Cytoskeleton最新文献

Temperature is a critical factor for living organisms. Many microorganisms migrate toward preferable temperatures, and this behavior is called thermotaxis. In this study, the molecular and physiological bases for thermotaxis are examined in Chlamydomonas reinhardtii. A mutant with knockout of a transient receptor potential (TRP) channel, trp2-3, showed defective thermotaxis. The swimming velocity and ciliary beat frequency of wild-type Chlamydomonas increase with temperature; however, this temperature-dependent enhancement of motility was almost absent in the trp2-3 mutant. Wild-type Chlamydomonas showed negative thermotaxis, but mutants deficient in the outer or inner dynein arm showed positive thermotaxis and a defect in temperature-dependent increase in swimming velocity, suggesting involvement of both dynein arms in thermotaxis.

A key step in regulation of Hippo pathway signaling in response to mechanical tension is recruitment of the LIM domain proteins TRIP6 and LIMD1 to adherens junctions. Mechanical tension also triggers TRIP6 and LIMD1 to bind and inhibit the Hippo pathway kinase LATS1. How TRIP6 and LIMD1 are recruited to adherens junctions in response to tension is not clear, but previous studies suggested that they could be regulated by the known mechanosensory proteins α-catenin and vinculin at adherens junctions. We found that the three LIM domains of TRIP6 and LIMD1 are necessary and sufficient for tension-dependent localization to adherens junctions. The LIM domains of TRIP6, LIMD1, and certain other LIM domain proteins have been shown to bind to actin networks under strain/tension. Consistent with this, we show that TRIP6 and LIMD1 colocalize with the ends of actin fibers at adherens junctions. Point mutations in a key conserved residue in each LIM domain that are predicted to impair binding to f-actin under strain inhibits TRIP6 and LIMD1 localization to adherens junctions and their ability to bind to and recruit LATS1 to adherens junctions. Together these results show that the ability of TRIP6 and LIMD1 to bind to strained actin underlies their ability to localize to adherens junctions and regulate LATS1 in response to mechanical tension.

The M-line of striated muscle is a complex structure that anchors myosin-containing thick filaments and also participates in signaling and proteostasis. While the physical associations among many M-line components have been defined, the mechanism of thick filament attachment is not completely understood. In Caenorhabditis elegans, myosin A is essential for viability and forms the site of M-line attachment at the center of the filament, whereas myosin B forms the filament arms. Using a mutant myosin A that forms ectopic filaments, we examined interactions between myosin A and M-line proteins in intact muscle cells. Ectopic myosin A recruits the giant kinase UNC-89/obscurin, a presumed scaffolding protein, in an interaction that requires the zinc-finger protein UNC-98, but not UNC-82/NUAK, UNC-97/PINCH, or UNC-96. In myosin A mutants, UNC-89/obscurin patterning is highly defective in embryos and adults. A chimeric myosin containing 169 residues of the myosin A C-terminal rod, coincident with the UNC-98/ZnF binding site, is sufficient for colocalization of UNC-89/obscurin and UNC-98/ZnF in M-line structures whereas a myosin chimera lacking these residues colocalizes with UNC-89/obscurin in M-lines that lack UNC-98. Thus, at least two myosin A rod regions contribute independently to M-line organization. We hypothesize that these M-line-organizing functions correspond to the essential “filament initiation function” performed by this isoform.

Metastasis-associated 1 (MTA1), a subunit of the nucleosome remodeling and histone deacetylation (NuRD) corepressor complex, was reported to be expressed in the cytoplasm of skeletal muscles. However, the exact subcellular localization and the functional implications of MTA1 in skeletal muscles have not been examined. This study aims to demonstrate the subcellular localization of MTA1 in skeletal muscles and reveal its possible roles in skeletal muscle pathogenesis. Striated muscles (skeletal and cardiac) from C57BL/6 mice of 4–5 weeks were collected to examine the expression of MTA1 by Western blotting and immunohistochemistry. Immunofluorescence and immunoelectron microscopy were performed for MTA1, α-actinin (a Z-disc marker protein), and SMN (survival of motor neuron) proteins. Gene Expression Omnibus (GEO) data sets were analyzed using the GEO2R online tool to explore the functional implications of MTA1 in skeletal muscles. MTA1 expression was detected by Western blotting and immunohistochemistry in skeletal and cardiac muscles. Subcellular localization of MTA1 was found in the Z-disc of sarcomeres, where α-actinin and SMN were expressed. Data mining of GEO profiles suggested that MTA1 dysregulation is associated with multiple skeletal muscle defects, such as Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, nemaline myopathy, and dermatomyositis. The GEO analysis also showed that MTA1 expression gradually decreased with age in mouse skeletal muscle precursor cells. The subcellular localization of MTA1 in sarcomeres of skeletal muscles implies its biological roles in sarcomere structures and its possible contribution to skeletal muscle pathology.

The microtubule cytoskeleton is a major structural element inside cells that directs self-organization using microtubule-associated proteins and motors. It has been shown that finite-sized, spindle-like microtubule organizations, called “tactoids,” can form in vitro spontaneously from mixtures of tubulin and the antiparallel crosslinker, MAP65, from the MAP65/PRC1/Ase family. Here, we probe the ability of MAP65 to form tactoids as a function of the ionic strength of the buffer to attempt to break the electrostatic interactions binding MAP65 to microtubules and inter-MAP65 binding. We observe that, with increasing monovalent salts, the organizations change from finite tactoids to unbounded length bundles, yet the MAP65 binding and crosslinking appear to stay intact. We further explore the effects of ionic strength on the dissociation constant of MAP65 using both microtubule pelleting and single-molecule binding assays. We find that salt can reduce the binding, yet salt never negates it. Instead, we believe that the salt is affecting the ability of the MAP65 to form phase-separated droplets, which cause the nucleation and growth of tactoids, as recently demonstrated.

ON THE FRONT COVER: Image of the myocardium. Microtubules (cyan), titin (magenta), and the nuclei (yellow) are fluorescently labeled and visualized by confocal microscopy.

Credit: Andreas Unger and Wolfgang A. Linke, Institute of Physiology II, University Hospital, Münster, Germany

ON THE INNER FRONT COVER: A molecular surface representation depicting Chlamydomonas non-conventional actin NAP1 (orange) with the actin depolymerization drug latrunculin-A (light blue spheres) and ATP (gray sticks).

Credit: Rikuri Morita, University of Tsukuba

ON THE BACK COVER: CLIK-1 (shown in green) is a calponin-related actin-binding protein that is predominantly expressed in the body wall muscle of the nematode Caenorhabditis elegans. Overlaps between CLIK-1 and actin filaments (shown in magenta) generate the whitish green appearance of the sarcomeres.

Credit: Shoichiro Ono, Winship Cancer Institute, Emory University School of Medicine

ON THE INNER BACK COVER: With liquid crystal polarized light microscopy, spindle microtubules exhibit striking birefringence, which is color enhanced using the ‘Red Hot’ lookup table in Image J software. This flattened spermatocyte failed to divide after meiosis I and now has both of the two spindles for meiosis II in its lone cytoplasmic space.

Credit: James LaFountain (University at Buffalo) and Rudolf Oldenbourg (Marine Biological Laboratory)

The nuclear lamina is widely recognized as the most crucial component in providing mechanical stability to the nucleus. However, it is still a significant challenge to model the mechanics of this multilayered protein network. We developed a constitutive model of the nuclear lamina network based on its microstructure, which accounts for the deformation phases at the dimer level, as well as the orientational arrangement and density of lamin filaments. Instead of relying on homology modeling in the previous studies, we conducted molecular simulations to predict the force-extension response of a highly accurate lamin dimer structure obtained through X-ray diffraction crystallography experimentation. Furthermore, we devised a semiflexible worm-like chain extension-force model of lamin dimer as a substitute, incorporating phases of initial stretching, uncoiling of the dimer coiled-coil, and transition of secondary structures. Subsequently, we developed a 2D network continuum model for the nuclear lamina by using our extension-force lamin dimer model and derived stress resultants. By comparing with experimentally measured lamina modulus, we found that the lamina network has sharp initial strain-hardening behavior. This also enabled us to carry out finite element simulations of the entire nucleus with an accurate microstructure-based nuclear lamina model. Finally, we conducted simulations of transendothelial transmigration of a nucleus and investigated the impact of varying network density and uncoiling constants on the critical force required for successful transmigration. The model allows us to incorporate the microstructure characteristics of the nuclear lamina into the nucleus model, thereby gaining insights into how laminopathies and mutations affect nuclear mechanics.