?(Fig.6)6) when comparing representative images of basal and stimulated muscle mass: (and and and and and and and by the total length of membrane counted (in m), and for rows and by the number of triads counted. translocation of GLUT4 to both plasma membrane and T tubules. Quantitation of the immunogold electron microscopy demonstrates the effects of insulin and contraction are additive and that every stimulus recruits GLUT4 from both large and small depots. Immunofluorescence double labeling for GLUT4 and transferrin receptor (TfR) demonstrates the small depots can be further subdivided into TfR-positive and TfR-negative elements. Interestingly, we observe that colocalization of TfR and GLUT4 is definitely improved by insulin and decreased by contractions. These results, supported by subcellular fractionation experiments, suggest that TfR-positive depots are only recruited by contractions. We do not find evidence for stimulation-induced unmasking of resident surface membrane GLUT4 transporters or for dilation of the T tubule system (Wang, W., P.A. Hansen, B.A. Marshall, J.O. Holloszy, and M. Mueckler. 1996. Axioskop (LSM 410 microscope equipped with an Axiovert TV microscope (and images are superimposed and viewed in color, their overlap, demonstrated in orphan constructions). However, in both areas, each of the large depots of GLUT4 has a TGN counterpart (some of the related elements). Bars, 5 m. Open in a separate window Number 10 Partial association of GLUT4 and transferrin receptor-containing constructions at the surface and in the core of basal materials. Single basal materials were stained simultaneously with an antibody to the transferrin receptor (and contours are differently formed. Bars, 5 m. Open in a separate window Number 11 Association of GLUT4 and TfR is definitely improved by insulin but decreased by contractions. Solitary materials from insulin- or contraction-stimulated muscle mass were stained and observed as explained in the story for Fig. ?Fig.10.10. Notice the improved overlap Cholecalciferol of the two stainings in insulin-stimulated materials and the Cholecalciferol decreased overlap in contraction-stimulated materials compared with basal materials (refer to Fig. ?Fig.10),10), suggesting that GLUT4+/TfR? elements are recruited by insulin and GLUT4+/TfR+ elements are recruited by contractions. Bars, 5 m. Open in a separate window Number 6 Confocal images reveal translocation of GLUT4 to the plasma membrane and T tubules of stimulated fibers.Teased solitary fibers Cholecalciferol from basal (and were taken from different fibers, the two types of patterns coexist and blend into one another in each solitary fiber (data not demonstrated). The dark channels are in fact blood vessels of the highly vascularized reddish soleus muscle mass (Ranvier, 1874). They follow a tortuous program, along and across the muscle mass fibers as can be seen by phase-contrast of whole mounts (data not shown). The two GLUT4 patterns correspond to dietary fiber segments without blood vessels (Fig. ?(Fig.11 = 3) of the total GLUT4 staining is associated with the superficial 3-m cytoplasmic coating and the remaining 32% with the deeper region of the dietary fiber, confirming the general impression from cryostat sections (Ralston and Ploug, 1996= 3) of the total GLUT4 staining, compared with 77% for the smaller, more symmetrical GLUT4-containing constructions. Large Clusters of GLUT4 Are Close to the TGN Earlier studies in the EM level have observed that portion of GLUT4 is definitely associated with the Golgi complex or TGN (Bornemann et al., 1992; Rodnick et al., 1992 and and and and and and they surround a nucleus (and the platinum grains are next to Golgi cisternae and to mutivesicular body, presumably endosomes (shows an abundantly labeled endosome in the myofibrillar IKK-beta core. Bars: (and the labeling seems excluded from the internal constructions whereas in they may be labeled. Notice also.