Isolation and characterization of distinct domains of sarcolemma and T-tubules from rat skeletal muscle

1. Several cell-surface domains of sarcolemma and T-tubule from skeletal-muscle fibre were isolated and characterized. 2. A protocol of subcellular fractionation was set up that involved the sequential low- and high-speed homogenization of rat skeletal muscle followed by KCl washing, Ca2+ loading and sucrose-density-gradient centrifugation. This protocol led to the separation of cell-surface membranes from membranes enriched in sarcoplasmic reticulum and intracellular GLUT4-containing vesicles. 3. Agglutination of cell-surface membranes using wheat-germ agglutinin allowed the isolation of three distinct cell-surface membrane domains: sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) and a T-tubule fraction enriched in protein tt28 and the alpha 2-component of dihydropyridine receptor. 4. Fractions SM1 and SM2 represented distinct sarcolemmal subcompartments based on different compositions of biochemical markers: SM2 was characterized by high levels of beta 1-integrin and dystrophin, and SM1 was enriched in beta 1-integrin but lacked dystrophin. 5. The caveolae-associated molecule caveolin was very abundant in SM1, SM2 and T-tubules, suggesting the presence of caveolae or caveolin-rich domains in these cell-surface membrane domains. In contrast, clathrin heavy chain was abundant in SM1 and T-tubules, but only trace levels were detected in SM2. 6. Immunoadsorption of T-tubule vesicles with antibodies against protein tt28 and against GLUT4 revealed the presence of GLUT4 in T-tubules under basal conditions and it also allowed the identification of two distinct pools of T-tubules showing different contents of tt28 and dihydropyridine receptors. 7. Our data on distribution of clathrin and dystrophin reveal the existence of subcompartments in sarcolemma from muscle fibre, featuring selective mutually exclusive components. T-tubules contain caveolin and clathrin suggesting that they contain caveolin- and clathrin-rich domains. Furthermore, evidence for the heterogeneous distribution of membrane proteins in T-tubules is also presented.

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Expression of dystrophin on the cell surface membrane of intrafusal fibers of human skeletal muscle

PROTOPLASMA ◽

10.1007/bf01323069 ◽

1989 ◽

Vol 152 (2-3) ◽

pp. 109-111 ◽

Cited By ~ 10

Author(s):

Hikaru Tanaka ◽

Mikiharu Yoshida ◽

Tsuneo Ishiguro ◽

Chikahiko Eguchi ◽

Ikuya Nonaka ◽

...

Keyword(s):

Skeletal Muscle ◽

Cell Surface ◽

Human Skeletal Muscle ◽

Surface Membrane ◽

Cell Surface Membrane ◽

Intrafusal Fibers

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The T-tubule is a cell-surface target for insulin-regulated recycling of membrane proteins in skeletal muscle

Biochemical Journal ◽

10.1042/bj3120393 ◽

1995 ◽

Vol 312 (2) ◽

pp. 393-400 ◽

Cited By ~ 28

Author(s):

P Muñoz ◽

M Rosemblatt ◽

X Testar ◽

M Palacín ◽

G Thoidis ◽

...

Keyword(s):

Skeletal Muscle ◽

Membrane Proteins ◽

Cell Surface ◽

Insulin Receptor ◽

Insulin Receptors ◽

Acute Administration ◽

Rat Skeletal Muscle ◽

Density Fractions ◽

A Cell ◽

T Tubules

(1) In this study we have determined the distribution of various membrane proteins involved in insulin-activated glucose transport in T-tubules and in sarcolemma from rat skeletal muscle. Two independent experimental approaches were used to determine the presence of membrane proteins in T-tubules: (i) the purification of T-tubules free from sarcolemmal membranes by lectin agglutination, and (ii) T-tubule vesicle immunoadsorption. These methods confirmed that T-tubules from rat skeletal muscle were enriched with dihydropyridine receptors and tt28 protein and did not contain the sarcolemmal markers dystrophin or beta 1-integrin. Both types of experiments revealed an abundant content of GLUT4 glucose carriers, insulin receptors and SCAMPs (secretory carrier membrane proteins) in T-tubule membranes. (2) Acute administration in vivo of insulin caused an increased abundance of GLUT4 in T-tubules and sarcolemma. On the contrary, insulin led to a 50% reduction in insulin receptors present in T-tubules and in sarcolemma, demonstrating that insulin-induced insulin receptor internalization affects T-tubules in the muscle fibre. The alteration in the content of GLUT4 and insulin receptors in T-tubules was a consequence of insulin-induced redistribution of these proteins. SCAMPs also redistributed in muscle membranes in response to insulin. They were recruited by insulin from intracellular high-density fractions to intracellular lighter-density fractions and to the cell surface, showing a pattern of insulin-induced cellular redistribution distinct from those of GLUT4 and the insulin receptor. (3) In conclusion, the T-tubule is a cell-surface target for membrane proteins involved in recycling such as SCAMPs or for membrane proteins that acutely redistribute in response to insulin such as GLUT4 or insulin receptors.

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The organization of cell surface membrane domains

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155992 ◽

1989 ◽

Vol 47 ◽

pp. 804-805

Author(s):

Michael Edidin

Keyword(s):

Cell Surface ◽

Membrane Lipids ◽

Surface Membrane ◽

Lateral Diffusion ◽

Cell Types ◽

Membrane Domains ◽

Membrane Biology ◽

Morphological Polarity ◽

Discrete Domains ◽

Membrane Components

Cell surface membranes are based on a fluid lipid bilayer and models of the membranes' organization have emphasised the possibilities for lateral motion of membrane lipids and proteins within the bilayer. Two recent trends in cell and membrane biology make us consider ways in which membrane organization works against its inherent fluidity, localizing both lipids and proteins into discrete domains. There is evidence for such domains, even in cells without obvious morphological polarity and organization [Table 1]. Cells that are morphologically polarised, for example epithelial cells, raise the issue of membrane domains in an accute form.The technique of fluorescence photobleaching and recovery, FPR, was developed to measure lateral diffusion of membrane components. It has also proven to be a powerful tool for the analysis of constraints to lateral mobility. FPR resolves several sorts of membrane domains, all on the micrometer scale, in several different cell types.

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