Cytoskeleton (Hoboken, N.J.)最新文献

Microtubule Associated Protein MAP1B is expressed at high levels during the early development of the nervous system, playing important roles in axonal growth, neuronal migration, and branching, as well as dendritic spine morphogenesis and synapse formation. MAP1B belongs to the MAP1 family, which includes MAP1A and MAP1S, as well as a known homolog in Drosophila (the Futsch gene). MAP1B is a polyprotein that undergoes proteolytic processing into heavy (HC) and light chains (LC1). It is composed of seven exons, including microtubule- and actin-binding domains, and conserved regions of both the N- and C-termini. In this Perspective, we investigated the structure of MAP1B from an evolutionary perspective, emphasizing the significance of conserved domains across different species. Through sequence analysis and alignment, exon structures, prediction of protein folding, and database searches, we identified key structural features of MAP1B and constructed a model based on these data. This approach allowed us to refine our understanding of known domains and uncover unrecognized, highly conserved domains that may have novel functions, providing valuable reference data for future research. In the process of searching for homolog proteins in vertebrates and invertebrates, we traced the deep roots of MAP1B as far back as the octopus, sea urchin, and Caenorhabditis elegans, underscoring the highly conserved properties of MAP1B. When compared to the other members of the MAP1 family, MAP1A and MAP1S, we found that they are far less conserved than MAP1B, even among vertebrates, supporting the conclusion that MAP1B represents the most ancient ancestral member of this family.

Capping protein regulator and myosin 1 Linker 1 (CARMIL1) is a multifunctional regulator of actin polymerization, ruffle formation, and lamellipodia development, making it essential for cell spreading and migration. While its protein-level functions are perceived, phospho-signaling of highly phosphorylated CARMIL1 remains unexplored. This study investigates CARMIL1 phosphorylation and its regulatory mechanisms. Global phosphoproteome datasets captured the most frequently detected and differentially regulated CARMIL1 phosphosites under different conditions to be in the CARMIL_C domain (T916, S968, and S1067). A coregulation-based method was employed to identify interactors and upstream kinases that are coregulated with the phosphorylation sites. These sites exhibited a consistent co-occurrence pattern including both positive and negative coregulation. The phosphosites of complex interactors showed positive and negative coregulation and were involved in cell cycle regulation and cell growth. AKT1, PAK2, and MYLK were identified as potential upstream kinases for CARMIL at S968, while WNK1 was predicted as a potential upstream kinase for S1067, suggesting distinct regulatory mechanisms for these phosphorylation sites. Phosphorylation at CDK1 S146, MAP4K2 S238, MINK1 S641, and TNIK S678 was found coregulated high with CARMIL T916 in human brain cancer. Notably, most coregulated proteins were associated with regulation of the actin cytoskeleton pathway. Our results show that phosphorylation of CARMIL1 in the C-terminal domain highly influences actin cytoskeletal organization. It offers new insights on CARMIL1-mediated cellular functions, deepening our comprehension of its involvement in cytoskeletal dynamics.

The microtubule cytoskeleton is a fundamental functional component of the cell. In vertebrate proliferating cells, centrosomes are the primary microtubule organizing center (MTOC), and their dysregulation has been linked to genomic instability and cancer. LZTS2, a known tumor suppressor, localizes to centrosomes and regulates microtubule severing. However, whether LZTS2 regulates centrosome structure and/or its function in microtubule organization or ciliation remains unknown. Here, we investigate the function of LZTS2 at the centrosome. Through fluorescence and electron microscopy assays, we observed that LZTS2 knockdown does not affect centriole biogenesis or structure, nor ciliation. Importantly, we show that LZTS2 depletion increases microtubule nucleation at the centrosome. Moreover, LZTS2 negatively regulates centrosomal levels of CEP135. Notably, depletion of LZTS2 can partially rescue the impaired centrosome microtubule nucleation caused by CEP135 knockdown. Taken together, our findings reveal a novel role for LZTS2 as a negative regulator of CEP135 and centrosomal microtubule nucleation, providing a potential mechanistic link to its tumor suppressor function.

The C2a projection of the central pair of flagella in Chlamydomonas reinhardtii harbours the A-kinase anchoring protein FAP65, FAP174, FAP147 and FAP70. FAP174, an RII-like protein with its N-terminal dimerization and docking domain, binds to the amphipathic helices of FAP65. Cryo-EM data do not reveal the entire sequences for FAP174 and FAP147. Hence, the interacting domains within this scaffold remain elusive. This study has identified the interacting domains of FAP174 with FAP147. The FAP147 protein and its MYCBPAP domain (129-639 a.a.) bind to the C-terminus of FAP174 (47-92 a.a.). In silico docking analyses using CABS-Dock to delineate the interaction identified several MYCBPAP-derived peptides, such as p3 (310-339), p4 (319-348), p9 (547-576), p13 (528-557) and p15 (350-379), to form stable interacting complexes with RMSD < 3 Å 2-3 times, and are potentially amphipathic. To gain atomistic details of the interaction, molecular dynamics (MD) simulations of the FAP147 MYCBPAP domain in complex with the FAP174 C-terminus were performed. It revealed stable interfacial contacts, a subset of which overlap with residues within the p15 peptide region of the MYCBPAP domain, while identifying G48, S49, P52, Y55, L79, Q80 and V83 as key interacting residues within the C-terminus of FAP174. Individual alanine substitution of the FAP174 residues, followed by overlay assay with FAP147, retained the interaction, indicating that the interaction does not depend solely on discrete amino acids but on broader interface interactions.

The study aimed to elucidate the mechanistic basis of impaired mechanosensitive pathways-YAP/TAZ and β-catenin signaling-in filamin C-deficient cells through investigation of transcriptomic profiles, actin cytoskeleton organization and focal adhesion structure. By using FlncKO-C2C12 cells and testing a number of inhibitors, we identified that mechanosensitive processes depend on filamin C function. We detected that filamin C deficiency leads to increased F/G-actin ratio and expansion of focal adhesion structures along with diminished nuclear accumulation of TAZ and β-catenin and decreased YAP/TAZ activity. Verteporfin-mediated YAP/TAZ inhibition caused adhesion enlargement and slight elevation of F/G-actin ratio in WT-C2C12 but had lower efficiency in FlncKO-C2C12. Of note, actin cytoskeletal stabilization through Jasplakinolide treatment rescued YAP/TAZ signaling specifically in filamin C-deficient cells, whereas inhibition of non-muscle myosin II with (-)Blebbistatin failed to recapitulate the signaling suppression observed in control cells. In addition, ROCK inhibition with Y-27632 demonstrated greater focal adhesion disassembly in FlncKO-C2C12 cells than in WT-C2C12 and produced a genotype-specific response, restoring β-catenin nuclear localization exclusively in FlncKO-C2C12 without affecting WT-C2C12 cells. In summary, we propose that in C2C12 muscle cells filamin C deficiency causes aberrant focal adhesion turnover and impairs actomyosin complex stabilization, which together compromise mechanosensitive pathways-YAP/TAZ and β-catenin-and affect differentiation potential of muscle cells already at the myoblast stage.