Cytoskeleton (Hoboken, N.J.)最新文献

Microtubules play essential roles in numerous cellular processes. All microtubules are built from the protein tubulin, yet individual microtubules can differ spatially and temporally due to their tubulin isotype composition and post-translational modifications (PTMs). The tubulin code hypothesis posits that these differences can regulate microtubule function. However, investigating the properties of specific tubulin PTMs in vitro has been challenging because most reconstitution assays rely on tubulin purified from brain tissue that contains highly heterogeneous and modified microtubules. In this study, we present an optimized method for the purification of milligram quantities of unmodified tubulin from large-scale cultures of HeLa S3 cells. We also describe steps for efficient chemical labeling of tubulin and the generation of controlled tubulin PTMs. These tubulins can be used in microscopy or biochemistry-based experiments to investigate how the tubulin code influences microtubule properties and functions. Overall, our method is easily adaptable, highly reproducible, and broadly accessible to labs with general equipment.

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.

Microtubule nucleation is a critical initiating step in microtubule assembly during the cell cycle. This fundamental process is primarily mediated by γ-tubulin-containing complexes, namely, the γ-tubulin small complex (γ-TuSC) and its higher-order assembly, the γ-tubulin ring complex (γ-TuRC). These complexes are recruited to specialized cellular structures called microtubule-organizing centers (MTOCs) through spatially regulated targeting factors. The centrosome represents the predominant MTOC in animal cells, with its functional counterpart in yeasts being the spindle pole body (SPB). Centrosome-derived microtubule networks form radial arrays that not only orchestrate spindle assembly but also govern intracellular organelle positioning. Intriguingly, plant cells and differentiated animal cells have developed noncentrosomal MTOCs (ncMTOCs) that exhibit cell type-specific localization patterns. These alternative nucleation sites, including the Golgi apparatus, the nuclear envelope, preexisting microtubules, and the cell cortex, play crucial roles in a wide range of cellular activities, including the regulation of cellular morphology, polarity establishment, and cell division. This review systematically examines the regulatory mechanisms underlying noncentrosomal microtubule nucleation from diverse ncMTOCs, with particular emphasis on their context-dependent functional specializations.

The Elongator complex has long been characterized for its role in tRNA modification and modulation of protein translation. Beyond these established functions, recent findings reveal an unexpected role for Elongator in directly interacting and tuning microtubule dynamics and properties, broadening its biological significance in eukaryotic cells. This places Elongator among a growing number of RNA-binding proteins, such EIF4A3, that have been shown to modulate the microtubule cytoskeleton.

Primary cilia are essential sensory organelles whose structural complexity has challenged detailed imaging analysis. Ultrastructure expansion microscopy (U-ExM) offers a promising approach by physically enlarging specimens in hydrogels, enabling nanoscale protein mapping. Here, we apply U-ExM to pancreatic islet cilia and demonstrate the conserved presence of all four axonemal dynein subtypes, including prominent localization of the intermediate chain DNAI1 in both primary cilia and centrioles. These findings suggest that U-ExM is a robust tool for ciliary studies and provide evidence that primary cilia may possess motor capabilities that could reshape our understanding of their function.

Pollen is a male gametophyte of angiosperms. Following meiosis, the microspore undergoes an asymmetric division called pollen mitosis I (PMI), which produces two cells of different sizes: a large vegetative cell and a small generative cell. Polarized nuclear migration and positioning during PMI are important for successful pollen development and cell differentiation. However, analyzing the pollen development process in real-time is challenging in many model plants with tricellular pollen, including Arabidopsis and rice. In this study, we established a method for live confocal imaging of microtubule and actin dynamics using suspension cultures with biolistic delivery of plasmid DNAs during PMI in Nicotiana benthamiana (Bentham's tobacco), containing bicellular pollen. Pharmacological studies have indicated that actin filaments are crucial for microspore nuclear positioning before PMI, cell plate expansion during cytokinesis, and chromatin dispersion in the vegetative cell nucleus after PMI. By contrast, the inhibition of microtubule assembly resulted in abnormal chromosome segregation and nuclear behavior after PMI, although nuclear positioning and asymmetric division were observed. Our in vitro live cell imaging system for PMI provides insights into the importance of cytoskeletal regulation in asymmetric division and differentiation during pollen development.

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 Kinesin superfamily of microtubule dependent motors is present in all eukaryotes. Not all of the subfamilies are represented in all kingdoms, and the ones that are do not always show conserved functions. Tight control of the cytoskeleton is essential for proper progression and completion of mitosis and cytokinesis, and key functions are carried out by kinesin motor proteins of various families. In this context, we take a closer look at plant kinesins involved in spindle formation and chromosome congression. Additionally, plant kinesins have been implicated in the deposition of cell plate material in the plane of cell division during cytokinesis in the angiosperm Arabidopsis thaliana and the moss Physcomitrium patens. In light of these recent discoveries, this mini-review aims to give an update on kinesins involved in plant cell division with brief reference to well-studied counterparts in other organisms.