We used sequence-specific antibodies to characterize two monocarboxylic acid transporters, MCT1 and MCT2, in astrocytes. Both proteins are expressed in primary cultures of cortical astrocytes, as indicated by immunoblotting and immunofluorescence. Both MCT1 and MCT2 are present in small, punctate structures in the cytoplasm and at the cell membrane. Cells showing very low levels of labeling for glial fibrillary acidic protein (GFAP) also label more dimly for MCT2, but not for MCT1. In vivo, double-label immunofluorescence studies coupled with confocal microscopy indicate that MCT1 and MCT2 are rare in astrocytes in the cortex. However, they are specifically labeled in astrocytes of the glial limiting membrane and in white matter tracts. Both transporters are also present in the microvasculature. Comparison of labeling for MCT1 and MCT2 with markers of the blood-brain barrier shows that the transporters are not always limited to the astrocytic endfeet in vivo. Our results suggest that the level of expression of monocarboxylic acid transporters MCT1 and MCT2 by cortical astrocytes in vivo is significantly lower than in vitro but that astrocytes in some other regions of the brain can express one or both proteins in significant amounts.

The possibility that certain integral plasma membrane (PM) proteins involved in Ca 2؉ homeostasis form junctional units with adjacent endoplasmic reticulum (ER) in neurons and glia was explored using immunoprecipitation and immunocytochemistry. Rat brain membranes were solubilized with the mild, non-ionic detergent, IGEPAL CA-630. Na ؉ /Ca 2؉ exchanger type 1 (NCX1), a key PM Ca 2؉ transporter, was immunoprecipitated from the detergent-soluble fraction. Several abundant PM proteins co-immunoprecipitated with NCX1, including the ␣2 and ␣3 isoforms of the Na ؉ pump catalytic (␣) subunit, and the ␣2 subunit of the dihydropyridine receptor. The adaptor protein, ankyrin 2 (Ank 2), and the cytoskeletal proteins, ␣-fodrin and ␤-spectrin, also selectively co-immunoprecipitated with NCX1, as did the ER proteins, Ca 2؉ pump type 2 (SERCA 2), and inositol-trisphosphate receptor type 1 (IP 3 R-1). In contrast, a number of other abundant PMs, adaptors, and cytoskeletal proteins did not co-immunoprecipitate with NCX1, including the Na ؉ pump ␣1 isoform, PM Ca 2؉ pump type 1 (PMCA1), ␤-fodrin, and Ank 3. In reciprocal experiments, immunoprecipitation with antibodies to the Na ؉ pump ␣2 and ␣3 isoforms, but not ␣1, co-immunoprecipitated NCX1; the antibodies to ␣1 did, however, co-immunoprecipitate PMCA1. Antibodies to Ank 2, ␣-fodrin, ␤-spectrin and IP 3 R-1 all co-immunoprecipitated NCX1. Immunocytochemistry revealed partial co-localization of ␤-spectrin with NCX1, Na ؉ pump ␣3, and IP 3 R-1 in neurons and of ␣-fodrin with NCX1 and SERCA2 in astrocytes. The data support the idea that in neurons and glia PM microdomains containing NCX1 and Na ؉ pumps with ␣2 or ␣3 subunits form Ca 2؉ signaling complexes with underlying ER containing SERCA2 and IP 3 R-1. These PM and ER components appear to be linked through the cytoskeletal spectrin network, to which they are probably tethered by Ank 2.

The sarcolemma of fast-twitch muscle is organized into "costameres," structures that are oriented transversely, over the Z and M lines of nearby myofibrils, and longitudinally, to form a rectilinear lattice. Here we examine the role of desmin, the major intermediate filament protein of muscle in organizing costameres. In control mouse muscle, desmin is enriched at the sarcolemmal domains that lie over nearby Z lines and that also contain ␤-spectrin. In tibialis anterior muscle from mice lacking desmin due to homologous recombination, most costameres are lost. In myofibers from desmin Ϫ/Ϫ quadriceps, by contrast, most costameric structures are stable. Alternatively, Z line domains may be lost, whereas domains oriented longitudinally or lying over M lines are retained. Experiments with pan-specific antibodies to intermediate filament proteins and to cytokeratins suggest that control and desmin Ϫ/Ϫ muscles express similar levels of cytokeratins. Cytokeratins concentrate at the sarcolemma at all three domains of costameres when the latter are retained in desmin Ϫ/Ϫ muscle and redistribute with ␤-spectrin at the sarcolemma when costameres are lost. Our results suggest that desmin associates with and selectively stabilizes the Z line domains of costameres, but that cytokeratins associate with all three domains of costameres, even in the absence of desmin. INTRODUCTIONDuchenne Muscular Dystrophy and related muscular dystrophies are caused by the mutation or loss of dystrophin and dystrophin-associated proteins (Campbell, 1995;Bonnemann et al., 1996;Straub and Campbell, 1997;Ozawa et al., 1998), respectively, but the functions of these proteins in healthy skeletal muscle are still poorly understood. Dystrophin, which is a member of the spectrin superfamily of membrane skeletal proteins (Davison and Critchley, 1988;Koenig et al., 1988;Dhermy, 1991;Ahn and Kunkel, 1993), accumulates in healthy muscle on the cytoplasmic face of the sarcolemma in linear structures that are oriented both longitudinally and transversely (Masuda et al., 1992;Minetti et al., 1992;Porter et al., 1992;Straub et al., 1992;Williams and Bloch, 1999b). The transverse structures, which lie at the sarcolemma over the Z and M lines of nearby myofibrils, are organized in a rib-like pattern and so are referred to as "costameres" (Pardo et al., 1983a). We also use this term to include the longitudinal elements, which, with the transverse domains, form a lattice-like network that underlies most of the skeletal muscle sarcolemma. All three costameric domains are enriched in dystrophin (Porter et al., 1992). We have found that, in the absence of dystrophin, the longitudinal and M line domains of costameres are more susceptible to disruption in Duchenne muscle and in muscle from the mdx mouse (Porter et al., 1992;Williams and Bloch, 1999b; see also Ehmer et al., 1997), suggesting that dystrophin functions more to stabilize these sarcolemmal domains than the domains that overlie Z lines. These studies also suggest that other structures associated with the sarco...

Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. We examined the contractile properties and morphology of fast-twitch skeletal muscle from mice lacking keratin 19. Tibialis anterior muscles of keratin-19-null mice showed a small but significant decrease in mean fiber diameter and in the specific force of tetanic contraction, as well as increased plasma creatine kinase levels. Costameres at the sarcolemma of keratin-19-null muscle, visualized with antibodies against spectrin or dystrophin, were disrupted and the sarcolemma was separated from adjacent myofibrils by a large gap in which mitochondria accumulated. The costameric dystrophin-dystroglycan complex, which co-purified with γ-actin, keratin 8 and keratin 19 from striated muscles of wild-type mice, co-purified with γ-actin but not keratin 8 in the mutant. Our results suggest that keratin 19 in fast-twitch skeletal muscle helps organize costameres and links them to the contractile apparatus, and that the absence of keratin 19 disrupts these structures, resulting in loss of contractile force, altered distribution of mitochondria and mild myopathy. This is the first demonstration of a mammalian phenotype associated with a genetic perturbation of keratin 19.

We used degenerate primers for the amino-and carboxyl-terminal ends of the rod domains of intermediate filament proteins in reverse transcriptase-PCR experiments to identify and clone cytokeratins 8 and 19 (K8 and K19) from cardiac muscle of the adult rat. Northern blots showed that K8 has a 2.2-kb transcript and K19 has a 1.9-kb transcript in both adult cardiac and skeletal muscles. Immunolocalization of the cytokeratins in adult cardiac muscle with isoform-specific antibodies for K8 and K19 showed labeling at Z-lines within the muscle fibers and at Z-line and M-line domains at costameres at the sarcolemmal membrane. Dystrophin and K19 could be co-immunoprecipitated and co-purified from extracts of cardiac muscle, suggesting a link between the cytokeratins and the dystrophin-based cytoskeleton at the sarcolemma. Furthermore, transfection experiments indicate that K8 and K19 may associate with dystrophin through a specific interaction with its actin-binding domain. Consistent with this observation, the cytokeratins are disrupted at the sarcolemmal membrane of skeletal muscle of the mdx mouse that lacks dystrophin. Together these results indicate that at least two cytokeratins are expressed in adult striated muscle, where they may contribute to the organization of both the myoplasm and sarcolemma.