ON THE BACK COVER: C2C12-myotubes. Immunofluorescence staining of TRPC1 channel (red) and phalloidin staining (green).
Credit: Klimenko E.S., Sukhareva K.S. (Almazov National Medical Research Centre, Institute of molecular biology and genetics, Saint-Petersburg, Russia)
ON THE INNER FRONT COVER: Zebrafish heart. Immunofluorescence staining of actinin-2 (red) and phalloidin staining (green).
Credit: Klimenko E.S., Makhnin I.A. (Almazov National Medical Research Centre, Institute of molecular biology and genetics, Saint-Petersburg, Russia)
Tropomyosin is an actin-binding protein that plays roles ranging from regulating muscle contraction to controlling cytokinesis and cell migration. The simple nematode Caenorhabditis elegans provides a useful model for studying the core functions of tropomyosin in an animal, having a relatively simple anatomy and a single tropomyosin gene, lev-11, that produces seven isoforms. Three higher molecular weight isoforms regulate the contraction of body wall and other muscles, but comparatively less is known of the functions of four lower molecular weight isoforms (LEV-11C, E, T, U). We demonstrate here that C. elegans can survive with a single low molecular weight isoform, LEV-11E. Mutants disrupted for LEV-11E die as young larvae, whereas mutants lacking all other short isoforms are viable, with no overt phenotype. Vertebrate low molecular weight tropomyosins are often considered “nonmuscle” isoforms, but we find LEV-11E localizes to sarcomeric thin filaments in pharyngeal muscle and co-precipitates from worm extracts with the formin FHOD-1, which is also associated with thin filaments in pharyngeal muscle. Pharyngeal sarcomere organization is grossly normal in larvae lacking LEV-11E, indicating that the tropomyosin is not required to stabilize thin filaments, but pharyngeal pumping is absent, suggesting LEV-11E regulates actomyosin activity similar to higher molecular weight sarcomeric tropomyosin isoforms.
ON THE INNER BACK COVER: Mouse embryonic fibroblasts cultured on fibronectin-coated stiff hydrogels for 1 hour.
Credit: Yongho Bae (Department of Pathology and Anatomical Sciences, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY)
ON THE FRONT COVER: C2C12-myoblasts. Immunofluorescence staining of smooth muscle actin (red) and phalloidin staining (green).
Credit: Klimenko E.S., Sukhareva K.S (Almazov National Medical Research Centre, Institute of molecular biology and genetics, Saint-Petersburg, Russia)
The organization of microtubules into a mitotic spindle is critical for animal cell proliferation and involves the cooperation of hundreds of proteins whose molecular roles and regulation are not fully understood. The protein product of the Drosophila gene abnormal spindle, Asp, is a microtubule-associated protein required for correct mitotic spindle formation. To better understand the contribution of Asp to microtubule organization during spindle formation, we reverse-engineered flies to express a version of Asp (AspLIE), predicted to have lost its ability to bind the phosphatase trimer PP2A-B56. We demonstrated that the AspLIE mutation reduced an interaction with the Drosophila PP2A-B56 regulatory subunit Widerborst (Wdb), as well as other proteins with known roles in spindle formation. AspLIE flies exhibited less robust microtubule minus-end cohesion at neural stem cell spindle poles, which was accompanied by a substantial developmental delay but no microcephaly. Predictive structural modeling suggests that the presence of Wdb alters the conformation of an Asp interaction with a tubulin dimer in a manner similar to that of the AspLIE mutation. Protein localization in the Drosophila embryo, in addition to in vitro microtubule organization experiments, suggests that a role of PP2A may be to prevent Asp from contributing to microtubule cross-linking at spindle microtubule plus ends. Together, these findings add new insights to mechanisms underlying microtubule organization within the mitotic spindle.
The primary cilium serves as an antenna of most vertebrate cells and is important for conveying cues from several signaling pathways into appropriate cellular responses during development and homeostasis. Cilia assembly and disassembly processes are thought to be strictly controlled; however, the precise nature of molecular events underlying this control still awaits full resolution. Through their enzymatic activity, kinases function as flexible yet highly controllable regulators of a vast variety of cellular processes. Their activity ranges from cell cycle control to regulation of cell motility, signal transduction, and metabolism. This review focuses on the emerging role of kinases in primary cilia biology. We underscore their functions in primary cilia formation, maintenance, and resorption while examining available models and the respective mechanisms of their actions.
Joint injuries are increasingly common and initiate a degenerative cascade in the cartilage extracellular matrix. Chondrocytes experience both intra- and extra-cellular changes during the initial phases of this process, including inflammatory activation and morphological change, initiating a catabolic feedback cycle that progresses toward osteoarthritis (OA). However, the link between this early morphological spreading and susceptibility to future inflammatory events is unclear. Thus, the objective of this study was to explore the implications of cellular spreading on early inflammatory activation. First, we treated bovine cartilage explants with control or degenerative media for 2 weeks and established early chondrocyte spreading and extracellular matrix loss around chondrocytes. Next, we either seeded chondrocytes on or encapsulated them within gelatin hydrogels of different stiffnesses to allow different degrees of spreading, followed by a short (2 h) inflammatory stimulus to measure inflammatory activation (NF-κB). We found in 2D that stiffer substrates led to greater chondrocyte spreading and NF-κB nuclear localization; however, in 3D, this trend was reversed, with the greatest spreading and activation in cells in the softest hydrogels. Finally, we investigated how hyaluronic acid hydrogel incorporation into these environments could impact this spreading-inflammtory activation relationship, showing that augmentation with HA reduced both facets. In conclusion, chondrocyte spreading, especially in 3D, is linked with reduced matrix stiffness, and this can make chondrocytes more susceptible to inflammation. Thus, future therapies should seek to address not only the inflammation in the joint but also to restore chondrocyte morphology and microenvironmental properties.
The actin cytoskeleton is central to force production in numerous cellular processes in eukaryotic cells. During clathrin-mediated endocytosis (CME), a dynamic actin meshwork is required to deform the membrane against high membrane tension or turgor pressure. Previous experimental work from our lab showed that several endocytic proteins, including actin and actin-interacting proteins, turn over several times during the formation of a vesicle during CME in yeast, and their dwell time distributions were reminiscent of gamma distributions with a peak around 1 s. However, the distribution for the filament cross-linking protein fimbrin contains a second peak around 0.5 s. To better understand the nature of these dwell time distributions, we developed a stochastic model for the dynamics of actin and its binding partners. Our model demonstrates that very fast actin filament disassembly is necessary to reproduce experimental dwell time distributions. Our model also predicts that actin-binding proteins bind rapidly to nascent filaments and filaments are fully decorated. Last, our model predicts that fimbrin detachment from actin endocytic structures is mechanosensitive to explain the extra peak observed in the dwell time distribution.
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