Cytoskeleton

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Actin cytoskeleton plays an important role in various aspects of atherosclerosis, a key driver of ischemic heart disease. Actin-binding protein Profilin1 (Pfn1) is overexpressed in atherosclerotic plaques in human disease, and Pfn1, when partially depleted globally in all cell types, confers atheroprotection in vivo. This study investigates the impact of endothelial cell (EC)-specific partial loss of Pfn1 expression in atherosclerosis development. We utilized mice engineered for conditional heterozygous knockout of the Pfn1 gene in ECs, with atherosclerosis induced by depletion of hepatic LDL receptor by gene delivery of PCSK9 combined with high-cholesterol diet...

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit from functional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin...

Post-translational modifications (PTMs) play a crucial role in regulating the function of many sarcomeric proteins, including myosin. Myosins comprise a family of motor proteins that play fundamental roles in cell motility in general and muscle contraction in particular. A myosin molecule consists of two myosin heavy chains (MyHCs) and two pairs of myosin light chains (MLCs); two MLCs are associated with the neck region of each MyHC's N-terminal head domain, while the two MyHC C-terminal tails form a coiled-coil that polymerizes with other MyHCs to form the thick filament backbone...

The axoneme is an intricate nanomachine responsible for generating the propulsive oscillations of cilia and flagella in an astonishing variety of organisms. New imaging techniques based on cryoelectron-tomography (cryo-ET) and subtomogram averaging have revealed the detailed structures of the axoneme and its components with sub-nm resolution, but the mechanical function of each component and how the assembly generates oscillations remains stubbornly unclear. Most explanations of oscillatory behavior rely on the dynamic regulation of dynein by some signal, but this may not be necessary if the system of dynein-driven slender filaments is dynamically unstable...

The cell's ability to change shape is a central feature in many cellular processes, including cytokinesis, motility, migration, and tissue formation. The cell constructs a network of contractile proteins underneath the cell membrane to form the cortex, and the reorganization of these components directly contributes to cellular shape changes. The desire to mimic these cell shape changes to aid in the creation of a synthetic cell has been increasing. Therefore, membrane-based reconstitution experiments have flourished, furthering our understanding of the minimal components the cell uses throughout these processes...

The mouse ocular lens is an excellent vertebrate model system for studying hexagonal cell packing and shape changes during tissue morphogenesis and differentiation. The lens is composed of two types of cells, epithelial and fiber cells. During the initiation of fiber cell differentiation, lens epithelial cells transform from randomly packed cells to hexagonally shaped and packed cells to form meridional row cells. The meridional row cells further differentiate and elongate into newly formed fiber cells that maintain hexagonal cell shape and ordered packing...

We investigated the impact of the phosphomimetic (Ser15 → Asp15) myosin regulatory light chain (S15D-RLC) on the Super-Relaxed (SRX) state of myosin using previously characterized transgenic (Tg) S15D-D166V rescue mice, comparing them to the Hypertrophic Cardiomyopathy (HCM) Tg-D166V model and wild-type (WT) RLC mice. In the Tg-D166V model, we observed a disruption of the SRX state, resulting in a transition from SRX to DRX (Disordered Relaxed) state, which explains the hypercontractility of D166V-mutated myosin motors...

Tropomyosin (TPM) is an essential sarcomeric component, stabilizing the thin filament and facilitating actin's interaction with myosin. In mammals, including humans, there are four TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing and using different promoters. In this study, we have examined the expression of transcripts as well as proteins of various sarcomeric TPM isoforms during human inducible pluripotent stem cell differentiation into cardiomyocytes...

Familial hypertrophic cardiomyopathy (HCM) affects .2% of the world's population and is inherited in an autosomal dominant manner. Mutations in cardiac α-actin are the cause in 1%-5% of all observed cases. Here, we describe the recombinant production, purification, and characterization of the HCM-linked cardiac α-actin variants p.A21V and p.D26N. Mass spectrometric analysis of the initially purified recombinant cardiac α-actin variants and wild-type protein revealed improper N-terminal processing in the Spodoptera frugiperda (Sf-9) insect cell system, compromising the labeling of the protein with fluorescent probes for biochemical studies...

Most cells tightly control the length of their cilia. The regulation likely involves intraflagellar transport (IFT), a bidirectional motility of multi-subunit particles organized into trains that deliver building blocks into the organelle. In Chlamydomonas, the anterograde IFT motor kinesin-2 consists of the motor subunits FLA8 and FLA10 and the nonmotor subunit KAP. KAP dissociates from IFT at the ciliary tip and diffuses back to the cell body. This observation led to the diffusion-as-a-ruler model of ciliary length control, which postulates that KAP is progressively sequestered into elongating cilia because its return to the cell body will require increasingly more time, limiting motor availability at the ciliary base, train assembly, building block supply, and ciliary growth...

Mechanical forces play a crucial role in diverse physiological processes, such as cell migration, cytokinesis, and morphogenesis. The actin cytoskeleton generates a large fraction of the mechanical forces via molecular interactions between actin filaments (F-actins) and myosin motors. Recent studies have shown that the common tendency of actomyosin networks to contract into a smaller structure deeply involves F-actin buckling induced by motor activities, fragmentation of F-actins, and the force-dependent unbinding of cross-linkers that inter-connect F-actins...

The accurate segregation of chromosomes during female meiosis relies on the precise assembly and function of the meiotic spindle, a dynamic structure primarily composed of microtubules. Despite the crucial role of microtubule dynamics in this process, the relationship between microtubule length and spindle size remains elusive. Leveraging Caenorhabditis elegans as a model system, we combined electron tomography and live imaging to investigate this correlation. Our analysis revealed significant changes in spindle length throughout meiosis, coupled with alterations in microtubule length...

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...

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...

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...

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...

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...

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...

No abstract text is available yet for this article.