Cytoskeleton (Hoboken, N.J.)最新文献

We analysed here the dynamic of the kinesin-like Pavarotti (Pav) during male gametogenesis of wild-type and Sas4 mutant flies. Pav localizes to the equatorial region and the inner central spindle of late anaphase wild-type spermatogonia and displays a strong concentration at the midbody during late telophase. At metaphase of the first meiotic division, Pav shows widespread localization on the equatorial region of the spermatocytes. This unusual distribution restricts and enhances during anaphase where antiparallel cortical microtubules overlap. Additional Pav staining is also found in the inner central spindle where the microtubules overlap between the segregating chromosomes. At late telophase, Pav accumulates to the midbody and on a weak ring that surround the cytoplasmic bridges. Pav localizes in an equatorial discontinuous ring of Sas4 spermatogonia where the non-centrosomal microtubules overlap, but the motor protein is absent in the interior central spindle where the inner microtubules are lacking. However, the anastral spindles properly support cell division, suggesting that astral microtubules are dispensable for Pav localization in the Sas4 spermatogonial cell cortex. This function is presumably replaced by the antiparallel cortical microtubules extending from the acentriolar polar regions. In contrast, the majority of the meiotic spindles in Sas4 mutant testes do not progress beyond late anaphase, and only a small fraction of the primary spermatocytes experienced an abnormal division with the assembly of aberrant telophase spindles. Pav accumulates around the chromatin clusters or enhanced at the plus ends of the antiparallel non-centrosomal cortical bundles of microtubules. However, these bundles are not arranged properly in the equatorial region of the cell and cytokinesis is abnormal or fails. Therefore, the observations in Sas4 mutant testes suggest that the spermatogonial mitoses correctly occur in the absence of astral microtubules, whereas meiotic divisions fail.

Optineurin (OPTN), a multifunctional cytosolic protein, is recognized as an autophagy adaptor. Its association with neurodegenerative diseases, like ALS, triggered extensive research. OPTN has been found in intracellular organelles, including the mitochondria, Golgi body, endosomes, microtubules, and the nucleus. The report of mitotic defects and delayed cell division in OPTN-depleted cells prompted us to explore OPTN's exact localization in the cell that could interfere with cell division assemblies. We used three distinct human cell lines, HeLa, HEK293, and SH-SY5Y, and probed them for OPTN localization using both centrosomal (Aurora A kinase, pericentrin, PCM1, Cep170, and γ-tubulin) and mitotic spindle markers (β-tubulin). Our immunofluorescence-based detection using wide-field fluorescence, confocal, and structured illumination microscopy (SIM) placed OPTN at the centrosome, which remained associated with the centriole after duplication and their migration during mitosis. OPTN was also observed at the junction of daughter cells during cytokinesis. Our finding reveals unmapped localizations of OPTN with key cytosolic assemblies that are directly involved in cell division.

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.