Cytoskeleton (Hoboken, N.J.)最新文献

Cilia are microtubule-based, highly specialized organelles that are indispensable for tissue and organ development. Failures in ciliogenesis underlie severe genetic disorders known as ciliopathies. Microfilaments, a major cytoskeletal component, maintain cell architecture and facilitate motility. Microfilaments are formed by the polymerization of actin. Actin not only regulates basal-body migration and docking at the plasma membrane but also interacts with motor proteins that mediate vesicular trafficking. Furthermore, remodeling of the actin lattice modulates microtubule growth and organization. In recent years, the contribution of microfilaments to ciliogenesis and ciliary homeostasis has garnered increasing interest. This review summarizes the regulatory mechanisms underlying actin cytoskeleton dynamics in ciliogenesis, ciliary elongation, and ciliary stability over recent years.

Single-molecule localization microscopy (SMLM) enables visualization of cytoskeletal architecture at nanoscale, uncovering ultrastructural details obscured in conventional imaging. In this study, we present a quantitative framework for characterizing microtubule continuity and integrity based on SMLM super-resolution imaging. We first applied this approach to evaluate the effects of various chemical fixation protocols on microtubule structural preservation. While conventional immunofluorescence imaging suggested intact microtubules after paraformaldehyde (PFA) fixation, SMLM revealed substantial fragmentation. To address this, we developed a computational algorithm that quantifies microtubule fragmentation using a defined fragmentation index (FI). Under identical 30-min fixation, quantitative analysis revealed a fragmentation hierarchy: 4% PFA > methanol > 1% glutaraldehyde (GA) ≈ 3% PFA + 0.1% GA, with the PFA-GA combination offering superior structural integrity and minimal background noise. Although prolonged PFA fixation improved preservation, it remained inferior to PFA-GA co-fixation. Notably, even a 10-min PFA-GA treatment was sufficient for effective stabilization. We further applied our framework to quantify microtubule length index (LI) in nocodazole-treated cells, revealing a drug-specific, dose-dependent microtubule disassembly. Together, we develop a quantitative pipeline based on SMLM, which establishes PFA-GA co-fixation as an optimal protocol for microtubule imaging and provides a scalable tool for super-resolution-based pharmacological screening.

Septins are conserved GTP-binding proteins that play key roles in cell division, mitochondrial dynamics and immune responses. Despite their importance to human health, pharmacological compounds to modify septins remain limited. Forchlorfenuron (FCF) was the first small molecule identified to modify septins, disrupting their organisation and promoting mitochondrial fragmentation. A more potent FCF analog (UR214-9) has recently been developed, but its effects on mitochondria were unknown. Here, we compare FCF and UR214-9 in vitro using macrophages and in vivo using zebrafish larvae. We demonstrate that both modifiers induce mitochondrial fragmentation in macrophages without altering mitochondrial mass or SEPT7 expression. Consistent with mitochondrial fragmentation, both modifiers trigger lytic cell death in a dose-dependent manner following lipopolysaccharide (LPS) priming. In vivo, both modifiers exhibit dose-dependent effects on the survival of zebrafish larvae, although UR214-9 was significantly more toxic. In agreement with in vitro results, we observed that FCF induces macrophage cell death and caspase-1 activity in zebrafish larvae. Together, our findings show that both septin modifiers impact mitochondrial integrity and macrophage survival. Understanding how septin modifiers regulate immune responses may have important implications for inflammatory disease research and could lead to the development of septin-based medicines for conditions characterised by dysregulated inflammation.

Primary ciliary dyskinesia (PCD) is a congenital disease caused by gene mutations linked to ciliary dysfunction. PCD causes different symptoms, including chronic sinusitis, infertility, situs inversus and hydrocephalus. Motile cilia on ventricular ependymal cells are a crucial factor in cerebrospinal fluid circulation, and dysfunction of these cells causes hydrocephalus. Deleted in primary ciliary dyskinesia (Dpcd) is one genetic abnormality known to cause PCD, and its knockout leads to hydrocephalus in mice. PCD occurs in Dpcd^-/- mice because of the lack of an inner dynein arm (IDA) in the motile cilia. However, how this deficiency is associated with the motility of ventricular ependymal motile cilia in Dpcd^-/- mice has not been demonstrated. Herein, we show that Dpcd induces partial defects in dyneins and aberrant motility in ventricular ependymal cilia. In Dpcd^-/- mice, the ependymal cilia demonstrated decreased amplitude, abnormal waveforms and low cerebrospinal fluid flow velocity. In addition, the amount of dynein axonemal heavy chains in some IDAs decreased in the ependymal cilia. In wild-type mice, Dpcd was localised in the cytoplasm and cilia of ependymal cells. Thus, abnormal ciliary movement in Dpcd^-/- mice is likely attributed to a defect in IDA assembly in the ependymal cilia.

Cytokinesis, the final step of cell division, necessitates precise coordination between the microtubule-based central spindle and the actomyosin contractile ring. KIF14, a member of the kinesin-3 family of motor proteins, has emerged as a crucial integrator of these cytoskeletal systems. This review consolidates recent advances in understanding KIF14's structural domains, its dual-binding capacity for microtubules and F-actin, and its mechanochemical characteristics. KIF14 collaborates with protein regulator of cytokinesis 1 (PRC1) to bundle and slide antiparallel microtubules, while phosphorylation mediated by NIMA related kinase 7 (Nek7) enables KIF14 to bind and transport Citron kinase (CIT-K) to the midbody. This process connects central spindle organization to RhoA-driven contractility. In addition, KIF14 interacts with centralspindlin components and actomyosin regulators, thereby reinforcing midzone integrity and promoting cleavage furrow ingression. Its persistent midbody localization and activity regulated by phosphorylation ensure the temporal coordination of late cytokinesis events. Collectively, these functions establish KIF14 as a dual-function integrator of spindle architecture and contractile-ring constriction, making it indispensable for successful cell division.

Post-translational modifications (PTMs) to tubulin subunits in microtubule filaments are thought to comprise a component of the tubulin code that specifies microtubule functions in cell physiology and animal development. Acetylation of Lysine-40 (K40) on α-tubulin (αTub-K40ac) and glutamylation of both α- and β-tubulin are two tubulin PTMs of interest to the field. Antibodies that recognize these PTMs have been indispensable tools to study the localization of these PTMs as well as their biological functions. Although widely used, these antibodies are procured from commercial sources and thus have drawbacks including availability, high cost, and lack of reproducibility. To mitigate these downsides, we report the protein sequences of GT335 (anti-glutamylation) and 6-11B-1 (anti-αTub-K40ac) monoclonal antibodies and describe the use of these sequences to generate recombinant monoclonal antibody (rMAb) versions of GT335 and 6-11B-1. We demonstrate through western blotting and immunofluorescence of cultured mammalian cells and Tetrahymena thermophila that rMAb-GT335 and rMAb-611B1 match the specific activity of the commercially available antibodies. Our work provides the field with a renewable source of antibodies with high specificity and affinity towards tubulin glutamylation and acetylation and opens the door to more reproducible and large-scale studies of the function and regulation these tubulin PTMs.

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.