Cytoskeleton (Hoboken, N.J.)最新文献

Dyneins are huge motor protein complexes that are essential for cell motility, cell division, and intracellular transport. Dyneins are classified into three major subfamilies, namely cytoplasmic, intraflagellar-transport (IFT), and ciliary dyneins, based on their intracellular localization and functions. Recently, several near-atomic resolution structures have been reported for cytoplasmic/IFT dyneins. In contrast, the structures of ciliary dyneins, as well as their regulatory mechanisms, have yet to be fully elucidated. Here, we isolated a heterodimeric ciliary dynein (IDA-f/I1) from Chlamydomonas reinhardtii, a ciliated green alga, and studied its structure in the presence or absence of ATP by negative-stain electron microscopy and single-particle analysis. Surprisingly, a population of IDA-f adopted a distinctive compact structure, which has been scarcely reported for ciliary dyneins but is very similar to the "phi-particle" structure widely recognized as the autoinhibited/inactivated conformation for cytoplasmic/IFT dyneins. Our results suggest that the inactivation mechanism of dimeric dyneins is conserved in all three dynein subfamilies, regardless of their cellular functions, highlighting the intriguing intrinsic regulatory mechanism that may have been acquired at an early stage in the evolution of dynein motors.

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.

The serine/threonine kinase polo-like kinase 1 (PLK1) is a master regulator of cell proliferation and contraction, but its physiological role in the lower urinary tract is unknown. We utilized transcriptomic programs of human bladder smooth muscle cells (hBSMCs), 3D bladder spheroid viability assays, and human ureterovesical junction contractility measurements to elucidate the impacts of PLK1 inhibition. This work reveals PLK1 reduction with the selective inhibitor TAK-960 (500 nM) suppresses high K+-evoked contractions of human urinary smooth muscle ex vivo while decreasing urothelial cell viability. Transcriptomic analysis of hBSMCs treated with TAK-960 shows modulation of cell cycle and contraction pathways, specifically through altered expression of Cys2/His2-type zinc finger transcription factors. In bladder spheroids, PLK1 inhibition also suppresses smooth muscle contraction protein filamin. Taken together, these findings establish PLK1 is a critical governor of urinary smooth muscle contraction and urothelial proliferation with implications for lower urinary tract disorders. Targeting PLK1 pharmacologically may therefore offer therapeutic potential to ameliorate hypercontractility and aberrant growth. Further elucidation of PLK1 signaling networks promises new insights into pathogenesis and much needed treatment advances for debilitating urinary symptoms.

The actin cytoskeleton is composed of both branched and unbranched actin filaments. In mammals, the unbranched actin filaments are primarily copolymers of actin and tropomyosin. Biochemical and imaging studies indicate that different tropomyosin isoforms are segregated to different actin filament populations in cells and tissues, providing isoform-specific functionality to the actin filament. Intrinsic to this model is the prediction that single-molecule imaging of tropomyosin isoforms would confirm homopolymer formation along the length of single actin filaments, a knowledge gap that remains unaddressed in the cellular environment. We combined chemical labeling of genetically engineered tropomyosin isoforms with electron tomography to locate individual tropomyosin molecules in fibroblasts. We find that the organization of two non-muscle tropomyosins, Tpm3.1 with Tpm4.2, can be distinguished from each other using light and electron microscopy. Visualization of single tropomyosin molecules associated with actin filaments supports the hypothesis that tropomyosins form continuous homopolymers, instead of heteropolymers, in the presence of all physiologically native actin-binding proteins. This is true for both isoforms tested. Furthermore, the data suggest that the tropomyosin molecules on one side of an actin filament may not be in register with those on the opposite side, indicating that each tropomyosin polymer may assembly independently.

The Ras-induced ERK pathway (Raf-MEK-ERK signaling cascade) regulates a variety of cellular responses including cell proliferation, survival, and migration. Activating mutations in RAS genes, particularly in the KRAS gene, constitutively activate the ERK pathway, resulting in tumorigenesis, cancer cell invasion, and metastasis. DA-Raf1 (DA-Raf) is a splicing isoform of A-Raf and contains the Ras-binding domain but lacks the kinase domain. Consequently, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative manner and can serve as a tumor suppressor that targets mutant Ras protein-induced tumorigenesis. We show here that MEK inhibitors and DA-Raf interfere with the in vitro collective cell migration and invasion of human KRAS-mutant carcinoma cell lines, the lung adenocarcinoma A549, colorectal carcinoma HCT116, and pancreatic carcinoma MIA PaCa-2 cells. DA-Raf expression was silenced in these cancer cell lines. All these cell lines had high collective migration abilities and invasion properties in Matrigel, compared with nontumor cells. Their migration and invasion abilities were impaired by suppressing the ERK pathway with the MEK inhibitors U0126 and trametinib, an approved anticancer drug. Expression of DA-Raf in MIA PaCa-2 cells reduced the ERK activity and hindered the migration and invasion abilities. Therefore, DA-Raf may function as an invasion suppressor protein in the KRAS-mutant cancer cells by blocking the Ras-ERK pathway when DA-Raf expression is induced in invasive cancer cells.

Focal adhesions serve as structural and signaling hubs, facilitating bidirectional communication at the cell-extracellular matrix interface. Paxillin and the related Hic-5 (TGFβ1i1) are adaptor/scaffold proteins that recruit numerous structural and regulatory proteins to focal adhesions, where they perform both overlapping and discrete functions. In this study, paxillin and Hic-5 were expressed in U2OS osteosarcoma cells as biotin ligase (BioID2) fusion proteins and used as bait proteins for proximity-dependent biotinylation in order to directly compare their respective interactomes. The fusion proteins localized to both focal adhesions and the centrosome, resulting in biotinylation of components of each of these structures. Biotinylated proteins were purified and analyzed by mass spectrometry. The list of proximity interactors for paxillin and Hic-5 comprised numerous shared core focal adhesion proteins that likely contribute to their similar functions in cell adhesion and migration, as well as proteins unique to paxillin and Hic-5 that have been previously localized to focal adhesions, the centrosome, or the nucleus. Western blotting confirmed biotinylation and enrichment of FAK and vinculin, known interactors of Hic-5 and paxillin, as well as several potentially unique proximity interactors of Hic-5 and paxillin, including septin 7 and ponsin, respectively. Further investigation into the functional relationship between the unique interactors and Hic-5 or paxillin may yield novel insights into their distinct roles in cell migration.