Cytoskeleton最新文献

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.

A key characteristic of cancer cells is their ability to induce changes in their microenvironment that render it permissive to tumor growth, invasion and metastasis. Indeed, these changes are required for tumor progression. Consequently, the tumor microenvironment is emerging as a key source of new targets against cancer, with novel therapies aimed at reversing tumor-promoting changes, reinstating a tumor-hostile microenvironment and suppressing disease progression. RHO-ROCK signaling, and consequent tension within the cellular actomyosin cytoskeleton, regulates a paracrine signaling cascade that establishes a tumor-promoting microenvironment. Here, we show that consistent with our observations in breast cancer, enhanced ROCK activity and consequent production of CRELD2 is associated with the recruitment and tumor-promoting polarization of cancer-associated fibroblasts in cutaneous squamous cell carcinoma. Our observations provide support for the notion that the role of RHO-ROCK signaling in establishing a tumor-promoting microenvironment may be conserved across patients and potentially also different cancer types.

Motile cilia have a so-called “9 + 2” structure, which consists of nine doublet microtubules and a central pair apparatus. The central pair apparatus (CA) is thought to interact mechanically with radial spokes and to control the flagellar beating. Recently, the components of the CA have been identified by proteomic and genomic analyses. Still, the mechanism of how the CA contributes to ciliary motility has much to be revealed. Here, we focused on one CA component with a large molecular weight: FAP47, and its relationship with two other CA components with large molecular weight: HYDIN, and CPC1. The analyses of motility of the Chlamydomonas mutants revealed that in contrast to cpc1 or hydin, which swam more slowly than the wild type, fap47 cells displayed wild-type swimming velocity and flagellar beat frequency, yet interestingly, fap47 cells have phototaxis defects and swim straighter than the wild-type cells. Furthermore, the double mutant fap47cpc1 and fap47hydin showed significantly slower swimming than cpc1 and hydin cells, and the motility defect of fap47cpc1 was rescued to the cpc1 level with GFP-tagged FAP47, indicating that the lack of FAP47 makes the motility defect of cpc1 worse. Cryo-electron tomography demonstrated that the fap47 lacks a part of the C1–C2 bridge of CA. Taken together, these observations indicate that FAP47 maintains the structural stiffness of the CA, which is important for flagellar regulation.

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.

ON THE FRONT COVER: MCF-7 cells treated with compound 2 (published in this issue) and Colchicine and stained with α-tubulin antibody for immunofluorescence (pseudo coloured). Credit: Jianhong Yang (Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy. West China Hospital, Sichuan University, Chengdu, China)

ON THE BACK COVER: HeLa cells expressing stable EGFP-α-tubulin were treated with 10 εM Cevipabulin for 1 min and monitored for tubulin using confocal fluorescence microscopy. Credit: Jianhong Yang (Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy. West China Hospital, Sichuan University, Chengdu, China)

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.