Modeling a disease-correlated tubulin mutation in budding yeast reveals insight into MAP-mediated dynein function.

Mutations in the genes that encode α- and ß-tubulin underlie many neurological diseases, most notably malformations in cortical development. In addition to revealing the molecular basis for disease etiology, studying such mutations can provide insight into microtubule function and the role of the large family of microtubule effectors. In this study, we use budding yeast to model one such mutation-Gly436Arg in α-tubulin, which is causative of malformations in cortical development-in order to understand how it impacts microtubule function in a simple eukaryotic system. Using a combination of in vitro and in vivo methodologies, including live cell imaging and electron tomography, we find that the mutant tubulin is incorporated into microtubules, causes a shift in α-tubulin isotype usage, and dramatically enhances dynein activity, which leads to spindle-positioning defects. We find that the basis for the latter phenotype is an impaired interaction between She1-a dynein inhibitor-and the mutant microtubules. In addition to revealing the natural balance of α-tubulin isotype utilization in cells, our results provide evidence of an impaired interaction between microtubules and a dynein regulator as a consequence of a tubulin mutation and sheds light on a mechanism that may be causative of neurodevelopmental diseases.

Suppression of nuclear oscillations in Saccharomyces cerevisiae expressing Glu tubulin.

In most eukaryotic cells, the C-terminal amino acid of alpha-tubulin is aromatic (Tyr in mammals and Phe in Saccharomyces cerevisiae) and is preceded by two glutamate residues. In mammals, the C-terminal Tyr of alpha-tubulin is subject to cyclic removal from the peptide chain by a carboxypeptidase and readdition to the chain by a tubulin-Tyr ligase. There is evidence that tubulin-Tyr ligase suppression and the resulting accumulation of detyrosinated (Glu) tubulin favor tumor growth, both in animal models and in human cancers. However, the molecular basis for this apparent stimulatory effect of Glu tubulin accumulation on tumor progression is unknown. Here we have developed S. cerevisiae strains expressing only Glu tubulin and used them as a model to assess the consequences of Glu tubulin accumulation in cells. We find that Glu tubulin strains show defects in nuclear oscillations. These defects are linked to a markedly decreased association of the yeast ortholog of CLIP170, Bik1p, with microtubule plus-ends. These results indicate that the accumulation of Glu tubulin in cells affects microtubule tip complexes that are important for microtubule interactions with the cell cortex.

Identification of novel bifunctional calmodulin-binding and microtubule-stabilizing motifs in STOP proteins.

Although microtubules are intrinsically labile tubulin assemblies, many cell types contain stable polymers, resisting depolymerizing conditions such as exposure to the cold or the drug nocodazole. This microtubule stabilization is largely due to polymer association with STOP proteins. There are several STOP variants, some with capacity to induce microtubule resistance to both the cold and nocodazole, others with microtubule cold stabilizing activity only. These microtubule-stabilizing effects of STOP proteins are inhibited by calmodulin and we now demonstrate that they are determined by two distinct kinds of repeated modular sequences (Mn and Mc), both containing a calmodulin-binding peptide, but displaying different microtubule stabilizing activities. Mn modules induce microtubule resistance to both the cold and nocodazole when expressed in cells. Mc modules, which correspond to the STOP central repeats, have microtubule cold stabilizing activity only. Mouse neuronal STOPs, which induce both cold and drug resistance in cellular microtubules, contain three Mn modules and four Mc modules. Compared with neuronal STOPs, the non-neuronal F-STOP lacks multiple Mn modules and this corresponds with an inability to induce nocodazole resistance. STOP modules represent novel bifunctional calmodulin-binding and microtubule-stabilizing sequences that may be essential for the generation of the different patterns of microtubule stabilization observed in cells.

STOP proteins are responsible for the high degree of microtubule stabilization observed in neuronal cells.

Neuronal differentiation and function require extensive stabilization of the microtubule cytoskeleton. Neurons contain a large proportion of microtubules that resist the cold and depolymerizing drugs and exhibit slow subunit turnover. The origin of this stabilization is unclear. Here we have examined the role of STOP, a calmodulin-regulated protein previously isolated from cold-stable brain microtubules. We find that neuronal cells express increasing levels of STOP and of STOP variants during differentiation. These STOP proteins are associated with a large proportion of microtubules in neuronal cells, and are concentrated on cold-stable, drug-resistant, and long-lived polymers. STOP inhibition abolishes microtubule cold and drug stability in established neurites and impairs neurite formation. Thus, STOP proteins are responsible for microtubule stabilization in neurons, and are apparently required for normal neurite formation.

Nonneuronal isoforms of STOP protein are responsible for microtubule cold stability in mammalian fibroblasts.

A number of cycling mammalian cells, such as NIH 3T3, contain abundant subsets of cold-stable microtubules. The origin of such microtubule stabilization in nonneuronal cells is unknown. We have previously described a neuronal protein, stable tubule-only polypeptide (STOP), that binds to microtubules and induces cold stability. We find that NIH 3T3 fibroblasts contain a major 42-kDa isoform of STOP (fibroblastic STOP, F-STOP). F-STOP contains the central repeats characteristic of brain STOP but shows extensive deletions of N- and C-terminal protein domains that are present in brain STOP. These deletions arise from differences in STOP RNA splicing. Despite such deletions, F-STOP has full microtubule stabilizing activity. F-STOP accumulates on cold-stable microtubules of interphase arrays and is present on stable microtubules within the mitotic spindle of NIH 3T3 cells. STOP inhibition by microinjection of affinity-purified STOP central repeat antibodies into NIH 3T3 cells abolishes both interphase and spindle microtubule cold stability. Similar results were obtained with Rat2 cells. These results show that STOP proteins have nonneuronal isoforms that are responsible for the microtubule cold stability observed in mammalian fibroblasts.

Genomic structure and chromosomal mapping of the mouse STOP gene (Mtap6).

The microtubule associated protein STOP (Stable Tubule Only Polypeptide) is a calmodulin-regulated protein able to induce a high degree of microtubule stability. STOP is abundant in neurons which contain large subpopulations of stable microtubules. Genomic clones spanning 67 kb and encompassing the mouse STOP gene (Mtap6) have been isolated and characterized. These clones derive from a single gene mapping to the E2-F1 region of mouse chromosome 7. The gene is composed of 4 exons that exhibit conventional vertebrate splicing sequences. Transcription of the gene initiate at multiple sites in a 85 nucleotide region located 530 bases upstream the translation initiation codon. Accordingly, the 5' flanking region of the gene lacks a TATA box or an initiator element at usual position. The protein encoded by the mouse STOP gene (Mtap6) is composed of 906 amino acids and presents a 91% identities with the rat brain STOP.

PCR cloning and sequence of the murine GPIIb gene promoter.

The promoter region of the murine GPIIb gene was cloned by PCR using oligonucleotides corresponding to the human gene promoter. Analysis of the sequence revealed homologies among species and the conservation of DNA motifs which could be implicated in the regulation of the gene. Transfection experiments show that the murine promoter contains informations for a tissue specific expression of the gene in megakaryocytic cell lines.

Gene regulation in megakaryocyte.

Analysis of the mechanisms which control the differentiation of the megakaryocytic lineage is a major commitment to understand the production of circulating blood platelets. One way to approach this question is to examine the promoter domain of a marker gene which is expressed exclusively in the megakaryocytic lineage and at an early stage of the differentiation process. For this purpose the gene coding for the platelet specific glycoprotein IIb was isolated and its promoter was analysed. This promoter contains positive and negative DNA responsive elements that are responsible for the cell specific expression of the gene. With this promoter region it is now possible to direct the expression of heterologous genes in vivo using the transgenic approach.

GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs.

Platelet GPIIbIIIa is only synthesized in megakaryocyte or in cell lines with megakaryocytic features. The sequence for GPIIb and GPIIIa have recently been derived from cDNAs obtained from HEL cells. The sequence of these proteins produced by the megakaryocyte, has however, not been determined yet. This study describes full length cDNAs for GPIIb and GPIIIa isolated from megakaryocyte cDNA libraries. The cDNA sequences indicate the presence of nucleotide differences, between the sequence of the GPIIIa cDNAs from HEL cells, endothelial cells and megakaryocytes. One difference was also observed between HEL and megakaryocyte GPIIb at position 633 where a cysteine in the megakaryocyte GPIIb, is replaced by a serine in the HEL sequence. The mRNA species for GPIIb (3.4 kb) and GPIIIa (6.1 kb) were of the same size in HEL cells and human megakaryocytes.

Isolation of the human platelet glycoprotein IIb gene and characterization of the 5' flanking region.

Platelet membrane glycoprotein (GP) IIbIIIa complex functions as a receptor for fibrinogen, von Willebrand factor and fibronectin, and mediates adhesive reactions of platelets. The gene for the GPIIb subunit is only active in megakaryocytic cell type. We have isolated this gene from a genomic library. The GPIIb gene was characterized by restriction mapping and sequencing of the 5' and 3' regions containing the first and the last exons. The transcription start site and the polyadenylation signal were identified. From these data we deduced that the gene spans a region of 22 kb and that the mRNA contains a leader sequence of 32 nucleotides. At the 3' end the last exon encodes the 19 amino acids corresponding to the cytoplasmic domain of the GPIIb light chain. Upstream the transcription start site, two sequences are homologous to consensus binding sites of the nuclear factors SP1 and CP2. Two inverted repeats were also identified in this region.

cDNA clones for human platelet GPIIb corresponding to mRNA from megakaryocytes and HEL cells. Evidence for an extensive homology to other Arg-Gly-Asp adhesion receptors.

Platelet glycoprotein (GP) IIb is one of the two subunits of the common platelet adhesion receptor, GPIIb-IIIa. The isolation, characterization and sequencing of cDNA clones encoding for the two polypeptide chains of GPIIb are described. A number of clones were isolated from lambda gt11 libraries constructed with mRNA from an erythroleukemic cell line, HEL, and human megakaryocytes. Two of these clones, lambda IIb1, from HEL cells, and lambda IIb2, from megakaryocytes, cross-hybridized and were selected for detailed analysis. The identification of these as authentic GPIIb clones was based on immunological criteria and confirmed by the presence of nucleotide sequences in each insert encoding for known protein sequences of platelet GPIIb. These clones contained inserts of 1.54 kb and 1.39 kb, respectively, with an overlapping sequence of 801 bp. The nucleotide sequence of the overlapping region was identical indicating that HEL cells produce a protein closely related, if not identical, to platelet GPIIb. The determined nucleotide sequence of two inserts included a coding sequence for 648 amino acid residues, a TAG stop codon and 185 nucleotides of 3' non-coding sequence followed by a poly(A) tail. The coding sequence contained a portion of the heavy chain, the junction between the heavy and light chains and the entire light chain including a potential transmembrane-spanning domain and a short cytoplasmic tail. When these cDNA were used to probe for GPIIb mRNA, a single mRNA species of 3.9 kb was identified in both HEL cells and human megakaryocytes. A comparison of the deduced amino acid sequence for GPIIb with those of the alpha subunit of the vitronectin and the fibronectin receptors revealed extensive homologies. These homologies further establish that GPIIb-IIIa from platelets, together with the vitronectin and the fibronectin receptors, are members of a supergene family of adhesion receptors with a recognition specificity for Arg-Gly-Asp amino acid sequences.