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J C Bulinski

Publications and source records attributed to J C Bulinski.

At least 37 records · Page 2Linked to original sources
PubMed1996

Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level.

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.

Animals↗
PubMed1995

Differential expression of alternatively spliced forms of MAP4: a repertoire of structurally different microtubule-binding domains.

We previously reported that the microtubule (MT)-binding domain of microtubule-associated protein 4 (MAP4) contains three 18-amino acid imperfect repeats that are homologous to the repeats found in the MT-binding domains of the neuronal MAPs, MAP2 and tau [Chapin, S. J., & Bulinski, J. C. (1991) J. Cell Sci. 98, 27-36]. Here we report the isolation of clones encoding additional isoforms of MAP4, which differ in the number of repeated elements contained within their MT-binding domains. In addition to clones encoding three repeats, we isolated clones encoding a four-repeat isoform, whose MT-binding domain bears a striking similarity to the four-repeat isoform of tau. Other MAP4 clones that we isolated encode five repeats. The additional repeat in the five-repeat isoform of MAP4 is quite unusual in its amino acid sequence; this unusual repeat was also found by Aizawa et al. [Aizawa, H., et al. (1990) J. Biol. Chem. 265, 13849-13855] among the repeats encoded by bovine MAP4 clones possessing four repeats. In humans, MAP4 was recently shown to be encoded by a single-copy gene [West, R. R., et al. (1991) J. Biol. Chem. 266, 21886-21896]; we demonstrated that the human MAP4 gene is located on human chromosome 3p21. Expression of multiple MAP4 isoforms from this gene, which appears to result from alternative RNA splicing, was investigated by RNase protection analysis of mammalian cell lines and rat tissues. The five-repeat isoform was the only form detectable in most cell lines, and it was the most abundant isoform expressed in rat lung, liver, kidney, spleen, and testis. However, in rat brain, heart, and skeletal muscle, although the five-repeat isoform was expressed at all developmental stages examined, the tau-like four-repeat isoform was also expressed, and its expression increased during development. The three-repeat isoform was expressed in heart and, to a lesser extent, in brain, skeletal muscle, and lung. Our results demonstrate that several different MAP4 isoforms are expressed in the rat in different tissues and at various developmental stages. Furthermore, our data suggest that differential expression of MAP4 isoforms possessing distinct MT-binding domains may be involved in the changes in MT dynamics or function that are known to accompany differentiation.

Alternative Splicing↗
PubMed1995

Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics.

We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.

Amino Acid Sequence↗
PubMed1994

Cellular microtubules heterogeneous in their content of microtubule-associated protein 4 (MAP4).

Previous immunolocalization studies using many primate cultured cell lines demonstrated that a microtubule-associated protein of M(r) approximately 210,000 which is now called MAP4, is present along the length of microtubules in interphase and mitotic cells [Bulinski and Borisy (1980) J. Cell Biol. 87:802-808; DeBrabander et al. (1981) J. Cell Biol. 91:438-455]. Since MAP4 has been implicated as a microtubule stabilizer, we asked whether all classes of microtubules possess an equal complement of MAP4. We have reexamined the cellular distribution of MAP4, using both conventional double-label immunofluorescence and an antibody blocking technique [Schulze and Kirschner (1987) J. Cell Biol. 104:277-288] to highlight microtubules lacking, or depleted in, MAP4. These techniques have revealed that thin processes extending from monkey kidney cells (TC-7), and those made by human neuroblastoma cells (IMR-32) in response to retinoic acid, are often deficient in MAP4 immunoreactivity. Since both types of cellular processes contain stable microtubules, which are enriched in detyrosinated (Glu) tubulin, we tested the ability of MAP4 to bind to microtubules made from pure Glu and pure tyrosinated (Tyr) tubulin in vitro. MAP4 bound to both types of microtubules, and the similar saturation level of MAP4 binding to Glu and Tyr microtubules suggested that differential binding to these forms of tubulin does not contribute directly to a mechanism for segregation of MAP4 on microtubules in vivo. In TC-7 cells, we also observed MAP4-depletion on single microtubules, distal regions of broad cytoplasmic extensions, and midbodies of dividing cells. MAP4 depletion may reflect recent, rapid growth of microtubules to which MAP4 has not yet bound, or the presence of other MAPs that may compete with MAP4 for binding sites on the MT. We suggest that different levels of MAP4 on microtubules may directly modulate microtubule dynamics within single cells, as well as other microtubule functions such as those involving microtubule motor activity.

Animals↗
PubMed1994

Purification and characterization of ensconsin, a novel microtubule stabilizing protein.

In previous studies (Bulinski and Borisy (1979). Proc. Nat. Acad. Sci. 76, 293-297; Weatherbee et al. (1980). Biochemistry 19, 4116-4123) a microtubule-associated protein (MAP) of M(r) approximately 125,000 was identified as a prominent MAP in HeLa cells. We set out to perform a biochemical characterization of this protein, and to determine its in vitro functions and in vivo distribution. We determined that, like the assembly-promoting MAPs, tau, MAP2 and MAP4, the 125 kDa MAP was both proteolytically sensitive and thermostable. An additional property of this MAP; namely, its unusually tight association with a calcium-insensitive population of MTs in the presence of taxol, was exploited in devising an efficient purification strategy. Because of the MAP's tenacious association with a stable population of MTs, and because it appeared to contribute to the stability of this population of MTs in vitro, we have named this protein ensconsin. We examined the binding of purified ensconsin to MTs; ensconsin exhibited binding that saturated its MT binding sites at an approximate molar ratio of 1:6 (ensconsin:tubulin). Unlike other MAPs characterized to date, ensconsin's binding to MTs was insensitive to moderate salt concentrations (< or = 0.6 M). We further characterized ensconsin in immunoblotting experiments using mouse polyclonal anti-ensconsin antibodies and antibodies reactive with previously described MAPs, such as high molecular mass tau isoforms, dynamin, STOP, CLIP-170 and kinesin. These experiments demonstrated that ensconsin is distinct from other proteins of similar M(r) that may be present in association with MTs. Immunofluorescence with anti-ensconsin antibodies demonstrated that ensconsin was detectable in association with most or all of the MTs of several lines of human epithelial, fibroblastic and muscle cells; its in vivo properties and distribution, especially in response to drug or other treatments of cells, were found to be different from those of MAP4, the predominant MAP found in these cell types. We conclude that ensconsin, a MAP found in a variety of human cells, is biochemically - and perhaps functionally - distinct from other MAPs present in non-neuronal cells.

Cell Extracts↗
PubMed1992

Regulation of cytoplasmic tubulin carboxypeptidase activity during neural and muscle differentiation: characterization using a microtubule-based assay.

A cycle of posttranslational modification of alpha-tubulin has previously been described in higher eukaryotes, in which a C-terminal tyrosine residue is removed and replaced by two complementary cytoplasmic enzymes. The activity of the detyrosinating enzyme, tubulin carboxypeptidase (TCP), and its potential for regulating the level of detyrosinated (Glu) subunits in microtubules (MTs) is of great interest, since TCP catalyzes the primary modification of tubulin and since the level of Glu alpha-tubulin in MTs increases during a variety of differentiative and morphogenetic events. As a first step in examining the role of TCP in cellular morphogenesis, it was necessary to develop an assay for TCP with sufficient sensitivity and specificity to detect TCP activity during these events. Unlike previously described assays for TCP, ours makes use of the affinity TCP exhibits for MTs. NGF-induced neurite outgrowth in PC-12 cells was accompanied by a moderate (approximately 2-fold) increase in TCP activity, while myogenesis of L6 cells resulted in an almost insignificant decrease in activity. Measurements of TCP activity during differentiation were correlated with the level of extract Tyr tubulin, which increased (approximately 37%) during neurite outgrowth and was unchanged during myogenic differentiation. Our results suggest that TCP activity is regulated relative to its substrate, Tyr tubulin, and that changes in MT dynamics, rather than enzymatic activities, are the primary determinants of MT posttranslational modification state during differentiation. In addition, the assay we have devised for TCP and the characterization of TCP during differentiation may allow the future delineation of the mechanism(s) of regulation of TCP and the role this enzyme plays in modulating MT function during differentiation.

Adrenal Gland Neoplasms↗
PubMed1991

Stabilization of post-translational modification of microtubules during cellular morphogenesis.

This review discusses the possible role of alpha-tubulin detyrosination, a reversible post-translational modification that occurs at the protein's C-terminus, in cellular morphogenesis. Higher eukaryotic cells possess a cyclic post-translational mechanism by which dynamic microtubules are differentiated from their more stable counterparts; a tubulin-specific carboxypeptidase detyrosinates tubulin protomers within microtubules, while the reverse reaction, tyrosination, is performed on the soluble protomer by a second tubulin-specific enzyme, tubulin tyrosine ligase. In general, the turnover of microtubules in undifferentiated, proliferating cells is so rapid that the microtubules accumulate very little detyrosinated tubulin; that is, they are enriched in tyrosinated tubulin. However, an early event common to at least three well-studied morphogenetic events--myogenesis, neuritogenesis, and directed cell motility--is the elaboration of a polarized array of stable microtubules that become enriched in detyrosinated tubulin. The formation of this specialized array of microtubules in specific locations in cells undergoing morphogenesis suggests that it plays an important role in generating cellular asymmetries.

Carboxypeptidases↗
PubMed1991

Chicken cardiac myofibrillogenesis studied with antibodies specific for titin and the muscle and nonmuscle isoforms of actin and tropomyosin.

Myofibrillogenesis was studied in cultured chick cardiomyocytes using indirect immunofluorescence microscopy and antibodies against alpha- and gamma-actin, muscle and nonmuscle tropomyosin, muscle myosin, and titin. Initially, cardiomyocytes, devoid of myofibrils, developed variable numbers of stress fiber-like structures with uniform staining for anti-muscle and nonmuscle actin and tropomyosin, and diffuse, weak staining with anti-titin. Anti-myosin labeled bundles of filaments that exhibited variable degrees of association with the stress fiber-like structures. Myofibrillogenesis occurred with a progressive, and generally simultaneous, longitudinal reorganization of stress fiber-like structures to form primitive sarcomeric units. Titin appeared to attain its mature pattern before the other major contractile proteins. Changes in the staining patterns of actin, tropomyosin, and myosin as myofibrils matured were interpreted as due to longitudinal filament alignment occurring before ordering in the axial direction. Non-muscle actin and tropomyosin were found with sarcomeric periodicity in the initial stages of sarcomere myofibrillogenesis, although their staining patterns were not identical. The localization of the "sarcomeric" proteins alpha-actin and muscle tropomyosin in stress fiber-like structures and the incorporation of non-muscle proteins in the initial stages of sarcomere organization bring into question the meaning of "sarcomeric" proteins in regard to myofibrillogenesis.

Actins↗
PubMed1991

Non-neuronal 210 x 10(3) Mr microtubule-associated protein (MAP4) contains a domain homologous to the microtubule-binding domains of neuronal MAP2 and tau.

A polyclonal antiserum raised against a HeLa cell microtubule-associated protein of Mr 210,000 (210 kD MAP or MAP4), an abundant non-neuronal MAP, was used to isolate cDNA clones encoding MAP4 from a human fetal brain lambda gt11 cDNA expression library. The largest of these clones, pMAP4.245, contains an insert of 4.1 kb and encodes a 245 kD beta-galactosidase fusion protein. Evidence that pMAP4.245 encodes MAP4 sequences includes immunoabsorption of MAP4 antibodies with the pMAP4.245 fusion protein, as well as identity of protein sequences obtained from HeLa 210 kD MAP4 with amino acid sequences encoded by pMAP4.245. The MAP4.245 cDNA hybridizes to several large (approximately 6-9 kb) transcripts on Northern blots of HeLa cell RNA. DNA sequencing of overlapping MAP4 cDNA clones revealed a long open reading frame containing a C-terminal region with three imperfect 18-amino acid repeats; this region is homologous to a motif present in the microtubule (MT)-binding domain of two prominent neuronal MAPs, MAP2 and tau. The pMAP4.245 sequence also encoded a series of unrelated repeats, located in the MAP's projection domain, N-terminal to the MT-binding domain. MAP4.245 fusion proteins bound to MTs in vitro, while fusion proteins that contained only the projection domain repeats failed to bind specifically to MTs. Thus, the major human non-neuronal MAP resembles two neuronal MAPs in its MT-binding domain, while most of the molecule has sequences, and presumably functions, distinct from those of the neuronal MAPs.

Amino Acid Sequence↗
PubMed1990

Immunochemical probing of the N-terminal segment on actin: the polymerization reaction.

The N-terminal segment of actin contains a cluster of acidic residues which are implicated in macromolecular interactions of this protein. In this work, the interrelationship between the N-terminal segment and the polymerization of actin was studied by using affinity-purified antibodies directed against the first seven N-terminal residues on alpha-skeletal actin (S alpha N). The Fab fragments of these antibodies showed equal affinities for G- and F-actin while the bivalent IgG bound preferentially to the polymerized actin. As monitored by pyrene fluorescence measurements, the binding of Fab to G-actin did not alter the kinetics of the MgCl2-induced polymerization; IgG accelerated this reaction considerably. Consistent with these observations, the binding of Fab to F-actin did not change its morphological appearance in electron micrographs and had no effect on the stability and the rate of dissociation of actin filaments. These results are discussed in terms of their implications to the spatial relationship between the N-terminal segment and the rest of the molecule and the context of the polymerization reaction of actin in vitro and in vivo.

Actins↗
PubMed1989

Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the alpha- and gamma-isoforms of actin.

The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogenic cultures and fluorescent-antibody staining. Efforts were made to compare the distribution and temporal sequence of incorporation of titin relative to that of the alpha- and gamma-isoforms of actin. The present observations suggested the following sequence of titin assembly: (1) newly synthesized titin molecules are distributed in a diffuse pattern throughout the sarcoplasm, (2) the titin molecules gradually associate with alpha- and gamma-actin-positive stress fiber-like structures (SFLS), (3) groups of titin molecules begin to segregate on the SFLS, and (4) titin molecules align in a mature doublet configuration in the sarcomeres of nascent myofibrils. Titin assembly on the SFLS often appeared prior to the onset of either alpha- or gamma-actin periodicity on nascent myofibrils; the latter result suggested a role for titin in sarcomeric organization. Actin distribution on SFLS and its periodicity on nascent myofibrils was usually identical between the alpha- and gamma-isoforms. This suggested that gamma-actin participated in myofibrillogenesis in a manner indistinguishable from that of alpha-actin. The transition seen from continuous actin staining of SFLS to the I-band staining pattern of mature myofibrils is discussed in relation to the corresponding reorganization of actin filaments and the molecular associations that this would entail.

Actins↗
PubMed1989

Generation of a stable, posttranslationally modified microtubule array is an early event in myogenic differentiation.

Microtubules (MTs) have been implicated to function in the change of cell shape and intracellular organization that occurs during myogenesis. However, the mechanism by which MTs are involved in these morphogenetic events is unclear. As a first step in elucidating the role of MTs in myogenesis, we have examined the accumulation and subcellular distribution of posttranslationally modified forms of tubulin in differentiating rat L6 muscle cells, using antibodies specific for tyrosinated (Tyr), detyrosinated (Glu), and acetylated (Ac) tubulin. Both Glu and Ac tubulin are components of stable MTs, whereas Tyr tubulin is the predominant constituent of dynamic MTs. In proliferating L6 myoblasts, as in other types of proliferating cells, the level of Glu tubulin was very low when compared with the level of Tyr tubulin. However, when we shifted proliferating L6 cells to differentiation media, we observed a rapid accumulation of Glu tubulin in cellular MTs. By immunofluorescence, the increase in Glu tubulin was first detected in MTs of prefusion myoblasts and was specifically localized to MTs that were associated with elongating portions of the cell. MTs in the multinucleated myotubes observed at later stages of differentiation maintained the elevated level of Glu tubulin that was observed in the prefusion myoblasts. When cells at early stages of differentiation (less than 1 d after switching the culture medium) were immunostained for Glu tubulin and the muscle-specific marker, muscle myosin, we found that the increase in Glu tubulin preceded the accumulation of muscle myosin. Thus, the elaboration of Glu MTs is one of the very early events in myogenesis. Ac tubulin also increased during L6 myogenesis; however, the increase in acetylation occurred later in myogenesis, after fusion had already occurred. Because detyrosination was temporally correlated with early events of myogenesis, we examined the mechanism responsible for the accumulation of Glu tubulin in the MTs of prefusion myoblasts. We found that an increase in the stability of L6 cell MTs occurred at the onset of differentiation, suggesting that the early increase in detyrosination that we observed is a manifestation of a decrease in MT dynamics in elongating myoblasts. We conclude that the establishment of an oriented array of microtubules heightened in its stability and its level of posttranslationally modified subunits may be involved in the subcellular remodeling that occurs during myogenesis.

Animals↗
PubMed1988

Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle.

In vertebrate skeletal muscle, the proliferating myoblasts synthesize nonmuscle isoforms of actin, and the cells begin to express muscle-specific actin isoforms during their myogenic differentiation. To study the distributions of the actin isoforms in myogenic cells and fully differentiated skeletal muscle, we prepared a peptide antibody specific for the skeletal alpha isoform of actin and used this antibody along with an antibody specifically reactive with nonmuscle gamma actin to stain cultured myotubes and adult skeletal myofibrils by double-indirect immunofluorescence. At this level of resolution, no differences in isoform localization were seen: Both muscle and nonmuscle actins were detected in the myotubes and in the striations of mature myofibrils. Myotubes were also double-stained using immunogold electron microscopy, and the isoform distributions were determined quantitatively by counting the two sizes of gold particles that corresponded to labeling with each antibody. A quantitative analysis of immunoreactivity revealed that, although both forms were present in all actin-containing structures, nonmuscle actin was relatively more prevalent along the edges (cortical microfilaments) of the myotubes, whereas the muscle isoform predominated in the interior regions (containing forming myofibrils). Thus, we have found evidence of a heterogeneous distribution of muscle and nonmuscle actin isoforms in differentiating myogenic cells, and we have demonstrated that a nonmuscle actin isoform is a component of the muscle contractile apparatus.

Actins↗
PubMed1988

Selective stabilization of microtubules oriented toward the direction of cell migration.

A small subset of the microtubule (MT) array in many cultured cells does not exhibit the rapid turnover (t 1/2 approximately equal to 10 min) shown by most cellular MTs. The function of the stable class of MTs is unknown and has been confounded by the apparent lack of organization of stable MTs within cells. Using an antibody against detyrosinated tubulin, a post-translationally modified form of tubulin that accumulates in stable MTs, we localized the stable MTs in mouse 3T3 cells induced to initiate directional migration by experimental wounding of confluent monolayers. Immediately after monolayer wounding, the distribution of stable MTs in cells at the wound edge resembled that in cells in the monolayer interior; most cells either contained randomly distributed stable MTs or lacked them entirely. However, by 20 min after wounding, cells at the wound margin began to generate an asymmetric MT array, with virtually all stable MTs oriented toward the cell edge in contact with the wound. Two hours after monolayer wounding, greater than or equal to 80% of cells at the wound margin had generated this polarized array of stable MTs, and the array was maintained for at least 12 hr. MTs in the polarized array showed enhanced resistance to depolymerization by nocodazole, thus providing an independent test of their stability. Formation of the polar array of stable MTs appeared to precede onset of cell migration and closely paralleled reorientation of the MT-organizing center. These results show that cultured cells can remodel their MT array rapidly in response to an extracellular signal and suggest that selective stabilization of MTs is an early event in the generation of cellular asymmetry.

Animals↗
PubMed1988

Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level.

Interphase cultured monkey kidney (TC-7) cells contain distinct subsets of cellular microtubules (MTs) enriched in posttranslationally detyrosinated (Glu) or tyrosinated (Tyr) alpha tubulin (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski. 1984. Cell. 38:779-789). To determine the relative stability of these subsets of MTs, we subjected TC-7 cells to treatments that slowly depolymerized MTs. We found Glu MTs to be more resistant than Tyr MTs to depolymerization by nocodazole in living cells, and to depolymerization by dilution in detergent-permeabilized cell models. However, in cold-treated cells, Glu and Tyr MTs did not differ significantly in their stability. Digestion of permeabilized cell models with pancreatic carboxypeptidase A, to generate Glu MTs from endogenous Tyr MTs, did not significantly alter the resistance of the endogenous Tyr MTs toward dilution-induced depolymerization. Furthermore, in human fibroblasts that contained no distinct Glu MTs, we observed a population of nocodazole-resistant MTs. These data suggest that Glu MTs possess enhanced stability against end-mediated depolymerization, yet detyrosination alone appears to be insufficient to confer this enhanced stability.

Animals↗