Collective cell migration is essential for development and tissue homeostasis, yet how integrin-extracellular matrix adhesion coordinates migratory force generation in vivo remains poorly understood. Here, we have used the zebrafish posterior lateral line primordium (pLLP) as a model for epithelial collective cell migration. We show that integrins α3 and α6b are expressed in different, yet overlapping, domains of the pLLP and function redundantly to support migration. The systematic combination of itga6b, itga3b and itga3a mutants disrupts integrin β1 localization, increases protrusive activity and impairs migration, revealing a spatially organized, partially redundant adhesion system. We further identify laminin α5 (Lama5) as a key component of the basement membrane (BM) underlying the migrating pLLP. While loss of Lama5 alone compromises BM integrity and pLLP morphology without impairing migration, simultaneous depletion of lama5 and itga6b leads to severe migration defects, with the formation of invadopodia-like structures and ultimately the stalling of migration. Together, these findings reveal a robust, redundant adhesion machinery that ensures persistent collective migration in vivo, and they establish fundamental principles of integrin-mediated adhesion that are relevant to development and disease.

Summary How developing organs acquire complex architectures while maintaining tissue integrity under mechanical stress is a fundamental question in mechanobiology. In the gill, pillar cells support the thin respiratory lamellae, but how they withstand hemodynamic forces is unknown. Here, we show that actomyosin-rich contractile apparatuses (CA) in zebrafish pillar cells act as load-bearing units that generate contractile forces and stabilize the lamellae through adhesion to and enclosure of extracellular collagen columns. CA number correlates with pillar cell constriction, linking subcellular architecture to tissue geometry. We further identify Shroom3 and Vinculin b as key regulators of CA assembly and force transmission: shroom3 deficiency or loss of vclb impairs CA formation and reduces pillar cell constriction, while loss of shroom3 causes lamellar collapse. Together, these findings support a mechanical threshold model in which lamellar integrity depends on a minimum number of CAs per pillar cell and establish the gill lamellae as a model for linking subcellular contractility to tissue-scale mechanical stability.

The Flower protein was suggested to couple the fusion of synaptic vesicles (SVs) to their recycling in different model organisms. It is supposed to trigger activity-dependent bulk endocytosis by conducting Ca 2+ at endocytic sites. However, this mode of action is debated. Here, we investigated the role of the Caenorhabditis elegans homologue FLWR-1 in neurotransmission. Our results confirm that FLWR-1 facilitates the recycling of SVs at the neuromuscular junction (NMJ). Ultrastructural analysis of synaptic boutons after hyperstimulation revealed an accumulation of large endocytic structures in flwr-1 mutants. These findings do not support a role of FLWR-1 in the formation of bulk endosomes but rather a function in their breakdown. Unexpectedly, the loss of FLWR-1 led to increased neuronal Ca 2+ levels in axon terminals during stimulation, particularly in GABAergic motor neurons, causing excitation-inhibition imbalance. We found that this increased NMJ transmission might be caused by deregulation of MCA-3, the nematode orthologue of the plasma membrane Ca 2+ ATPase (PMCA). In vivo molecular interactions indicated that FLWR-1 may be a positive regulator of the PMCA and might influence its recycling through modification of plasma membrane levels of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ).

Legionella pneumophila is an intracellular pathogen that causes Legionnaires' disease. The bacteria release effector proteins, some of which remodel host autophagic-lysosomal pathways. One such effector is RavZ, which delipidates ATG8 proteins, making compromising autophagy in Legionella-infected cells. Here we show that SidE effectors also affect these pathways, by mediating phosphoribosyl-ubiquitination (PR-Ub) of the autophagic SNARE proteins STX17 and SNAP29. STX17 modification induces recruitment of STX17-positive membranes from the endoplasmic reticulum to Legionella-containing phagosomes, forming replicative vacuoles. Using proximity labeling, biochemistry and Legionella infection studies, we define a mechanism by which autophagy is hijacked by bacteria to recruit ER membranes to the bacterial vacuole, via a structure bearing autophagy markers but not fusing with lysosomes. Mass-spectrometric identification of PR-Ub sites and mutational studies show that phosphoribosyl-ubiquitination of STX17 alters its interaction with ATG14L, which causes ER membranes to be recruited to the bacterial vacuole in a PI3K-dependent manner. On the other hand, phosphoribosyl-ubiquitination of SNAP29 inhibits the formation of the autophagosomal SNARE complex (STX17-SNAP29-VAMP8) via steric hindrance, thus preventing the fusion of bacterial vacuoles with lysosomes.

RNA splicing enables the functional adaptation of cells to changing contexts. Impaired splicing has been associated with diseases, including retinitis pigmentosa, but the underlying molecular mechanisms and cellular responses remain poorly understood. In this work, we report that deficiency of ubiquitin-specific protease 39 (USP39) in human cell lines, zebrafish larvae, and mice led to impaired spliceosome assembly and a cytotoxic splicing profile characterized by the use of cryptic 5 ' splice sites. Disruptive cryptic variants evaded messenger RNA (mRNA) surveillance pathways and were translated into misfolded proteins, which caused proteotoxic aggregates, endoplasmic reticulum (ER) stress, and, ultimately, cell death. The detrimental consequence of splicing-induced proteotoxicity could be mitigated by up-regulating the ubiquitin-proteasome system and selective autophagy. Our findings provide insight into the molecular pathogenesis of spliceosome-associated diseases.

Among the various signals governing autophagy, ubiquitination plays a critical role both by controlling the stability of upstream regulators or components of macroautophagy/autophagy pathways and by facilitating the recruitment of cargo to autophagy receptors. As such, modulators of ubiquitin signaling can influence autophagic substrate degradation. Recently, we identified a non-proteolytic ubiquitin signal at the Ragulator complex subunit LAMTOR1 that is reversed by the deubiquitinase USP32. Loss of USP32 promotes ubiquitination within the unstructured N-terminal region of LAMTOR1 and prevents its efficient interaction with the vacuolar-type H+-ATPase, a prerequisite for full activation of MTORC1 at lysosomes. Consequently, MTORC1 activity is decreased and autophagy is upregulated in USP32 knockout cells. This phenotype is conserved in Caenorhabditis elegans. Depletion of USP32 homolog CYK-3 in worms results in LET-363/MTOR inhibition and autophagy induction. Based on our data, we propose an additional control layer of the MTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.

ABSTRACT Autophagy is a catabolic process during which cytosolic material is enwrapped in a newly formed double-membrane structure called the autophagosome, and subsequently targeted for degradation in the lytic compartment of the cell. The fusion of autophagosomes with the lytic compartment is a tightly regulated step and involves membrane-bound SNARE proteins. These play a crucial role as they promote lipid mixing and fusion of the opposing membranes. Among the SNARE proteins implicated in autophagy, the essential SNARE protein YKT6 is the only SNARE protein that is evolutionarily conserved from yeast to humans. Here, we show that alterations in YKT6 function, in both mammalian cells and nematodes, produce early and late autophagy defects that result in reduced survival. Moreover, mammalian autophagosomal YKT6 is phospho-regulated by the ULK1 kinase, preventing premature bundling with the lysosomal SNARE proteins and thereby inhibiting autophagosome–lysosome fusion. Together, our findings reveal that timely regulation of the YKT6 phosphorylation status is crucial throughout autophagy progression and cell survival.

The selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and, thereby, disease maintenance and progression in acute myeloid leukemia (AML). Mitophagy is orchestrated by a variety of mitophagy receptors whose interplay is not well understood. Here, we established a pairwise multiplexed CRISPR screen targeting mitophagy receptors to elucidate redundancies and gain a deeper understanding of the functional interactome governing mitophagy in AML. We identified OPTN (optineurin) as sole non-redundant mitophagy receptor and characterized its unique role in AML. Knockdown and overexpression experiments demonstrated that OPTN expression is rate-limiting for AML cell proliferation. In a MN1-driven murine transplantation model, loss of OPTN prolonged overall median survival by 7 days (+21%). Mechanistically, we found broadly impaired mitochondrial respiration and function with increased mitochondrial ROS, that most likely caused the proliferation defect. Our results decipher the intertwined network of mitophagy receptors in AML for both ubiquitin-dependent and receptor-mediated mitophagy, identify OPTN as a non-redundant tool to study mitophagy in the context of leukemia and suggest OPTN inhibition as an attractive therapeutic strategy.Abbreviations: AML: acute myeloid leukemia; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; CTRL: control; DFP: deferiprone; GI: genetic interaction; KD: knockdown; KO: knockout; ldMBM, lineage-depleted murine bone marrow; LFC: log2 fold change; LIR: LC3-interacting region; LSC: leukemic stem cell; MAGeCK: Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout; MDIVI-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MOM: mitochondrial outer membrane; NAC: N-acetyl-L-cysteine; OA: oligomycin-antimycin A; OCR: oxygen consumption rate; OE: overexpression; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; ROS: reactive oxygen species; SEM: standard error of the mean; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; UBD: ubiquitin-binding domain; WT: wild type

Autophagosome isolation enables the thorough investigation of structural components and engulfed materials. Recently, we introduced a novel antibody-based FACS-mediated method for isolation of native macroautophagic/autophagic vesicles and confirmed the quality of the preparations. We performed phospholipidomic and proteomic analyses to characterize autophagic vesicle-associated phospholipids and protein cargoes under different autophagy conditions. Lipidomic analyses identified phosphoglycerides and sphingomyelins within autophagic vesicles and revealed that the lipid composition was unaffected by different rates of autophagosome formation. Proteomic analyses identified more than 4500 potential autophagy substrates and showed that in comparison to autophagic vesicles isolated under basal autophagy conditions, starvation only marginally affected the cargo profile. Proteasome inhibition, however, resulted in the enhanced degradation of ubiquitin-proteasome system components. Taken together, the novel isolation method enriched large quantities of autophagic vesicles and enabled detailed analyses of their lipid and cargo composition.

Accumulation of misfolded proteins or perturbation of calcium homeostasis leads to endoplasmic reticulum (ER) stress and is linked to the pathogenesis of neurodegenerative diseases. Hence, understanding the ability of neuronal cells to cope with chronic ER stress is of fundamental interest. Interestingly, several brain areas uphold functions that enable them to resist challenges associated with neurodegeneration. Here, we established novel clonal mouse hippocampal (HT22) cell lines that are resistant to prolonged (chronic) ER stress induced by thapsigargin (TgR) or tunicamycin (TmR) as in vitro models to study the adaption to ER stress. Morphologically, we observed a significant increase in vesicular und autophagosomal structures in both resistant lines and ‘giant lysosomes’, especially striking in TgR cells. While autophagic activity increased under ER stress, lysosomal function appeared slightly impaired; in both cell lines, we observed enhanced ER-phagy. However, proteomic analyses revealed that various protein clusters and signaling pathways were differentially regulated in TgR versus TmR cells in response to chronic ER stress. Additionally, bioenergetic analyses in both resistant cell lines showed a shift toward aerobic glycolysis (‘Warburg effect’) and a defective complex I of the oxidative phosphorylation (OXPHOS) machinery. Furthermore, ER stress-resistant cells differentially activated the unfolded protein response (UPR) comprising IRE1α and ATF6 pathways. These findings display the wide portfolio of adaptive responses of neuronal cells to chronic ER stress. ER stress-resistant neuronal cells could be the basis to uncover molecular modulators of adaptation, resistance, and neuroprotection as potential pharmacological targets for preventing neurodegeneration.

Autophagy is a central eukaryotic catabolic pathway responsible for clearance and recycling of an extensive portfolio of cargoes, which are packed in vesicles, called autophagosomes, and are delivered to lysosomes for degradation. Besides basal autophagy, which constantly degrades cellular material, the pathway is highly responsive to several stress conditions. However, the exact protein content and phospholipid composition of autophagosomes under changing autophagy conditions remain elusive so far. Here, we introduce a FACS-based approach for isolation of native unmanipulated autophagosomes and ensure the quality of the preparations. Employing quantitative proteomics and phospholipidomics, we obtained a profound cargo and lipid profile of autophagosomes purified upon basal autophagy conditions, nutrient deprivation, and proteasome inhibition. Indeed, starvation only mildly affected the content profile, while interference with proteasome activity showed stronger effects and specifically altered autophagosome cargoes. Interestingly, the phospholipid composition of autophagosomes was unaffected by the different treatments. Thus, the novel isolation method enables purification of intact autophagosomes in large quantities and allows protein content and phospholipid profiling without the requirement of exhaustive cellular fractionation or genetic manipulation.

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid-binding features of αS through the design of site-directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane-bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death. BRIEF STATEMENT OUTLINING SIGNIFICANCE: Modulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane-bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS.

The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.

Mitochondria are dynamic eukaryotic organelles involved in a variety of essential cellular processes including the generation of adenosine triphosphate (ATP) and reactive oxygen species (ROS) as well as in the control of apoptosis and autophagy. Impairments of mitochondrial functions lead to aging and disease. Previous works with the ascomycete Podospora anserina demonstrated that mitochondrial morphotype as well as mitochondrial ultrastructure change during aging. The latter goes along with an age-dependent reorganization of the inner mitochondrial membrane leading to a change from lamellar cristae to vesicular structures. Especially from studies with yeast it is known that beside the F 1 F o -ATP-synthase and the phospholipid cardiolipin also the ‘mitochondrial contact site and cristae organizing system’ (MICOS) complex, existing of the Mic60- and Mic10-subcomplex, is essential for proper cristae formation. In the present study, we aimed to understand the mechanistic basis of age-related changes of the mitochondrial ultrastructure. We observed that MICOS subunits are co-regulated at the posttranscriptional level. This regulation partially depends on the mitochondrial iAAA-protease PaIAP. Most surprisingly, we made the counterintuitive observation that, despite the loss of lamellar cristae and of mitochondrial impairments, the ablation of MICOS subunits (except of PaMIC12) leads to a pronounced lifespan extension. Moreover, simultaneous ablation of subunits of both MICOS subcomplexes synergistically increases lifespan, providing formal genetic evidence that both subcomplexes affect lifespan by different and at least partially independent pathways. At the molecular level we found that ablation of Mic10-subcomplex components leads to a mitohormesis-induced lifespan extension, while lifespan extension of Mic60-subcomplex mutants seems to be controlled by another pathway. Overall, our data demonstrate that both MICOS subcomplexes have different functions and play distinct roles in the aging process of P. anserina .

Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity.

Autophagy is dependent on appropriate lipid supply for autophagosome formation. The regulation of lipid acquisition and the autophagy network response to lipid-limiting conditions are mostly elusive. Here, we show that the knockout of the RAB GTPase RAB18 interferes with lipid droplet catabolism, causing an impaired fatty acid release. The resulting reduced lipid-droplet-derived lipid availability influences autophagy and provokes adaptive modifications of the autophagy network. These adjustments include increased expression and phosphorylation of ATG2B as well as augmented formation of the ATG12-ATG5 conjugate. Moreover, ATG9A shows an enhanced phosphorylation at amino acid residues tyrosine 8 and serine 14, resulting in an increased ATG9A trafficking. Via pharmacological inhibition of Y8 phosphorylation, we demonstrate that this ATG9A modification is important to maintain basal autophagy under RAB18 knockout conditions. However, while the network adaptations are sufficient to maintain basal autophagic activity, they are incapable of ensuring autophagy induction upon starvation, which is characterized by an enhanced lipid demand. Thus, here, we define the molecular role of RAB18 in connecting lipid droplets and autophagy, emphasize the significance of lipid droplets as lipid sources for the degradative pathway, and uncover a remarkable autophagy network plasticity, including phosphorylation-dependent ATG9A activation, to compensate reduced lipid availability in order to rescue basal autophagic activity.

Autophagy is a lysosomal degradation pathway that mediates protein and organelle turnover and maintains cellular homeostasis. Autophagosomes transport cargo to lysosomes and their formation is dependent on an appropriate lipid supply. Here, we show that the knockout of the RAB GTPase RAB18 interferes with lipid droplet (LD) metabolism, resulting in an impaired fatty acid mobilization. The reduced LD-derived lipid availability influences autophagy and provokes adaptive modifications of the autophagy network, which include increased ATG2B expression and ATG12-ATG5 conjugate formation as well as enhanced ATG2B and ATG9A phosphorylation. Phosphorylation of ATG9A directs this transmembrane protein to the site of autophagosome formation and this particular modification is sufficient to rescue autophagic activity under basal conditions in the absence of RAB18. However, it is incapable of enabling an increased autophagy under inductive conditions. Thus, we illustrate the role of RAB18 in connecting LDs and autophagy, further emphasize the importance of LD-derived lipids for the degradative pathway, and characterize an ATG9A phosphorylation-dependent autophagy rescue mechanism as an adaptive response that maintains autophagy under conditions of reduced LD-derived lipid availability.

Retromer is an endosomal multi-protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans-Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer-associated RAB7-specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer-dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin-mediated mitophagy.

Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of Epb41l5 resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrix-dependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression.

A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism.