A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the alpha- and beta-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V beta-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V beta-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V beta-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V beta-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.     

The direct interaction of phospholipase C-gamma 1 with phospholipase D2 is important for epidermal growth factor signaling

The epidermal growth factor (EGF) receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-gamma1 is regarded to be critical for EGF-induced mitogenesis. In this study, we report for the first time a phospholipase complex composed of PLC-gamma1 and phospholipase D2 (PLD2). PLC-gamma1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and co-immunoprecipitation analysis in COS-7 cells show that the Src homology (SH) 3 domain of PLC-gamma1 binds to the proline-rich motif within the Phox homology (PX) domain of PLD2. The interaction between PLC-gamma1 and PLD2 is EGF stimulation-dependent and potentiates EGF-induced inositol 1,4,5-trisphosphate (IP(3)) formation and Ca(2+) increase. Mutating Pro-145 and Pro-148 within the PX domain of PLD2 to leucines disrupts the interaction between PLC-gamma1 and PLD2 and fails to potentiate EGF-induced IP(3) formation and Ca(2+) increase. However, neither PLD2 wild type nor PLD2 mutant affects the EGF-induced tyrosine phosphorylation of PLC-gamma1. These findings suggest that, upon EGF stimulation, PLC-gamma1 directly interacts with PLD2 and this interaction is important for PLC-gamma1 activity.     

Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity

The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.     

Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2.

Upon binding to its cell surface receptor, platelet-derived growth factor (PDGF) causes the tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and stimulates the production of diacylglycerol and inositol 1,4,5-triphosphate. We showed that following stimulation by PDGF, rat-2 cells overexpressing PLC-gamma 1 display an increase in the levels of both tyrosine-phosphorylated PLC-gamma 1 and inositol phosphates compared with the parental rat-2 cells. This increased responsiveness to PDGF is a direct effect of PLC-gamma 1 overexpression, as a cell line expressing similar levels of an enzymatically inactive point mutant of PLC-gamma 1, PLC-gamma 1 335Q, did not show elevated inositol phosphate production in response to PDGF. Hematopoietic cells express PLC-gamma 2, a PLC isoform that is closely related to PLC-gamma 1. When rat-2 cells overexpressing PLC-gamma 2 were treated with PDGF, an increase in both the tyrosine phosphorylation and the in vivo activity of PLC-gamma 2 was observed. Aluminum fluoride (AIF4-), a universal activator of PLC linked to G-proteins, did not produce an increase in the levels of inositol phosphates in either of the overexpressing cell lines compared with parental rat-2 cells, demonstrating that PLC-gamma isoforms respond specifically to a receptor with tyrosine kinase activity.     

Cbl competitively inhibits epidermal growth factor-induced activation of phospholipase C-gamma1

Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation through its two Src homology (SH) 2 domains and its single SH3 domain, which interact with signaling molecules in response to various growth factors and hormones. However, the role of the SH domains in the growth factor-induced regulation of PLC-gamma1 is unclear. By peptide-mass fingerprinting analysis we have identified Cbl as a binding protein for the SH3 domain of PLC-gamma1 from rat pheochromatocyte PC12 cells. Association of Cbl with PLC-gamma1 was induced by epidermal growth factor (EGF) but not by nerve growth factor (NGF). Upon EGF stimulation, both Cbl and PLC-gamma1 were recruited to the activated EGF receptor through their SH2 domains. Mutation of the SH2 domains of either Cbl or PLC-gamma1 abrogated the EGF-induced interaction of PLC-gamma1 with Cbl, indicating that SH2-mediated translocation is essential for the association of PLC-gamma1 and Cbl. Overexpression of Cbl attenuated EGF-induced tyrosine phosphorylation and the subsequent activation of PLC-gamma1 by interfering competitively with the interaction between PLC-gamma1 and EGFR. Taken together, these results provide the first indications that Cbl may be a negative regulator of intracellular signaling following EGF-induced PLC-gamma1 activation.     

Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules

Mammalian cells express a spectrum of tubulin isotypes whose relationship to the diversity of microtubule function is unknown. To examine whether different isotypes are segregated into functionally distinct microtubules, we generated immune sera capable of discriminating among the various naturally occurring beta-tubulin isotypes. Cloned fusion proteins encoding each isotype were used first to tolerogenize animals against shared epitopes, and then as immunogens to elicit a specific response. In experiments using these sera, we show that there is neither complete nor partial segregation of beta-tubulin isotypes: both interphase cytoskeletal and mitotic spindle microtubules are mixed copolymers of all expressed beta-tubulin isotypes. Indeed, a highly divergent isotype normally expressed only in certain hematopoietic cells is also indiscriminately assembled into all microtubules both in their normal context and when transfected into HeLa cells.     

Structural characterization of the split pleckstrin homology domain in phospholipase C-gamma1 and its interaction with TRPC3

Phospholipase C (PLC)-gamma is unique among the PLC enzymes because each PLC-gamma isozyme contains a split pleckstrin homology (PH) domain with an SH2SH2SH3 tandem repeat insertion (where SH indicates Src homology domain) in the middle of its sequence. Split PH domains exist in a number of other proteins that play crucial signaling roles. However, little is known about the structure and function of split PH domains. The C-terminal half of the PLC-gamma split PH domain has been implicated to interact directly with the TRPC3 calcium channel, thereby providing a direct coupling mechanism between PLC-gamma and agonist-induced calcium entry. However, this interaction has not been proved by direct biochemical or structural studies. Here we determined the three-dimensional structure of the split PH domain of PLC-gamma1, and we found that the split PH domain of the enzyme folds into a canonical PH domain fold with high thermostability. The SH2SH2SH3 insertion between the beta3 and beta4 strands does not change the structure of the split PH domain. In contrast to the majority of phospholipid-binding PH domains, the PLC-gamma1 split PH domain lacks the signature lipid-binding motif located between the beta1 and beta2 strands. Consistent with this structural feature, the split PH domain of PLC-gamma1 does not bind to phospholipids. Multiple biochemical and biophysical experiments have argued against a direct interaction between TRPC3 and the C-terminal half of the PLC-gamma1 split PH domain. Our data pointed to the existence of a yet to be elucidated interaction mechanism between TRPC3 and PLC-gamma1.     

Grb2 negatively regulates epidermal growth factor-induced phospholipase C-gamma1 activity through the direct interaction with tyrosine-phosphorylated phospholipase C-gamma1

Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation. Upon the stimulation of growth factors and hormones, PLC-gamma1 is rapidly phosphorylated at three known sites; Tyr771, Tyr783 and Tyr1254 and its enzymatic activity is up-regulated. In this study, we demonstrate for the first time that Grb2, an adaptor protein, specifically interacts with tyrosine-phosphorylated PLC-gamma1 at Tyr783. The association of Grb2 with PLC-gamma1 was induced by the treatment with epidermal growth factor (EGF). Replacement of Tyr783 with Phe completely blocked EGF-induced interaction of PLC-gamma1 with Grb2, indicating that tyrosine phosphorylation of PLC-gamma1 at Tyr783 is essential for the interaction with Grb2. Interestingly, the depletion of Grb2 from HEK-293 cells by RNA interference significantly enhanced increased EGF-induced PLC-gamma1 enzymatic activity and mobilization of the intracellular Ca2+, while it did not affect EGF-induced tyrosine phosphorylation of PLC-gamma1. Furthermore, overexpression of Grb2 inhibited PLC-gamma1 enzymatic activity. Taken together, these results suggest Grb2, in addition to its key function in signaling through Ras, may have a negatively regulatory role on EGF-induced PLC-gamma1 activation.     

EGF-dependent association of phospholipase C-gamma1 with c-Cbl

The structure of phospholipase Cgamma1 (PLC-gamma1) contains two SH2 domains and one SH3 domain. While the function of the SH2 domains in PLC-gamma1 are well described, to date no growth factor-dependent function for the SH3 domain has been presented. To assess SH3 domain function in the context of the full-length PLC-gamma1, this domain was deleted and the mutant was stably expressed in Plcg1 null mouse embryonic fibroblasts. Following EGF treatment of cells, the PLC-gamma1DeltaSH3 mutant displayed the same increased level of tyrosine phosphorylation and association with EGF receptor as wild-type PLC-gamma1. Also, the SH3 mutant demonstrated membrane translocation and mediated the mobilization of intracellular Ca(2+) in response to EGF. c-Cbl is shown to associate with tyrosine phosphorylated PLC-gamma1 in an EGF-dependent manner, but no association was detected with the PLC-gamma1DeltaSH3 mutant. Interestingly, PDGF, which also tyrosine phosphorylates PLC-gamma1, failed to induce c-Cbl association with PLC-gamma1 and also provoked no c-Cbl tyrosine phosphorylation. This suggests that c-Cbl tyrosine phosphorylation is necessary for its interaction with PLC-gamma1. Evidence of a direct association of c-Cbl with PLC-gamma1 was provided by pull-down and overlay experiments, using glutathione S-transferase fusion proteins that contain the SH3 domain of PLC-gamma1. The data, therefore, show an EGF-inducible direct association of PLC-gamma1 with c-Cbl in vivo that is mediated by the SH3 domain of PLC-gamma1.     

Src homology domains in phospholipase C-gamma1 mediate its anti-apoptotic action through regulating the enzymatic activity

Phospholipase-gamma1 (PLC-gamma1) prevents programmed cell death, for which the enzymatic activity has been implicated. However, the biological function of Src homology (SH) domains of PLC-gamma1 in promoting cell survival remains elusive. Here, we showed that deletion of the N-SH2 domain or both N-SH2 and C-SH2 domains, but not the SH3 domain, abolished the anti-apoptotic activity of PLC-gamma1. Surprisingly, removal of the whole SH domain inhibited apoptosis. The lipase-inactive PLC-gamma1 mutant (LIM) failed to suppress apoptosis. Moreover, the phospholipase activity in SH3- or whole SH domain-deleted cells was comparable to that of wild-type cells. By contrast, the enzymatic activity was substantially ablated in SH2 domain-deleted or LIM cells. A pharmacological inhibitor of PLC-gamma1 robustly diminished the anti-apoptotic action in wild-type, SH3- or whole SH domain-deleted cells, whereas pretreatment of SH2 domain-deleted or LIM cells with agents activating PKC and calcium mobilization markedly promoted cell survival. These results indicate that SH domains in PLC-gamma1 might mediate its anti-apoptotic action by regulating the enzymatic activity.     

Tubulin composition and microtubule nucleation of a griseofulvin-resistant Chinese hamster ovary cell mutant with abnormal spindles

A griseofulvin-resistant Chinese hamster ovary (CHO) mutant (Grs-2) which has an altered beta-tubulin subunit as well as wild-type beta-tubulin is temperature-sensitive (ts) for growth at 40.5 degrees C. This growth defect appears to result from the formation of abnormal mitotic spindles at the non-permissive temperature (Abraham, I et al., J cell biol 97 (1983) 1055) [19]. Light microscopy of spindles isolated from mutant cells cultured at the permissive temperature showed a typical bipolar morphology, whereas spindles isolated at the non-permissive temperature were multipolar. In order to study the role of tubulin in spindle formation, we analyzed the tubulin composition of the multipolar spindles. Two-dimensional gels and immunoblotting analysis of one-dimensional electrophoretic gels stained with monoclonal anti-Chinese hamster brain beta-tubulin antibody revealed that both mutant and wild-type beta-tubulins were present in similar proportions in both bipolar spindles at 37 degrees C and multipolar spindles at 40.5 degrees C. The ratio between wild-type and mutant tubulin in spindles was also found to be the same as in the cytoplasmic microtubule network in interphase cells, providing evidence that the mutant beta-tubulin appeared to be incorporated in a similar manner into both interphase and mitotic microtubule structures. In vitro microtubule polymerization onto centrosomes prepared from mutant Grs-2 demonstrated that 80% of the sites for microtubule nucleation were without centrioles, suggesting fragmentation of pericentriolar material away from centrioles. This may be one of the causes of multipolar spindle formation in the mutant cells. These results, therefore, suggest that abnormal formation of spindles in mutant cells is due not to the presence of the mutant tubulin per se, but to the abnormal behavior of this mutant tubulin in the cellular environment during mitosis or abnormal interaction with other components in the spindle at 40.5 degrees C.     

Cofactor D functions as a centrosomal protein and is required for the recruitment of the gamma-tubulin ring complex at centrosomes and organization of the mitotic spindle

Microtubules are highly dynamic structures, composed of alpha/beta-tubulin heterodimers. Biosynthesis of the functional dimer involves the participation of several chaperones, termed cofactors A-E, that act on folding intermediates downstream of the cytosolic chaperonin CCT (1, 2). We show that cofactor D is also a centrosomal protein and that overexpression of either the full-length protein or either of two centrosome localization domains leads to the loss of anchoring of the gamma-tubulin ring complex and of nucleation of microtubule growth at centrosomes. In contrast, depletion of cofactor D by short interfering RNA results in mitotic spindle defects. Because none of these changes in cofactor D activity produced a change in the levels of alpha-or beta-tubulin, we conclude that these newly discovered functions for cofactor D are distinct from its previously described role in tubulin folding. Thus, we describe a new role for cofactor D at centrosomes that is important to its function in polymerization of tubulin and organization of the mitotic spindle.     

Taxol-dependent mutants of Chinese hamster ovary cells with alterations in alpha- and beta-tubulin

Chinese hamster ovary cell mutants resistant to the microtubule stabilizing drug taxol were isolated in a single step. Of these 139 drug-resistant mutants, 59 exhibit an absolute requirement for taxol for normal growth and division, 13 have a partial requirement, and 69 grow normally without the drug. Two-dimensional gel analysis of whole cell proteins reveal "extra" spots representing altered tubulins in 13 of the mutants. Six of these have an altered alpha-tubulin and seven have an altered beta-tubulin. Cells with an absolute dependence on taxol become large and multinucleated when deprived of the drug. In contrast, partially dependent cells exhibit some multinucleation, but most cells appear normal. In one mutant that has an absolute dependence on taxol, the cells appear to die more quickly and their nuclei do not increase in size or number. As previously found for another taxol-dependent mutant (Cabral, F., 1983, J. Cell. Biol., 97:22-29), the taxol dependence of the mutants described in this paper behaves recessively in somatic cell hybrids, and the cells are more susceptible to being killed by colcemid than are the wild-type parental cells. When compared with wild-type cells, taxol-dependent mutants have normal arrays of cytoplasmic microtubules but form much smaller mitotic spindles in the presence of taxol. When deprived of the drug, however, these mutants cannot complete assembly of the mitotic spindle apparatus, as judged by tubulin immunofluorescence. Thus, the defects leading to taxol dependence in these mutants with defined alterations in alpha- and beta-tubulin appear to result from the cell's inability to form a functional mitotic spindle. Reversion analysis indicates that the properties of at least one alpha-tubulin mutant are conferred by the altered tubulin seen on two-dimensional gels.     

Differential roles of the Src homology 2 domains of phospholipase C-gamma1 (PLC-gamma1) in platelet-derived growth factor-induced activation of PLC-gamma1 in intact cells

Upon stimulation of cells with platelet-derived growth factor (PDGF), phospholipase C-gamma1 (PLC-gamma1) binds to the tyrosine-phosphorylated PDGF receptor through one or both of its Src homology 2 (SH2) domains, is phosphorylated by the receptor kinase, and is thereby activated to hydrolyze phosphatidylinositol 4, 5-bisphosphate. Association of PLC-gamma1 with the insoluble subcellular fraction is also enhanced in PDGF-stimulated cells. The individual roles of the two SH2 domains of PLC-gamma1 in mediating the interaction between the enzyme and the PDGF receptor have now been investigated by functionally disabling each domain. A critical Arg residue in each SH2 domain was mutated to Ala. Both wild-type and mutant PLC-gamma1 proteins were transiently expressed in a PLC-gamma1-deficient fibroblast cell line, and these transfected cells were stimulated with PDGF. The mutant protein in which the COOH-terminal SH2 domain was disabled bound to the PDGF receptor. Accordingly, it was phosphorylated by the receptor, catalyzed the production of inositol phosphates, and mobilized intracellular calcium to extents similar to (but slightly less than) those observed with the wild-type enzyme. In contrast, the mutant in which the NH(2)-terminal SH2 domain was impaired did not bind to the PDGF receptor and consequently was neither phosphorylated nor activated. These results suggest that the NH(2)-terminal SH2 domain, but not the COOH-terminal SH2 domain, of PLC-gamma1 is required for PDGF-induced activation of PLC-gamma1. Functional impairment of the SH2 domains did not affect the PDGF-induced redistribution of PLC-gamma1, suggesting that recruitment of PLC-gamma1 to the particulate fraction does not involve the SH2 domains.     

Subcellular sequestration of an antigenically unique beta-tubulin

Tubulin from Trypanosoma brucei was characterized by Western blotting using well defined monoclonal antibodies reacting with alpha- or beta-tubulin and a new monoclonal antibody, 1B41, raised against a microtubule-enriched fraction of T. brucei, which specifically reacts with the beta-subunit of tubulin from either T. brucei or rat brain. This antibody has been used to examine the subcellular distribution of the corresponding antigen in T. brucei by indirect immunofluorescence. The epitope recognized by 1B41 is restricted to a thin line extending from the basal body region to the anterior end of the cell body. To determine the relationship between the immunoreactive zone and the flagellum, double-label immunofluorescence was performed in both interphase and mitotic cells with 1B41 and a flagellar marker, the monoclonal antibody 5E9, specific for the paraflagellar rod polypeptides of trypanosomes. These experiments revealed that the immunoreactive tubulin was contained in a part of the subpellicular cytoskeleton that remained in a constant spatial correspondence with the flagellum throughout the cell division cycle. The beta-tubulin recognized by 1B41 may be segregated into the microtubular structures associated with a cisterna of the endoplasmic reticulum forming the subflagellar microtubule quartet (SFMQ). These results suggest that the presence of an antigenically unique beta-tubulin defines a subpopulation of microtubules possessing specific dynamic properties that may be involved in the morphogenesis of daughter cells during the division of T. brucei.     

Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry

Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.     

Mutation in the beta-tubulin signature motif suppresses microtubule GTPase activity and dynamics, and slows mitosis.

We introduced a threonine-to-glycine point mutation at position 143 in the "tubulin signature motif" 140Gly-Gly-Gly-Thr-Gly-Ser-Gly146 of Saccharomyces cerevisiae beta-tubulin. In an electron diffraction model of the tubulin dimer, this sequence comes close to the phosphates of a guanine nucleotide bound in the beta-tubulin exchangeable E site. Both the GTP-binding affinity and the microtubule (MT)-dependent GTPase activity of tubulin isolated from haploid tub2-T143G mutant cells were reduced by at least 15-fold, compared to tubulin isolated from control wild-type cells. The growing and shortening dynamics of MTs assembled from alphabeta:Thr143Gly-mutated dimers were also strongly suppressed, compared to control MTs. The in vitro properties of the mutated MTs (slower growing and more stable) are consistent with the effects of the tub2-T143G mutation in haploid cells. The average length of MT spindles in large-budded mutant cells was only 3.7 +/- 0.2 microm, approximately half of the size of MT arrays in large-budded wild-type cells (average length = 7.1 +/- 0.4 microm), suggesting that there is a delay in mitosis in the mutant cells. There was also a higher proportion of large-budded cells with unsegregated nuclei in mutant cultures (30% versus 12% for wild-type cells), again suggesting such a delay. The results show that beta:Thr143 of the tubulin signature motif plays an important role in GTP binding and hydrolysis by the beta-tubulin E site and support the idea that tubulins belong to a family of proteins within the GTPase superfamily that are structurally distinct from the classic GTPases, such as EF-Tu and p21(ras). The data also suggest that MT dynamics are critical for MT function in yeast cells and that spindle MT assembly and disassembly could be coordinated with other cell-cycle events by regulating beta-tubulin GTPase activity.     

Polyamines are required for phospholipase C-gamma1 expression promoting intestinal epithelial restitution after wounding

Intestinal mucosal restitution occurs by epithelial cell migration, rather than by proliferation, to reseal superficial wounds after injury. Polyamines are essential for the stimulation of intestinal epithelial cell (IEC) migration during restitution in association with their ability to regulate Ca2+ homeostasis, but the exact mechanism by which polyamines induce cytosolic free Ca2+ concentration ([Ca2+]cyt) remains unclear. Phospholipase C (PLC)-gamma1 catalyzes the formation of inositol (1,4,5)-trisphosphate (IP3), which is implicated in the regulation of [Ca2+]cyt by modulating Ca2+ store mobilization and Ca2+ influx. The present study tested the hypothesis that polyamines are involved in PLC-gamma1 activity, regulating [Ca2+]cyt and cell migration after wounding. Depletion of cellular polyamines by alpha-difluoromethylornithine inhibited PLC-gamma1 expression in differentiated IECs (stable Cdx2-transfected IEC-6 cells), as indicated by substantial decreases in levels of PLC-gamma1 mRNA and protein and its enzyme product IP3. Polyamine-deficient cells also displayed decreased [Ca2+]cyt and inhibited cell migration. Decreased levels of PLC-gamma1 by treatment with U-73122 or transfection with short interfering RNA specifically targeting PLC-gamma1 also decreased IP3, reduced resting [Ca2+]cyt and Ca2+ influx after store depletion, and suppressed cell migration in control cells. In contrast, stimulation of PLC-gamma1 by 2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)-benzenesulfonamide induced IP3, increased [Ca2+]cyt, and promoted cell migration in polyamine-deficient cells. These results indicate that polyamines are absolutely required for PLC-gamma1 expression in IECs and that polyamine-mediated PLC-gamma1 signaling stimulates cell migration during restitution as a result of increased [Ca2+]cyt.     

STU1, a suppressor of a beta-tubulin mutation, encodes a novel and essential component of the yeast mitotic spindle

We have isolated a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in S. cerevisiae, that confers a specific defect in spindle microtubule function. At 14 degrees C, tub2-406 cells lack a normal bipolar spindle but do assemble functional cytoplasmic microtubules. In an attempt to identify proteins that are important for spindle assembly, we screened for suppressors of the cold-sensitivity of tub2-406 and obtained four alleles of a novel gene, STU1. Genetic interactions between stu1 alleles and alleles of TUB1 and TUB2 suggest that Stu1p specifically interacts with microtubules. STU1 is essential for growth and disruption of STU1 causes defects in spindle assembly that are similar to those produced by the tub2-406 mutation. The nucleotide sequence of the STU1 gene predicts a protein product of 174 kD with no significant similarity to known proteins. An epitope-tagged Stulp colocalizes with microtubules in the mitotic spindle of yeast. These results demonstrate that Stulp is an essential component of the yeast mitotic spindle.     

Stu1p is physically associated with beta-tubulin and is required for structural integrity of the mitotic spindle

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1-5 mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1-5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin-related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with beta-tubulin and identify the domains required for this interaction on both Stu1p and beta-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.