Randall Division of Cell and Molecular Biophysics

About: Randall Division of Cell and Molecular Biophysics is a based out in . It is known for research contribution in the topics: Actin cytoskeleton & Skeletal muscle. The organization has 576 authors who have published 1229 publications receiving 78279 citations.

Large Intragenic Deletion in DSTYK Underlies Autosomal-Recessive Complicated Spastic Paraparesis, SPG23

Abstract: SPG23 is an autosomal-recessive neurodegenerative subtype of lower limb spastic paraparesis with additional diffuse skin and hair dyspigmentation at birth followed by further patchy pigment loss during childhood. Previously, genome-wide linkage in an Arab-Israeli pedigree mapped the gene to an approximately 25 cM locus on chromosome 1q24–q32. By using whole-exome sequencing in a further Palestinian-Jordanian SPG23 pedigree, we identified a complex homozygous 4-kb deletion/20-bp insertion in DSTYK (dual serine-threonine and tyrosine protein kinase) in all four affected family members. DSTYK is located within the established linkage region and we also found the same mutation in the previously reported pedigree and another Israeli pedigree (total of ten affected individuals from three different families). The mutation removes the last two exons and part of the 3′ UTR of DSTYK . Skin biopsies revealed reduced DSTYK protein levels along with focal loss of melanocytes. Ultrastructurally, swollen mitochondria and cytoplasmic vacuoles were also noted in remaining melanocytes and some keratinocytes and fibroblasts. Cultured keratinocytes and fibroblasts from an affected individual, as well as knockdown of Dstyk in mouse melanocytes, keratinocytes, and fibroblasts, were associated with increased cell death after ultraviolet irradiation. Keratinocytes from an affected individual showed loss of kinase activity upon stimulation with fibroblast growth factor. Previously, dominant mutations in DSTYK were implicated in congenital urological developmental disorders, but our study identifies different phenotypic consequences for a recurrent autosomal-recessive deletion mutation in revealing the genetic basis of SPG23.

N-terminus-mediated dimerization of ROCK-I is required for RhoE binding and actin reorganization

Abstract: ROCK-I (Rho-associated kinase 1) is a serine/threonine kinase that can be activated by RhoA and inhibited by RhoE. ROCK-I has an N-terminal kinase domain, a central coiled-coil region and a RhoA-binding domain near the C-terminus. We have previously shown that RhoE binds to the N-terminal 420 amino acids of ROCK-I, which includes the kinase domain as well as N-terminal and C-terminal extensions. In the present study, we show that N-terminus-mediated dimerization of ROCK-I is required for RhoE binding. The central coiled-coil domain can also dimerize ROCK-I in cells, but this is insufficient in the absence of the N-terminus to allow RhoE binding. The kinase activity of ROCK-I(1-420) is required for dimerization and RhoE binding; however, inclusion of part of the coiled-coil domain compensates for lack of kinase activity, allowing RhoE to bind. N-terminus-mediated dimerization is also required for ROCK-I to induce the formation of stellate actin stress fibres in cells. These results indicate that dimerization via the N-terminus is critical for ROCK-I function in cells and for its regulation by RhoE.

Targeted disruption of the mouse Csrp2 gene encoding the cysteine-and glycine-rich LIM domain protein CRP2 result in subtle alteration of cardiac ultrastructure

Abstract: Background The cysteine and glycine rich protein 2 (CRP2) encoded by the Csrp2 gene is a LIM domain protein expressed in the vascular system, particularly in smooth muscle cells. It exhibits a bimodal subcellular distribution, accumulating at actin-based filaments in the cytosol and in the nucleus. In order to analyze the function of CRP2 in vivo, we disrupted the Csrp2 gene in mice and analysed the resulting phenotype.

Cellular, biochemical and molecular changes in muscles from patients with X-linked myotubular myopathy due to MTM1 mutations.

Abstract: Centronuclear myopathies are early-onset muscle diseases caused by mutations in several genes including MTM1, DNM2, BIN1, RYR1 and TTN. The most severe and often fatal X-linked form of myotubular myopathy (XLMTM) is caused by mutations in the gene encoding the ubiquitous lipid phosphatase myotubularin, an enzyme specifically dephosphorylating phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate. Because XLMTM patients have a predominantly muscle-specific phenotype a number of pathogenic mechanisms have been proposed, including a direct effect of the accumulated lipid on the skeletal muscle calcium channel ryanodine receptor 1, a negative effect on the structure of intracellular organelles and defective autophagy. Animal models knocked out for MTM1 show severe reduction of ryanodine receptor 1 mediated calcium release but, since knocking out genes in animal models does not necessarily replicate the human phenotype, we considered it important to study directly the effect of MTM1 mutations on patient muscle cells. The results of the present study show that at the level of myotubes MTM1 mutations do not dramatically affect calcium homeostasis and calcium release mediated through the ryanodine receptor 1, though they do affect myotube size and nuclear content. On the other hand, mature muscles such as those obtained from patient muscle biopsies exhibit a significant decrease in expression of the ryanodine receptor 1, a decrease in muscle-specific microRNAs and a considerable up-regulation of histone deacetylase-4. We hypothesize that the latter events consequent to the primary genetic mutation, are the cause of the severe decrease in muscle strength that characterizes these patients.

Dual color single particle tracking via nanobodies

Abstract: Single particle tracking is a powerful tool to investigate the function of biological molecules by following their motion in space. However, the simultaneous tracking of two different species of molecules is still difficult to realize without compromising the length or density of trajectories, the localization accuracy or the simplicity of the assay. Here, we demonstrate a simple dual color single particle tracking assay using small, bright, high-affinity labeling via nanobodies of accessible targets with widely available instrumentation. We furthermore apply a ratiometric step-size analysis method to visualize differences in apparent membrane viscosity.