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Mucolipidosis type IV is a lysosomal storage disease that is characterized by delayed psychomotor development and retinal degeneration due to cell death, in addition to other symptoms that are due to aberrant functions of live tissues. Caenorhabditis elegans CUP-5 is the orthologue of human TRPML1, the protein that is dysfunctional in Mucolipidosis type IV patients. Mirroring Mucolipidosis type IV pathology, loss of C. elegans CUP-5 results in developing intestinal cell death in embryos leading to embryonic lethality, while other tissues in adults lacking CUP-5 are alive but dysfunctional. We had previously shown that ESCRT-Associated proteins and the ATP-Binding Cassette Transporter MRP-4 are necessary for acquiring aberrant and poorly functional lysosomes in the absence of CUP-5. In this study, we show that the aberrant lysosomes permeabilize or rupture, thus releasing lysosomal degradative enzymes that kill cells in the absence of CUP-5. We also show that the autophagy-related protein ATG-9 mediates, in an autophagy-independent manner, this lysosomal permeabilization. We finally propose phenotypic and biochemical models linking CUP-5 to lysosomal defects and cell death.

Rab GTPase cascades drive endosomal membrane maturation by sequentially activating and inactivating Rab proteins. These transitions in Rab signaling require the coordinated actions of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The yeast VINE complex is an endosomal VPS9-family GEF that stimulates a GAP to inactivate the Rab5 homolog Vps21, suggesting a role for VINE in coordinating Rab transitions. Here we report that VINE acts through its catalytic GEF domain to promote signaling by the Rab5-related GTPase Ypt10 and establish a pool of Ypt10 at late endosomes. Ypt10 activation occurs downstream of Vps21 activity, placing Ypt10 within a late endosomal Rab cascade. Genome-wide protein proximity screens revealed a VINE-dependent interaction between Ypt10 and the GEF Mon1-Ccz1. Our data suggest that VINE and Ypt10 regulate late endosomal recruitment of Mon1-Ccz1 to enhance the activation of its substrate, the Rab7 homolog Ypt7. Together, these findings define a Vps21-VINE-Ypt10 regulatory module that adds a layer of control within the late endosomal Vps21-to-Ypt7 cascade and establish VINE as a dual Rab regulator. Through opposing activities on Vps21 and Ypt10, VINE may couple Rab5 inactivation to Mon1-Ccz1 recruitment to provide more precise control of degradative protein traffic to the vacuole. Significance statementFour Rab5-family GTPases direct protein sorting and membrane maturation in the yeast endolysosomal system, yet their individual functions, and the role of the little-studied Rab Ypt10, are unclear. Using genome-wide proximity screens, we find that the GEF complex VINE establishes a pool of Ypt10 at late endosomes downstream of Vps21, where Ypt10 recruits Mon1-Ccz1, the activator of the Rab7 homolog Ypt7. Because VINE also drives GAP-mediated suppression of Vps21, our results suggest it acts as a dual Rab regulator, coupling Vps21 inactivation to Ypt10 activation to fine-tune the endosomal Rab cascade.

Bacterial deubiquitinases (DUBs) are important virulence effectors that manipulate host ubiquitin signaling during infection. ElaD, a CE-clan DUB expressed by enterohemorrhagic Escherichia coli, preferentially cleaves K63-linked ubiquitin chains, yet its effects on conserved cellular stress responses remain poorly understood. We demonstrate that ElaD exhibits redox-dependent DUB activity in vitro. In addition, we identified the molecular basis underlying the selective recognition of substrate proteins, ubiquitin and NEDD8 by ElaD. Structural and mutational analyses reveal that, beyond the conserved catalytic site, ElaD engages ubiquitin through a combination of electrostatic and hydrophobic interactions. Using Saccharomyces cerevisiae as a heterologous model system, we show that wild-type ElaD rescues the proteotoxic stress phenotype of ubp2{Delta} yeast cells, whereas specific ElaD mutants fail to confer a similar response. Furthermore, expression of ElaD suppresses oxidative stress-induced accumulation of K63-linked polyubiquitin and may perturb stress-associated translational regulation linked to K63 ubiquitin signaling. Consequently, cells expressing ElaD exhibit altered stress adaptation and diminished fitness during prolonged oxidative stress. Collectively, these findings indicate that ElaD perturbs ubiquitin-mediated stress signaling by counteracting K63-linked ubiquitination events that support adaptive cellular responses. Our study highlights how a bacterial DUB can reprogram conserved ubiquitin-dependent pathways and exploit host ubiquitin signaling networks to modulate cellular stress responses and protein homeostasis. These findings further suggest potential host targets of bacterial DUBs during infection.

Cilia are assembled by intraflagellar transport (IFT), which relies on two protein complexes: IFT-A and IFT-B. It is generally assumed that IFT-B and IFT-A are critical for anterograde and retrograde transport, respectively. However, full deletion of IFT-A genes in several organisms suggests a possible contribution to anterograde transport. In many species, cilia collapse when IFT is altered, hindering functional studies. Here, we investigated the role of IFT-A in the protist Trypanosoma brucei, where IFT is not required for cilium maintenance. Following the inducible knockdown of IFT88 (an IFT-B member) or IFT140 (an IFT-A member), we monitored the fate of several IFT proteins in preassembled cilia using live imaging and evaluated the consequences on train formation by volumetric electron microscopy. Surprisingly, both IFT88 and IFT140 turned out to be essential for anterograde train assembly. Their depletion initially led to the formation of shorter trains and subsequently to an inhibition of train injection. We propose a model to reconcile the diverging phenotypes reported in the literature.

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for mitochondrial proteins. Approximately 99 % of mitochondrial proteins are synthesized as precursor proteins (preproteins) in the cytosol and subsequently translocated into mitochondria through the TOM complex. The TOM complex exists in a dynamic equilibrium among multiple assembly states through spatial rearrangements of its subunits. The recent cryo-electron microscopy (cryo-EM) studies revealed near-atomic structures of the TOM core dimer, whereas previous biochemical studies indicated the TOM complex functions as a trimer in intact mitochondria. However, the relationship between the core dimer and the functional trimer remains unclear. In the present study, we analyzed the dynamics of the TOM complex using high-speed atomic force microscopy (HS-AFM) to investigate the assembly states and conformation transitions of the TOM complexes. We demonstrated that purified yeast TOM complexes predominantly adopt a trimeric organization but dynamically dissociate into dimeric and monomeric states during HS-AFM observation. The trimeric particles observed by HS-AFM exhibited spherical molecular shapes consistent with a trimeric structural model proposed from previous crosslinking analyses. In contrast, the dissociated dimeric particles closely resembled the dimensions of the TOM core-dimer structures determined by cryo-EM. Furthermore, HS-AFM analyses provided insight into the spatial arrangement of the Tom20 receptor, consistent with previous models of the trimeric TOM complex. These observations enabled characterization of the trimeric TOM complex in vitro and provide a foundation for future structural and functional analyses of TOM complex assembly.

Dsn1 and Ndc80 are essential proteins of the kinetochore complex and required for chromosome segregation in dividing cells and for regulating development and microtubule dynamics in postmitotic neurons. With conditional deletion of floxed alleles, we here show that Dsn1 and Ndc80 are also required for the viability of postmitotic neurons, both in cultures of hippocampal and cortical neurons and in vivo in the retina. Loss of these proteins triggers apoptosis, as indicated by caspase cleavage and an increase in nuclear DNA breakage. The pro-survival function of the kinetochore components is distinct from that which was previously demonstrated for regulation of neuronal synaptogenesis. The microtubule-binding domain of Ndc80 is required for the synaptogenic functions but Ndc80 lacking this domain can nonetheless rescue the viability of neurons from which Ndc80 has been deleted. Similarly, whereas the synaptogenic function involves regulation of microtubules in axons and dendrites, a nucleus-localized Dsn1 is sufficient to rescue the viability of neurons from which Dsn1 has been deleted. Thus, postmitotic neurons retain a nuclear requirement for components of the kinetochore in order to prevent apoptosis.

In eukaryotes, our understanding of clathrin-independent endocytosis (CIE) lags far behind that of clathrin-mediated endocytosis (CME). CIE plays key roles in internalizing receptors, viruses, bacterial toxins, and pathogens; thus, deeper mechanistic insights are critical for understanding cellular strategies for plasma membrane regulation. Yeast CIE requires a signal relay between the stress sensor Mid2, the guanine nucleotide exchange factor (GEF) Rom1, the Rho1 GTPase, and the formin Bni1. While GEFs promote GTPase activity, GTPase-activating proteins (GAPs) conversely stimulate nucleotide hydrolysis and GTPase inactivation. Here, we provide new insight into CIE, adding the RhoGAP Sac7 as a regulator. SAC7 deletion in CME-deficient cells improved cargo internalization, and Sac7 localizes primarily to the mother cortex. Cells lacking SAC7 accumulate active Rho1 and retain Bni1 at the plasma membrane, where Bni1 retention may subsequently enhance actin assembly needed for CIE. Our results thus demonstrate that Sac7 negatively regulates CIE by restricting cortical Rho1 activity.

Microproteins translated from short open reading frames are increasingly understood to play important roles in cell biology and development. Here, we describe a microprotein in Drosophila that is expressed in ovarian follicle cells which surround the developing oocyte. The Dafcin microprotein is predicted to form an amphipathic alpha-helix, a structure known to interact with lipid bilayers. The structure of Dafcin most resembles the influenza HA fusion peptide, which induces negative curvature of endosomal membranes. Dafcin tagged with GFP localizes to the Golgi and is ultimately secreted from the follicle cells. Remarkably, this occurs without the microprotein having a secretory signal sequence. The protein is taken up into the oocyte by endocytosis, localizing to the inner face of storage lysosomes called yolk granules. Mutant analysis shows that Dafcin is required to limit the size of yolk granules. This may occur by inducing negative membrane curvature like HA peptide. In support, liposomes formed in vitro with both Dafcin and HA peptides are smaller in size.

Quantitative structural analysis by Photoactivated Localization Microscopy (PALM) on the nanoscale is often restricted to fixed cells because motion during prolonged data acquisition distorts image reconstruction. Here, we develop motion-corrected PALM (mcPALM), a live-cell super-resolution approach combining a conventional fluorescence channel with PALM to correct motion-induced spreading of localizations. We further introduce a photoactivation-based correction to estimate molecule numbers from incomplete trajectories. Using PI3P-marked endosomes in yeast as a dynamic model system, we show that mcPALM recovers a live-cell maturation trajectory linking motion-corrected endosome size and calibrated PI3P content, consistent with fixed-cell benchmarks. Unlike fixed-cell PALM, mcPALM preserves endosome dynamics, revealing stage-dependent directed transport and maturation-associated motility shift. Thus, mcPALM extends PALM from static structural measurements in fixed samples to integrated quantification of nanoscale structure, molecular composition and dynamics in living cells. This framework is broadly applicable to other mobile organelles and biomolecular assemblies, enabling live-cell studies on how molecular organization and dynamics are coupled to biological function.

Neuronal loss is a hallmark of neurodegenerative diseases. Phosphatidylserine (PS), a key eat me signal, is exposed on stressed viable neurons, triggering their premature phagocytosis by activated glia. We investigated whether PS masking could serve as a universal strategy to prevent neuronal loss in two distinct Drosophila models of neurodegeneration: an adult-stage-specific knockdown of skpA and a Huntingtons disease model initiated during embryogenesis. Both models exhibit neuronal loss, motor dysfunction, and reduced lifespan. To mask PS, we used a truncated form of MFG-E8, a glycoprotein that binds PS without promoting engulfment. PS masking preserved two neuronal populations in both models, indicating that these neurons were eliminated alive via phagoptosis. Motor function and lifespan were improved to varying degrees, depending on the timing and severity of neuronal damage. These findings reveal that aberrant glial phagocytosis contributes to neuronal vulnerability and identify PS masking as a promising therapeutic approach for neurodegenerative diseases. Significance StatementNeuronal loss is a defining feature of neurodegenerative diseases, yet its underlying mechanisms remain incompletely understood. Here, we demonstrate in two Drosophila models of neurodegeneration that stressed but viable neurons are prematurely eliminated by glial phagocytosis through phosphatidylserine (PS) exposure. By masking PS with a truncated form of MFG-E8, we prevented neuronal loss, improved motor performance, and extended lifespan, highlighting PS-dependent removal of live neurons as a critical contributor to neurodegeneration. Our findings provide the first in vivo evidence that PS masking protects neurons in distinct neurodegenerative contexts, offering a broadly applicable strategy for therapeutic intervention. This work positions aberrant glial phagocytosis as a disease-driving mechanism and establishes Drosophila as a powerful model for dissecting neuron-glia interactions in neurodegeneration.

Entry of intracellular pathogenic bacteria is widely considered an actin-driven process, potentially overlooking contributions of the microtubule cytoskeleton. Here, we identify a host microtubule state as a determinant of early infection efficiency by Chlamydia pneumoniae. Human cells enriched in acetylated microtubules are preferentially infected, whereas detyrosinated microtubules show no such association. Pharmacological stabilization of microtubules via Taxol enhances infection, while selective elevation of acetylation with Tubacin does not, indicating that microtubule stability rather than acetylation alone is critical. Thus, a pre-existing interphase microtubule architecture supports C. pneumoniae entry. Consistently, mitotic cells, characterized by a reorganized microtubule architecture, remain permissive but show severely reduced infection efficiency. In addition, infection induces a dose-dependent increase in microtubule acetylation that requires bacterial viability and is not observed during uptake of Yersinia pseudotuberculosis effector protein Invasin-coated beads, indicating that entry/internalization alone is insufficient to trigger this response. To probe how early chlamydial secreted effectors might engage the microtubule cytoskeleton, we focused on the conserved TarP family member CPn0572, an actin and microtubule regulator, which increases microtubule acetylation when ectopically expressed in human cells. Controlled expression of microtubule-localized CPn0572 in the yeast Schizosaccharomyces pombe leads to altered microtubule dynamic and mechanical behaviour, promoting force-bearing microtubules. Together, these findings show that distinct interphase microtubules define a permissive cellular state for bacterial entry and suggest that early chlamydial effector activities might promote a specific microtubule persistence phenotype.

Fission is essential for proper mitochondrial function and for cellular homeostasis. Dysfunction in mitochondrial fission is associated with several neurological disorders, including the rare and lethal encephalopathy EMPF1, which is caused by de novo heterozygous DNM1L variants. DNM1L encodes the mitochondrial fission mechanoenzyme DRP1, which can intrinsically self-assemble and induce membrane scission. Wild-type DRP1 puncta that appear throughout the cytoplasm are thought to be pre-scission complexes of well-ordered oligomeric assemblies. Immunofluorescence imaging of patient-derived EMPF1 fibroblasts carrying assembly-deficient DNM1L variants reveals elongated mitochondrial networks consistent with impaired fission. Despite this loss-of-function phenotype, these cells retain essentially wild-type numbers of DRP1 puncta. We confirmed the previously reported inability of purified pathogenic DRP1 variants p.Gly363Asp and p.Gly401Ser to assemble under conditions in which WT DRP1 forms helical polymers. Under macromolecular crowding conditions, however, both wild-type and mutant DRP1 access condensed states whose formation depends on protein concentration and solution conditions. Acute treatment of EMPF1 fibroblasts with 1,6-hexanediol preferentially alters DRP1 puncta fluorescence intensity and distribution in mutant cells relative to wild type, indicating genotype-dependent differences in puncta material properties. Together, these findings support a model in which DRP1 puncta occupy a continuum of condensed states, only a subset of which mature into fission-competent assemblies, revealing biomolecular condensation as a previously unrecognized layer of DRP1 regulation. Biasing DRP1 along this continuum may provide a mechanistic basis for impaired fission in EMPF1 and suggest opportunities to restore productive assembly in select pathogenic contexts.

Centrosome amplification is frequently associated with chromosomal instability and tumor progression, but how cells coordinate centriole assembly with the control of centrosome numbers and quality remains poorly understood. TIAM1 is a RAC1 guanine nucleotide exchange factor previously implicated in centrosome-associated signaling and {beta}TrCP-dependent control of PLK4 abundance. Here, we examined how Tiam1 regulates autophagy-lysosome homeostasis in mouse embryonic fibroblasts induced to overexpress PLK4. In contrast to a previous model in which Tiam1 loss promotes productive centriole overduplication, we found, by super-resolution imaging and expansion microscopy, an abnormal distribution of PLK4 on the centrioles centriole-associated structures following TIAM1 depletion, suggesting that TIAM1 may support the organization or maturation of centrioles. TIAM1 depletion also resulted in increased LC3B-positive puncta and enlarged LAMP1-positive compartments, but this was not accompanied by increased LC3B-II accumulation after bafilomycin A1 treatment. These findings suggest that TIAM1 may act at the interface between centriole assembly and endolysosomal/autolysosomal organization, linking TIAM1 to lysosome-associated centrosome quality-control pathways.

Autophagy is a catabolic process that degrades damaged organelles and aggregation-prone proteins and plays key roles during development and in maintaining cellular homeostasis. It can be induced by stress including starvation, oxidative stress, or accumulation of misfolded proteins. Autophagy declines with age and there is great interest in manipulating autophagy to improve neurodegenerative diseases, as its stimulation shows promise to improve diseases including Huntington, Alzheimer, and Parkinson. Endosomal microautophagy (e-MI) is a type of autophagy in which cytosolic proteins are delivered to late endosomes and degraded upon incorporation into intraluminal vesicles of multivesicular bodies. Here, we report that the actin nucleation-promoting factors (NPFs) known to activate the Arp2/3 complex to promote branched actin assembly can alter the dynamics of e-MI. We found that upon stress exposure, overexpression of the NPFs WASp, Wash, or SCAR results in an expedited induction of e-MI. Strikingly, Wash is uniquely required for physiological e-MI induction implying that NPFs are not functionally redundant for e-MI. We show that the WASH complex regulates e-MI on late endosomes acting via Arp2/3 and thus likely branched actin. Surprisingly, the regulation of e-MI by Wash is independent of retromer that is known to recruit Wash to early endosomes for its role in recycling of membrane proteins and rather reflects a novel degradative aspect of Wash function. Taken together, we identified a novel function of NPFs as upstream regulators of e-MI that could be used to activate e-MI ectopically to improve aggregate clearance during neurodegeneration.

The insertase Oxa1 is required for protein insertion into the inner mitochondrial membrane and for the biogenesis of oxidative phosphorylation complexes. While most eukaryotes encode one or two Oxa1 proteins, we identified three paralogs in Trypanosoma brucei: TbOxa1-1, TbOxa1-2, and TbOxa1-3. Knock-out of individual paralogs followed by phenotypic analyses and proteomic characterization of submitochondrial fractions revealed distinct functions. Respiratory chain complexes I and IV are primarily affected by loss of TbOxa1-1, whereas complex III and ATP synthase depend on TbOxa1-2; the ablation of TbOxa1-3 results in minor phenotypes in culture. In TbOxa1-2-depleted cells, ATP synthase biogenesis is compromised by the defective import or processing of the nuclear-encoded subunit-c, which also requires a rhomboid peptidase-like protein. Further, the ablation of TbOxa1-2 triggers accumulation of membrane proteins in the matrix, supporting its role in conservative sorting. Together, our results demonstrate that the trypanosomal Oxa1 machinery evolved a paralog-specific division of labor to manage a highly divergent mitochondrial membrane proteome.

The nuclear envelope (NE) surrounds the genetic material and is continuous with the endoplasmic reticulum (ER). In yeast and other organisms undergoing closed mitosis, nuclear envelope expansion (NME) is strictly required to accommodate spindle elongation and ensure proper chromosome segregation within a single nuclear compartment. Failure to expand the NE during mitosis leads to chromosome missegregation. Here, we show that deletion of the unstructured N-terminal domain of Rgf3, a Rho1-specific guanine nucleotide exchange factor (GEF), causes early mitotic defects that produce the characteristic "cut" phenotype of untimely cell division. The rgf3{Delta}N2 mutant displays spindle buckling, a hallmark of anaphase nuclei unable to properly expand the NE. From yeast to mammals, phosphatidic acid (PA)--a key precursor in phospholipid biosynthesis--is metabolized via two competing pathways, the cytidine diphosphate-diacylglycerol (CDP-DAG) and the Kennedy pathways, both contributing to lipid membrane homeostasis. We provide evidence that impaired Rho1 activation in rgf3{Delta}N2 selectively disrupts phospholipid synthesis through the CDP-choline branch of the Kennedy pathway. Thus, Rho1 promotes mitotic progression by modulating phospholipid biosynthesis to enable efficient NME during anaphase. HighlightsThe N-terminus of Rgf3 is required for proper nuclear envelope expansion (NME) during anaphase. The structurally flexible N-terminal domain of Rgf3 is essential for localized Rho1 activation. Active Rho1 drives mitotic membrane growth by modulating phospholipid synthesis through the Kennedy pathway.

Quantitative image analysis of subcellular organization requires sufficient spatial resolution to resolve individual organelles and sample size to capture heterogeneity both within cells and between cells. Existing imaging approaches often force a tradeoff between spatial resolution and throughput, limiting the ability to measure organelle-level phenotypes across cell populations. Here, we establish high-throughputs expansion microscopy (HiExM) as a scalable pipeline for single-organelle analysis. As a benchmark, we focus on mapping late endosomes and lysosomes (LELs), a heterogeneous organelle class whose small size, dense intracellular distribution, and functional diversity make it difficult to quantify accurately using conventional light microscopy. HiExM increases effective spatial resolution while preserving compatibility with large-scale image acquisition, enabling robust segmentation and quantitative profiling of individual LELs across large cell populations. Using this pipeline, we identified differences in intracellular trafficking behavior among anti-transferrin receptor antibodies that could not be captured by conventional colocalization analysis alone. We further integrate spatial and morphological features with learned image-based representations that can define relationships between LEL morphology and subcellular position as well as how these relationships respond to perturbations. Together, our work establishes HiExM as a generalizable platform for scalable single-organelle profiling, enabling an analytical framework for quantifying discrete organelles across cells and conditions.

Synaptojanin 1 is a brain enriched phosphoinositide phosphatase implicated in endocytosis at the synapse. A mutation (R258Q) that selectively impairs its Sac1 phosphatase domain causes early onset familial Parkinsonism. Neurons of mice with this mutation display synaptic vesicle traffic defects across the brain, but selective dystrophic changes in a subset of dopaminergic axons in the dorsolateral striatum. Using correlative light microscopy-FIB-SEM of mutant mouse striata to visualize in 3D these abnormal structures we show that they represent clusters of focal axonal dilations harboring massive, onion-like DAT enriched plasma membrane infoldings, generally localized next to cell bodies of neighboring cells, often engulfing evaginations of such cells. This dysmorphia was associated with a deficit in dopamine release in the same striatal region. Given the involvement of Synj1 in endocytic mechanisms, these structures may reflect an imbalance between exocytosis and endocytosis. Their occurrence only in a subset of axons suggest a vulnerability threshold of these axons beyond which the expansion of the plasma membrane is not counteracted by compensatory mechanisms.

Macrophages destroy pathogens by engulfing them into phagosomes that mature into degradative phagolysosomes via lysosome fusion. Leishmania parasites subvert this antimicrobial pathway to establish intracellular infection and cause leishmaniasis. We previously identified the SNARE protein syntaxin-2 (Stx2) as a promoter of phagolysosome biogenesis that simultaneously limits particle binding and uptake. Consistent with this dual role, Stx2-depleted macrophages (Stx2-KD) show enhanced binding and internalization of Leishmania major. Stx2-KD macrophages also sustain higher intracellular parasite loads. We find that L. major actively targets macrophage Stx2 by selectively depleting Stx2 from phagosomes through its virulence metalloprotease GP63. Phagosomes containing GP63-deficient L. major retain Stx2 and acquire increased levels of lysosomal hydrolases and v-ATPase, restoring degradative capacity. In BALB/c mice, L. major infection markedly reduces Stx2 in infected tissues in a GP63-dependent manner. Collectively, our findings identify GP63-mediated Stx2 depletion as a key virulence strategy of L. major, positioning the GP63-Stx2 axis as a promising therapeutic target for leishmaniasis.

Efferocytosis, the recognition, engulfment, and degradation of apoptotic cells by phagocytes, is essential for tissue homeostasis and development, and its failure contributes to chronic inflammation and neurodegeneration. The amyloid precursor protein (APP), a central pathogenic factor in Alzheimers disease, retains physiological functions independent of amyloid production that remain poorly understood. Here, we identify a conserved, non-amyloidogenic role for APP in regulating apoptotic cell degradation via the endolysosomal pathway. Using Drosophila APPL as a model, structure-function analysis demonstrated that the intracellular internalization domain of APPL, but not its secreted ectodomain, is required for efficient apoptotic cell degradation. Immunoprecipitation coupled with mass spectrometry revealed a physical interaction between APPL and the microtubule severing ATPase Spastin, mediated by the microtubule-interacting and trafficking domain of Spastin. APPL interacts with Spastin on endosomal microtubules and modulates the dynamics of the Spastin-ESCRT-III complex, enabling Spastin to sever microtubules and promote endosomal tubule fission. Loss of APPL disrupts this process, causing aberrant endosomal tubulation and impaired lysosome biogenesis. Furthermore, it compromises the function of residual lysosomes, characterized by reduced acidity, diminished proteolytic activity, and increased lysosomal damage, which ultimately impairs the degradation of engulfed apoptotic cells. Critically, this phenotype is evolutionarily conserved in C. elegans and mice. Together, these findings establish a conserved APP-Spastin axis that regulates endolysosomal homeostasis and apoptotic cargo digestion. This reveals a critical non-amyloidogenic function of APP in maintaining tissue homeostasis through efficient efferocytosis, with broad implications for inflammatory and neurodegenerative disorders that warrant further investigation.