Journal of Cell Biology

The class III phosphatidylinositol 3-kinase complex (PI3K(III)) generates phosphatidylinositol-3-phosphate (PI(3)P), a lipid that defines endosomal membrane identity. Two PI3K(III) complexes share core subunits but differ in their fourth component: the Atg14-containing complex I functions in autophagy, whereas the Uvrag-containing complex II is required for endosomal maturation. Despite this, the mechanism by which complex II promotes lysosomal function remains unclear. Using Drosophila nephrocytes, we show that PI(3)P is enriched on Rab7-positive late endosomes and that the Hsp70 chaperone Hsc70-4 binds phosphoinositides. Loss of PI3K complex II disrupts endolysosomal organization and phenocopies Hsc70-4 inhibition. In both cases, clathrin accumulates on intracellular, often endosomal membranes, Rab7 compartments are disorganized, and abnormal endolysosomal structures form. These defects are accompanied by impaired HOPS recruitment, lysosomal dysfunction, and secretion of endolysosomal content. Importantly, clathrin depletion partially rescues these defects. Together, our findings identify a role for PI3K complex II in promoting clathrin removal from endosomal membranes and link PI(3)P and Hsc70-4 activity to lysosomal maturation.

Telomere-led rapid prophase chromosome movements (RPMs) during meiotic prophase are critical for homologous chromosome pairing and proper meiotic progression. These movements are generated by the cytoskeleton and are transmitted to the telomeres via the LINC complex, yet the cytoplasmic components that generate these forces remain poorly defined. Among candidates of microtubule-associated motor proteins in mouse primary spermatocytes, we confirmed KIF5B as a specific interactor of the KASH5-LINC complex. Total internal reflection fluorescence microscopy and microtubule sedimentation assays performed with purified recombinant proteins suggest a direct interaction between KASH5 and KIF5B on microtubules, enhanced by MAP7, a known KIF5B-recruiting and activating cofactor. Mapping the KIF5B-binding surface of KASH5 revealed that KASH5 N-terminal EF-hand domains mediate the interaction. Further, in vivo KIF5B-KASH5 interaction and KIF5B role in RPMs are evidenced as (1) KIF5B is recruited by KASH5-SUN1 to the nuclear envelope in two different cultured somatic cell models, (2) KIF5B is telomere-associated and colocalizes with KASH5, and microtubules associated with the nuclear envelope in mouse spermatocytes, and (3) chemical inhibition of KIF5B reduces telomere-led chromosome motions. Altogether, our findings identify the KIF5B kinesin as a previously unrecognized component of the force-generating machinery that drives chromosome movement during meiotic prophase I, acting through KASH5 as a specific nuclear membrane adaptor.

Synaptic vesicle proteins (SVPs) are synthesised in the neuronal soma trafficked as precursor synaptic vesicles (pre-SVs) on route to synapses. While pre-SVs are known to have heterogeneous protein composition and can co-traffic with lysosomal proteins. In this study, we assess the trafficking routes and kinetics of Synatobrevin-1 (SNB-1) released from the ER using the RUSH system in vivo in C. elegans touch receptor neurons. We showed that ER-released SNB-1 follows at least two temporally distinct trafficking routes. A predominantly anterogradely moving population of SNB-1 carrying vesicles appeared early, within 20 minutes of ER release in the axon without overlap with lysosomal proteins. Another SNB-1 population at 45 minutes post-ER release overlapped with endolysosomal compartments in both the cell body and the axon. Early SNB-1 carrying vesicles co-migrate with a transmembrane synaptic vesicle protein Synaptogyrin (SNG-1) and RAB-27 but fewer with RAB-3, suggesting that SVPs can be co-sorted into the same carriers prior to overlap with lysosomal proteins. The SV-lysosomal protein overlap occurs even when SNB-1 endocytosis on the plasma membrane is reduced in unc-11/ap180 mutants. Finally, we identified SAM-4/Myrlysin, a subunit of the BORC complex, as a regulator of both the trafficking kinetics of Synaptobrevin-1 intermediates and the cargo composition of pre-SVs. Loss of SAM-4 accelerated SV-lysosomal protein overlap and reduced co-transport of SNG-1 with SNB-1 in early pre-SVs in the axon. Together, these findings reveal heterogeneity in pre-SV biogenesis routes and identify SAM-4 as a key regulator of both the kinetics and cargo composition of synaptic vesicle precursors.

In metazoans, mitochondria optimally distribute to sites of need through long-range transport events on microtubules. The prevailing model for this trafficking mechanism is that the tail-anchored calcium-binding GTPase, Miro, recruits cytosolic TRAK and associated molecular motors to the outer mitochondrial membrane. Therefore, Miro is proposed to be an obligate adaptor for TRAK required for bulk mitochondrial transport, a process that is considered particularly important for long-range trafficking in neurons, and thus, for viability. Here, we impaired Miro-TRAK interaction in vivo by introducing a point mutation into the Drosophila TRAK orthologue Milton, that impairs its interaction with Miro, based on recent structural evidence. Flies harbouring this point mutation are viable to adulthood. Moreover, neurons carrying this mutation exhibit little to no observable reduction in axonal mitochondria. Mutant flies, however, display progressive loss of motor function with age and reduced lifespan. We therefore call into question the long-standing view that Miro plays an obligatory role in mitochondrial trafficking and challenge the canonical model for mitochondrial transport.

During maturation of a Golgi cisterna, multiple vesicular transport pathways recycle resident Golgi proteins. Recycling vesicles are captured by Golgi-associated tethers. To assign individual tethers to specific recycling pathways in Saccharomyces cerevisiae, we examined tether arrival and departure using kinetic mapping, and we examined tether function using an ectopic tether localization assay. Those approaches yielded mutually consistent results. Our analysis focused on two coiled coil golgin tethers and the multi-subunit tether GARP. At an intermediate stage of cisternal maturation, the golgin Sgm1 tethers proteins that follow an intra-Golgi recycling pathway dependent on COPI. At a late stage of cisternal maturation, GARP and the golgin Imh1 tether trans- Golgi network (TGN) proteins that follow an intra-Golgi recycling pathway dependent on the AP-1 and Ent5 clathrin adaptors. This involvement of GARP in intra-Golgi recycling had not previously been documented. Imh1 also tethers proteins that recycle from prevacuolar endosome compartments to the TGN. Our findings contribute to an integrated model of Golgi membrane traffic.

Membrane contact sites (MCSs) enable communication between organelles and play central roles in lipid metabolism. In budding yeast, the nucleus-vacuole junction (NVJ) functions as a dynamic platform that integrates lipid metabolism and stress responses. However, it remains unclear whether NVJ structure and function are broadly conserved across eukaryotes, particularly because Nvj1, the key membrane tethering factor that mediates NVJ formation in budding yeast, is absent in higher eukaryotes. Here, we investigated whether an MCS analogous to the NVJ in budding yeast exists in fission yeast (Schizosaccharomyces pombe), which lacks Nvj1. We show that an NVJ is present in fission yeast and serves as a platform for the accumulation of sterol synthesis factors, including the HMG-CoA reductase Hmg1 and the INSIG homolog Ins1. We further demonstrate that the localization of these factors depends on the membrane protein insertase Snd302 and is dynamically regulated by nutrient conditions. Our findings reveal that, despite the absence of Nvj1, the NVJ is functionally conserved as a site for sterol synthesis in fission yeast, suggesting a conserved role of spatial organization in lipid metabolism.

The spindle midzone, an array of overlapping, antiparallel microtubules, contributes to chromosome segregation and cytokinesis. As cells exit mitosis, midzone microtubules reorganize to form the midbody, the location of cell abscission. The mechanisms governing microtubule dynamics during this transition remain incompletely understood. The microtubule depolymerase, Kif2a, has been shown to contribute to midzone microtubule length control (Uehara et al., 2013), but how the depolymerase is regulated is not understood. Since CAMSAPs govern minus-end microtubule dynamics, we examined their role in midzone microtubule behavior. CAMSAP2, the major CAMSAP in HeLa cells, localized to the minus-ends of midzone microtubules and cells depleted of CAMSAP2, showed similar phenotypes as cells depleted of Kif2a, including elongated and bent midzones and enlarged asters. Next, we localized Kif2a in CAMSAP2-depleted cells and vice versa. CAMSAP2 remained present and extended along elongated midzone microtubules in Kif2a-depleted cells. In contrast Kif2a localization was no longer present at microtubule minus-ends but retained at plus-ends in CAMSAP2-depleted cells. In long-term live-cell movies of CAMSAP2-depleted cells abscission at the midbody was not detected, although two daughter cells formed. Markers for abscission including ESCRT-III component CHMP2A and Spastin were mislocalized, and midzone overlap zones, marked by PRC1, were extended. Together, our results demonstrate that CAMSAP2 is essential for midzone microtubule organization and dynamics, ultimately impacting cell abscission.

After endocytosis, transmembrane cargo reaches sorting endosomes where it is partitioned into physically distinct recycling or degradative microdomains. While the J-domain protein RME-8/DNAJC13 is known to maintain these boundaries by actively removing degradative machinery from the recycling microdomain, other factors that contribute to this spatial organization remain poorly defined. Here, we identify the conserved tetraspan protein SCM-1/SCAMP as a key microdomain organizer, discovered through RME-8 proximity-dependent biotinylation screens in C. elegans and human cells. Leveraging the large endosomes of C. elegans coelomocytes, we show that SCM-1 is selectively enriched within the recycling microdomain. In scm-1 mutants, recycling and degradative microdomains still assemble but fail to remain spatially distinct, resulting in inappropriate microdomain overlap. This loss of boundary integrity occurs without increasing the recruitment of sorting machineries, indicating a mechanism distinct from the RME-8-mediated uncoating pathway. scm-1 mutants exhibit significant sorting defects, including misrouting of recycling cargo MIG-14/Wls and v-SNARE SNB-2/VAMP3 to late endosomes and lysosomes. We find that snb-2 mutants themselves missort MIG-14 to late endosomes and lysosomes, suggesting that SNB-2 sorting is key for recycling function. Our data suggest that both microdomains lose efficiency in scm-1 mutants, as cargo missorted into late endosomes and lysosomes is not depleted overall, and degradation of an independent ESCRT-dependent cargo is delayed. We conclude that SCM-1 ensures endosomal sorting fidelity by stabilizing microdomain boundary integrity, a process required for efficient recycling and degradation of transmembrane cargo.

The plasma membrane is laterally organised into dynamic nanoscale and mesoscale domains. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) form actomyosin dependent nanoclusters at the outer leaflet of the plasma membrane via transbilayer coupling to inner leaflet lipids. These nanoclusters are nucleated downstream of integrin activation in a mechanochemical fashion and require vinculin and myosin 1 activity. Here, we identify an antagonistic relationship between vinculin and a negative regulator of myosin 1, tropomyosin, in regulating GPI-AP nanoclustering. We show vinculin mediated restoration of clustering is myosin-1 sensitive. The actin and lipid-binding vinculin tail is sufficient to restore clustering, indicating that vinculin, in particular its actin and lipid-proximal tail promote nanoclustering. Furthermore, re-expression of the non-muscle tropomyosin isoform Tpm2.1 in Tpm2-deficient MDA-MB-231 cells suppress GPI-AP nanoclustering while Tpm2.1 mutants defective in stable actin-filament association do not. Structural superposition of actin-bound vinculin tail and tropomyosin assemblies suggests that vinculin and tropomyosin engage overlapping or closely adjacent surfaces on actin filaments. Consistent with this, depletion of tropomyosin in vinculin-null cells restores GPI-AP nanoclustering. Together these results suggest that competitive interactions between tropomyosin and vinculin regulate actin availability for myosin 1 and hence contribute to the regulation of GPI-AP nanoclusters.

The Ezrin, Radixin, and Moesin (ERM) family of proteins anchors the actin cytoskeleton to the plasma membrane for the purpose of either stabilizing or altering cell shape. In Caenorhabditis elegans, ERM-1, is essential for cell polarity, signaling, intestine development, and larval viability. Interestingly, ERM-1 proteins are produced by erm-1 mRNA transcripts that concentrate at the plasma membrane in embryos. The localization of erm-1 mRNA to the plasma membrane occurs in a 3UTR-independent, translation-dependent manner, directed by the PH-subdomain within ERM-1s N-terminal FERM domain. This has led to the model that erm-1 mRNA, its associated ribosome, and its emerging nascent peptide are all transported together to the plasma membrane as a complex. Here, we characterize the transport mechanism. Using a microscopy approach, we observed that the localizations of erm-1 mRNA and ERM-1 protein to the plasma membrane were disrupted by nocodazole treatment, illustrating a microtubule role. Furthermore, erm-1 mRNA and ERM-1 protein localized to the plasma membrane independently of myosin and dynein motors, but dependent on the kinesin bmk-1 (bmk-1), a plus-end-directed, Kinesin-5 family motor protein. Loss of bmk-1 did not reduce the total number of erm-1 mRNA molecules in the cell, arguing against a diffusion- and protection-based mechanism of mRNA localization. Together, these findings suggest that erm-1 mRNA is localized via an active transport pathway mediated by a plus-end-directed kinesin adapter. Interestingly, loss of bmk-1 led to diffuse localization of ERM-1 protein along the plasma membrane and reduced ERM-1 protein levels at the site of abscission, the midbody, and the midbody remnant. This suggests that ERM-1 local translation at the plasma membrane is critical for its proteins ultimate spatial patterning in the cell.

Recent evidence indicates that mitochondria, through the activity of the E3 Ub ligase MARCH5, are critical for de novo peroxisome biogenesis. Here we report that peroxisome biogenesis factor Pex26 is a MARCH5 client protein. In peroxisome-containing cells, MARCH5 interacts with Pex26 and facilitates the transfer of newly synthesized Pex26 from the OMM to peroxisomes. MARCH5 also controls peroxisomal delivery of other candidate peroxins in peroxisome-containing cells. On the other hand, in peroxisome-deficient cells, the turnover rate of Pex26 is dramatically increased, and MARCH5 targets this protein for p97-dependent proteasomal degradation. Both activities are mediated by MARCH5-dependent Pex26 ubiquitination. Knockout of Pex26 induces the accumulation of cells containing Tom20-positive, Catalase-deficient pre-peroxisomes. Further supporting the critical role of MARCH5 in peroxisome biogenesis, these structures are absent in Pex26/MARCH5 double knockout cells. The data support the model, where in peroxisome-containing cells, MARCH5 acts as a peroxisome biogenesis factor, while with defective peroxisome biogenesis, as in Zellweger syndrome cells, it protects mitochondria from potentially toxic accumulation of peroxins on the OMM.

Understanding the behavior of - and {beta}-cells within intact human islets is essential for elucidating mechanisms of metabolic control in diabetes. Current cell-type identification strategies rely on destructive labeling or on advanced imaging modalities such as Fluorescence Lifetime Imaging Microscopy (FLIM), which provide rich metabolic information but require specialized instrumentation and acquisition protocols. Here we show that structured intracellular intensity patterns derived from endogenous autofluorescence are sufficient to discriminate and {beta} cells in living human islets. Using rotation-invariant Local Ternary Pattern (LTP) descriptors combined with morphological features, we achieve highly accurate classification (AUC = 0.92), improving upon previously reported benchmarks. The resulting framework is lightweight, interpretable, and compatible with standard imaging configurations, enabling accessible and scalable analysis of label-free microscopy data. Interpretability analyses demonstrate that discrimination is driven predominantly by fine-scale intracellular intensity organization rather than global morphology. In the spectral window employed, cytoplasmic autofluorescence is prominently shaped by lipofuscin-rich granules. Consistent with prior reports of higher lipofuscin accumulation in {beta}-cells, the dominant features identified here likely reflect differences in granule abundance and spatial organization between endocrine cell types. These findings indicate that endogenous intensity patterns encode sufficient structural information for reliable /{beta} discrimination, providing a biologically grounded and fully non-destructive framework for the identification of pancreatic islet cell types.

Antisense oligonucleotides (ASOs) enter cells efficiently, but the compartment from which productive escape occurs remains uncertain. We used live-cell microscopy, ratiometric pH measurements and 3D focused ion beam scanning electron microscopy (FIB-SEM) in U2OS cells to track a Malat1-targeting ASO from uptake to delivery. The ASO entered by endocytosis and accumulated in late endosomes, endolysosomes and lysosomes, where it induced luminal neutralization without galectin-3 recruitment or limiting-membrane rupture. Under conditions that reduced Malat1-RNA by >90%, quantitative imaging showed that less than 4% of internalized ASOs reached the nucleus. L-leucyl-L-leucine methyl ester (LLOMe)-induced membrane damage released co-internalized dextran but not ASOs, showing that ASOs remain sequestered even in damaged late endocytic compartments. In apilimod-expanded organelles, ASOs concentrated at limiting membranes and intraluminal foci with constrained motion, consistent with association with membrane and luminal structures. Although G3BP1/2 has been proposed to plug damaged endocytic membranes, we detected no recruitment of G3BP1 to endosomes or lysosomes; loss of G3BP1 and G3BP2 increased functional delivery modestly. We therefore propose that productive escape occurs earlier in endocytosis, most likely in early or recycling endosomes, where ASOs would still be unbound within the lumen and where membrane fusion and fission could generate perforations permitting release.

The ability of epithelial cells to cope with injury and undergo regeneration depends on tightly coordinated cellular responses. IFRD1 is a stress-responsive protein that is evolutionarily conserved and required for the cellular regeneration program paligenosis; however, how IFRD1 works in paligenosis is not known. Here we demonstrate that IFRD1 is primarily a cytosolic ribosome-binding protein, specifically binding 80S monosomes that are not actively engaged in translation. Using multiple in vivo and in vitro injury models, including cerulein-induced pancreatitis in mice and tunicamycin-induced ER stress in cell culture, we demonstrate that IFRD1 acts as a ribosome-salvaging factor, preventing ribosomes from degradation. In the absence of IFRD1 during ER stress, non-translating 80S ribosomes were unstable and prone to disassembly and selective degradation. The resulting accumulation of degraded ribosomal subunits overwhelmed cellular autophagic machinery, as evidenced by accumulation of the autophagy-tagging protein p62, even though overall autophagic flux remained unaffected. Ultimately, cells lacking IFRD1 showed reduced mTORC1 activity followed by increased cell death, consistent with patterns observed in cells lacking IFRD1 during paligenosis. Thus, we detail a previously unrecognized cellular function for IFRD1 in stabilizing and preserving the mature ribosome pool during metabolic and translational transitions such as paligenosis.

Centromeres are epigenetically specified chromosomal sites that support kinetochore assembly and often embedded within large satellite DNA arrays. Recent telomere to telomere genome assemblies have revealed extensive variation in centromeric satellite arrays between chromosomes and between individuals, but the functional significance of this variation remains unclear. To determine how satellite array size influences centromere function, we generated hybrid mouse models in which homologous chromosomes with different array sizes are paired in meiosis I, creating array size asymmetry across each meiotic bivalent. When an extremely small array is paired with a moderate size array, we find that array size asymmetry leads to functional asymmetry in both centromere chromatin and interactions with spindle microtubules, lagging chromosomes in anaphase I, and increased aneuploidy in MII eggs. In contrast, pairing an extremely large array with a moderate array does not lead to functional centromere asymmetry. Together, these results suggest a threshold model in which centromere array size is tolerated across a broad range, but minimal arrays become functionally limiting when paired with larger arrays in meiosis.

Chromosome segregation fidelity during meiosis is critical for genome integrity, with aneuploidy causing infertility, miscarriages, and congenital anomalies. In the oocytes of many species, spindle assembly occurs in the absence of centrosomes that normally function as microtubule-organizing centers at the poles. Such acentrosomal spindles are believed to pose significant challenges for accurate chromosome segregation compared to centrosomal organized spindles. Previous work in Drosophila has shown that the chromosomal passenger complex (CPC) is required for acentrosomal spindle assembly. We found that heterochromatin protein-1 (HP1) plays a critical role in regulating CPC localization and spindle assembly. Furthermore, HP1 moves to the microtubules, where it has roles in building a functional spindle and interacts with the CPC to regulate chromosome biorientation. These results indicate that spindle assembly is mediated by multiple interactions between the CPC, HP1, and the chromosomes, and provide insights into the mechanisms that restricts spindle assembly to the chromosomes in Drosophila oocytes.

The mechanistic target of Rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to nutrient availability. mTORC1 recruitment to lysosomes by the Rag dimer complex (RagA/B and RagC/D) is a crucial step in mTORC1 signaling. Substrates of the MiT/TFE transcription factor family, like TFEB and TFE3, directly interact with the Rag complex when activated by folliculin (FLCN). This selective recruitment and subsequent phosphorylation of MiT/TFE substrates leads to a non-canonical mTORC1 signaling pathway regulating lysosomal biogenesis and autophagy. Recently, we showed that compound heterozygous mutations in VPS41 specifically impair non-canonical mTORC1 signaling. VPS41, as part of the HOPS complex, is required for fusion of lysosomes with endosomes and autophagosomes. Here we addressed the mechanism by which VPS41/HOPS complex regulates mTORC1 activity. We show that multiple HOPS subunits interact with the Rag dimers as well as with FLCN. Depletion of HOPS subunits results in reduced lysosomal localization of both Rags and FLCN, and the nuclear translocation of TFE3/TFEB. The VPS41-Rag interactions required the RING domain of VPS41, but were independent of RHEB, Rag activity or presence of other HOPS components. We conclude that HOPS is necessary for the recruitment of crucial components of the non-canonical mTORC1 signaling pathway onto lysosomal membranes. These data further our molecular understanding of disease-causing VPS41/HOPS mutations and indicate a crucial role for HOPS in connecting lysosomal trafficking to lysosomal signaling.

Faithful chromosome segregation is essential for producing viable gametes during meiosis, a specialized type of cell division compared to mitosis. Unlike mitosis, meiosis involves two consecutive chromosome segregation events without an intervening round of DNA replication. Here we identify Gim3, a subunit of the ubiquitously expressed and conserved prefoldin complex, as a critical regulator of meiotic but not mitotic chromosome segregation in budding yeast. Loss of Gim3 causes profound defects in chromosome segregation and gamete viability through reduced tubulin protein levels, which are also associated with reduced spindle length. In mitosis, however, GIM3 deletion minimally affects spindle length and chromosome segregation, despite similarly reduced tubulin levels in both contexts, highlighting a previously unrecognized difference between the sensitivity of meiotic and mitotic spindles to tubulin abundance. In addition to chromosome segregation defects, gim3{Delta} cells exhibit aberrant meiotic cellular remodeling, including defects in exclusion of age-associated protein aggregates from newly forming gametes. Importantly, experimentally induced meiotic chromosome mis-segregation similarly disturbs cellular remodeling. Together, our findings identify Gim3 as a key factor required for maintaining chromosome segregation integrity during meiosis and reveal a previously unrecognized link between chromosome segregation and meiotic cellular remodeling.

Lysosomes exhibit spatial heterogeneity, but establishing causal relationships remains challenging with existing relocalization strategies. We present a modular toolkit that rapidly repositions lysosomes on demand by recruiting inducible motors. This decouples the location of organelles from systemic stress. We demonstrate that peripheral lysosomes adapt quickly by exhibiting luminal alkalinization and reduced proteolytic capacity. Furthermore, peripheral sequestration impairs autophagic flux by creating a spatial "trafficking bottleneck". We use this system to provide an unbiased proteomic characterization of spatially distinct lysosomal populations using mass spectrometry. Our findings reveal a distinct set of proteins and complexes that are spatially partitioned between perinuclear and peripheral lysosomes. Perinuclear lysosomes are configured for metabolic recycling. They have a high density of V1 V-ATPase subunits and contain the nucleoside transporter SLC29A3. Conversely, peripheral lysosomes serve as secretory outposts that are enriched in cathepsin Z and the SPG11/SPG15/AP5 complex. These validated cell lines and extensive datasets provide a flexible framework for investigating the functional specialization of different lysosome populations.

In Caenorhabditis elegans embryos, the nuclear transport receptor IMB-2 (Importin Beta Family-2, a karyopherin {beta}2) preferentially localizes to the nuclear envelope along with its encoding mRNA. This suggests that imb-2 mRNA is locally translated at the nuclear envelope. To test whether imb-2s two putative human orthologs, Transportin 1 (TNPO1) and Transportin 2 (TNPO2), exhibited similar mRNA localization and local translation, we performed smiFISH and microscopy in U2OS, HeLa, and human pluripotent stem cells. Neither human TNPO1 nor TNPO2 mRNA localized to the nuclear envelope in any tested human cell type. However, the human TNPO1 protein and the C. elegans IMB-2 protein both localized to the nucleus and its periphery. This suggests that despite their shared functional roles and high amino acid sequence identities (52% and 51%, respectively), these karyopherins differed in their translational dynamics.