Browsing by Subject "Cytoplasm"

Now showing 1 - 5 of 5
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    Association with endoplasmic reticulum promotes proteasomal degradation of GADD34 protein.
    (The Journal of biological chemistry, 2011-06) Zhou, Wei; Brush, Matthew H; Choy, Meng S; Shenolikar, Shirish
    Stress-induced endogenous and ectopically expressed GADD34 proteins were present both in the cytoplasm and in membranes, with their membrane association showing similar biochemical properties. Deletion of N-terminal sequences in GADD34-GFP proteins highlighted an amphipathic helix, whose hydrophobic surface, specifically valine 25 and leucine 29, mediated endoplasmic reticulum (ER) localization. Substitution of leucines for three arginines on the polar surface indicated that the same helix also mediated the association of GADD34 with mitochondria. Fluorescence protease protection and chemical modification of cysteines substituted in the membrane-binding domain pointed to a monotopic insertion of GADD34 into the outer layer of the ER membrane. Fluorescence recovery after photobleaching showed that ER association retards the mobility of GADD34 in living cells. Both WT GADD34 and the mutant, V25R, effectively scaffolded the α-isoform of protein phosphatase-1 (PP1α) and enabled eIF2α dephosphorylation. However, the largely cytosolic V25R protein displayed a reduced rate of proteasomal degradation, and unlike WT GADD34, whose ectopic expression resulted in a dilated or distended ER, V25R did not modify ER morphology. These studies suggested that the association of with ER modulates intracellular trafficking and proteasomal degradation of GADD34, and in turn, its ability to modify ER morphology.
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    Dysferlin, annexin A1, and mitsugumin 53 are upregulated in muscular dystrophy and localize to longitudinal tubules of the T-system with stretch.
    (Journal of neuropathology and experimental neurology, 2011-04) Waddell, LB; Lemckert, FA; Zheng, XF; Tran, J; Evesson, FJ; Hawkes, JM; Lek, A; Street, NE; Lin, P; Clarke, NF; Landstrom, AP; Ackerman, MJ; Weisleder, N; Ma, J; North, KN; Cooper, ST
    Mutations in dysferlin cause an inherited muscular dystrophy because of defective membrane repair. Three interacting partners of dysferlin are also implicated in membrane resealing: caveolin-3 (in limb girdle muscular dystrophy type 1C), annexin A1, and the newly identified protein mitsugumin 53 (MG53). Mitsugumin 53 accumulates at sites of membrane damage, and MG53-knockout mice display a progressive muscular dystrophy. This study explored the expression and localization of MG53 in human skeletal muscle, how membrane repair proteins are modulated in various forms of muscular dystrophy, and whether MG53 is a primary cause of human muscle disease. Mitsugumin 53 showed variable sarcolemmal and/or cytoplasmic immunolabeling in control human muscle and elevated levels in dystrophic patients. No pathogenic MG53 mutations were identified in 50 muscular dystrophy patients, suggesting that MG53 is unlikely to be a common cause of muscular dystrophy in Australia. Western blot analysis confirmed upregulation of MG53, as well as of dysferlin, annexin A1, and caveolin-3 to different degrees, in different muscular dystrophies. Importantly, MG53, annexin A1, and dysferlin localize to the t-tubule network and show enriched labeling at longitudinal tubules of the t-system in overstretch. Our results suggest that longitudinal tubules of the t-system may represent sites of physiological membrane damage targeted by this membrane repair complex.
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    Measurement of intracellular strain on deformable substrates with texture correlation.
    (J Biomech, 2007) Gilchrist, Christopher L; Witvoet-Braam, Sietske W; Guilak, Farshid; Setton, Lori A
    Mechanical stimuli are important factors that regulate cell proliferation, survival, metabolism and motility in a variety of cell types. The relationship between mechanical deformation of the extracellular matrix and intracellular deformation of cellular sub-regions and organelles has not been fully elucidated, but may provide new insight into the mechanisms involved in transducing mechanical stimuli to biological responses. In this study, a novel fluorescence microscopy and image analysis method was applied to examine the hypothesis that mechanical strains are fully transferred from a planar, deformable substrate to cytoplasmic and intranuclear regions within attached cells. Intracellular strains were measured in cells derived from the anulus fibrosus of the intervertebral disc when attached to an elastic silicone membrane that was subjected to tensile stretch. Measurements indicated cytoplasmic strains were similar to those of the underlying substrate, with a strain transfer ratio (STR) of 0.79. In contrast, nuclear strains were much smaller than those of the substrate, with an STR of 0.17. These findings are consistent with previous studies indicating nuclear stiffness is significantly greater than cytoplasmic stiffness, as measured using other methods. This study provides a novel method for the study of cellular mechanics, including a new technique for measuring intranuclear deformations, with evidence of differential magnitudes and patterns of strain transferred from the substrate to cell cytoplasm and nucleus.
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    Porcine endothelial cells cocultured with smooth muscle cells became procoagulant in vitro.
    (Tissue Eng Part A, 2010-06) Pang, Zhengyu; Niklason, Laura E; Truskey, George A
    Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.
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    The genetics of sex ratio distortion by cytoplasmic infection under maternal and contagious transmission: an epidemiological study.
    (Theoretical population biology, 1978-12) Uyenoyama, MK; Feldman, MW
    Nuclear and cytoplasmic determinants jointly influence the sex ratio in several organisms. A mathematical model of a maternally inherited extra-chromosomal agent that affects the fitness of its carriers and distorts the sex ratio in their broods is analyzed. The agent is transmitted through the cytoplasm from mother to daughter, or it may pass contagiously among females of the same generation. It is shown that under natural selection the deviation between the population sex ratio and Fisher's optimum value evolves to a minimum. © 1978.