back on the transcriptional program of the muscle type.<sup>7</sup> Hence, the morphogenesis of mitochondria and myofibrils must be tightly coordinated.

Here, we investigated the mechanism of how mitochondria
intercalate in between the developing myofibrils in flight mus-
cles. We found that this intercalation occurs within only 2 h
from 26 to 28 h APF. Interestingly, this intercalation coincides
with the time of myofibril assembly: at 30 h APF, periodic imma-
ture sarcomeres are present in all of the 2,000 myofibrils that
have simultaneously assembled in each flight muscle fiber.<sup>13,35</sup>

Thus, the intercalation between these assembled myofibrils
happens homogenously throughout the cell, resulting in 2,000
myofibrils, each of which is surrounded by long tubular mito-
chondria.²⁴,²⁵ We revealed a similar simultaneous change in
mitochondria localization during the assembly of the cross-stri-
ated myofibrils in developing mouse muscle fibers. This argues
for a conserved mechanism.

We discovered an active transport mechanism of the mito-
chondria into the myofibril bundles by the microtubule cytoskel-
eton. The evidence for this transport mechanism is fivefold: first,
we found that microtubules are closely associated with the actin
filaments or the assembling myofibrils during the stage of
mitochondria intercalation. Second, we documented that after
intercalation, mitochondria are transported at longer distances
along the orientation of the microtubule axis, both in flight and
mammalian muscles. Third, we showed that severing microtu-
bules blocks actin bundle formation and mitochondria redistribu-
tion. Fourth, we identified kinesin motor proteins as essential for
mitochondria intercalation, both in flies and mice. Finally, we
showed directly that kinesin is required for the long-range mito-
chondria transport after myofibril assembly. Hence, these obser-
vations provide strong evidence for our hypothesis that the
microtubule cytoskeleton directs the mitochondria into the prox-
imity of individual myofibrils, both in *Drosophila* and mammalian
muscles. The increased longitudinal translocation distances
covered and the anti-parallel microtubule organization, as evi-
denced by the bidirectional spread of a local tubulin signal,
ensure an equal distribution of the mitochondria within each of
the myofibril bundles: as mitochondria continuously move back
and forth along the myofibril axis, they stay distributed.

How is the transport initiated, and how is kinesin recognizing
the mitochondria to be transported? The standard kinesin-mito-
chondria adaptors Milton and Miro, required for transport in neu-
rons,<sup>32,36</sup> are likely linking mitochondria with the kinesin motor in
leg muscles, as *Miro* or *kinesin* know-down result in identical
phenotypes. However, the molecular linker in flight muscles re-
mains unclear to date. Furthermore, we do not know how the
mitochondria intercalation is specifically triggered only at 26 h
APF, when a large number of microtubules are already present
within the actin filament bundles. A plausible mechanism could
be that the early actin bundles are forming a branched mesh
with a small pore size, and hence the rather large mitochondria
cannot intercalate and remain at the bundle surface. When the
actin filaments in each bundle condense to defined myofibrils,
enough space between the individual myofibrils is generated,
allowing the kinesin motors to drag mitochondria in between
the assembling myofibrils. This hypothesis is supported by the
observation that induced hyperfusion, resulting in larger mito-
chondria, blocks the intercalation entirely.<sup>7</sup>

What is the physiological advantage for muscles to apply such
an intercalation mechanism? We see at least three advantages.
First, it ensures that each myofibril is surrounded by a maximal
number of mitochondria. As these will grow in volume when

(F) Cultured myofibers were co-transfected with 4xmts-NeonGreen to label mitochondria and with KIF5B siRNA-1, KIF5B siRNA-2, or control siRNA to test kinesin function. Representative images of mature striated myofibers for control or kinesin knockdown are shown with normalized plot profiles for actin in magenta and mitochondria in green. Note mitochondria are clustered in KIF5B knockdown myofibers.

(G) Quantification of the mitochondria clustering index from data in (F). (N = 7, 6, 6, respectively; see Data S1.) Tukey's multiple comparisons test: "*p* < 0.001, "*p* < 0.05. Scale bars represent 10 µm in (A) and 5 µm in (C) and (D). See also Figure S5, Video S8, and Data S1.
development proceeds, each mature sarcomere is in intimate
proximity to an ATP source. Second, the rapid transport along
the microtubules next to the myofibrils ensures that mitochon-
dria are equally distributed around all 2,000 myofibrils per flight
muscle cell and along the cross-striated myofibrils of mamma-
lian fibers. If mitochondria would stay clustered, ATP support
would not be suboptimal. Third and most importantly, the inter-
calation allows limiting the diameter growth of the myofibrils by
physically blocking the space in between them until a maximum
pressure is reached.⁷,³⁷ Thus, the sarcomeres can maintain an
optimal diameter compatible with ATP supply. This is supported
by our finding that the myofibril diameter in flight muscles in-
creases and displays a larger variation when mitochondria
intercalation was blocked. In the extreme scenario of blocking
mitochondria intercalation entirely by dramatically increasing
mitochondria fusion, flight muscle fate can even be partially
transformed to a cross-striated leg muscle fate.⁷ We have not
observed such a dramatic transcriptional change when blocking
mitochondria intercalation by inhibiting kinesin-dependent
transport, likely because the mechanical environment around
the flight muscle nuclei is more preserved compared with flight
muscles housing mitochondria with changed fusion rates,
many of which show signs of degeneration.⁷ As flight muscle
nuclei sense their mechanical environment to adapt the tran-
scriptional program,³⁷ the coordination of mitochondria with
myofibril morphogenesis during development is essential.

The proposed transport mechanism requires that every
individual myofibril be surrounded by several microtubules ex-
tending along its length. This has been found to be the case for
flight muscles at 32 h APF using high-resolution electron micro-
scopy.²⁴,²⁵ The here shown super-resolution imaging of flight
muscle cross-sections and developing mammalian muscles sup-
ports that microtubules progressively surround the nascent myo-
fibrils. Mitochondria use the few microtubules still present outside
the myofibril bundles to translocate into the bundles. Once inside,
the high concentration of microtubules provides a highway for the
transport and achieves a homogenous distribution of mitochon-
dria within the developing and growing muscles.

How do microtubules organize themselves in proximity to the
actin filament bundles? At the moment, we can only speculate. It
is well established that microtubules are organized around the
many nuclei of the muscle fibers.¹⁸,³⁸ However, at the intercala-
tion stage, the nuclei are located outside of the myofibril bundles:
at the surface of mature mammalian fibers and in between the
actin bundles in flight muscles, thus not where most microtu-
bules are found. Hence, it is more likely that the assembling
myofibrils recruit or organize the microtubules and vice versa,
as supported by the dramatic actin filament defects found within
2 h after microtubule severing. One candidate for such linker pro-
tein between actin and microtubules is Shortstop (spektrapla-
kin).³⁹⁻⁴¹ Microtubules could then act as an orientation cue and
help actin polymerization along the long axis of the muscle, as
had been suggested.⁴² However, all prevalent models of myofi-
brillogenesis—the tension-driven self-organization model,¹³ the
pre-myofibril model,⁴³ or the stress fiber-based model—would
need significant updates to integrate a function for microtubules
in assembling long and straight myofibrils that span through
large muscle cells. Microtubules then ensure that each myofibril
will be surrounded by the energy-producing mitochondria.

The detailed myofibril and mitochondria arrangements will be adjusted to the muscle type. As the mammalian myofibrils are cross-striated, the ensheathing of each myofibril by the mitochondria is less complete compared with the flight muscle. Some mitochondria only send thin protrusions to the mature mammalian sarcomeres at defined positions of the sarcomere.¹ A similar observation was made in cross-striated *Drosophila* leg muscles, with their mitochondria projecting thin extensions to the sarcomere Z-discs.²,⁷ Even the formation of these extensions requires kinesin function, at least in *Drosophila* leg muscles, suggesting that microtubule-based mitochondria transport is a continuous and widespread phenomenon during muscle development and possibly also during muscle maintenance.

**Limitations of the study**

Our results defined a mechanism for how mitochondria are
transported into the vicinity of the assembling myofibrils. Howev-
er, we do not yet know how the microtubules, responsible for this
transport, are specifically located in proximity to the myofibrils.
Furthermore, we do not understand how the microtubules pro-
vide a directional cue to the assembling myofibrils to run only
along the long axis of the muscle fiber. Future experiments will
be needed to pinpoint the mechanistic role of microtubules for
myofibril assembly or orientation.

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be
directed to and will be fulfilled by the lead contact, Frank Schnorrer (frank.
schnorrer@univ-amu.fr).

**Materials availability**

Unique materials generated in this study are available from the **lead contact** without restriction.

**Data and code availability**

All data generated in this study are available from the **lead contact** without restriction.

ACKNOWLEDGMENTS

We thank Manos Mavrakis for generously sharing the UAS-LAD9-GFP line before publication. We are grateful to Dirk Görlich, Yohanns Bellaïche, Valdimir Gelfand, Thomas Rival, Ferenc Jankovics, the Vienna *Drosophila* Resource Centre (VDRC), and the Bloomington *Drosophila* Stock Center for fly strains and nanobodies. We are grateful to Christophe Pitaval for excellent technical support and the entire Schnorrer and Gomes labs for insightful discussions on this manuscript. We are indebted to the IBDM imaging and fly facilities for help with image acquisition, maintenance of the microscopes, and production of fly food. We thank the Bioimaging and Rodent facilities of GIMM for the technical support.

This work was supported by Aix-Marseille University (PhD fellowship. J.A.),
by the Fondation pour la Recherche Médicale (FDT202106012964, J.A.), the
Centre National de la Recherche Scientifique (CNRS, F.S., and N.M.L.), the
European Research Council under the European Union's Horizon 2020
Programme (ERC-2019-SyG 856118 F.S. and ERC-2018 810207 E.R.G.), the
excellence initiative Aix-Marseille University A*MIDEX (ANR-11-IDEX-0001-
02, F.S.), the French National Research Agency with ANR-ACHN MITO-DY-
NAMICS (ANR-18-CE45-0016-01, F.S.), the France-Biolmaging national
research infrastructure (ANR-10-INBS-04-01) and by funding from France
2030, the French Government program managed by the French National
Research Agency (ANR-16-CONV-0001), and from Excellence Initiative of
Aix-Marseille University – A*MIDEX (Turing Centre for Living Systems).
# **Developmental Cell**
## **Article**

CellPress
OPEN ACCESS

## AUTHOR CONTRIBUTIONS

Conceptualization: F.S., N.M.L., J.A., and E.R.G.; methods: J.A., N.M.L., D.C., and A.d.R.S.; investigation and validation: J.A., N.M.L., D.C., and A.d.R.S.; visualization: J.A., N.M.L., A.d.R.S., E.R.G., and F.S.; funding acquisition: F.S. and E.R.G.; supervision: F.S., N.M.L., E.R.G., and A.d.R.S.; writing – original draft: F.S., N.M.L., J.A., A.d.R.S., and E.R.G.; writing – review & editing: F.S., N.M.L., J.A., A.d.R.S., and E.R.G.;

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* *Drosophila* husbandry and strains

* Cell culture of primary myofibers

* METHOD DETAILS

* Immunostaining of *Drosophila* flight muscle and image processing

* Immunostaining of in vitro cultures of mouse myofibers

* Quantification of myofibrils width

* Plasmids and transfections of mouse myofibers

* Live imaging and photo-conversion

* Live imaging and photo-conversion of in vitro cultures of mouse myofibers

* Quantification of the photoconverted Dendra2 signal spread

* Quantification of the individual mitochondria dynamics

* Quantification of the mitochondria clustering index

* Data Visualization

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.devcel.2025.06.033.

Received: September 9, 2024

Revised: March 20, 2025

Accepted: June 23, 2025

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# Developmental Cell
## Article

CellPress
OPEN ACCESS

## STAR★METHODS

## KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>goat anti-mouse IgG Alexa Fluor 488</td><td>Molecular Probes</td><td>Cat# A-11001, RRID: AB_2534069</td></tr><tr><td>goat anti-mouse IgG Alexa Fluor 568</td><td>Molecular Probes</td><td>Cat# A-11031, RRID: AB_144696</td></tr><tr><td>goat anti-rat IgG Alexa Fluor 555</td><td>Life Technologies</td><td>Cat# 4417, RRID: AB_10696896</td></tr><tr><td>GFP nanobody Alexa Fluor 488</td><td>Dirk Görlich laboratory</td><td>Nanobody-ID SB19</td></tr><tr><td>mouse monoclonal anti-α-tubulin</td><td>Sigma-Aldrich</td><td>Cat# CP06, RRID: AB_2617116</td></tr><tr><td>rat anti-tyrosinated α-tubulin (YL1/2)</td><td>ECACC</td><td>Cat# 92092402</td></tr><tr><td>mouse monoclonal anti-ATP5</td><td>Abcam</td><td>Cat# ab14748, RRID: AB301447</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>VECTASHIELD plus DAPI</td><td>Vector</td><td>SKU: H-1200-10</td></tr><tr><td>VECTASHIELD</td><td>Vector</td><td>SKU: H-1000-10</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>Cat#32670</td></tr><tr><td>phalloidin-rhodamine</td><td>Thermo Fisher Scientific</td><td>Cat# R415</td></tr><tr><td>Phalloidin Alexa Fluor 647</td><td>Life Technologies</td><td>Cat#A22287</td></tr><tr><td>collagenase type V</td><td>Sigma-Aldrich</td><td>Cat#C9263</td></tr><tr><td>dispase II</td><td>Gibco</td><td>Cat#17105-041</td></tr><tr><td>DPBS</td><td>Gibco</td><td>Cat#14040133</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>Cat#31980022</td></tr><tr><td>FBS</td><td>Eurobio Scientific</td><td>Cat#CVFSVF00-01</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>Cat#15140-122</td></tr><tr><td>Iscove's Modified Dulbecco's Medium (IMDM) with GlutaMAX</td><td>Invitrogen</td><td>Cat #31980022</td></tr><tr><td>Matrigel</td><td>Corning Inc.</td><td>Cat#356231</td></tr><tr><td>horse serum</td><td>HyClone</td><td>Cat#HYCLSH30074.02</td></tr><tr><td>Fibroblast Growth Factor</td><td>Sigma-Aldrich</td><td>Cat#F3133</td></tr><tr><td>nocodazole</td><td>Sigma-Aldrich</td><td>Cat#M1404-2MG</td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>Cat#11668-019</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>GIBCO</td><td>Cat #31985070</td></tr><tr><td>recombinante Rat agrin</td><td>R&amp;D Systems</td><td>Cat#550-AG-100</td></tr><tr><td>Goat serum</td><td>Sigma-Aldrich</td><td>Cat #G9023</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>Cat #A7906</td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>Cat #47036</td></tr><tr><td>Fluoromount-G mounting medium</td><td>Southern Biotechnology</td><td>Cat#0100-01</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>D. melanogaster: w1118</td><td>BDSC</td><td>RRID: BDSC_3605</td></tr><tr><td>D. melanogaster: Mef2-GAL4</td><td>Schnorrer et al.<sup>31</sup></td><td>RRID: BDSC_27390</td></tr><tr><td>D. melanogaster: Shine-GAL4</td><td>BDSC, Di Pietro et al.<sup>30</sup></td><td>RRID: BDSC_94199</td></tr><tr><td>D. melanogaster: UAS-mit-GFP III</td><td>BDSC</td><td>RRID: BDSC_8443</td></tr><tr><td>D. melanogaster: UAS-mit-GFP II</td><td>BDSC</td><td>RRID: BDSC_8442</td></tr><tr><td>D. melanogaster: UAS-mit-Dendra2 III</td><td>El Fissi et al.<sup>44</sup></td><td>gift of Thomas Rival</td></tr><tr><td>D. melanogaster: UAS-mit-Dendra2 II</td><td>El Fissi et al.<sup>44</sup></td><td>gift of Thomas Rival</td></tr><tr><td>D. melanogaster: UAS-mit-Kate2</td><td>Poliacikova et al.<sup>15</sup></td><td>gift of Thomas Rival</td></tr><tr><td>D. melanogaster: UAS-LAD9-GFP (affirmer binding actin)</td><td>Martins et al.<sup>45</sup></td><td>gift of Manos Mavrakis</td></tr><tr><td>D. melanogaster: UAS-Lifeact-Ruby</td><td>Hatan et al.<sup>44</sup></td><td>RRID: BDSC_35545</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>D. melanogaster: Act88F-GFP (fTRG10028)</td><td>Sarov et al.<sup>46</sup></td><td>RRID: VDRC_318362</td></tr><tr><td>D. melanogaster: Flightin-GFP (fTRG876)</td><td>Sarov et al.<sup>46</sup></td><td>RRID: VDRC_318238</td></tr><tr><td>D. melanogaster: β-tubulin60D-GFP</td><td>Sarov et al.<sup>46</sup></td><td>RRID: VDRC_318309</td></tr><tr><td>D. melanogaster: Mhc-Tau-GFP</td><td>Chen and Olson<sup>45</sup></td><td>RRID: BDSC_53739</td></tr><tr><td>D. melanogaster: UAS-tubulin-EOS</td><td>Lu et al.<sup>26</sup></td><td>gift of Vladimir Gelfand</td></tr><tr><td>D. melanogaster: UAS-spastin-eGFP</td><td>Jankovics and Brunner<sup>27</sup></td><td>Gift of Ferenc Jankovics</td></tr><tr><td>D. melanogaster: UAS-khc-RNAi</td><td>Dietzl et al.<sup>47</sup></td><td>RRID: VDRC_GD44337</td></tr><tr><td>D. melanogaster: UAS-miro-RNAi</td><td>Dietzl et al.<sup>47</sup></td><td>RRID: VDRC_KK106683</td></tr><tr><td>D. melanogaster: UAS-Drp1</td><td>BDSC</td><td>RRID: BDSC_51647</td></tr><tr><td>Mus Musculus: C57BL/6</td><td>Charles River</td><td>Strain code: 027</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>KIF5B siRNA#1</td><td>Thermo Fisher Scientific</td><td>ID s68782</td></tr><tr><td>KIF5B siRNA#2</td><td>Thermo Fisher Scientific</td><td>ID s68781</td></tr><tr><td>Silencer Select Negative Control</td><td>Thermo Fisher Scientific</td><td>Cat#4390843</td></tr><tr><td colspan="3"><strong>Recombinant DNA</strong></td></tr><tr><td>mMito-dendra2</td><td>AddGene</td><td>Plasmid #55796</td></tr><tr><td>4xmts-mNeonGreen</td><td>AddGene</td><td>Plasmid #98876</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>Fiji (ImageJ)</td><td>Schindelin et al.<sup>48</sup></td><td>https://imagej.net/software/fiji/</td></tr><tr><td>Prism 9 (version 9.5.1)</td><td>GraphPad Software</td><td>https://www.graphpad.com</td></tr><tr><td>MTrackJ (ImageJ plugin)</td><td>Image Science</td><td>https://imagej.net/plugins/mtrackj</td></tr><tr><td>Python</td><td>Python Software Foundation</td><td>https://www.python.org</td></tr><tr><td colspan="3"><strong>Other</strong></td></tr><tr><td>μ-Slide 8 Well ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized</td><td>Ibidi</td><td>Cat #80826</td></tr><tr><td>Zeiss LSM 980 with Airyscan 2</td><td>Zeiss</td><td>N/A</td></tr><tr><td>Objective Plan-Apochromat 63x/1.4 Oil DIC M27</td><td>Zeiss</td><td>Item no.:420782-9900-799</td></tr></tbody></table>

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Drosophila husbandry and strains

All crosses were carried out at 27°C under standard conditions.⁷ Flight muscle development movies used *Mef2*-GAL4, UAS-mit-Kate2; UAS-LAD9GFP (affimer binding actin, gift of Manos Mavrakis⁴⁹) pupae for double labelling of mitochondria and actin. *In vivo* double labelling of mitochondria and microtubules was performed with *Mef2*-GAL4, UAS-mit-Kate2¹⁵; *Mhc*-TauGFP (BL53739)⁴⁵ pupae. *In vivo* double labelling of actin and microtubules was performed with *Mef2*-GAL4, UAS-LifeAct-Ruby (BL35545)⁵⁰; *Mhc*-TauGFP pupae. In photoconversion experiments, *Mef2*-GAL4, UAS-mit-Dendra²¹⁴ pupae were used to study mitochondrial dynamics and *Mef2*-GAL4, UAS-td-EOS-α-Tubulin²⁶ pupae were used to study microtubule dynamics (gift of Vladimir Gelfand). Spastin induction experiments were performed with *shine*-GAL4³⁰; UAS-*spastin*-EGFP.²⁷ Experiments using fixed flight muscles were performed with *Mef2*-GAL4, UAS-mit-GFP for Figure 1 or *βTub60D*-GFP fosmid flies⁴⁶ for Figure 3 or *Mef2*-GAL4, UAS-mit-Kate2; *βTub60D*-GFP flies for Figure 4. Kinesin knockdown experiments were performed with *Mef2*-GAL4, UAS-mitDendra2 crossed to *w[1118]* control or UAS-*khc-IR* (VDRC: GD44337).⁴⁷ Miro knockdown experiments were performed with *Mef2*-GAL4 UAS-mitDendra2 crossed to *w[1118]* control or UAS-*miro-IR* (VDRC: KK106683) pupa. The fosmid expression comparison experiment were performed with *Mef2*-GAL4, *Act88F-GFP* (fTRG10028) or *Mef2*-GAL4, *flightin-GFP* (fTRG876)⁴⁶ cross to *w[1118]* control or UAS-*khc-IR* (VDRC: GD44337).⁴⁷

Cell culture of primary myofibers

All procedures using animals were approved by the institutional ethics committee and followed the guidelines of the National Research Council Guide for the Care and Use of Laboratory Animals.

*In vitro* myofibers were differentiated as previously described⁵¹ from primary myoblasts isolated from dorsal hind-limb muscles of 5-7-day-old C57BL/6 mice. Briefly, muscles were dissected, minced mechanically with curved scissors and digested with collagenase type V (Sigma-Aldrich #C9263) and dispase II (Gibco #17105-041) in DPBS (Gibco #14040133) for 90 min at 37°C with agitation.
# Developmental Cell

## Article

<figure><img src="image_44.png" /></figure>

CellPress

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The digestion was stopped by mixing IMDM with GlutaMAX (Gibco #31980022), 10% FBS (Eurobio Scientific #CVFSVF00-01) and 1% penicillin-streptomycin (Gibco #15140-122). The tissue homogenate was centrifuged for 5 min at 75 x g to remove undigested tissue. Supernatant was collected and centrifuged for 5 min at 350 x g. After centrifugation, cells were resuspended in the same medium, passed through a 40 µm cell strainer and pre-plated for 4 hours to allow for fibroblasts adherence. Non-attached cells were collected, centrifuged for 5 min at 350 x g and plated at a density of 220,000 cells/ml in IMDM with GlutaMAX 20% FBS, 1% Pen/Strep, 1% chicken embryo extract. Cells were seeded in µ-dish 35 mm dishes (Ibidi; 80136) pre-coated with 1% Matrigel (Corning Inc. #356231). After 3 days of proliferation, medium was replaced to induce differentiation with IMDM with GlutaMAX, 2% horse serum (HyClone #HYCLSH30074.02) and 1% Pen/Strep. The following day, a coating of 50% Matrigel was placed on top of the seeded cells. After upper coating, the medium was supplemented with 10 µg/ml recombinant agrin (R&D Systems #550-AG-100) and fibroblast growth factor (Sigma-Aldrich #F3133). **Drug treatments:** To depolymerize microtubules, 10 µg/mL nocodazole (Sigma-Aldrich #M1404-2MG) was added to cells 5 min before live imaging acquisition.

## METHOD DETAILS

### Immunostaining of *Drosophila* flight muscle and image processing

Flight muscles were dissected at the indicated stage of development. Pupae were freed from their pupal case with forceps and pierced 3 times in the abdomen with a dissection needle to punch holes. Pupae were then fixed in 4% PFA in PBS + 0.3% Triton (PBST) for 30 min at room-temperature under agitation. Each fixed pupa was then dissected into two hemi-thoraxes according to a published protocol.⁵² Briefly, fixed pupae were placed in a silicon petri dish filled with PBS and immobilised with insect pins. The ventral side of the pupa was removed with scissors by cutting from head to abdomen along the ventral midline to expose the flight muscle. The trachea and fat body were removed with forceps and gentle pipetting before the two halves of the thorax were cut. The hemi-thoraxes were incubated 2 hours in 24-well plates for actin staining with phalloidin rhodamine (1/500, Invitrogen R415) or GFP nanobody (1/2000, gift of Dirk Görlich) to visualise mitochondria and microtubules that were labelled with *Mef2-GAL4*, UAS-mit-GFP or *βTub60D*-GFP fosmid. UAS-mit-Dendra2 labelling mitochondria were visualised without additional antibody.

In pupae from the *shine-GAL4* experiments the mitochondria were visualised by a two-step immunostaining. After fixation and dissection, which was done as noted above, blocking was performed for 1h RT in 5% normal goat serum (NGS) in PBS. Samples were then incubated in anti-ATP5α antibody (1/500, Abcam #ab14748) overnight, followed by secondary antibody (goat anti-mouse Alexa Fluor 488) for 2 h at room temperature. Between each step, the pupae were washed 3 times for 1, 5 and 15 min in PBST, respectively. The hemi-thoraxes were mounted in Vectashield containing DAPI with two coverslips on each side as spacers. Images from fixed flight muscles were acquired with a Zeiss LSM880 confocal with or without AiryScan super-resolution and processed with Fiji (ImageJ).⁴⁸

### Immunostaining of in vitro cultures of mouse myofibers

Mouse myofibers were fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature and permeabilized with 0.5% Triton X-100 for 5 minutes. Cells were blocked in a solution containing 10% goat serum, 5% BSA and MOM block (R&D Systems). Primary and secondary antibodies were diluted unblocking solution and 0.1% saponin. Cells were incubated with primary antibodies overnight at 4°C and washed three times for 10 minutes with PBS. Cells were incubated with secondary antibodies for 1 hour at room temperature and washed three times for 10 minutes with PBS. Cells were mounted with Fluoromount-G (Invitrogen). Antibodies used for immunostaining were anti-tyrosinated α-tubulin (YL1/2) (1/25 ECACC) and anti-rat Alexa Fluor 555 secondary antibody (1/400, Life Technologies). Phalloidin conjugated with Alexa Fluor 647 (1/200, Life Technologies, A22287) was used to stain actin. DAPI was used to stain the nucleus (1/10,000; Sigma-Aldrich).

Image acquisition was performed on a Zeiss LSM 980 confocal microscope equipped with the ZEN gray edition software, using a Plan-Apochromat 63x Oil DIC objective (numerical aperture: 1.40). All images were processed in Fiji.⁴⁸ For nocodazole treated myofibers, to account for cell drift, the Fast4Dreg Fiji plugin drift correction tool was used.⁵³ GraphPad Prism version 8.0.0 (San Diego, CA, USA) was used for data fitting. Data analysis was performed in Microsoft Excel and GraphPad Prism.

### Quantification of myofibrils width

Myofibers developing at 90h APF from *Mef2-GAL4* or *Mef2-Gal4*, UAS-*khc-IR* flies are imaged longitudinally. Stacks are used to reconstruct a virtual cross section. Circles containing cross cut myofibrils were manually generated and the diameter of each circle was measured to quantify the mean myofibril diameter and its variability (standard deviation) for each animal.

### Plasmids and transfections of mouse myofibers

*In vitro* myofibers were transfected 3 days after plating using Lipofectamine 2000 (Thermo Fisher Scientific; 11668-019) following the manufacturer's instructions: 1 µg of DNA per ibidi 35-mm dish of mMito-dendra2 construct (AddGene; Plasmid #55796) was used to label photoconvertible mitochondria, while 4xmts-mNeonGreen(AddGene; Plasmid #98876) was employed to label mitochondria for immunostaining; for kinesin-1 (KIF5B) depletion, cells were co-transfected with 0.5 µg of 4xmts-mNeonGreen (AddGene; Plasmid #98876) and 10 nM of KIF5B siRNA#1 (ID s68782) or siRNA#2 (ID s68781) (Thermo Fisher Scientific). Silencer Select Negative Control (Thermo Fisher Scientific; #4390843) was used as the control.
## Live imaging and photo-conversion

Live imaging in *Drosophila*: pupae were mounted for *in vivo* imaging according to the published protocol⁵⁴ and imaged with a spinning disc confocal microscope (Olympus). Briefly, scissors were used to cut a small window in the pupal case facing one side of the thorax. Pupae were then placed in a custom-made microscope slide with a groove and slightly tilted sideways (about 20 degrees) such that the window in the pupal case and the flight muscles below face the microscope objective. Coverslips as spacer were applied to both sides of the pupae and an additional coverslip with a drop of 50% glycerol was placed on top of the pupa to mount it. Tape was placed on both sides to hold the mounting. During acquisition, samples were kept at 27°C in an incubation chamber at the microscope.

Dendra2 photo-conversion in *Drosophila*: photo-conversion was performed with a 405 nm laser for 30 sec at 10% laser power on a square region of interest located in the middle of the flight muscle at 25 h, 27 h or 32 h APF. A dual-colour 488 nm / 561 nm acquisition was launched about 1 minute after the start of photo-conversion to track both the non-converted and the converted Dendra2 signals each 2 minutes for 30 minutes.

## Live imaging and photo-conversion of in vitro cultures of mouse myofibers

Dendra2 photoconversion in mammalian cell culture: Photoconversion experiments were performed on a Zeiss LSM 980 confocal microscope equipped with the ZEN gray edition software, using a Plan-Apochromat 63x Oil DIC objective (numerical aperture: 1.40). Live imaging was performed using a large cage incubator to maintain cultures at 37°C and 5% CO₂. Fluorescence photoconversion was conducted using a 405 nm wavelength laser at 100% laser power for one iteration. The photoconverted area consisted of a rectangle with 15 µm of width. Each fiber was imaged during 30 min periods at 2 min intervals, with one frame being acquired before photoconversion.

## Quantification of the photoconverted Dendra2 signal spread

### Flight muscles

A line 100 µm long and 10.83 µm thick was drawn from the photoconverted area along the long axis and a line 50 µm long along the short axis. The plot profiles of the lines were obtained for each time point. Intensity values of the photoconverted Dendra2 signal for each position along the line were normalised to the average intensity for the corresponding time point. Normalised values greater than 1-fold were considered as true signals, hence, the signal front was determined as the position at which the photoconverted signal reduces to or below 1-fold. The shift of the distance for the signal front at t=0 minutes and t=20 minutes was measured (one value per animal).

### Cultured myofibres

A line 50 µm long and 2.12 µm thick was drawn from the photoconverted area towards the end of the myofibre to include most of the photo-converted signal. The background signal was subtracted from the intensity values of the photo-converted Dendra2 signal for each position along the line. Background signal was defined as the maximum photoconverted Dendra2 value at a position included in the non-photoconverted area (from 20 µm to 50 µm position along the line) at time 0. The intensity values for each position along the line, corrected for background greater than 0, were used to determine the front of the signal. The shift of the distance of the signal front at t=0 minutes and t=30 minutes was measured (one value per muscle cell).

## Quantification of the individual mitochondria dynamics

High-speed (200 msec) *in vivo* movies of developing flight muscle at 26–27 h APF from intact pupae expressing two copies of *UAS-mitGFP* driven by *Mef2-GAL4* were used to manually track individual mitochondria transport using the MTrackJ 1.5.1 ImageJ plugin. Only mitochondria that were isolated enough to be observed were tracked. Each track is tracking a single mitochondrion continuously along one direction. From each track, the distance, the speed and the orientation of the transport were quantified using the initial and final position of the mitochondrion track. The orientation of each track (in degrees) was measured as deviation from to the long axis (0 degree) and is reported between 0 and 90 degrees. Transport of mitochondria initially located inside or outside of bundles were compared for the quantification.

## Quantification of the mitochondria clustering index

Cultured myofibres: A 60 µm long and 3.53 µm thick line was drawn along the long axis of each myofibre. The intensity values of the mitochondria and actin signals for each position along the line were normalised to their average intensity and plotted. The ratio (normalised mitochondria value – normalised actin value) was measured for each position. The mitochondria clustering index was defined as the standard deviation of all ratio values along the line. Quantification was performed on cross-striated mature myofibres at day 4 transfected with control RNAi or KIF5B RNAi.

## Data Visualization

Line profiles of mitochondria and actin intensities along the y axis shown in Figure 1B, or photoconverted mitochondria in Figure 2B-C and Figure 7C-D were plotted using the Python package Matplotlib, after normalization of raw data (available in the Data S1 table). Binning with a rolling window size of 20 (Figure 1B, Figure 2B-C) or 40 (Figure 7C-D) was done via SciPy lfilter to smooth the intensity profiles for visualization purposes. Box plots were done using GraphPad Prism 8.0.2.
**Developmental Cell**
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<figure><img src="image_45.png" /></figure>

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QUANTIFICATION AND STATISTICAL ANALYSIS

Quantification and statistical analyses were performed using GraphPad Prism 8.0.2 and are reported in Data S1. For all plots, the median was used for the line, the first and last quartile for the box, the minimum and maximum value for the whiskers, and the points represent n = animals, except for Figure2 H-J where n = track of individual mitochondria. Each quantification was performed on a minimum of four animals per condition, see figure legends and Data S1. Mann Whitney or Tukey's multiple comparisons tests were conducted using GraphPad Prism 8.0.2. Statistical significance was represented as follows: ns (not significant): *P* > 0.05; *: *P* ≤ 0.05; **: *P* ≤ 0.01; ***: *P* ≤ 0.001; ****: *P* ≤ 0.0001.