Journal of Cell Science

Microtubules, composed of - and {beta}-tubulin polymers, are essential components of the eukaryotic cytoskeleton. They maintain cellular shape and structural integrity and play critical roles in cell division and in intracellular vesicular transport. In Plasmodium, the parasite that causes malaria, nuclear replication during the liver stage is among the fastest known in eukaryotic cells and relies heavily on microtubules for DNA segregation and cytoskeletal organization. Despite their importance, the role of microtubules in liver stage development remains largely unexplored. Here, we investigated microtubule dynamics during liver stage development using a combination of cell and molecular biology techniques, expansion microscopy, and live-cell imaging. By employing antibodies specific for {beta}-tubulin post-translational modifications (PTMs), we found that the Plasmodium sporozoites subpellicular microtubules (SSPM) persist during liver infection, giving rise to liver stage parasite microtubule bundles (LSPMB). These LSPMB form multimeric tubulin structures within hepatocytes and are redistributed to the hemi-spindle poles of parasite nuclei during schizogony. Deletion of the C-terminal region encompassing all known Plasmodium -tubulin PTM sites prevented sporozoite migration from the mosquito midgut to the salivary glands, effectively blocking parasite transmission. Using Plasmodium microtubule-specific depolymerisation drugs, we found that while LSPMB are stable in sporozoites, they exhibit dynamic behavior during hepatocyte infection. Given the regulatory role of PTMs in microtubule dynamics, we generated parasite mutants by substituting and deleting key {beta}-tubulin C-terminal residues involved in PTMs. Substitution of the polyglutamylation site with alanine and deletion of the C-terminal tyrosination/detyrosination motifs impaired parasite growth during liver infection. Together, our findings reveal extensive microtubule remodeling during liver stage development and establish -tubulin C-terminal modifications as critical regulators of both intracellular development and parasite transmission of Plasmodium parasites.

Spatiotemporal allocation and dynamic redistribution of centrosomal proteins are critical for coordinating the structural and molecular events that ensure successful cell division. While depletion of the multifunctional protein Optineurin (OPTN) impairs cell-cycle progression, our previous report of the presence of OPTN at key cell-division assemblies raised the question of how its trafficking is coordinated during cell division. In this study, using cell-cycle synchronisation coupled with flow cytometric analysis and confocal microscopy, we report that OPTN progressively dissociates from centrosomes during mitosis, redistributes to cytokinetic structures, accumulates at the spindle midzone, and then concentrates at the midbody during cytokinesis. Moreover, following this transition, OPTN progressively re-emerges at centrosomes during G1 of the subsequent cell cycle. Further, microtubule perturbation and regrowth assays provide evidence that OPTN recruitment and retention at the pericentriolar matrix (PCM) can occur independently of microtubules, whereas its exit is microtubule-dependent. Collectively, this study depicts, for the first time, a detailed movement trail of OPTN, highlighting its centrosome-linked redistribution and microtubule dependency. This context-dependent localisation and trafficking of OPTN across the centrosome-midbody axis establish a definitive spatiotemporal roadmap that can be strategically utilised to study its functional role in cell division.

One of the first genes identified to regulate the spacing of nuclei in the multinucleated myofiber was Kinesin-1. However, the mechanism by which Kinesin-1 or other kinesins regulate myonuclear spacing is not known. Critically, the myofiber lacks centrosomes, and the many myonuclei act as the primary microtubule organizing centers of the cell. Because of this unique re-structuring, we hypothesized that the kinesins that drive centrosomes apart during mitotic spindle elongation may play a similar role in spacing myonuclei. We found that the bipolar Kinesin-5 (Klp61f) and the (-)-end directed Kinesin-14 (ncd) were both necessary for myonuclear spacing at different times, with both being necessary during embryogenesis, but only ncd being necessary in the fully differentiated myofiber. To investigate the shared mechanisms during embryogenesis, we used live-imaging and found that, similar to the mitotic spindle, Klp61f acts as an accelerator for myonuclear movement, whereas ncd acts as a brake contrary to this movement. To investigate these mechanisms and test the hypothesis that this is dependent on microtubule-sliding, we used super-resolution microscopy to visualize and quantify the microtubule network in animals with disrupted Klp61f or ncd. We found that in both cases, there was a decrease in the amount of microtubule overlap between neighboring myonuclei. Furthermore, we found that disrupting ncd led to severe changes in microtubule network organization, supporting our hypotheses that microtubule-sliding is necessary to space myonuclei, and that ncd likely functions through a unique mechanism in the differentiated myofiber to maintain myonuclear spacing. Together, our data supports a model where myonuclear spacing is regulated by a counteracting force generated by different kinesins during embryonic development. Furthermore, one kinesin, ncd, is repurposed in the differentiated myofiber to dynamically crosslink microtubules, a function necessary to anchor nuclei in place. Thus, kinesin motors regulate myonuclear spacing across developmental time by leveraging opposing forces through diverse mechanisms.

Bipolar spindle assembly is essential for accurate chromosome segregation. KIFC1, a conserved Ran- regulated minus-end-directed kinesin-14 motor, accumulates in the nucleus during interphase and promotes chromatin-mediated spindle assembly during mitosis and meiosis. In human oocytes, reduced KIFC1 levels destabilize meiotic spindles, a defect that can be rescued by increasing KIFC1 expression. However, how KIFC1 expression levels affect mitotic spindle stability during cleavage divisions in vertebrates remains unclear. Here, we show that whereas an approximately 50% reduction in KIFC1 causes no detectable defects in spindle assembly, approximately 10-fold overexpression of KIFC1 induces monopolar spindle formation, leading to chromosome mis-segregation and embryonic lethality in medaka early embryos. KIFC1 overexpression results in ectopic centrosomal localization during interphase, impairing the separation of duplicated centrosomes before mitotic entry. Analyses of KIFC1 mutants demonstrated that these centrosome separation defects require KIFC1s microtubule-binding and motor activities and are further enhanced by deletion of KIFC1s nuclear localization sequences. Together, our findings demonstrate that tight regulation of KIFC1 expression and its nuclear sequestration is essential for the proper separation and positioning of duplicated centrosomes before mitotic entry, thereby ensuring efficient bipolar spindle assembly during the rapid cleavage divisions of vertebrate embryos. HighlightsO_LIKIFC1 accumulates in the nucleus and at the embryonic spindle midplane via the Ran pathway. C_LIO_LIPartial KIFC1 depletion does not impair spindle assembly in medaka early embryos. C_LIO_LIKIFC1 overexpression induces monopolar spindles by preventing centrosome separation. C_LIO_LICentrosome separation defects require KIFC1 microtubule-binding and motor activity. C_LI

Cell surface receptors are regulated via a variety of mechanisms including proteolytic inactivation by metalloproteases. Cells often co-express both the receptor and its cognate protease simultaneously on the cell surface, often with the proteases in an active state. Despite this, receptor cleavage is generally not constitutive, and rather must be induced. How a receptor and its inactivating protease can both be present in their active form on the cell surface with no cleavage of the target receptor is unclear. Using the efferocytic receptor MERTK and its inactivating protease ADAM17 as a model, along with super-resolution microscopy approaches, we demonstrate that MERTK and ADAM17 are localized to distinct sub-regions of the plasma membrane. These microdomains are distinct subcellular structures, with MERTK contained within actin-based corrals and ADAM17 contained within caveolae. Induction of MERTK cleavage by PMA results in the reorganization of the membrane-proximal actin cytoskeleton that enables MERTK to diffuse into ADAM17-containing microdomains. Only after localizing to ADAM17-containing microdomains is MERTK cleaved. These results demonstrate that the spatial organization of the plasma membrane is dynamically regulated, with reorganization of membrane microdomains playing a role in the regulation of ADAM17 activity.

Actin is critical for cell integrity and nuclear processes, but methods to visualise how environmental and intracellular signals affect cellular monomeric (G-) actin levels are lacking. We present the GASPs (G-actin sensing proteins), genetically encoded G-actin FRET sensors. Using cytoplasmic- and nuclear-localised GASPs, we show that in fibroblasts integrity of focal adhesions plays a critical role in maintaining low G-actin levels, and that cytoplasmic and nuclear G-actin levels increase at high cell densities and when spreading is restricted. Upon serum stimulation, it is depletion of the cytoplasmic G-actin pool that correlates with rapid nuclear accumulation of the MRTF transcription factors, whose subcellular localisation is controlled by G-actin. Stimuli that induce rapid increases or decreases in cytoplasmic G-actin level induce similar but slower changes in nuclear G-actin level, indicating that fluctuations in nuclear G-actin levels are largely subservient to cytoplasmic actin dynamics.

Budding yeast cells can transition from vegetative to filamentous growth under specific environmental stimuli. Saccharomyces cerevisiae has served as a model to understand this process, which is linked to the virulence of many fungal pathogens. One manifestation of filamentation in S. cerevisiae is the formation of pseudohyphal cells, which are elongated and divide in a polarized manner. Here, we established a live-cell imaging assay to monitor this morphogenetic transition and developed a set of fluorescent expression reporters to probe the activity of various signaling pathways during the first 10 hours of this transition. This approach allowed us to identify the stimulus that activates the filamentous growth Mitogen-Activated Protein Kinase (fgMAPK) pathway, which has long evaded identification. Our results indicate that the fgMAPK cascade senses mechanical cues and becomes activated when cells grow on a solid surface. In addition, we have observed that the stimulation of the transcription factor Gln3 regulated by the TOR pathway in low ammonium condition correlates with the elongation of the cells.

RASAL3 acts as a negative regulator of small cellular GTPases in hematopoietic cells. In immune cells, it primarily modulates the RAS/MAPK signaling pathway and affects cellular events including proliferation, differentiation, survival, and migration. Due to its inhibitory role in T cells, RASAL3 may represent a potential modulatory target for improving therapeutic strategies such as cell-based immunotherapy. However, most existing knowledge about RASAL3 function is derived from murine models, and its role in human T-cell signaling remains insufficiently characterized. To address this gap, we systematically investigated the function of RASAL3 in human primary T cells and T-cell line. For this purpose, we employed RASAL3 overexpression, CRISPR/Cas9-mediated deletion, and siRNA-mediated knockdown to thoroughly analyze the effects of RASAL3 on T-cell signaling, proliferation, and migration. Our data demonstrate that RASAL3 modulates primarily CDC42 and RAC1/RAC2 GTPases activity, SAPK/JNK phosphorylation, c-Fos and c-Jun expression, and IL-2 gene promoter activation. In addition, RASAL3 regulates actin polymerization and T-cell migration. Notably, loss of RASAL3 increases Jurkat T cells motility in vivo and potentiates their homing to the spleen. Collectively, these findings identify RASAL3 as an important regulator of human T-cell activation and motility and highlight its application potential for improving CAR-T cell therapy.

Transfection of cells with DNA plasmids typically involves the uptake of lipoparticles by endocytosis, followed by the inefficient escape of these particles from endosomes into the cytoplasm. We found that the expression of transfected plasmids was reduced in cells depleted of either SEPTIN 9 or proteins in the endosomal sorting complexes required for transport (ESCRT) pathway. The reduction in plasmid expression could not be fully explained by effects on endocytosis. SEPTIN 9 depletion appeared to reduce the acidification of plasmid-containing compartments, suggesting that it primarily affects the pH-sensitive escape of plasmids from endosomes. Depletion of the ESCRT proteins VPS36 or ALIX resulted in especially dramatic reductions in transfected plasmid expression, which were accompanied by reduced colocalization between the transfected DNA and CHMP4, an ESCRT protein important for endosomal membrane remodeling during intraluminal vesicle formation. Finally, transfected plasmid DNA was strongly colocalized with LC3B, suggesting that the default pathway for transfected material is autophagy.

Gasdermins (GSDMs) are a family of pore forming protein that trigger pyroptosis by permeabilizing cell membranes. Pyroptotic cells often release the proinflammatory cytokines interleukin-1{beta} (IL-1{beta}), and IL-18, thereby promoting an inflammatory response. GSDMs are typically cleaved by caspases or granzymes, which enable their translocation to the membrane. Here, we showed GSDMA and GSMDB localize to the cytoskeletal fraction of keratinocytes. Disruption of cell junctions causes gasdermin A and B (GSDMA and GSDMB) to translocate to the membrane fraction in the absence of cleavage. Cell junction disrupted keratinocytes release post-translationally modified keratins, but not IL-1{beta} or IL-18. These events depend on endocytic mechanisms associated with recycling of cell junctional proteins. Our study suggests that cell junction disruption can drive translocation of GSDMA and GSDMB from cytoskeleton to plasma membrane in keratinocytes, however there may be a subsequent trigger that causes the confirmational change allowing these gasdermins to form open pores.

Malaria parasite invasion of red blood cells is powered by Myosin A (MyoA), the actomyosin motor which is a core component of the glideosome. Our previous phosphoproteomic study identified a cyclic GMP-dependent phosphorylation site on MyoA at Ser19, suggesting that cyclic nucleotide signalling may directly regulate motor activity during invasion. However, the functional significance of this phosphorylation site remains unclear. Here, we show that Plasmodium falciparum MyoA phosphorylation at Ser19 is dynamically regulated during the intraerythrocytic cycle, peaking in extracellular merozoites and rapidly declining following invasion. Using conditional depletion of MyoA, we show that MyoA is essential for red blood cell invasion but dispensable for gametocyte development. Conditional complementation with mutant versions of MyoA that either mimic (S19D) or ablate phosphorylation (S19A) reveal markedly impaired asexual parasite proliferation, highlighting the importance of precise regulation of MyoA phosphorylation at Ser19. In vitro motility assays using parasite-derived MyoA showed that ablating phosphorylation either pharmacologically or by introducing an S19A mutation, reduces motor speed. Conversely, introducing a phosphomimetic S19D mutation restores motility levels comparable to phosphorylated wild type MyoA. Collectively, these results establish MyoA Ser19 phosphorylation as a regulatory switch for actomyosin motor activity.

Desmin is the major intermediate filament protein of muscle, yet its potential nuclear functions remain poorly understood. Here, we investigated the nuclear localization and organization of endogenous desmin in human skeletal muscle cells using quantitative confocal microscopy, biochemical fractionation, targeted MRM/SRM proteomics, and computational structural modeling. Desmin localized to the nuclear compartment and exhibited significantly stronger colocalization with emerin and lamin A/C than with lamin B1. Nuclear fractionation and targeted proteomics independently confirmed endogenous nuclear desmin, while protein-protein docking predicted structurally plausible interactions with all three nuclear envelope proteins, supporting a context-dependent nuclear interaction network. Together, these findings identify the nuclear envelope as a functional platform for desmin and suggest that nuclear desmin contributes to nucleo-cytoskeletal communication and the organization of mechanically responsive nuclear architecture during myogenesis. This work expands the current understanding of desmin biology and provides a framework for investigating its role in desmin-related muscle disease.

Although MAP1LC3B/LC3B (LC3) is best known as a cytoplasmic marker of autophagosome biogenesis, a substantial pool of LC3 resides in the nucleus and shuttles dynamically between nuclear and cytoplasmic compartments, yet the composition and regulation of this nuclear interactome remain poorly defined. Using mass spectrometry-based proteomic profiling of nuclear GFP-LC3 immunoprecipitates from primary human trabecular meshwork (TM) cells, a mechanosensitive ocular cell type, we identified a reproducible nuclear LC3 interactome enriched for proteins containing LC3-interacting region (LIR) and expanded LIR (xLIR) motifs and reported nuclear localization. Among these, clathrin heavy chain (CLTC) emerged as a previously unrecognized nuclear LC3 partner that localizes to the nucleus and colocalizes with nuclear LC3 puncta. CLTC depletion reduced basal LC3-II levels, consistent with a role in autophagosome biogenesis, and markedly impaired nuclear LC3 accumulation induced by both nuclear export blockade with leptomycin B and cyclic mechanical stretch, without altering total CLTC abundance, indicating that CLTC actively promotes LC3 nuclear translocation rather than serving as a passive scaffold. Strikingly, mechanical stress-induced nuclear LC3 trafficking, but not its basal component, was selectively impaired in TM cells derived from glaucoma patients, despite comparable CLTC levels, pointing to a defect in coupling this trafficking pathway to mechanotransduction rather than in the core transport machinery itself. Together, these findings establish nuclear LC3 trafficking as an actively regulated, CLTC-dependent process linked to cytoskeletal and vesicular machinery, and implicate its dysregulation in mechanically stressed glaucomatous cells, providing a framework for understanding how autophagy intersects with nuclear homeostasis, mechanotransduction, and glaucoma pathogenesis.

As the main microtubule network organisers of animal cells, centrosomes orchestrate diverse cellular processes. Centrosomes have been linked to cancer when numerically or structurally aberrant, yet their precise roles at different cancer stages remains to be defined. Here, we study centrosome function in the epithelial-to-mesenchymal transition (EMT), a key driver of cancer initiation, progression and metastasis. We first depleted centrosomes in a breast cancer mesenchymal-like model, resulting in insignificant cellular or nuclear rearrangements. We then investigated non-carcinogenic breast cells undergoing the EMT via TGF{beta} signalling, and removed the centrosomes before triggering this transition. Stimulated epithelial cells acquired a mesenchymal phenotype and rearranged their cytoskeleton. By contrast, stimulated cells lacking centrosomes retained epithelial morphology, showed little cytoskeletal remodelling, yet still altered nuclear shape and upregulated key EMT markers. Transcriptome analysis revealed reduced expression of important genes involved in extracellular matrix organisation and a downregulation of cell junction components when lacking centrosomes; further analysis revealed paxillin showing reduced colocalisation with actin focal adhesions. Centrosomes thus prove essential for cytoskeletal remodelling in the EMT of breast cancer cells.

Endosomal trafficking is a major pathway that delivers cell-surface proteins, including glutamate receptors, to support neurotransmission and normal brain functions. Activity-dependent insertion of glutamate receptors is essential for synaptic plasticity, learning and memory. Copine-6 is a neuronal-specific calcium (Ca2+) binding protein that mediates activity-induced exocytosis of -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors. The activation of N-methyl-D-aspartate (NMDA) receptors triggers Ca2+-dependent translocation of Copine-6 to intracellular endosomal compartments. However, the mechanisms underlying the activity-dependent accumulation of Copine-6 in endosomes remain unknown. Here, we show that Copine-6 exhibits Ca2+-dependent binding to phosphatidylinositol-3-phosphate (PI(3)P) through the C2B domain and displays enhanced interaction with active Rab11a in a Ca2+-independent manner via the vWA domain. Mutations in the C2B that inhibit binding to PI(3)P not only block the activity-induced translocation of Copine-6 to early endosomes, but it also causes an aberrant accumulation of Copine-6 in recycling endosomes. Consequently, loss of Copine-6 expression impairs the efficient coupling of early and recycling endosomes and blocks activity-dependent delivery of both AMPA and NMDA receptors onto the neuronal plasma membrane. These defects can be restored by re-expressing wild-type Copine-6, but not the C2B phospholipid-binding mutant. Together, our findings establish Copine-6 as a molecular bridge that enhances coupling between the early and recycling endosomal membranes, thereby facilitating the activity-dependent forward trafficking of glutamate receptors to the neuronal plasma membrane to maintain synaptic potentiation.

Vacuolar H+ ATPases (v-ATPases) are conserved proton pumps that support diverse biological functions through acidification of cellular organelles. The protozoan parasite Leishmania requires its v-ATPase for survival in the sand fly vector and mammalian host, but genetic mutants remain viable in vitro. To gain further insight into this conditionally lethal phenotype, we first mapped organellar localization of the v-ATPase by co-localisation imaging of fluorescently tagged v-ATPase subunits and organelle markers. The v-ATPase signal was strongest in the flagellar pocket region, consistent with enrichment in the contractile vacuole complex (CVC). To define the conditions that require a functional v-ATPase, deletion mutants were exposed to different stresses (pH, temperature, osmolality, dense culture). All tested deviations from standard culture conditions affected the mutants' growth rate, viability or both. Despite differences in phenotype severity, all stressors triggered the formation of a large autolysosome, positive for the autophagy marker protein ATG8 and the lysosomal enzyme cysteine peptidase A, indicating an arrest at the final step of autophagy. Measurements with the pH sensor pHLuorin2 showed that the luminal pH of the lysosomes was 5.6 in unperturbed promastigotes and 7.1 in v-ATPase mutants. These data support a canonical function for the Leishmania v-ATPase in lysosome acidification and autophagy, which is essential for parasite differentiation, and identify the poorly characterized Leishmania CVC as another major site of v-ATPase concentration.

Interleukin-15 (IL-15) trans-presentation (TP) by B cells is an important mechanism of T-cell activation; however, the spatial organisation of interleukin-15 receptor (IL-15R) relative to major histocompatibility complex (MHC) molecules on B-cell microvilli remains poorly understood. As microvilli protrude from the B-cell surface and may serve as sites of initial B cell-T-cell contact, the distribution of IL-15R and MHC molecules within these structures may be important during the earliest stages of T-cell recognition and activation. Here, we investigated the spatial association and molecular proximity of IL-15R with MHC class I and class II molecules on B-cell microvilli before immunological synapse formation, using confocal microscopy, stimulated emission depletion (STED) microscopy, stochastic optical reconstruction microscopy (STORM), and fluorescence lifetime imaging microscopy-based Forster resonance energy transfer (FLIM-FRET). Both MHC class I and class II molecules showed significant spatial association with IL-15R; however, the extent of colocalisation decreased as spatial resolution increased. STED microscopy revealed significant colocalisation between IL-15R and MHC class I, whereas STORM did not detect this association. In contrast, IL-15R and MHC class II remained significantly colocalised at both resolutions. FLIM-FRET further demonstrated molecular proximity between IL-15R and both MHC class I and class II molecules, with higher FRET efficiency observed for MHC class II. Collectively, these findings indicate that IL-15R is spatially organised in proximity to both MHC class I and class II molecules on B-cell microvilli before immunological synapse formation. This arrangement at potential sites of initial B-cell-T-cell contact may facilitate the coordination of IL-15 trans-presentation and antigen presentation during the earliest stages of B-cell-T-cell interactions.

Rhodopsin trafficking from the photoreceptor inner segment to the outer segment is essential for photoreceptor function, yet the molecular mechanism(s) regulating this process remain incompletely understood. MYO1C is an actin-based motor protein implicated in intracellular cargo trafficking. Here, we investigated its role in rhodopsin trafficking and photoreceptor cell homeostasis. In-silico docking identified a putative interaction between the MYO1C C-terminal region and the C-terminal region of rhodopsin containing the conserved VxPx ciliary trafficking motif. Biochemical studies confirmed that full-length MYO1C interacts with rhodopsin, whereas deletion of the MYO1C C-terminal domain abolished this interaction. Live-cell imaging, ciliary localization, and fluorescence recovery after photobleaching in hTERT-RPE1 cells demonstrated that the MYO1C C-terminal region is required for efficient rhodopsin trafficking, membrane localization, and ciliary targeting. In native murine rod photoreceptors MYO1C localized to both inner and outer segments. Global Myo1c deficiency in mice caused age-dependent rhodopsin mislocalization, apo-opsin accumulation and progressive retinal dysfunction, characterized primarily by reduced scotopic ERG responses and delayed a-wave recovery, beginning at 6-months, while photopic responses were relatively preserved. Rod-specific Myo1c deletion similarly caused progressive scotopic dysfunction and reduced a-wave recovery following light stimulation. In contrast, cone-specific Myo1c deletion preserved photopic function and a-wave recovery. Together, these findings identify MYO1C as an important regulator of rhodopsin trafficking and demonstrate a preferential, cell-autonomous requirement for MYO1C in maintaining rod photoreceptor homeostasis and phototransduction recovery. These findings establish a mechanistic link between MYO1C-dependent rhodopsin trafficking and age-dependent rod photoreceptor cell dysfunction.

To maintain genome stability, it is crucial that cells do not initiate cytokinesis until chromosomes have been properly segregated. In the model budding yeast Saccharomyces cerevisiae, a surveillance mechanism called the Spindle Position Checkpoint (SPoC) ensures this coordination by regulating the Mitotic Exit Network (MEN) to couple exit from mitosis and cytokinesis to spindle position. The MEN is conserved in Ascomycota where the orthologous pathway in the fission yeast Schizosaccharomyces pombe, the Septation Initiation Network (SIN), regulates cytokinesis in response to defects in spindle elongation. Here, we show that the MEN/SIN pathway is conserved in the basidiomycetous budding yeast and human pathogen, Cryptococcus neoformans, and controls cytokinesis. However, spindle position or elongation does not regulate pathway activation or cell cycle progression in C. neoformans. In essence, there appears to be no SPoC in this organism to delay cytokinesis upon defects in mitosis. We speculate that while increasing the risk of genome instability, the lack of a SPoC might facilitate C. neoformans's ability to change ploidy in the host.

The sarcoplasmic reticulum (SR) is an elaborate membrane network and the primary intracellular Ca2+ store for muscle contraction. SR abundance, morphology, and spatial organization must be remodeled during muscle atrophy and hypertrophy, yet the mechanisms underlying this remodeling remain poorly understood. Autophagy has been implicated in SR homeostasis and may contribute to this process. We previously showed that a subset of Drosophila larval muscles remodels into adult dorsal internal oblique muscles through an autophagy-dependent process accompanied by extensive SR reorganization. Here, we show that autophagy is essential for maintaining SR abundance and architecture during muscle remodeling. Blocking autophagosome formation caused the accumulation of large aggregates of SR membranes, whereas blocking autophagosome-lysosome fusion led to the accumulation of autophagosomes with SR transmembrane proteins localized to their membranes. Moreover, disrupting LC3-interacting region (LIR)-dependent cargo recognition did not impair SR remodeling, arguing against a major contribution from selective autophagic degradation of the SR. Instead, our findings suggest that SR membrane components are incorporated into autophagosomal membranes during muscle remodeling. We propose that this membrane flow couples the removal of excess SR membrane to autophagosome biogenesis during atrophy, thereby facilitating large-scale SR reorganization during muscle remodeling.