Cytoskeleton (Hoboken, N.J.)最新文献

Long-distance intracellular transport of ionotropic glutamate receptors (iGluRs) is essential for proper excitatory synaptic function underlying learning and memory. Many neuropsychiatric and neurodegenerative conditions have abnormal iGluR transport and trafficking, leading to an intense interest in the mechanisms and factors regulating these processes. Although iGluRs and synaptic protein transport have been studied in cultured neurons, in vitro systems lack the specific connectivity of native circuits essential for the organization and regulation of compartmentalized synaptic signaling. Here, we describe an in vivo imaging approach that leverages the optical transparency of C. elegans to measure the transport of glutamate receptors in a fully intact neural system. Our workflow includes a standardized protocol for worm mounting, high-resolution imaging, and quantification of motor-driven iGluR transport in C. elegans. We discuss critical parameters for optimal signal-to-noise ratio, analysis, and reproducibility. Through years of optimization, we have established which fluorophores and genetic tools are the most effective and reproducible for in vivo transport imaging. These results provide a refined and reproducible framework for studying motor-driven iGluR transport in an intact nervous system and highlight important technical variables that can affect in vivo transport imaging.

Cooperativity between cytoskeletal proteins is crucial for spatiotemporal coordination in biological processes, like oogenesis. In mammalian and Drosophila oogenesis, proper assembly and function of actin networks require coordination between actin assembly factors Spire and formins, as well as actin-associated proteins like myosins and Rab GTPases. Here, we investigate the interaction between Spire and Myosin V (MyoV) in Drosophila oogenesis. We combine in vitro biochemical assays with immunofluorescence and genetics to probe the interaction and its impact on polarity establishment and the actin mesh that fills the oocyte during mid-oogenesis. Expressed Spire and MyoV constructs colocalize in punctae during mid oogenesis, with considerable enrichment near the oocyte cortex. Purified constructs interact directly in vitro, and we find that Spire can weakly activate MyoV ATPase activity. Cytoplasmic flows, critical for polarity establishment, are faster and more coordinated in the absence of MyoV, although not to the extent of fast streaming. This intermediate streaming has not been observed before. Interestingly, this MyoV-dependent change in ooplasm dynamics is sensitive to Spire levels. Despite this interplay, the actin mesh and polarity establishment appear normal when binding mutants of Spire and MyoV are expressed in the Drosophila germline. These findings suggest that direct interaction between Spire and MyoV is not essential for their primary roles at this stage of development.

Kinesin-1 is a member of the kinesin superfamily that plays an essential role in intracellular cargo transport. In the absence of cargo, Kinesin-1 exhibits low motor activity due to autoinhibition. Multiple studies have demonstrated that adaptor proteins, which link cargos to Kinesin-1, can activate Kinesin-1 by releasing the autoinhibition. To elucidate the molecular mechanism of adaptor-mediated activation, in vitro reconstitution of the Kinesin-1 complex has proven to be a powerful approach. We have shown that the binding of an adaptor protein, Nesprin-4, is sufficient to activate Kinesin-1 motility in vitro. Here, we present protocols for the observation and assessment of adaptor-mediated Kinesin-1 activation. Using C. elegans and human Kinesin-1 as examples, we describe steps and critical considerations for preparing the Kinesin-1 complex. We also detail how to mix it with an adaptor protein and visualize the resulting motility using total internal reflection fluorescence (TIRF) microscopy. Finally, we describe analytical methods for assessing motor activation. This protocol will be valuable for future studies aiming to evaluate the activation of Kinesin-1 by known or unknown activator molecules.

The migration of osteoblasts (OBs) is crucial for bone formation, remodeling, and healing. This requires the coordinated activity of cytoskeletal components, including microtubules (MTs). MTs complement actin filaments by regulating focal adhesion turnover and facilitating the delivery of essential proteins and cargo. However, the roles and regulation of MTs during OB migration remain unclear. Previous studies show that Runt-related transcription factor-2 (Runx2), a master regulator of OB differentiation, promotes MT stability in pathological contexts, such as breast cancer metastasis. In this study, we investigated the effects of Runx2 deficiency on OB migration and MT dynamics using wild-type and Runx2-deficient calvarial OBs. To assess MT function more precisely, we treated cells with microtubule-targeting agents (MTAs) that differentially affect dynamic and stable MT populations. Measurements of K40 on α-tubulins were utilized to mark longer-lived and stable MTs. Our findings revealed distinct differences in the dynamics and regulation of MTs respective to Runx2 status. Runx2-deficient OBs demonstrated increased levels of acetyl-α-tub as measured by whole cell lysate. During nutrient stress, such as glucose starvation, Runx2-deficient OBs exhibit a more rapid increase in acetyl-α-tub. However, these cells are also more sensitive to losing this stable MT fraction, notably upon exposure to MTA vinblastine. Confocal microscopy of the enzymes regulating acetyl-α-tub, ATAT1 and HDAC6, reveals striking differences in subcellular localization and colocalization to α-tubulins. Interestingly, wound-healing assays suggest Runx2-deficient OBs possess enhanced migratory capacity under both basal conditions and following MT disruption. Altogether, these findings uncover a novel role for Runx2 in regulating MT dynamics and suggest that, in specific contexts, Runx2 may suppress OB migration.

Before muscle specific proteins are expressed in precursor cells of cardiac muscle, only the nonmuscle myosin II isoform of myosin II is present in the cells. It provides contractile force for the cell divisions that occur, and it is present together with actin in fibers in the cytoplasm. When expression of muscle proteins occurs, both muscle and nonmuscle isoforms of myosin II co-exist in the same cells. Nonmuscle myosin II isoforms are organized in minisarcomeric bands in premyofibrils with alternating bands of muscle-specific alpha-actinin. In the same cell, muscle myosin II is present in mature myofibrils that are unassociated with nonmuscle myosin II isoforms. There is an intermediate group of fibrils, i.e., nascent myofibrils, in which both isoforms of myosin II are present in the cardiomyocytes. Since muscle and nonmuscle myosin II form copolymers in solution, we asked whether any of the myofibril proteins that interact with muscle myosin could prevent nonmuscle and muscle myosin from copolymerizing, thus explaining the absence of nonmuscle myosin II in mature myofibrils. We examined the effects of two myosin-binding proteins involved in cardiomyopathies: C-Protein (myosin binding protein or MyBP-C) and titin, on the filament forming properties of the two different types of myosin II. In filament forming conditions, neither C-protein nor titin bind nonmuscle myosin II. Both C-protein and titin, as expected, bind muscle myosin II. C-Protein does not inhibit the copolymerization of the two different types of myosin IIs. Co-polymerization of nonmuscle and muscle myosin IIs is prevented in the presence of either full length titin isolated from cardiac muscles or a bacterially expressed titin peptide containing just one myosin-binding region. In the presence of titin, paracrystals of Light Meromyosin (LMM) change their normal 14 nm periodicities in pure LMM paracrystals to 42 nm in copolymerization of LMM and full-length titin. Our experiments suggest four novel roles of titin in myofibrillogenesis: one: prevention of copolymerization of nonmuscle myosin II and muscle myosin II in mature myofibrils, and two: formation of thick filaments with linear cross-bridges separated by 42 nm repeats in the absence of C-Protein. Two additional properties of titin in myofibrillogenesis would be titin's capture and transport of copolymers of myosin filaments to premyofibrils to form nascent myofibrils, and the start of release of nonmuscle myosin II from copolymeric myosin II filaments resulting in mature myofibrils lacking nonmuscle myosins.

Plant cells create a plasma membrane-associated network of microtubules that are nucleated by γ-tubulin ring complexes primarily through microtubule-dependent microtubule nucleation (MDMN). This dynamic array organizes into specific patterns in response to developmental and environmental cues to influence primary cell wall construction. The molecular mechanisms directing the creation of cortical microtubule array patterns are largely unknown. The hetero-octameric AUGMIN complex facilitates mitotic spindle formation by associating γ-tubulin ring complexes with existing spindle microtubules and creating parallel branched microtubules through MDMN. AUGMIN8, the key linker protein connecting the AUGMIN complex to the parent microtubule, is encoded by a paralogous family of QWRF genes in flowering plants. Members of the QWRF family are distinguished by an unstructured N-terminal half encoded in a single 5' exon. We hypothesize that the QWRF paralogs form interchangeable AUGMIN microtubule binding subunits that confer specific roles to the AUGMIN complex in mitotic and non-mitotic microtubule arrays. We identify four QWRF family members expressed in Arabidopsis hypocotyl cells and investigate the sites of QWRF interaction with cortical microtubules using transient transformation of fluorescently tagged constructs in the heterologous Nicotiana benthamiana system. We show that full-length QWRF8 and QWRF4 associate with non-mitotic, cortical microtubules as distributed puncta where QWRF8 shows evidence for two independent sites of microtubule association. Sequence comparisons and in vivo assay with homologous fragments from QWRF1, 2, 4, and 5 define a shared N-terminal conserved microtubule association domain. We additionally identify protein regions leading to the formation of microtubule-associated "QWRF bodies" potentially linked to discontinuous localization on microtubules. We identify the "QWRF" protein motif as a conserved domain associating the AUGMIN8 paralogs with AUGMIN6, part of the larger AUGMIN complex.

Microtubules are critical components of the cytoskeleton that are extensively involved in various cellular and biological processes. The execution of these functions is intricately linked to post-translational modifications of tubulin. Post-translational modifications of tubulin include acetylation, tyrosination, de-tyrosination, glutamylation, SUMOylation, and so on. These modifications are closely associated with a wide range of biological processes. Accumulating evidence indicates that aberrant microtubule modifications are implicated in various diseases, including cancer, Alzheimer's disease, neurodevelopmental disorders, cardiac atrial hypertrophy, and even infertility. Aneuploid oocytes are a common cause of infertility, spontaneous abortion, trisomy syndrome, and other congenital abnormalities. The occurrence of aneuploidy is often closely associated with defects in spindle assembly, which are influenced by a series of tubulin modifications. In this review, we aimed to summarize the factors that affect tubulin modification and explore the key mechanisms underlying aneuploidy in human oocytes, thereby providing new insights and strategies for the treatment of infertility and prevention of congenital defects in newborns.