Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.

The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here, we show that its lysosomal degradation is dependent on ubiquitylation at lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4, and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells, and cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive nor a localization-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4 or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.

Untargeted Metabolomic Profiling of Extracellular Vesicles Isolated from Human Seminal Plasma.

Seminal extracellular vesicles (SemEVs) are repositories of biomolecules, including metabolites involved in the regulation of sperm function. The correlation between the metabolite profile of SemEVs and semen parameters, along with their role in regulating sperm function, is an unexplored area. This preliminary study evaluated the metabolomic content of SemEVs. Semen samples were obtained from 18 healthy men, and SemEVs were extracted from seminal plasma using the size exclusion chromatography qEV Gen 2-35 nm column coupled with an automatic fraction collector. The physical characterization of SemEVs was carried out with the ZetaView PMX-430-Z QUATT laser system. EV protein markers were detected using Western blot. In addition, these SemEVs were used for metabolomic profiling and functional bioinformatic analysis. The mean concentration of isolated SemEVs was 1.7 ± 1.1 × 1011/mL of seminal plasma, whereas SemEVs size and zeta potential were 129.5 ± 5.5 nm and -40.03 ± 3.99 mV, respectively. Western blot analysis confirmed the presence of EV specific markers such as CD81, ALIX, and TSG101. A total of 107 metabolites were identified using this untargeted metabolomic approach in SemEVs. Bioinformatics analysis further revealed that metabolites associated with tyrosine metabolism were highly enriched in these SemEVs. Ingenuity Pathway Analysis (IPA) also indicated that these metabolites present in SemEVs were involved in the regulation of the free radical scavenging pathway. Furthermore, our metabolomic results suggest that these SemEV-associated metabolites may play a pivotal role in the maintenance of seminal plasma redox homeostasis.

Endosomal membrane budding patterns in plants.

Multivesicular endosomes (MVEs) sequester membrane proteins destined for degradation within intralumenal vesicles (ILVs), a process mediated by the membrane-remodeling action of Endosomal Sorting Complex Required for Transport (ESCRT) proteins. In Arabidopsis, endosomal membrane constriction and scission are uncoupled, resulting in the formation of extensive concatenated ILV networks and enhancing cargo sequestration efficiency. Here, we used a combination of electron tomography, computer simulations, and mathematical modeling to address the questions of when concatenated ILV networks evolved in plants and what drives their formation. Through morphometric analyses of tomographic reconstructions of endosomes across yeast, algae, and various land plants, we have found that ILV concatenation is widespread within plant species, but only prevalent in seed plants, especially in flowering plants. Multiple budding sites that require the formation of pores in the limiting membrane were only identified in hornworts and seed plants, suggesting that this mechanism has evolved independently in both plant lineages. To identify the conditions under which these multiple budding sites can arise, we used particle-based molecular dynamics simulations and found that changes in ESCRT filament properties, such as filament curvature and membrane binding energy, can generate the membrane shapes observed in multiple budding sites. To understand the relationship between membrane budding activity and ILV network topology, we performed computational simulations and identified a set of membrane remodeling parameters that can recapitulate our tomographic datasets.

The Dsc ubiquitin ligase complex identifies transmembrane degrons to degrade orphaned proteins at the Golgi.

The Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for endosome and Golgi associated proteasomal degradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition.

Tissue-specific knockout in the Drosophila neuromuscular system reveals ESCRT's role in formation of synapse-derived extracellular vesicles.

Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions during development. However, this approach has not been successfully applied to most Drosophila tissues, including the Drosophila neuromuscular junction (NMJ). To expand tissue-specific CRISPR to this powerful model system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of CRISPR-TRiM by knocking out multiple genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. We used CRISPR-TRiM to discover an essential role for SNARE components in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to release extracellular vesicles at the NMJ. Thus, we have successfully developed an NMJ CRISPR mutagenesis approach which we used to reveal genes important for NMJ structural plasticity.

Tsg101 UEV Interaction with Nedd4 HECT Relieves E3 Ligase Auto-Inhibition, Promoting HIV-1 Assembly and CA-SP1 Maturation Cleavage.

Tsg101, a component of the endosomal sorting complex required for transport (ESCRT), is responsible for recognition of events requiring the machinery, as signaled by cargo tagging with ubiquitin (Ub), and for recruitment of downstream acting subunits to the site. Although much is known about the latter function, little is known about its role in the earlier event. The N-terminal domain of Tsg101 is a structural homologue of Ub conjugases (E2 enzymes) and the protein associates with Ub ligases (E3 enzymes) that regulate several cellular processes including virus budding. A pocket in the domain recognizes a motif, PT/SAP, that permits its recruitment. PT/SAP disruption makes budding dependent on Nedd4L E3 ligases. Using HIV-1 encoding a PT/SAP mutation that makes budding Nedd4L-dependent, we identified as critical for rescue the residues in the catalytic (HECT) domain of the E3 enzyme that lie in proximity to sites in Tsg101 that bind Ub non-covalently. Mutation of these residues impaired rescue by Nedd4L but the same mutations had no apparent effect in the context of a Nedd4 isomer, Nedd4-2s, whose N-terminal (C2) domain is naturally truncated, precluding C2-HECT auto-inhibition. Surprisingly, like small molecules that disrupt Tsg101 Ub-binding, small molecules that interfered with Nedd4 substrate recognition arrested budding at an early stage, supporting the conclusion that Tsg101-Ub-Nedd4 interaction promotes enzyme activation and regulates Nedd4 signaling for viral egress. Tsg101 regulation of E3 ligases may underlie its broad ability to function as an effector in various cellular activities, including viral particle assembly and budding.

SNX27:Retromer:ESCPE-1-mediated early endosomal tubulation impacts cytomegalovirus replication.

Introduction: Cytomegaloviruses (CMVs) extensively reorganize the membrane system of the cell and establish a new structure as large as the cell nucleus called the assembly compartment (AC). Our previous studies on murine CMV (MCMV)-infected fibroblasts indicated that the inner part of the AC contains rearranged early endosomes, recycling endosomes, endosomal recycling compartments and trans-Golgi membrane structures that are extensively tubulated, including the expansion and retention of tubular Rab10 elements. An essential process that initiates Rab10-associated tubulation is cargo sorting and retrieval mediated by SNX27, Retromer, and ESCPE-1 (endosomal SNX-BAR sorting complex for promoting exit 1) complexes. Objective: The aim of this study was to investigate the role of SNX27:Retromer:ESCPE-1 complexes in the biogenesis of pre-AC in MCMV-infected cells and subsequently their role in secondary envelopment and release of infectious virions. Results: Here we show that SNX27:Retromer:ESCPE1-mediated tubulation is essential for the establishment of a Rab10-decorated subset of membranes within the pre-AC, a function that requires an intact F3 subdomain of the SNX27 FERM domain. Suppression of SNX27-mediated functions resulted in an almost tenfold decrease in the release of infectious virions. However, these effects cannot be directly linked to the contribution of SNX27:Retromer:ESCPE-1-dependent tubulation to the secondary envelopment, as suppression of these components, including the F3-FERM domain, led to a decrease in MCMV protein expression and inhibited the progression of the replication cycle. Conclusion: This study demonstrates a novel and important function of membrane tubulation within the pre-AC associated with the control of viral protein expression.

EhVps35, a retromer component, is a key factor in secretion, motility, and tissue invasion by Entamoeba histolytica.

In humans and Drosophila melanogaster, the functional convergence of the endosomal sorting complex required for transport (ESCRT) machinery that is in charge of selecting ubiquitinated proteins for sorting into multivesicular bodies, and the retromer, that is the complex responsible for protein recycling to the plasma membrane and Golgi apparatus. ESCRT and retromer complexes are codependent for protein sorting recycling, degradation, and secretion. In this article, we studied the EhVps35 C isoform (referred to as EhVps35), that is the central member of the Entamoeba histolytica retromer, and its relation with the ESCRT machinery during sorting and protein recycling events and their involvement virulence. Our findings revealed that EhVps35 interacts with at least 300 proteins that participate in multiple cellular processes. Laser confocal and transmission electronic microscopy images, as well as secretion assays, revealed that EhVps35 is secreted in vesicles together with EhVps23 and EhADH (both ESCRT machinery proteins). In addition, immunoprecipitation, immunofluorescence, and molecular docking assays revealed the relationship among EhVps35 and other ESCRT machinery proteins. Red blood cell stimulus increased EhVps35 secretion, and the knockdown of the Ehvps35 gene in trophozoites reduced their capacity to migrate and invade tissues. This also impacts the cellular localization of ubiquitin, EhVps23 (ESCRT-I), and EhVps32 (ESCRT-III) proteins, strongly suggesting their functional relationship. Our results, taken together, give evidence that EhVps35 is a key factor in E. histolytica virulence mechanisms and that it, together with the ESCRT machinery components and other regulatory proteins, is involved in vesicle trafficking, secretion, migration, and cell proliferation.

The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation.

Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic components. The final degradation step is essential for clearing autophagic cargo and recycling macromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conserved transmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.

Interaction between ESCRT-III proteins and the yeast SERINC homolog Tms1.

The endosomal sorting complex required for transport (ESCRT)-III is involved in membrane remodeling and abscission during intraluminal vesicle (ILV) formation at endosomes. Our data now suggest that ESCRT-III function could be connected to lipid remodeling of the endosomal membrane. This notion is based on our finding that ESCRT-III proteins bind to the yeast serine incorporator (SERINC) homolog Tms1. Human SERINC3 and SERINC5 are HIV-1 restriction factors and have been shown to act as scramblases, flipping phospholipids between membrane leaflets. Due to the extraordinarily high sequence conservation between Tms1 and human SERINCs, it is likely that Tms1 is also a scramblase. While deletion of TMS1 had only a moderate effect on the sorting of multivesicular body (MVB) cargo proteins, the simultaneous deletion of a component of the Vps55/Vps68 complex led to a strong synergistic phenotype. This pronounced synergism suggests that Tms1 and Vps55/Vps68 perform a parallel function at endosomes. Vps55/Vps68 loosely resembles Tms1 in its overall structure. Thus, it is possible that Vps55/Vps68 is also a scramblase. Since both Vps55 and Tms1 physically interact with ESCRT-III proteins, we propose that the recruitment of a scramblase plays a crucial role in ESCRT-III-dependent membrane remodeling at endosomes.

NEDD4 family ubiquitin ligase AIP4 interacts with Alix to enable HBV naked capsid egress in an Alix ubiquitination-independent manner.

Hepatitis B virus (HBV) exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for virion budding. In addition to enveloped virions, HBV-replicating cells nonlytically release non-enveloped (naked) capsids independent of the integral ESCRT machinery, but the exact secretory mechanism remains elusive. Here, we provide more detailed information about the existence and characteristics of naked capsid, as well as the viral and host regulations of naked capsid egress. HBV capsid/core protein has two highly conserved Lysine residues (K7/K96) that potentially undergo various types of posttranslational modifications for subsequent biological events. Mutagenesis study revealed that the K96 residue is critical for naked capsid egress, and the intracellular egress-competent capsids are associated with ubiquitinated host proteins. Consistent with a previous report, the ESCRT-III-binding protein Alix and its Bro1 domain are required for naked capsid secretion through binding to intracellular capsid, and we further found that the ubiquitinated Alix binds to wild type capsid but not K96R mutant. Moreover, screening of NEDD4 E3 ubiquitin ligase family members revealed that AIP4 stimulates the release of naked capsid, which relies on AIP4 protein integrity and E3 ligase activity. We further demonstrated that AIP4 interacts with Alix and promotes its ubiquitination, and AIP4 is essential for Alix-mediated naked capsid secretion. However, the Bro1 domain of Alix is non-ubiquitinated, indicating that Alix ubiquitination is not absolutely required for AIP4-induced naked capsid secretion. Taken together, our study sheds new light on the mechanism of HBV naked capsid egress in viral life cycle.

HRS Facilitates Newcastle Disease Virus Replication in Tumor Cells by Promoting Viral Budding.

Newcastle disease virus (NDV) is a highly pathogenic avian infectious disease agent and also a promising oncolytic virus with broad application prospects. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery has been increasingly recognized for its crucial role in the life cycles of enveloped viruses, influencing processes such as viral entry, replication, and budding. In this study, we employed an RNA interference screening approach to identify key ESCRT components that regulate NDV replication in tumor cells. qPCR, immunofluorescence, and Western blot assays demonstrated that knockdown of HRS, CHMP4A, CHMP4B, and CHMP4C significantly impaired NDV replication in HeLa cells, with HRS exhibiting the most pronounced inhibitory effect. Additionally, HRS knockout significantly inhibited viral budding and suppressed NDV-induced cell death in HeLa cells. Notably, NDV infection was shown to significantly upregulate HRS gene and protein expression in a time-dependent manner. In conclusion, this study systematically identifies critical ESCRT components involved in NDV replication within tumor cells, with a particular focus on the role of HRS in promoting NDV's replication by promoting viral budding, offering new insights for the development of NDV-based oncolytic therapies.

The Rubicon-WIPI axis regulates exosome biogenesis during ageing.

Cells release intraluminal vesicles in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNA interference screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for intraluminal vesicle formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for age-dependent changes in exosome quantity and quality.

An internal linker and pH biosensing by phosphatidylinositol 5-phosphate regulate the function of the ESCRT-0 component TOM1.

Target of Myb1 (TOM1) facilitates the transport of endosomal ubiquitinated proteins destined for lysosomal degradation; however, the mechanisms regulating TOM1 during this process remain unknown. Here, we identified an adjacent DXXLL motif-containing region to the TOM1 VHS domain, which enhances its affinity for ubiquitin and can be modulated by phosphorylation. TOM1 is an endosomal phosphatidylinositol 5-phosphate (PtdIns5P) effector under Shigella flexneri infection. We pinpointed a consensus PtdIns5P-binding motif in the VHS domain. We show that PtdIns5P binding by TOM1 is pH-dependent, similarly observed in its binding partner TOLLIP. Under acidic conditions, TOM1 retained its complex formation with TOLLIP, but was unable to bind ubiquitin. S. flexneri infection inhibits pH-dependent endosomal maturation, leading to reduced protein degradation. We propose a model wherein pumping of H+ to the cytosolic side of endosomes contributes to the accumulation of TOM1, and possibly TOLLIP, at these sites, thereby promoting PtdIns5P- and pH-dependent signaling, facilitating bacterial survival.

Ubiquitin-mediated endosomal stress: A novel organelle stress of early endosomes that initiates cellular signaling pathways: USP8 serves as a gatekeeper of ubiquitin-mediated endosomal stress to counteract the activation of cellular signaling pathways.

Cells utilize diverse organelles to maintain homeostasis and to respond to extracellular stimuli. Recently, multifaceted aspects of organelle stress caused by various factors have been emerging. The endosome is an essential organelle, functioning as the central hub for membrane trafficking in cooperation with the ubiquitin system. However, knowledge regarding endosomal stress, which refers to organelle stress of the endosome, is currently limited. We recently revealed ubiquitin-mediated endosomal stress of early endosomes (EEs) and its responsive signaling pathways. These findings shed light on the relevance of ubiquitin-mediated endosomal stress to physiological and pathological processes. Here, we present a hypothesis that ubiquitin-mediated endosomal stress may have significant roles in biological contexts and that ubiquitin-specific protease 8 is a key regulator of ubiquitin clearance from EEs.

Identification of Neighboring Proteins of Endosomal Regulators by Using TurboID-Based Proximity Labeling.

In the endomembrane system, multivesicular bodies (MVBs) play a crucial role in sorting ubiquitinated membrane proteins into intraluminal vesicles for degradation upon fusion with vacuoles or lysosomes. This process involves regulations by multiprotein complexes, including endosomal sorting complex required for transport (ESCRT) I-III, and accessory proteins. Although many organellar proteomes have been identified in plant cells, the information of specific proteomes associated with regulators engaged in MVB biogenesis remains limited. Here, using the ESCRT component FREE1 as an example, we describe a method to identify neighboring proteins of endosomal regulators by using an approach of TurboID-based proximity labeling.

Preserving Genome Integrity: Unveiling the Roles of ESCRT Machinery.

The endosomal sorting complex required for transport (ESCRT) machinery is composed of an articulated architecture of proteins that assemble at multiple cellular sites. The ESCRT machinery is involved in pathways that are pivotal for the physiology of the cell, including vesicle transport, cell division, and membrane repair. The subunits of the ESCRT I complex are mainly responsible for anchoring the machinery to the action site. The ESCRT II subunits function to bridge and recruit the ESCRT III subunits. The latter are responsible for finalizing operations that, independently of the action site, involve the repair and fusion of membrane edges. In this review, we report on the data related to the activity of the ESCRT machinery at two sites: the nuclear membrane and the midbody and the bridge linking cells in the final stages of cytokinesis. In these contexts, the machinery plays a significant role for the protection of genome integrity by contributing to the control of the abscission checkpoint and to nuclear envelope reorganization and correlated resilience. Consistently, several studies show how the dysfunction of the ESCRT machinery causes genome damage and is a codriver of pathologies, such as laminopathies and cancer.

Exploring the clinical and biological significance of the cell cycle-related gene CHMP4C in prostate cancer.

BACKGROUND: Prostate cancer (PCa) stands as the second most prevalent malignancy impacting male health, and the disease's evolutionary course presents formidable challenges in the context of patient treatment and prognostic management. Charged multivesicular body protein 4 C (CHMP4C) participates in the development of several cancers by regulating cell cycle functions. However, the role of CHMP4C in prostate cancer remains unclear. METHODS: In terms of bioinformatics, multiple PCa datasets were employed to scrutinize the expression of CHMP4C. Survival analysis coupled with a nomogram approach was employed to probe into the prognostic significance of CHMP4C. Gene set enrichment analysis (GSEA) was conducted to interrogate the functional implications of CHMP4C. In terms of cellular experimentation, the verification of RNA and protein expression levels was executed through the utilization of qRT-PCR and Western blotting. Upon the establishment of a cell line featuring stable CHMP4C knockdown, a battery of assays, including Cell Counting Kit-8 (CCK-8), wound healing, Transwell, and flow cytometry, were employed to discern the impact of CHMP4C on the proliferation, migration, invasion, and cell cycle function of PCa cells. RESULTS: The expression of CHMP4C exhibited upregulation in both PCa cells and tissues, and patients demonstrating elevated CHMP4C expression levels experienced a notably inferior prognosis. The nomogram, constructed using CHMP4C along with clinicopathological features, demonstrated a commendable capacity for prognostic prediction. CHMP4C knockdown significantly inhibited the proliferation, migration, and invasion of PCa cells (LNcaP and PC3). CHMP4C could impact the advancement of the PCa cell cycle, and its expression might be regulated by berberine. Divergent CHMP4C expression among PCa patients could induce alterations in immune cell infiltration and gene mutation frequency. CONCLUSIONS: Our findings suggest that CHMP4C might be a prognostic biomarker in PCa, potentially offering novel perspectives for the advancement of precision therapy for PCa.

Loss of HD-PTP function results in lipodystrophy, defective cellular signaling and altered lipid homeostasis.

His domain protein tyrosine phosphatase (HD-PTP; also known as PTPN23) facilitates function of the endosomal sorting complexes required for transport (ESCRTs) during multivesicular body (MVB) formation. To uncover its role in physiological homeostasis, embryonic lethality caused by a complete lack of HD-PTP was bypassed through generation of hypomorphic mice expressing reduced protein, resulting in animals that are viable into adulthood. These mice exhibited marked lipodystrophy and decreased receptor-mediated signaling within white adipose tissue (WAT), involving multiple prominent pathways including RAS/MAPK, phosphoinositide 3-kinase (PI3K)/AKT and receptor tyrosine kinases (RTKs), such as EGFR. EGFR signaling was dissected in vitro to assess the nature of defective signaling, revealing decreased trans-autophosphorylation and downstream effector activation, despite normal EGF binding. This corresponds to decreased plasma membrane cholesterol and increased lysosomal cholesterol, likely resulting from defective endosomal maturation necessary for cholesterol trafficking and homeostasis. The ESCRT components Vps4 and Hrs have previously been implicated in cholesterol homeostasis; thus, these findings expand knowledge on which ESCRT subunits are involved in cholesterol homeostasis and highlight a non-canonical role for HD-PTP in signal regulation and adipose tissue homeostasis.

A p62-dependent rheostat dictates micronuclei catastrophe and chromosome rearrangements.

Chromosomal instability (CIN) generates micronuclei-aberrant extranuclear structures that catalyze the acquisition of complex chromosomal rearrangements present in cancer. Micronuclei are characterized by persistent DNA damage and catastrophic nuclear envelope collapse, which exposes DNA to the cytoplasm. We found that the autophagic receptor p62/SQSTM1 modulates micronuclear stability, influencing chromosome fragmentation and rearrangements. Mechanistically, proximity of micronuclei to mitochondria led to oxidation-driven homo-oligomerization of p62, limiting endosomal sorting complex required for transport (ESCRT)-dependent micronuclear envelope repair by triggering autophagic degradation. We also found that p62 levels correlate with increased chromothripsis across human cancer cell lines and with increased CIN in colorectal tumors. Thus, p62 acts as a regulator of micronuclei and may serve as a prognostic marker for tumors with high CIN.