Journal of cell science最新文献

In the nematode Caenorhabditis elegans, sperm-derived mitochondria and membranous organelles (MOs) are selectively degraded by autophagy in embryos in a process termed allophagy. For this process, ALLO-1 functions as an autophagy adaptor. The allo-1 gene encodes two splice isoforms, ALLO-1a and ALLO-1b, which have different C-terminal sequences and are predominantly targeted to MOs and paternal mitochondria, respectively. However, the mechanism by which ALLO-1 targets the paternal organelles remains unknown. In this study, X-ray crystallography analysis reveals that the C-terminal region of ALLO-1a forms a parallel coiled-coil structure. In addition, AlphaFold2-Multimer predicts that this region directly interacts with ubiquitin. We showed that ALLO-1a interacts with K48- and K63-linked polyubiquitin in vitro and found that the D355 residue of ALLO-1a at the predicted interface with ubiquitin is important for its ubiquitin binding in vitro and also for its MO targeting and MO degradation in embryos. These results suggest that ubiquitin is a marker for the recognition of MOs by the autophagy machinery in C. elegans embryos.

MYO1F, a long-tailed myosin of class I, is selectively expressed in immune cells and upregulated in microglia associated with neurodegenerative pathogenesis. Myosin motor functions are regulated by adaptor proteins that mediate cargo attachment and motor recruitment. To define the MYO1F interactome, we used in situ proximity labelling and proteomics in human myeloid cells. We identified a distinct SH3-domain-dependent adaptor module comprising CD2AP, ASAP1, SH3BP2 and SH3KBP1 (herein termed the CASS group of proteins). Interestingly, CD2AP is an Alzheimer's disease (AD) risk gene upregulated in the microglia of individuals with AD, which are implicated in phagocytic responses to amyloid-β. Structural modelling and mutagenesis confirmed multivalent proline-rich motif interactions between the CASS group of proteins and the MYO1F SH3 domain. Additional binding partners associate with the MYO1F pleckstrin homology (PH) domain. Immunofluorescence revealed colocalisation of MYO1F and the CASS group of proteins at actin-rich podosomes and phagocytic cups in macrophages and microglia. Functional assays demonstrated that MYO1F recruitment to the phagocytic cup requires motor activity and intact PH and SH3 domains. We provide the first MYO1F interactome identifying adaptor proteins for MYO1F in podosomes and during phagocytosis, offering new insights into its function in disease-associated microglia during neurodegeneration.

Serum response factor (SRF) and its cofactors, myocardin-related transcription factors A and B (MRTF-A and MRTF-B, respectively), regulate transcription of numerous cytoskeletal structural and regulatory genes, and most MRTF/SRF inactivation phenotypes reflect deficits in cytoskeletal dynamics. We show that MRTF-SRF activity is required for effective proliferation of both primary and immortalised fibroblast and epithelial cells. Cells lacking the MRTFs or SRF proliferate very slowly, express elevated levels of senescence-associated secretory phenotype (SASP) factors and senescence-associated β-galactosidase activity, and inhibit proliferation of co-cultured primary wild-type cells. They exhibit decreased levels of CDK1 and CKS2 proteins, and elevated levels of CDK inhibitors, usually p27 (also known as CDKN1B). These phenotypes, which can be fully reversed by re-expression of MRTF-A, are also seen in wild-type cells arrested by serum deprivation. Moreover, in wild-type cells direct interference with cytoskeletal dynamics through inhibition of Rho kinases (ROCKs) or myosin ATPase induces a similar proliferative defect to that seen in MRTF-null cells. MRTF-null cells exhibit multiple cytoskeletal defects and markedly reduced contractility. We propose that MRTF-SRF signalling will be required for cell proliferation in cell types and environments where physical progression through cell cycle transitions requires high contractility.

Recent advances in spatiotemporally resolved imaging and single-molecule labeling technologies have provided new mechanistic insight into the very early phases of insulin-responsive GLUT4 (also known as SLC2A4) trafficking. Live-cell assays combining quantum dot tracking and fluorescence-based fusion reporters have uncovered a previously overlooked, insulin-induced initial and fundamental step - that static GLUT4 vesicles undergo heterotypic fusion with transferrin receptor-positive endosomes. This insulin-induced fusion functions as a molecular gateway - termed 'fusion-guided GLUT4 entry' (FGG4E) - that enables GLUT4 molecules, originally sequestered in static vesicles, to circulate within a dynamic endosomal network when insulin is present, escaping the trafficking itinerary that leads to static retention. Through this pathway, insulin-stimulated GLUT4 is efficiently delivered to the plasma membrane and continues to recycle dynamically between the plasma membrane and endosomal compartments. After insulin withdrawal, GLUT4 molecules are retrieved from the endosomal system and returned to the static pool. In this Opinion article, we propose that this revised model highlights a key regulatory role for heterotypic vesicle fusion as a gateway linking basal retention with dynamic mobilization and recycling, and redefines GLUT4 trafficking beyond the classical view of vesicle mobilization.

Peroxisomes are single-membrane-bound organelles essential for human health, yet the mechanisms of peroxisome biogenesis are not fully understood. Here using a systematic double screening approach, we identified ribosome-binding protein 1 (RRBP1) as a novel peroxisome biogenesis factor in human cells. Deletion of RRBP1 in HEK293T cells led to a reduction in both peroxisome number and peroxisomal protein levels as well as in defects in processing of peroxisomal matrix proteins, such as ACOX1 and thiolase. However, cell proliferation and protein translation were not altered in cells lacking RRBP1. RRBP1 depletion did not affect peroxisome-endoplasmic reticulum (ER) contact sites, and pexophagy did not contribute to the reduction of peroxisomes in RRBP1 knockout cells. Instead, in the absence of RRBP1, peroxisomal proteins were processed by proteasomal degradation, suggesting that RRBP1 plays a role in the insertion of these proteins into ER membranes and their stabilization. Altogether, our results show that RRBP1 promotes peroxisome biogenesis in human cells, highlighting the power of systematic approaches in discovering novel factors of organellar biogenesis.

Integrin-based adhesion complexes serve as primary sites for actomyosin force transmission to the extracellular matrix, providing traction that drives cell mechanical responses including adhesion, migration and mechano-signaling. Talin (herein referring generically unless specified) is the principal force-transmission protein that orchestrates molecular events underlying adhesion mechanosensing. Although talin has been an effective target for chemogenetic and optogenetic manipulation of integrin-based adhesions, existing approaches relied on dual-construct heterodimerization, creating challenges in maintaining consistent stoichiometric balance of each component and multiplexing with additional genetically encoded probes. To overcome these limitations, we develop a single-construct optogenetic talin utilizing pdDronpa1.2 for light-inducible C-terminal homodimerization. We demonstrate its application by dissecting overlapping roles of dimerization and actin binding mediated by the native C-terminal region of talin, showing that artificial light-induced homodimerization is sufficient to promote talin recruitment to adhesion sites, adhesion formation, actin retrograde flow engagement and downstream mechanosignaling, thereby underscoring the crucial importance of talin dimer. Multiplexing of our single-construct optodimerizable talin with quantitative actin dynamics imaging or super-resolution single-molecule tracking is also showcased, establishing its versatility in spatiotemporally precise manipulation of mechanobiological processes.

The thrombopoietin receptor (TpoR; also known as MPL) is essential to the production of platelets. Activation of the surface receptor by its ligand thrombopoietin (TPO; also known as THPO) leads to the lineage-specific differentiation of the haematopoietic stem and myeloid progenitors into megakaryocytes and platelets. Moreover, platelet surface TpoR scavenges the serum TPO, creating a feedback mechanism that regulates the availability of TPO, ensuring platelet homeostasis. Therefore, the surface expression of the receptor must be tightly regulated during the process of megakaryopoiesis. Megakaryopoiesis is accompanied by alterations in the expression of the two non-muscle myosin isoforms IIA and IIB. Using cell line models of megakaryopoiesis and COS7 cells that preferentially express the NMIIB isoform, the traffic, surface expression and signalling of TpoR was found to be augmented by the expression of NMIIA. This was attributed to NMIIA-dependent remodelling of the cortical actin network. Consequently, ROCK inhibition generated an altered cortical actin network along with reduced surface expression and signalling of TpoR. Thus, our study demonstrated that megakaryopoiesis-dependent alteration in NMIIA expression contributed to enhanced TpoR surface expression and signalling.