EMBO Reports

Schmitt, Daniel; Bozkurt, Süleyman; Henning‐Domres, Pascale; Huesmann, Heike; Eimer, Stefan; Bindila, Laura; Behrends, Christian; Boyle, Emily; Wilfling, Florian; Tascher, Georg; Münch, Christian; Behl, Christian; Kern, Andreas

doi: 10.15252/embr.202153065pmid: 36215690

Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody‐based FACS‐mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin–proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses.

Aweida, Dina; Cohen, Shenhav

doi: 10.15252/embr.202255175pmid: 36278411

Maintenance of desmin intermediate filaments (IF) is vital for muscle plasticity and function, and their perturbed integrity due to accelerated loss or aggregation causes atrophy and myopathies. Calpain‐1‐mediated disassembly of ubiquitinated desmin IF is a prerequisite for desmin loss, myofibril breakdown, and atrophy. Because calpain‐1 does not harbor a bona fide ubiquitin‐binding domain, the precise mechanism for desmin IF disassembly remains unknown. Here, we demonstrate that the AAA‐ATPase, ATAD1, is required to facilitate disassembly and turnover of ubiquitinated desmin IF. We identified PLAA and UBXN4 as ATAD1's interacting partners, and their downregulation attenuated desmin loss upon denervation. The ATAD1‐PLAA‐UBXN4 complex binds desmin filaments and promotes a release of phosphorylated and ubiquitinated species into the cytosol, presenting ATAD1 as the only known AAA‐ATPase that preferentially acts on phosphorylated substrates. Desmin filaments disassembly was accelerated by the coordinated functions of Atad1 and calpain‐1, which interact in muscle. Thus, by extracting ubiquitinated desmin from the insoluble filament, ATAD1 may expose calpain‐1 cleavage sites on desmin, consequently enhancing desmin solubilization and degradation in the cytosol.

O'Brien, Siobhan; Kelso, Susan; Steinhart, Zachary; Orlicky, Stephen; Mis, Monika; Kim, Yunhye; Lin, Sichun; Sicheri, Frank; Angers, Stephane

doi: 10.15252/embr.202255044pmid: 36278408

FBXW7, which encodes a substrate‐specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post‐translational stability of various proteins involved in cellular proliferation. Here, using genome‐wide CRISPR screens, we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF‐FBXW7 E3 ligase. Further analysis showed that the CCNL1–CDK11 complex is critical at the G2‐M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss‐of‐function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.

Meschichi, Anis; Zhao, Lihua; Reeck, Svenja; White, Charles; Da Ines, Olivier; Sicard, Adrien; Pontvianne, Frédéric; Rosa, Stefanie

doi: 10.15252/embr.202254736pmid: 36278395

Homologous recombination (HR) is a conservative DNA repair pathway in which intact homologous sequences are used as a template for repair. How the homology search happens in the crowded space of the cell nucleus is, however, still poorly understood. Here, we measure chromosome and double‐strand break (DSB) site mobility in Arabidopsis thaliana, using lacO/LacI lines and two GFP‐tagged HR reporters. We observe an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. This increase in mobility is lost in the sog1‐1 mutant, a central transcription factor of the DNA damage response in plants. Also, DSB sites show particularly high mobility levels and their enhanced mobility requires the HR factor RAD54. Our data suggest that repair mechanisms promote chromatin mobility upon DNA damage, implying a role of this process in the early steps of the DNA damage response.

Hu, Liang; Brichalli, Wyatt; Li, Naren; Chen, Shifan; Cheng, Yaqing; Liu, Qinfang; Xiong, Yulan; Yu, Jianzhong

doi: 10.15252/embr.202255851pmid: 36285521

The Hippo pathway is an evolutionarily conserved developmental pathway that controls organ size by integrating diverse regulatory inputs, including actomyosin‐mediated cytoskeletal tension. Despite established connections between the actomyosin cytoskeleton and the Hippo pathway, the upstream regulation of actomyosin in the Hippo pathway is less defined. Here, we identify the phosphoinositide‐3‐phosphatase Myotubularin (Mtm) as a novel upstream regulator of actomyosin that functions synergistically with the Hippo pathway during growth control. Mechanistically, Mtm regulates membrane phospholipid PI(3)P dynamics, which, in turn, modulates actomyosin activity through Rab11‐mediated vesicular trafficking. We reveal PI(3)P dynamics as a novel mode of upstream regulation of actomyosin and establish Rab11‐mediated vesicular trafficking as a functional link between membrane lipid dynamics and actomyosin activation in the context of growth control. Our study also shows that MTMR2, the human counterpart of Drosophila Mtm, has conserved functions in regulating actomyosin activity and tissue growth, providing new insights into the molecular basis of MTMR2‐related peripheral nerve myelination and human disorders.

Herman, Neta; Kadener, Sebastian; Shifman, Sagiv

doi: 10.15252/embr.202254720pmid: 36245419

Insulator proteins located at the boundaries of topological associated domains (TAD) are involved in higher‐order chromatin organization and transcription regulation. However, it is still not clear how long‐range contacts contribute to transcriptional regulation. Here, we show that relative‐of‐WOC (ROW) is essential for the long‐range transcription regulation mediated by the boundary element‐associated factor of 32kD (BEAF‐32). We find that ROW physically interacts with heterochromatin proteins (HP1b and HP1c) and the insulator protein (BEAF‐32). These proteins interact at TAD boundaries where ROW, through its AT‐hook motifs, binds AT‐rich sequences flanked by BEAF‐32‐binding sites and motifs. Knockdown of row downregulates genes that are long‐range targets of BEAF‐32 and bound indirectly by ROW (without binding motif). Analyses of high‐throughput chromosome conformation capture (Hi‐C) data reveal long‐range interactions between promoters of housekeeping genes bound directly by ROW and promoters of developmental genes bound indirectly by ROW. Thus, our results show cooperation between BEAF‐32 and the ROW complex, including HP1 proteins, to regulate the transcription of developmental and inducible genes through long‐range interactions.

Park, Kwangjin; Leroux, Michel R

doi: 10.15252/embr.202255420pmid: 36408840

The cilium evolved to provide the ancestral eukaryote with the ability to move and sense its environment. Acquiring these functions required the compartmentalization of a dynein‐based motility apparatus and signaling proteins within a discrete subcellular organelle contiguous with the cytosol. Here, we explore the potential molecular mechanisms for how the proximal‐most region of the cilium, termed transition zone (TZ), acts as a diffusion barrier for both membrane and soluble proteins and helps to ensure ciliary autonomy and homeostasis. These include a unique complement and spatial organization of proteins that span from the microtubule‐based axoneme to the ciliary membrane; a protein picket fence; a specialized lipid microdomain; differential membrane curvature and thickness; and lastly, a size‐selective molecular sieve. In addition, the TZ must be permissive for, and functionally integrates with, ciliary trafficking systems (including intraflagellar transport) that cross the barrier and make the ciliary compartment dynamic. The quest to understand the TZ continues and promises to not only illuminate essential aspects of human cell signaling, physiology, and development, but also to unravel how TZ dysfunction contributes to ciliopathies that affect multiple organ systems, including eyes, kidney, and brain.

Raja, Erna; Changarathil, Gopakumar; Oinam, Lalhaba; Tsunezumi, Jun; Ngo, Yen Xuan; Ishii, Ryutaro; Sasaki, Takako; Imanaka‐Yoshida, Kyoko; Yanagisawa, Hiromi; Sada, Aiko

doi: 10.15252/embr.202255478pmid: 36278510

Sakai, Yoshiki; Hanafusa, Hiroshi; Hisamoto, Naoki; Matsumoto, Kunihiro

doi: 10.15252/embr.202255076pmid: 36278516

Histidine phosphorylation is an emerging noncanonical protein phosphorylation in animals, yet its physiological role remains largely unexplored. The protein histidine phosphatase (PHPT1) was recently identified for the first time in mammals. Here, we report that PHIP‐1, an ortholog of PHPT1 in Caenorhabditis elegans, promotes axon regeneration by dephosphorylating GPB‐1 Gβ at His‐266 and inactivating GOA‐1 Goα signaling, a negative regulator of axon regeneration. Overexpression of the histidine kinase NDK‐1 also inhibits axon regeneration via GPB‐1 His‐266 phosphorylation. Thus, His‐phosphorylation plays an antiregenerative role in C. elegans. Furthermore, we identify a conserved UNC‐51/ULK kinase that functions in autophagy as a PHIP‐1‐binding protein. We demonstrate that UNC‐51 phosphorylates PHIP‐1 at Ser‐112 and activates its catalytic activity and that this phosphorylation is required for PHIP‐1‐mediated axon regeneration. This study reveals a molecular link from ULK to protein histidine phosphatase, which facilitates axon regeneration by inhibiting trimeric G protein signaling.

Raja, Erna; Changarathil, Gopakumar; Oinam, Lalhaba; Tsunezumi, Jun; Ngo, Yen Xuan; Ishii, Ryutaro; Sasaki, Takako; Imanaka‐Yoshida, Kyoko; Yanagisawa, Hiromi; Sada, Aiko

doi: 10.15252/embr.202255478pmid: 36278510

Tissue stem cells (SCs) divide infrequently as a protective mechanism against internal and external stresses associated with aging. Here, we demonstrate that slow‐ and fast‐cycling SCs in the mouse skin epidermis undergo distinct aging processes. Two years of lineage tracing reveals that Dlx1+ slow‐cycling clones expand into the fast‐cycling SC territory, while the number of Slc1a3+ fast‐cycling clones gradually declines. Transcriptome analysis further indicate that the molecular properties of each SC population are altered with age. Mice lacking fibulin 7, an extracellular matrix (ECM) protein, show early impairments resembling epidermal SC aging, such as the loss of fast‐cycling clones, delayed wound healing, and increased expression of inflammation‐ and differentiation‐related genes. Fibulin 7 interacts with structural ECM and matricellular proteins, and the overexpression of fibulin 7 in primary keratinocytes results in slower proliferation and suppresses differentiation. These results suggest that fibulin 7 plays a crucial role in maintaining tissue resilience and epidermal SC heterogeneity during skin aging.