Detection of protein kinase activity specifically activated at metaphase-anaphase transition

We have previously reported that Ser13 and Ser34 on glial fibrillary acidic protein (GFAP) in the cleavage furrow of glioma cells are phosphorylated during late mitotic phase (Matsuoka, Y., K. Nishizawa, T. Yano, M. Shibata, S. Ando, T. Takahashi, and M. Inagaki. 1992, EMBO (Eur. Mol. Biol. Organ.) J. 11:2895-2902). This observation implies a possibility that there is a protein kinase specifically activated at metaphase-anaphase transition. To further analyze the cell cycle- dependent GFAP phosphorylation, we prepared monoclonal antibodies KT13 and KT34 which recognize the phosphorylation of GFAP at Ser13 and Ser34, respectively. Immunocytochemical studies with KT13 and KT34 revealed that the GFAP phosphorylation in the cleavage furrow during late mitotic phase occurred not only in glioma cells but also in human SW-13 and mouse Ltk- cells in which GFAP was ectopically expressed, thus the phosphorylation can be monitored in a wide range of cell types. Furthermore, we detected kinase activity which phosphorylates GFAP at Ser13 and Ser34 in the lysates of late mitotic cells but not in those of interphase cells or early mitotic cells. These results suggest that there exists a protein kinase which is specifically activated at the transition of metaphase to anaphase not only in GFAP-expressing cells but also in cells without GFAP.     

Two different protein kinases act on a different time schedule as glial filament kinases during mitosis.

Glial fibrillary acidic protein (GFAP) is a component of glial filaments specific to astroglia. We now report the spatial and temporal distributions of four phosphorylated sites in the GFAP molecule during mitosis of astroglial cells, determined by antibodies which can distinguish phosphorylated epitopes from non-phosphorylated-epitopes. Immunofluorescence microscopy showed that the Ser8 residues in the entire cytoplasmic glial filament system are initially phosphorylated when the cells enter mitosis. In cytokinesis, the phosphoSer8 residues become dephosphorylated, whereas Thr7, Ser13 and Ser34 in glial filaments at the cleavage furrow become the preferred sites of phosphorylation. The cdc2 kinase purified from mitotic cells can phosphorylate GFAP at Ser8 but not at Thr7, Ser13 or Ser34, in vitro. These results suggest that cdc2 kinase acts as a glial filament kinase only at the G2-M phase transition while other glial filament kinases are probably activated at the cleavage furrow before final separation of the daughter cells.     

Assembly regulatory domain of glial fibrillary acidic protein. A single phosphorylation diminishes its assembly-accelerating property.

Phosphorylation of glial fibrillary acidic protein (GFAP) induces disassembly of the filaments. An amino-terminal fragment of bovine GFAP (G-Hf) was produced by lysylendopeptidase digestion. G-Hf formed ribbon-like filaments in the presence of GFAP even in low ionic strength, whereas the fragment itself did not form any structures. Only one (PK3) of the five V8 protease fragments of G-Hf accelerated GFAP assembly to the same degree as G-Hf did, whereas the other fragments did not. When PK3 was cleaved into two fragments, it lost the assembly-accelerating property. The sequence of PK3 was determined as RRRVTSATRRSYVSSSE, which corresponded to residues 3-19 of porcine GFAP. It was concluded that PK3 contains a sequence indispensable for GFAP assembly and that neither PK1 (RRRVTS) nor PK2 (ATRRSYVSSSE) included all of the sequence. A single phosphorylation of PK3 by cyclic AMP-dependent protein kinase diminished its assembly-accelerating property. The phosphorylation site was determined as Ser-12 of porcine GFAP. It was shown that single phosphorylation of the amino-terminal head domain, which contains an indispensable sequence for GFAP assembly, might be sufficient for GFAP disassembly.     

Tricyclic antidepressant amitriptyline activates fibroblast growth factor receptor signaling in glial cells: involvement in glial cell line-derived neurotrophic factor production

Recently, both clinical and animal studies demonstrated neuronal and glial plasticity to be important for the therapeutic action of antidepressants. Antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production through monoamine-independent protein-tyrosine kinase, extracellular signal-regulated kinase (ERK), and cAMP responsive element-binding protein (CREB) activation in glial cells (Hisaoka, K., Takebayashi, M., Tsuchioka, M., Maeda, N., Nakata, Y., and Yamawaki, S. (2007) J. Pharmacol. Exp. Ther. 321, 148-157; Hisaoka, K., Maeda, N., Tsuchioka, M., and Takebayashi, M. (2008) Brain Res. 1196, 53-58). This study clarifies the type of tyrosine kinase and mechanism of antidepressant-induced GDNF production in C6 glioma cells and normal human astrocytes. The amitriptyline (a tricyclic antidepressant)-induced ERK activation was specifically and completely inhibited by fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors and siRNA for FGFR1 and -2. Treatment with amitriptyline or several different classes of antidepressants, but not non-antidepressants, acutely increased the phosphorylation of FGFRs and FGFR substrate 2α (FRS2α). Amitriptyline-induced CREB phosphorylation and GDNF production were blocked by FGFR-tyrosine kinase inhibitors. Therefore, antidepressants activate the FGFR/FRS2α/ERK/CREB signaling cascade, thus resulting in GDNF production. Furthermore, we attempted to elucidate how antidepressants activate FGFR signaling. The effect of amitriptyline was inhibited by heparin, non-permeant FGF-2 neutralizing antibodies, and matrix metalloproteinase (MMP) inhibitors. Serotonin (5-HT) also increased GDNF production through FGFR2 (Tsuchioka, M., Takebayashi, M., Hisaoka, K., Maeda, N., and Nakata, Y. (2008) J. Neurochem. 106, 244-257); however, the effect of 5-HT was not inhibited by heparin and MMP inhibitors. These results suggest that amitriptyline-induced FGFR activation might occur through an extracellular pathway, in contrast to that of 5-HT. The current data show that amitriptyline-induced FGFR activation might occur by the MMP-dependent shedding of FGFR ligands, such as FGF-2, thus resulting in GDNF production.     

Identification of major ERK-related phosphorylation sites in Gab1

Gab1 (Grb2-associated binder1) belongs to a family of multifunctional docking proteins that play a central role in the integration of receptor tyrosine kinase (RTK) signaling, i.e., mediating cellular growth response, transformation, and apoptosis. In addition to RTK-specific tyrosine phosphorylation, these docking proteins also can be phosphorylated on serine/threonine residues affecting signal transduction. Since serine and threonine phosphorylation are capable of modulating the initial signal one major task to elucidate signal transduction via Gab1 is to determine the exact localization of distinct phosphorylation sites. To address this question in this report we examined extracellular signal-regulated kinases 1/2 (ERK) specific serine/threonine phosphorylation of the entire Gab1 engaged in insulin signaling in more detail in vitro. To elucidate the ERK1/2-specific phosphorylation pattern of Gab1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Subsequently, phosphorylated serine/threonine residues were identified by sequencing the separated phosphopeptides using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that ERK1/2 phosphorylate Gab1 at six serine/threonine residues (T312, S381, S454, T476, S581, S597) in consensus motifs for MAP kinase phosphorylation. Serine residues S454, S581, S597, and threonine residue T476 represent nearly 80% of overall incorporated phosphate. These sites are located adjacent to src homology region-2 (SH2) binding motifs (YVPM-motif: Y447, Y472, Y619) specific for the phosphatidylinositol 3kinase (PI3K). The biological role of identified phosphorylation sites was proven by PI3K and Akt activity in intact cells. These data demonstrate that ERK1/2 modulate insulin action via Gab1 by targeting serine and threonine residues beside YXXM motifs. Accordingly, insulin signaling is blocked at the level of PI3K.     

S100B-Mediated Inhibition of the Phosphorylation of GFAP Is Prevented by TRTK-12

S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.     

Phospholipase D1 is phosphorylated and activated by protein kinase C in caveolin-enriched microdomains within the plasma membrane

Activities of phospholipase D (PLD) in diverse subcellular organelles have been identified but the details of regulatory mechanisms in such locations are unknown. Protein kinase C (PKC) is a major regulator of PLD. Serine 2, threonine 147, and serine 561 residues of phospholipase D1 (PLD1) were determined as sites of phosphorylation by PKC (Kim, Y., Han, J. M., Park, J. B., Lee, S. D., Oh, Y. S., Chung, C., Lee, T. G., Kim, J. H., Park, S. K., Yoo, J. S., Suh, P. G., Ryu, S. H. (1999) Biochemistry 38, 10344-10351). In our present study, a triple mutation of these phosphorylation sites diminished markedly phorbol 12-myristate 13-acetate (PMA)-induced PLD1 activity in COS-7 cells. We looked at the location of the PLD1 phosphorylation by PKC by observing PMA induced band shifts and by use of anti-phospho-PLD1 monoclonal antibody. The shifted PMA-induced proteins and the immunoreactivity of the anti-phospho-PLD1 antibody were mainly found in the caveolin-enriched membrane (CEM) fraction. Depletion of cellular cholesterol led to a loss of this compartmentalization of phosphorylated PLD1 in the CEM. Replacement of the cellular cholesterol led to the restoration of phosphorylated PLD1 in the CEM. Immunocytochemical studies of COS-7 cells revealed that PLD1 was localized in the plasma membrane as well as in the vesicular structures in the cytoplasm, but the phosphorylation of PLD1 occurred only in the plasma membrane. Our results, therefore, show that phosphorylation, and thereby activation, of PLD1 by PKC occurs in the caveolin and cholesterol-enriched low density domain of the plasma membrane in COS-7 cells.     

Phosphorylation sites linked to glial filament disassembly in vitro locate in a non-alpha-helical head domain

Glial fibrillary acidic protein (GFAP), the intermediate filament component of astroglial cells, can serve as an excellent substrate for both cAMP-dependent protein kinase and protein kinase C, in vitro. GFAP phosphorylated by each protein kinase does not polymerize, and the filaments that do polymerize tend to depolymerize after phosphorylation. Dephosphorylation of phospho-GFAP by phosphatase led to a recovery of the polymerization competence of GFAP. Most of the phosphorylation sites for cAMP-dependent protein kinase and protein kinase C on GFAP are the same, Ser-8, Ser-13, and Ser-34. cAMP-dependent protein kinase has one additional phosphorylation site, Thr-7. All the sites are located within the amino-terminal non-alpha-helical head domain of GFAP. These observations pave the way for in vivo studies on organization of glial filaments.     

P2Y12 receptor stimulation inhibits beta-adrenergic receptor-induced differentiation by reversing the cyclic AMP-dependent inhibition of protein kinase B

Cyclic AMP-dependent induction of differentiation by activation of the beta-adrenergic receptor is correlated with inhibition of protein kinase B activity concomitant with growth arrest and increase in glial fibrillary acidic protein (GFAP) synthesis in rat C6 glioma cells. Costimulation of the beta-adrenergic receptor with purinergic receptors activated by 2-methylthio-adenosine-5'-diphosphate (2MeSADP) increased protein kinase B (PKB) phosphorylation above the level measured in non-stimulated cells and abolished cAMP-dependent differentiation. Transfection of cells with constitutively active PKB confirmed that reactivation of PKB is involved in the 2MeSADP-dependent inhibition of GFAP synthesis. The P2Y(12) and P2Y(13) receptor antagonist AR-C69931MX [N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloro-methylene ATP] decreased PKB phosphorylation to the level in non-stimulated cells, whereas the P2Y(13) antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and P(1),P(3)-di(adenosine-5') tetraphosphate (Ap(4)A) did not alter the 2MeSADP-induced phosphorylation of PKB, showing that enhanced PKB activity and subsequent phosphorylation of glycogen synthase kinase-3 is due to stimulation of the P2Y(12) receptor. In addition, experiments in the presence of pertussis toxin and phosphatidylinositol 3-kinase (PI 3-K) activity assays demonstrated that the P2Y(12) receptor-mediated increase in PKB phosphorylation is G(i) protein- and PI 3-K-dependent. The presented data demonstrated that a cAMP-dependent inhibition of PKB induces differentiation of C6 glioma cells and that inhibition of adenylate cyclase and reactivation of the PI 3-K/PKB pathway by the P2Y(12) receptor reverses differentiation into enhanced proliferation.     

Roles of Rho-associated Kinase in Cytokinesis; Mutations in Rho-associated Kinase Phosphorylation Sites Impair Cytokinetic Segregation of Glial Filaments

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.     

Abnormal Tau phosphorylation of the Alzheimer-type also occurs during mitosis

In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into filaments and it may be related to the reactivation of mitotic mechanisms. In order to investigate the link between Tau phosphorylation and mitosis, Xenopus laevis oocytes in which most of the M-phase regulators have been discovered were used as a cell model. The human Tau isoform htau412 (2+3-10+) was microinjected into prophase I oocytes that were then stimulated by progesterone that activate cyclin-dependent kinase pathways. Hyperphosphorylation of the Tau isoform, which is characterized by a decrease of its electrophoretic mobility and its labelling by a number of phosphorylation-dependent antibodies, was observed at the time of germinal vesicle breakdown. Surprisingly, Tau immunoreactivity, considered as typical of Alzheimer's pathology (AT100 and phospho-Ser422), was observed in meiosis II. Because meiosis II is considered as a mitosis-like phase, we investigated if our observation was also relevant to a neurone-like model. Abnormal Tau phosphorylation was detected in mitotic human neuroblastoma SY5Y cells overexpressing Tau. Regarding AT100-immunoreactivity and phospho-Ser422, we suggest that phosphatase 2A inhibition and a phosphorylation combination of mitotic kinases may lead to this Alzheimer-type phosphorylation. Our results not only demonstrate the involvement of mitotic kinases in Alzheimer-type Tau phosphorylation but also indicate that Xenopus oocyte could be a useful model to identify the kinases involved in this process.     

Roles of Rho-associated kinase and myosin light chain kinase in morphological and migratory defects of focal adhesion kinase-null cells

Fibroblasts derived from focal adhesion kinase (FAK)-null mouse embryos have a reduced migration rate and an increase in the number and size of peripherally localized adhesions (Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., and Yamamoto, T. (1995) Nature 377, 539-544). In this study, we have found that Y27632, a specific inhibitor for Rho-associated kinase (Rho-kinase), dramatically reversed the round cell morphology of FAK(-/-) cells to a spread fibroblast-like shape in 30 min and significantly enhanced their motility. The effects of Y27632 on the FAK(-/-) cell morphology and motility were concomitant with reorganization of the actin cytoskeleton and redistribution of focal adhesions. Conversely, the expression of the constitutively active Rho-kinase in FAK(+/+) cells led to round cell shape and inhibition of cell motility. Furthermore, coincident with the formation of cortical actin filaments, myosin light chain (MLC), Ser-19-phosphorylated MLC, and MLC kinase mainly accumulated at the FAK(-/-) cell periphery. We found that the disruption of actin filaments by cytochalasin D prevented the peripheral accumulation of MLC kinase and that inhibition of myosin-mediated contractility by 2,3-butanedione monoxime induced FAK(-/-) cells to spread. Taken together, our results suggest that Rho-kinase may mediate the formation of cortical actomyosin filaments at the FAK(-/-) cell periphery, which further recruits MLC kinase to the cell periphery and generates a non-polar contractile force surrounding the cell, leading to cell rounding and decreased motility.     

Phosphorylation of Mcm4 at specific sites by cyclin-dependent kinase leads to loss of Mcm4,6,7 helicase activity.

Mcm proteins that play an essential role in eukaryotic DNA replication are phosphorylated in vivo, and cyclin-dependent protein kinase is at least in part responsible for the phosphorylation of Mcm4. Our group reported that the DNA helicase activity of Mcm4,6,7 complex, which may be involved in initiation of DNA replication, is inhibited following phosphorylation by Cdk2/cyclin A in vitro. Here, we further examined the interplay between mouse Mcm4,6,7 complex and cyclin-dependent kinases and determined the sites required for the phosphorylation of Mcm4. Six Ser and Thr residues, in all, were required for the phosphorylation. Inhibition of Mcm4,6,7 helicase activity by Cdk2/cyclin A was largely relieved by introducing mutations in these residues of Mcm4. Anti-phosphothreonine antibodies raised against one of these sites reacted with Mcm4 prepared from HeLa cells at mitotic phase but did not bind to those at G(1) and G(1)/S, suggesting that this site is mainly phosphorylated in the mitotic phase. Mcm4,6,7 complex purified from HeLa cells at the mitotic phase exhibited a low level of DNA helicase activity, compared with the complexes prepared from cells at other phases. These results suggest that phosphorylation of Mcm4 at specific sites leads to loss of Mcm4,6,7 DNA helicase activity.     

In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells

Phosphorylation of the regulatory light chain of myosin II (MLC) controls the contractility of actomyosin in nonmuscle and muscle cells. It has been reported that cdc2 phosphorylates MLC in vitro at Ser-1 or Ser-2 and Thr-9 which protein kinase C phosphorylates (Satterwhite, L. L., M. J. Lohka, K. L. Wilson, T. Y. Scherson, L. K. Cisek, J. L. Corden, and T. D. Pollard. 1992 J. Cell Biol. 118:595-605). We have examined in vivo phosphorylation of MLC during mitosis and after the release of mitotic arrest. Phosphate incorporation of MLC in mitotic cells is found to be 6-12 times greater than that in nonmitotic cells. Phosphopeptide maps have revealed that the MLC from mitotic cells is phosphorylated at Ser-1 and/or Ser-2 (Ser-1/2), but not at Thr-9. MLC is also phosphorylated to a much lesser extent at Ser-19 which myosin light chain kinase phosphorylates. On the other hand, MLC of nonmitotic cells is phosphorylated at Ser-19 but not at Ser-1/2. The extent of phosphate incorporation is doubled at 30 min after the release of mitotic arrest when some cells start cytokinesis. Phosphopeptide analyses have revealed that the phosphorylation at Ser-19 is increased 20 times, while the phosphorylation at Ser-1/2 is decreased by half. This high extent of MLC phosphorylation at Ser-19 is maintained for another 30 min and gradually decreased to near the level of interphase cells as cells complete spreading at 180 min. On the other hand, phosphorylation at Ser-1/2 is decreased to 18% at 60 min, and is practically undetectable at 180 min after the release of mitotic arrest. The stoichiometry of MLC phosphorylation has been determined by quantitation of phosphorylated and unphosphorylated forms of MLC separated on 2D gels. The molar ratio of phosphorylated MLC to total MLC is found to be 0.16 +/- 0.06 and 0.31 +/- 0.05 in interphase and mitotic cells, respectively. The ratio is increased to 0.49 +/- 0.05 at 30 min after the release of mitotic arrest. These results suggest that the change in the phosphorylation site from Ser-1/2 to Ser-19 plays an important role in signaling cytokinesis.     

Expression of Y53A-Actin in Dictyostelium Disrupts the Cytoskeleton and Inhibits Intracellular and Intercellular Chemotactic Signaling

We showed previously that phosphorylation of Tyr⁵³, or its mutation to Ala, inhibits actin polymerization in vitro with formation of aggregates of short filaments, and that expression of Y53A-actin in Dictyostelium blocks differentiation and development at the mound stage (Liu, X., Shu, S., Hong, M. S., Levine, R. L., and Korn, E. D. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 13694–13699; Liu, X., Shu, S., Hong, M. S., Yu, B., and Korn, E. D. (2010) J. Biol. Chem. 285, 9729–9739). We now show that expression of Y53A-actin, which does not affect cell growth, phagocytosis, or pinocytosis, inhibits the formation of head-to-tail cell streams during cAMP-induced aggregation, although individual amoebae chemotax normally. We show that expression of Y53A-actin causes a 50% reduction of cell surface cAMP receptors, and inhibits cAMP-induced increases in adenylyl cyclase A activity, phosphorylation of ERK2, and actin polymerization. Trafficking of vesicles containing adenylyl cyclase A to the rear of the cell and secretion of the ACA vesicles are also inhibited. The actin cytoskeleton of cells expressing Y53A-actin is characterized by numerous short filaments, and bundled and aggregated filaments similar to the structures formed by copolymerization of purified Y53A-actin and wild-type actin in vitro. This disorganized actin cytoskeleton may be responsible for the inhibition of intracellular and intercellular cAMP signaling in cells expressing F-Y53A-actin.     

Distribution and ontogeny of glial fibrillary acidic protein in the snail Megalobulimus abbreviatus

Glial fibrillary acidic protein (GFAP) is the major component of intermediate glial filaments in the central nervous system of many vertebrates and invertebrates. In vertebrates, this protein is mainly expressed in mature astrocytes and provides structural cell stability. The highly conserved structure and glial specificity of this protein have allowed studies of ontogeny and phylogeny using antibodies. The present study investigated the ontogenetic profile and molecular weight of GFAP in the snail, Megalobulimus abbreviatus, particularly in cerebral ganglia and subesophageal mass, by immunohistochemistry and immunoblotting. Our results confirm and extend previous studies about glial intermediate filaments in snails, showing: (i) a higher GFAP content in cerebral ganglia than in subesophageal mass; (ii) a developmental increase of GFAP immunocontent in cerebral ganglia, as described in Vertebrates; and (iii) an electrophoretic band for GFAP of approximately 55 kDa.     

Association of glycosphingolipids with intermediate filaments of mesenchymal, epithelial, glial, and muscle cells.

We reported recently that two glycosphingolipids (GSLs), globoside (Gb4) and ganglioside GM3, colocalized with vimentin intermediate filaments of human umbilical vein endothelial cells. To determine whether this association is unique to endothelial cells or to vimentin, we analyzed a variety of cell types. Double-label immunofluorescent staining of fixed, permeabilized cells, with and without colcemid treatment, was performed with antibodies against glycolipids and intermediate filaments. Globoside colocalized with vimentin in human and mouse fibroblasts, with desmin in smooth muscle cells, with keratin in keratinocytes and hepatoma cells, and with glial fibrillary acidic protein (GFAP) in glial cells. Globoside colocalization was detected only with vimentin in MDCK and HeLa cells, which contain separate vimentin and keratin networks. GM3 ganglioside also colocalized with vimentin in human fibroblasts. Association of other GSLs with intermediate filaments was not detected by immunofluorescence, but all cell GSLs were detected in cytoskeletal fractions of metabolically labelled endothelial cells. These observations indicate that globoside colocalizes with vimentin, desmin, kertain and GFAP, with a preference for vimentin in cells that contain both vimentin and keratin networks. The nature of the association is not yet known. Globoside and GM3 may be present in vesicles associated with intermediate filaments (IF), or bound directly to IF or IF associated proteins. The prevalence of this association suggests that colocalization of globoside with the intermediate filament network has functional significance. We are investigating the possibility that intermediate filaments participate in the intracellular transport and sorting of glycosphingolipids.     

Connectin filaments link thick filaments and Z lines in frog skeletal muscle as revealed by immunoelectron microscopy

In an earlier study connectin, an elastic protein of striated muscle, was found to be associated with "gap filaments" originating from the thick filaments in the myofibril, but it was not clear whether it extends to Z lines or not (Maruyama, K., H. Sawada, S. Kimura, K. Ohashi, H. Higuchi, and Y. Umazume, 1984, J. Cell Biol., 99:1391-1397). In the present immunoelectron microscopic study using polyclonal antibodies against native connectin, we have concluded that the connectin structures are directly linked to Z lines from the thick (myosin) filaments in myofibrils of skinned fibers of frog skeletal muscle. There were five distinct antibody-binding stripes in each half of the A band and two stripes in the A-I junction region. Deposits of antibodies were recognized in I bands and Z lines. We suggest that connectin filaments run alongside the thick filaments, starting from a region approximately 0.15 micron from the center of the A band.