Characterization of Subcellular Dynamics of Sterol Methyltransferases Clarifies Defective Cell Division in smt2 smt3, a C-24 Ethyl Sterol-Deficient Mutant of Arabidopsis.

An Arabidopsis sterol mutant, smt2 smt3, defective in sterolmethyltransferase2 (SMT2), exhibits severe growth abnormalities. The loss of C-24 ethyl sterols, maintaining the biosynthesis of C-24 methyl sterols and brassinosteroids, suggests specific roles of C-24 ethyl sterols. We characterized the subcellular localizations of fluorescent protein-fused sterol biosynthetic enzymes, such as SMT2-GFP, and found these enzymes in the endoplasmic reticulum during interphase and identified their movement to the division plane during cytokinesis. The mobilization of endoplasmic reticulum-localized SMT2-GFP was independent of the polarized transport of cytokinetic vesicles to the division plane. In smt2 smt3, SMT2-GFP moved to the abnormal division plane, and unclear cell plate ends were surrounded by hazy structures from SMT2-GFP fluorescent signals and unincorporated cellulose debris. Unusual cortical microtubule organization and impaired cytoskeletal function accompanied the failure to determine the cortical division site and division plane formation. These results indicated that both endoplasmic reticulum membrane remodeling and cytokinetic vesicle transport during cytokinesis were impaired, resulting in the defects of cell wall generation. The cell wall integrity was compromised in the daughter cells, preventing the correct determination of the subsequent cell division site. We discuss the possible roles of C-24 ethyl sterols in the interaction between the cytoskeletal network and the plasma membrane.

Poxvirus A51R Proteins Negatively Regulate Microtubule-Dependent Transport by Kinesin-1.

Microtubule (MT)-dependent transport is a critical means of intracellular movement of cellular cargo by kinesin and dynein motors. MT-dependent transport is tightly regulated by cellular MT-associated proteins (MAPs) that directly bind to MTs and either promote or impede motor protein function. Viruses have been widely shown to usurp MT-dependent transport to facilitate their virion movement to sites of replication and/or for exit from the cell. However, it is unclear if viruses also negatively regulate MT-dependent transport. Using single-molecule motility and cellular transport assays, we show that the vaccinia virus (VV)-encoded MAP, A51R, inhibits kinesin-1-dependent transport along MTs in vitro and in cells. This inhibition is selective as the function of kinesin-3 is largely unaffected by VV A51R. Interestingly, we show that A51R promotes the perinuclear accumulation of cellular cargo transported by kinesin-1 such as lysosomes and mitochondria during infection. Moreover, A51R also regulates the release of specialized VV virions that exit the cell using kinesin-1-dependent movement. Using a fluorescently tagged rigor mutant of kinesin-1, we show that these motors accumulate on A51R-stabilized MTs, suggesting these stabilized MTs may form a "kinesin-1 sink" to regulate MT-dependent transport in the cell. Collectively, our findings uncover a new mechanism by which viruses regulate host cytoskeletal processes.

Vesicle transport of matrix metalloproteinases.

Multicellular organisms consist of cells and extracellular matrix (ECM). ECM creates a cellular microenvironment, and cells locally degrade the ECM according to their cellular activity. A major group of enzymes that modify ECM belongs to matrix metalloproteinases (MMPs) and play major roles in various pathophysiological events. ECM degradation by MMPs does not occur in all cellular surroundings but only where it is necessary, and cells achieve this by directionally secreting these proteolytic enzymes. Recent studies have indicated that such enzyme secretion is achieved by targeted vesicle transport along the microtubules, and several kinesin superfamily proteins (KIFs) have been identified as responsible motor proteins involved in the processes. This chapter discusses recent findings of the vesicle transport of MMPs and their roles.

A model of microtubule depolymerization by kinesin-8 motor proteins.

The dimeric kinesin-8 motors have the biological function of depolymerizing microtubules (MTs) from the plus end. However, the molecular mechanism of the depolymerization promoted by the kinesin-8 motors is still undetermined. Here, a model is proposed for the MT depolymerization by the kinesin-8 motors. Based on the model, the dynamics of depolymerization in the presence of the single motor at the MT plus end under no load and under load on the motor is studied theoretically. The dynamics of depolymerization in the presence of multiple motors at the MT plus end is also analyzed. The theoretical results explain well the available experimental data. The studies can also be applicable to other families of kinesin motors such as kinesin-13 mitotic centromere-associated kinesin motors that have the ability to depolymerize MTs.

A novel variant in GAS2 is associated with autosomal dominant nonsyndromic hearing impairment in a Chinese family.

Knockout of GAS2 (growth arrest-specific protein 2), causes disorganization and destabilization of microtubule bundles in supporting cells of the cochlear duct, leading to hearing loss in vivo. However, the molecular mechanism through which GAS2 variant results in hearing loss remains unknown. By Whole-exome sequencing, we identified a novel heterozygous splicing variant in GAS2 (c.616-2 A > G) as the only candidate mutation segregating with late-onset and progressive nonsyndromic hearing loss (NSHL) in a large dominant family. This splicing mutation causes an intron retention and produces a C-terminal truncated protein (named GAS2mu). Mechanistically, the degradation of GAS2mu via the ubiquitin-proteasome pathway is enhanced, and cells expressing GAS2mu exhibit disorganized microtubule bundles. Additionally, GAS2mu further promotes apoptosis by increasing the Bcl-xS/Bcl-xL ratio instead of through the p53-dependent pathway as wild-type GAS2 does, indicating that GAS2mu acts as a toxic molecule to exacerbate apoptosis. Our findings demonstrate that this novel variant of GAS2 promotes its own protein degradation, microtubule disorganization and cellular apoptosis, leading to hearing loss in carriers. This study expands the spectrum of GAS2 variants and elucidates the underlying pathogenic mechanisms, providing a foundation for future investigations of new therapeutic strategies to prevent GAS2-associated progressive hearing loss.

Keeping it fresh: ribosomal protein SA sustains sarcomeric function via localized translation.

Mechanical stress from cardiomyocyte contraction causes misfolded sarcomeric protein replacement. Sarcomeric maintenance utilizes localized pools of mRNAs and translation machinery, yet the importance of localized translation remains unclear. In this issue of the JCI, Haddad et al. identify the Z-line as a critical site for localized translation of sarcomeric proteins, mediated by ribosomal protein SA (RPSA). RPSA localized ribosomes at Z-lines and was trafficked via microtubules. Cardiomyocyte-specific loss of RPSA in mice resulted in mislocalized protein translation and caused structural dilation from myocyte atrophy. These findings demonstrate the necessity of RPSA-dependent spatially localized translation for sarcomere maintenance and cardiac structure and function.

Editing of endogenous tubulins reveals varying effects of tubulin posttranslational modifications on axonal growth and regeneration.

Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of tubulin PTMs were often revealed indirectly through the deletion of modifying enzymes or the overexpression of tubulin mutants. In this study, we directly edited the endogenous tubulin loci to install PTM-mimicking or -disabling mutations and studied their effects on microtubule stability, neurite outgrowth, axonal regeneration, cargo transport, and sensory functions in the touch receptor neurons of Caenorhabditis elegans. We found that the status of ß-tubulin S172 phosphorylation and K252 acetylation strongly affected microtubule dynamics, neurite growth, and regeneration, whereas α-tubulin K40 acetylation had little influence. Polyglutamylation and detyrosination in the tubulin C-terminal tail had more subtle effects on microtubule stability likely by modulating the interaction with kinesin-13. Overall, our study systematically assessed and compared several tubulin PTMs for their impacts on neuronal differentiation and regeneration and established an in vivo platform to test the function of tubulin PTMs in neurons.

Kinesin-1-transported liposomes prefer to go straight in 3D microtubule intersections by a mechanism shared by other molecular motors.

Kinesin-1 ensembles maneuver vesicular cargoes through the three-dimensional (3D) intracellular microtubule (MT) network. To define how such cargoes navigate MT intersections, we first determined how many kinesins from an ensemble on a lipid-based cargo simultaneously engage a MT, and then determined the directional outcomes (straight, turn, terminate) for liposome cargoes at perpendicular MT intersections. Run lengths of 350-nm diameter liposomes decorated with up to 20, constitutively active, truncated kinesin-1 KIF5B (K543) were longer than single motor transported cargo, suggesting multiple motor engagement. However, detachment forces of lipid-coated beads with ~20 kinesins, measured using an optical trap, showed no more than three simultaneously engaged motors, with a single engaged kinesin predominating, indicating anticooperative MT binding. At two-dimensional (2D) and 3D in vitro MT intersections, liposomes frequently paused (~2 s), suggesting kinesins simultaneously bind both MTs and engage in a tug-of-war. Liposomes showed no directional outcome bias in 2D (1.1 straight:turn ratio) but preferentially went straight (1.8 straight:turn ratio) in 3D intersections. To explain these data, we developed a mathematical model of liposome transport incorporating the known mechanochemistry of kinesins, which diffuse on the liposome surface, and have stiff tails in both compression and extension that impact how motors engage the intersecting MTs. Our model predicts the ~3 engaged motor limit observed in the optical trap and the bias toward going straight in 3D intersections. The striking similarity of these results to our previous study of liposome transport by myosin Va suggests a "universal" mechanism by which cargoes navigate 3D intersections.

Revealing the dynamic whole transcriptome landscape of Clonorchis sinensis: Insights into the regulatory roles of noncoding RNAs and microtubule-related genes in development.

Clonorchis sinensis is a significant zoonotic food-borne parasite that causes a range of hepatobiliary diseases, which in severe cases can even lead to cholangiocarcinoma. To explore new diagnostic and treatment strategies, the dynamic RNA regulatory processes across different developmental stages of C. sinensis were analyzed by using whole-transcriptome sequencing. The chromosomal-level genome of C. sinensis was used for sequence alignment and annotation. In this study, we identified a total of 59,103 RNAs in the whole genome, including 2,384 miRNAs, 25,459 mRNAs, 27,564 lncRNAs and 3,696 circRNAs. Differential expression analysis identified 6,556 differentially expressed mRNAs, 2,231 lncRNAs, 877 miRNAs and 20 circRNAs at different developmental stages. Functional enrichment analysis highlighted the critical role of microtubule-related biological processes in the growth and development of C. sinensis. And coexpression analysis revealed 97 lncRNAs and 85 circRNAs that were coexpressed with 42 differentially expressed mRNAs that associated with microtubules at different developmental stages of C. sinensis. The expression of the microtubule-related genes dynein light chain 2 (DLC2) and dynein light chain 4 (DLC4) increased with C. sinensis development, and DLC2/4 could be inhibited by albendazole. Finally, by constructing competing endogenous RNA (ceRNA) networks, the lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA regulatory relationships were constructed, and the ceRNA networks of MSTRG.14258.5-novel_miR_2287-newGene_28215 and MSTRG.14258.5-novel_miR_2216-CSKR_109340 were verified. This study suggests, through whole transcriptome sequencing, that the context of microtubule regulation may play an essential role in the development and growth of C. sinensis.

Localisation and function of key axonemal microtubule inner proteins and dynein docking complex members reveal extensive diversity among vertebrate motile cilia.

Vertebrate motile cilia are classified as (9+2) or (9+0), based on the presence or absence of the central pair apparatus, respectively. Cryogenic electron microscopy analyses of (9+2) cilia have uncovered an elaborate axonemal protein composition. The extent to which these features are conserved in (9+0) cilia remains unclear. CFAP53, a key axonemal filamentous microtubule inner protein (fMIP) and a centriolar satellites component, is essential for motility of (9+0), but not (9+2) cilia. Here, we show that in (9+2) cilia, CFAP53 functions redundantly with a paralogous fMIP, MNS1. MNS1 localises to ciliary axonemes, and combined loss of both proteins in zebrafish and mice caused severe outer dynein arm loss from (9+2) cilia, significantly affecting their motility. Using immunoprecipitation, we demonstrate that, whereas MNS1 can associate with itself and CFAP53, CFAP53 is unable to self-associate. We also show that additional axonemal dynein-interacting proteins, two outer dynein arm docking (ODAD) complex members, show differential localisation between types of motile cilia. Together, our findings clarify how paralogous fMIPs, CFAP53 and MNS1, function in regulating (9+2) versus (9+0) cilia motility, and further emphasise extensive structural diversity among these organelles.

Modelling intracellular transport in crowded environments: effects of motor association to cargos.

In intracellular transports, motor proteins transport macromolecules as cargos to desired locations by moving on biopolymers such as microtubules. Recent experiments suggest that, while moving in crowded environments, cargos that can associate motor proteins during their translocation have larger run-length and association time compared to free motors. Here, we model the dynamics of a cargo that can associate at the most m free motors present on the microtubule track as obstacles to its motion. The proposed models display competing effects of association and crowding, leading to a peak in the run-length with the free-motor density. For m = 2 and 3, we show that this feature is governed by the largest eigenvalue of the transition matrix describing the cargo dynamics. In all the above cases, free motors are assumed to be present on the microtubule as stalled obstacles. We finally compare simulation results for the run-length for general scenarios where the free motors undergo processive motion in addition to binding and unbinding to or from the microtubule.

Nanoparticle-assisted tubulin assembly is environment dependent.

Nanomaterials acquire a biomolecular corona upon introduction to biological media, leading to biological transformations such as changes in protein function, unmasking of epitopes, and protein fibrilization. Ex vivo studies to investigate the effect of nanoparticles on protein-protein interactions are typically performed in buffer and are rarely measured quantitatively in live cells. Here, we measure the differential effect of silica nanoparticles on protein association in vitro vs. in mammalian cells. BtubA and BtubB are a pair of bacterial tubulin proteins identified in Prosthecobacter strains that self-assemble like eukaryotic tubulin, first into dimers and then into microtubules in vitro or in vivo. Förster resonance energy transfer labeling of each of the Btub monomers with a donor (mEGFP) and acceptor (mRuby3) fluorescent protein provides a quantitative tool to measure their binding interactions in the presence of unfunctionalized silica nanoparticles in buffer and in cells using fluorescence spectroscopy and microscopy. We show that silica nanoparticles enhance BtubAB dimerization in buffer due to protein corona formation. However, these nanoparticles have little effect on bacterial tubulin self-assembly in the complex mammalian cellular environment. Thus, the effect of nanomaterials on protein-protein interactions may not be readily translated from the test tube to the cell in the absence of particle surface functionalization that can enable targeted protein-nanoparticle interactions to withstand competitive binding in the nanoparticle corona from other biomolecules.

Centrosome age breaks spindle size symmetry even in cells thought to divide symmetrically.

Centrosomes are the main microtubule-organizing centers in animal cells. Due to the semiconservative nature of centrosome duplication, the two centrosomes differ in age. In asymmetric stem cell divisions, centrosome age can induce an asymmetry in half-spindle lengths. However, whether centrosome age affects the symmetry of the two half-spindles in tissue culture cells thought to divide symmetrically is unknown. Here, we show that in human epithelial and fibroblastic cell lines centrosome age imposes a mild spindle asymmetry that leads to asymmetric cell daughter sizes. At the mechanistic level, we show that this asymmetry depends on a cenexin-bound pool of the mitotic kinase Plk1, which favors the preferential accumulation on old centrosomes of the microtubule nucleation-organizing proteins pericentrin, γ-tubulin, and Cdk5Rap2, and microtubule regulators TPX2 and ch-TOG. Consistently, we find that old centrosomes have a higher microtubule nucleation capacity. We postulate that centrosome age breaks spindle size symmetry via microtubule nucleation even in cells thought to divide symmetrically.

CryoET reveals actin filaments within platelet microtubules.

Crosstalk between the actin and microtubule cytoskeletons is important for many cellular processes. Recent studies have shown that microtubules and F-actin can assemble to form a composite structure where F-actin occupies the microtubule lumen. Whether these cytoskeletal hybrids exist in physiological settings and how they are formed is unclear. Here, we show that the short-crossover Class I actin filament previously identified inside microtubules in human HAP1 cells is cofilin-bound F-actin. Lumenal F-actin can be reconstituted in vitro, but cofilin is not essential. Moreover, actin filaments with both cofilin-bound and canonical morphologies reside within human platelet microtubules under physiological conditions. We propose that stress placed upon the microtubule network during motor-driven microtubule looping and sliding may facilitate the incorporation of actin into microtubules.

Microtubules and actin filaments direct nuclear movement during the polarisation of Marchantia spore cells.

The multicellular haploid stage of land plants develops from a single haploid cell produced by meiosis - the spore. Starting from a non-polar state, these spores develop polarity, divide asymmetrically and establish the first axis of symmetry. Here, we show that the nucleus migrates from the cell centroid to the basal pole during polarisation of the Marchantia polymorpha spore cell. A microtubule organising centre on the leading edge of the nucleus initiates a microtubule array between the nuclear surface and the cortex at the basal pole. Simultaneously, cortical microtubules disappear from the apical hemisphere but persist in the basal hemisphere. This is accompanied by the formation a dense network of fine actin filaments between the nucleus and the basal pole cortex. Experimental depolymerisation of either microtubules or actin filaments disrupts cellular asymmetry. These data demonstrate that the cytoskeleton reorganises during spore polarisation and controls the directed migration of the nucleus to the basal pole. The presence of the nucleus at the basal pole provides the cellular asymmetry for the asymmetric cell division that establishes the apical-basal axis of the plant.

Immunodetection of tubulin and centromeric histone H3 (CENH3) proteins in Glycine species.

BACKGROUND: The centromeres appear as primary constrictions on monocentric metaphase chromosomes; where sister chromatids are held together and assemble the proteinaceous kitechore complex at which microtubule proteins attach during nuclear divisions for pulling sister chromatids to opposite cell poles. The movement of chromosomes is usually governed by structural proteins that are either species-specific or highly conserved, such as the centromere-specific histone H3 (CENH3) and tubulin proteins, respectively. METHODS AND RESULTS: We aimed to detect these proteins across eight different Glycine species by an immunofluorescence assay using specific antibodies. Furthermore, with the α-tubulin antibody we traced the dynamics of microtubules during the mitotic cell cycle in Glycine max. With two-color immunofluorescence staining, we showed that both proteins interact during nuclear division. CONCLUSIONS: Finally, we proved that in different diploid and tetraploid Glycine species CENH3 can be detected in functional centromeres with spatial proximity of microtubule proteins.

A force-sensitive mutation reveals a non-canonical role for dynein in anaphase progression.

The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.

Tether-scanning the kinesin motor domain reveals a core mechanical action.

Natural kinesin motors are tethered to their cargoes via short C-terminal or N-terminal linkers, whose docking against the core motor domain generates directional force. It remains unclear whether linker docking is the only process contributing directional force or whether linker docking is coupled to and amplifies an underlying, more fundamental force-generating mechanical cycle of the kinesin motor domain. Here, we show that kinesin motor domains tethered via double-stranded DNAs (dsDNAs) attached to surface loops drive robust microtubule (MT) gliding. Tethering using dsDNA attached to surface loops disconnects the C-terminal neck-linker and the N-terminal cover strand so that their dock-undock cycle cannot exert force. The most effective attachment positions for the dsDNA tether are loop 2 or loop 10, which lie closest to the MT plus and minus ends, respectively. In three cases, we observed minus-end-directed motility. Our findings demonstrate an underlying, potentially ancient, force-generating core mechanical action of the kinesin motor domain, which drives, and is amplified by, linker docking.

Discovery of Novel Diaryl-Substituted Fused Heterocycles Targeting Katanin and Tubulin with Potent Antitumor and Antimultidrug Resistance Efficacy.

Disrupting microtubule dynamics has emerged as a promising strategy for cancer treatment. However, drug resistance remains a challenge hindering the development of microtubule-targeting agents. In this work, a novel class of diaryl substituted fused heterocycles were designed, synthesized, and evaluated, which were demonstrated as effective dual katanin and tubulin regulators with antitumor activity. Following three rounds of stepwise optimization, compound 21b, featuring a 3H-imidazo[4,5-b]pyridine core, displayed excellent targeting capabilities on katanin and tubulin, along with notable antiproliferative and antimetastatic effects. Mechanistic studies revealed that 21b disrupts the microtubule network in tumor cells, leading to G2/M cell cycle arrest and apoptosis induction. Importantly, 21b exhibited significant inhibition of tumor growth in MDA-MB-231 and A549/T xenograft tumor models without evident toxicity and side effects. In conclusion, compound 21b presents a novel mechanism for disrupting microtubule dynamics, warranting further investigation as a dual-targeted antitumor agent with potential antimultidrug resistance properties.