scholarly journals An essential role for α4A-tubulin in platelet biogenesis

2019 ◽  
Vol 2 (1) ◽  
pp. e201900309 ◽  
Author(s):  
Catherine Strassel ◽  
Maria M Magiera ◽  
Arnaud Dupuis ◽  
Morgane Batzenschlager ◽  
Agnès Hovasse ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Essential Role ◽  
Missense Mutations ◽  
Single Plane ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
Marginal Band ◽  
Late Stages ◽  
Megakaryocyte Differentiation ◽  
Platelet Formation

During platelet biogenesis, microtubules (MTs) are arranged into submembranous structures (the marginal band) that encircle the cell in a single plane. This unique MT array has no equivalent in any other mammalian cell, and the mechanisms responsible for this particular mode of assembly are not fully understood. One possibility is that platelet MTs are composed of a particular set of tubulin isotypes that carry specific posttranslational modifications. Although β1-tubulin is known to be essential, no equivalent roles of α-tubulin isotypes in platelet formation or function have so far been reported. Here, we identify α4A-tubulin as a predominant α-tubulin isotype in platelets. Similar to β1-tubulin, α4A-tubulin expression is up-regulated during the late stages of megakaryocyte differentiation. Missense mutations in the α4A-tubulin gene cause macrothrombocytopenia in mice and humans. Defects in α4A-tubulin lead to changes in tubulin tyrosination status of the platelet tubulin pool. Ultrastructural defects include reduced numbers and misarranged MT coils in the platelet marginal band. We further observed defects in megakaryocyte maturation and proplatelet formation inTuba4a-mutant mice. We have, thus, discovered an α-tubulin isotype with specific and essential roles in platelet biogenesis.

scholarly journals Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans

2017 ◽  
Author(s):  
Chaogu Zheng ◽  
Margarete Diaz-Cuadros ◽  
Ken C.Q. Nguyen ◽  
David H. Hall ◽  
Martin Chalfie
Keyword(s):  
Function Analysis ◽  
Critical Role ◽  
Neurite Growth ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Post Translational Modification ◽  
Exterior Surface ◽  
Tubulin Isotypes ◽  
C Elegans ◽  
Tubulin Isotype

AbstractTubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the C. elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an α-tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization.Table of Content (TOC) Highlight SummaryDifferent tubulin isotypes perform different functions in the regulation of MT structure and neurite growth, and missense mutations of tubulin genes have three types of distinct effects on MT stability and neurite growth. One α-tubulin isotype appears to induce relative instability due to the lack of potential post-translational modification sites.

Selected Expression and Functional Importance of α4a-Tubulin in Platelet Biogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1360-1360
Author(s):  
Catherine Strassel ◽  
Agnes Hovasse ◽  
Sylvie Moog ◽  
Magda Mageira ◽  
Morgane Batzenschlager ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Human Platelets ◽  
Band Formation ◽  
Acetylated Tubulin ◽  
New Information ◽  
Tubulin Isotype ◽  
Tubulin Isoforms ◽  
Marginal Band ◽  
Platelet Formation

Abstract Platelets are produced from mature megakaryocytes (MK) following a profound cellular reorganization. This includes the assembly of microtubules (MT) into a unique submenbranous coiled structure, the marginal band (MB). This process is thought to depend on a specific αβ-tubulin isotype repertoire. The MK-restricted-β1-tubulin, the predominant isoform of the MB, is already known to be important for platelet biogenesis but the implication of other isotypes is currently unknown. Our goal was to establish the αβ-tubulin repertoire in platelets and during megakaryopoiesis and to evaluate the implication of selected isotypes in platelet formation. To establish an exhaustive list of the tubulin isotypes, we used combination of RT PCR and proteomic analyses to quantify the expression of each isotype in human platelets and in human MK differentiated in culture from CD34+ hematopoietic progenitors. Information gained on the hierarchical combination of tubulin isoforms in the course of platelet biogenesis has been extended at the functional level to investigate both their role in marginal band formation and platelet functions β6-, β5- and α1c-tubulin transcripts were already present in CD34+ cells and decreased during the final stages of megakaryopoiesis. On the other hand, β1-, α4A- and α8-tubulin transcripts were only observed later during MK differentiation and in platelets. Quantitative LC-SRM mass spectrometry confirmed the predominant expression of β1 and α4A-isotypes in platelets. A functional role of the newly identified α4a-tubulin was supported by the thrombocytopenia and enlarged platelets with a decreased number of MT coils (1-3) comprising less-acetylated tubulin in mice carrying a point mutation in tuba4a. Additionally, a tendency to increased responses to several agonists was observed in these platelets. This study reveals new information on the evolution of the tubulin isotype repertoire in platelet formation pointing to a role of less-widely expressed α-isotypes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Six mouse alpha-tubulin mRNAs encode five distinct isotypes: testis-specific expression of two sister genes.

1986 ◽  
Vol 6 (7) ◽  
pp. 2409-2419 ◽  
Author(s):  
A Villasante ◽  
D Wang ◽  
P Dobner ◽  
P Dolph ◽  
S A Lewis ◽  
...  
Keyword(s):  
Duplication Event ◽  
Developmental Expression ◽  
Untranslated Regions ◽  
Coding Region ◽  
Specific Expression ◽  
Translational Initiation ◽  
Alpha Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
Genes Encoding

Five mouse alpha-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the alpha-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (M alpha 3 and M alpha 7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of alpha-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that the duplication event leading to a pair of genes encoding a testis-specific alpha-tubulin isotype predated the mammalian radiation, and both members of the duplicated sequence have been maintained since species divergence. A second alpha-tubulin gene, M alpha 6, is expressed ubiquitously at a low level, whereas a third gene, M alpha 4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct alpha-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various alpha-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules.

Thromboxane Synthase Has the Same Pattern of Expression as Platelet Specific Glycoproteins during Human Megakaryocyte Differentiation

2000 ◽  
Vol 83 (05) ◽  
pp. 759-768 ◽  
Author(s):  
Natacha Vitrat ◽  
Rémi Letestu ◽  
Aline Massé ◽  
Vladimir Lazar ◽  
Najet Debili ◽  
...  
Keyword(s):  
Formation Process ◽  
The Other ◽  
Rt Pcr ◽  
Thromboxane Synthase ◽  
Platelet Production ◽  
Cd34 Cells ◽  
Specific Proteins ◽  
Deficient Mice ◽  
Megakaryocyte Differentiation ◽  
Platelet Formation

SummaryRegulation of the platelet formation process is poorly understood. It has been shown that p45NF-E2 deficient mice have a profound defect in platelet formation and recently the first platelet/megakaryocytic gene regulated by NF-E2, thromboxane synthase (TXS), has been identified. In this study, we investigated TXS expression as a model of a gene regulated by NF-E2 during MK differentiation. Megakaryocytic cells derived from blood CD34+ cells were purified according to their stage of maturation on the basis of expression of CD34, CD41a and CD42a, permitting to define different stages in MK differentiation. By means of real-time quantitative RT-PCR, we could determine that the level of TXS increased during differentiation in parallel with the expression of c-mpl and GPIIb (CD41). However, amounts of TXS transcripts increased about 1.6-fold more than that of GPIIb or c-mpl transcripts during maturation. Expression of TXS and MK specific proteins such as CD41a, CD42a and vWF was also correlated in maturing MKs. In addition, staining by anti-TXS antibody of proplatelet bearing MKs was not increased in comparison to that observed in mature MK, suggesting that TXS is not upregulated during platelet formation. In addition, we investigated whether TXS and cyclooxygenase could be involved in platelet formation by adding aspirin into the cultures. No significant decrease of platelet production was observed.In conclusion, this study shows that TXS is coordinately expressed with the other platelet proteins during MK differentiation but is not directly involved in platelet formation.

scholarly journals The Caenorhabditis elegans Microtubule-severing Complex MEI-1/MEI-2 Katanin Interacts Differently with Two Superficially Redundant β-Tubulin Isotypes

2004 ◽  
Vol 15 (1) ◽  
pp. 142-150 ◽  
Author(s):  
Chenggang Lu ◽  
Martin Srayko ◽  
Paul E. Mains
Keyword(s):  
Caenorhabditis Elegans ◽  
Genetic Background ◽  
Morphological Changes ◽  
Meiotic Spindle ◽  
Tubulin Isotypes ◽  
Microtubule Severing ◽  
Tubulin Isotype ◽  
Embryonic Viability ◽  
Β Tubulin

The microtubule-severing protein complex katanin is required for a variety of important microtubule-base morphological changes in both animals and plants. Caenorhabditis elegans katanin is encoded by the mei-1 and mei-2 genes and is required for oocyte meiotic spindle formation and must be inactivated before the first mitotic cleavage. We identified a mutation, sb26, in the tbb-2 β-tubulin gene that partially inhibits MEI-1/MEI-2 activity: sb26 rescues lethality caused by ectopic MEI-1/MEI-2 expression during mitosis, and sb26 increases meiotic defects in a genetic background where MEI-1/MEI-2 activity is lower than normal. sb26 does not interfere with MEI-1/MEI-2 microtubule localization, suggesting that this mutation likely interferes with severing. Tubulin deletion alleles and RNA-mediated interference revealed that TBB-2 and the other germline enriched β-tubulin isotype, TBB-1, are redundant for embryonic viability. However, limiting MEI-1/MEI-2 activity in these experiments revealed that MEI-1/MEI-2 preferentially interacts with TBB-2–containing microtubules. Our results demonstrate that these two superficially redundant β-tubulin isotypes have functionally distinct roles in vivo.

scholarly journals Generation of antisera that discriminate among mammalian alpha-tubulins: introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function.

1988 ◽  
Vol 106 (6) ◽  
pp. 2011-2022 ◽  
Author(s):  
W Gu ◽  
S A Lewis ◽  
N J Cowan
Keyword(s):  
Immune Response ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Alpha Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
Carboxy Terminal ◽  
Nih 3T3 ◽  
Shared Epitopes

To assay the functional significance of the multiple but closely related alpha-tubulin polypeptides that are expressed in mammalian cells, we generated three specific immune sera, each of which uniquely recognizes a distinct alpha-tubulin isotype. All three isotypes are expressed in a tissue-restricted manner: one (M alpha 3/7) only in mature testis, one (M alpha 4) mainly in muscle and brain, and the third (M alpha 6) in several tissues at a very low level. A fourth specific antiserum was also generated that distinguishes between the tyrosinated and nontyrosinated form of a single alpha-tubulin isotype. Because individual tubulin isotypes cannot be purified biochemically, these sera were raised using cloned fusion proteins purified from host Escherichia coli cells. To suppress the immune response to shared epitopes, animals were first rendered tolerant to fusion proteins encoding all but one of the known mammalian alpha-tubulin isotypes. Subsequent challenge with the remaining fusion protein then resulted in the elicitation of an immune response to unique epitopes. Three criteria were used to establish the specificity of the resulting sera: (a) their ability to discriminate among cloned fusion proteins representing all the known mammalian alpha-tubulin isotypes; (b) their ability to uniquely detect alpha-tubulin in whole extracts of tissues; and (c) their capacity to stain microtubules in fixed preparations of cells transfected with sequences encoding the corresponding isotype. The transfection experiments served to demonstrate (a) the coassembly of M alpha 3/7, M alpha 4, and M alpha 6 into both interphase and spindle microtubules in HeLa cells and NIH 3T3 cells, and (b) that the M alpha 4 isotype, which is unique among mammalian alpha-tubulins in that it lacks an encoded carboxy-terminal tyrosine residue, behaves like other alpha-tubulin isotypes with respect to the cycle of tyrosination/detyrosination that occurs in most cultured cells.

scholarly journals The Roles of β-Tubulin Mutations and Isotype Expression in Acquired Drug Resistance

2007 ◽  
Vol 3 ◽  
pp. 117693510700300 ◽  
Author(s):  
J. Torin Huzil ◽  
Ke Chen ◽  
Lukasz Kurgan ◽  
Jack A. Tuszynski
Keyword(s):  
Drug Resistance ◽  
Rational Design ◽  
Resistant Cell ◽  
Microtubule Assembly ◽  
Acquired Drug Resistance ◽  
Tubulin Isotypes ◽  
Microtubule Disruption ◽  
Tubulin Isotype ◽  
Β Tubulin ◽  
Isotype Expression

The antitumor drug paclitaxel stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and eventually apoptosis. Upon assembly of the α/β-tubulin heterodimer, GTP becomes bound to both the α and β-tubulin monomers. During microtubule assembly, the GTP bound to β-tubulin is hydrolyzed to GDP, eventually reaching steady-state equilibrium between free tubulin dimers and those polymerized into microtubules. Tubulin-binding drugs such as paclitaxel interact with β-tubulin, resulting in the disruption of this equilibrium. In spite of several crystal structures of tubulin, there is little biochemical insight into the mechanism by which anti-tubulin drugs target microtubules and alter their normal behavior. The mechanism of drug action is further complicated, as the description of altered β-tubulin isotype expression and/or mutations in tubulin genes may lead to drug resistance as has been described in the literature. Because of the relationship between β-tubulin isotype expression and mutations within β-tubulin, both leading to resistance, we examined the properties of altered residues within the taxane, colchicine and Vinca binding sites. The amount of data now available, allows us to investigate common patterns that lead to microtubule disruption and may provide a guide to the rational design of novel compounds that can inhibit microtubule dynamics for specific tubulin isotypes or, indeed resistant cell lines. Because of the vast amount of data published to date, we will only provide a broad overview of the mutational results and how these correlate with differences between tubulin isotypes. We also note that clinical studies describe a number of predictive factors for the response to anti-tubulin drugs and attempt to develop an understanding of the features within tubulin that may help explain how they may affect both microtubule assembly and stability.

scholarly journals Complex regulation and functional versatility of mammalian alpha- and beta-tubulin isotypes during the differentiation of testis and muscle cells.

1988 ◽  
Vol 106 (6) ◽  
pp. 2023-2033 ◽  
Author(s):  
S A Lewis ◽  
N J Cowan
Keyword(s):  
Cell Types ◽  
Beta Tubulin ◽  
Specific Expression ◽  
Alpha Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
A Cell ◽  
Complex Regulation ◽  
Carboxy Terminal ◽  
Different Cell Types

In the accompanying paper (Gu, W., S. A. Lewis, and N. J. Cowan. 1988. J. Cell Biol. 106: 2011-2022), we report the generation of three antisera, each of which uniquely recognizes a different mammalian alpha-tubulin isotype, plus a fourth antibody that distinguishes between microtubules containing the tyrosinated and nontyrosinated form of the only known mammalian alpha-tubulin gene product that lacks an encoded carboxy-terminal tyrosine residue. These sera, together with five sera we raised that distinguish among the known mammalian beta-tubulin isotypes, have been used to study patterns of tubulin isotype-specific expression in muscle and testis, two tissues in which characteristic developmental changes are accompanied by dramatic rearrangements in microtubule structures. As in the case of cells in culture, there is no evidence to suggest that there is subcellular sorting of different tubulin isotypes among different kinds of microtubule, even in a cell type (the developing spermatid) that simultaneously contains such functionally distinct structures as the manchette and the flagellum. On the other hand, the patterns of expression of the various tubulin isotypes show marked and distinctive differences in different cell types and, in at least one case, evidence is presented for regulation at the translational or posttranslational level. The significance of these observations is discussed in terms of the existence of the mammalian alpha- and beta-tubulin multigene families.

ADF/n-cofilin–dependent actin turnover determines platelet formation and sizing

Blood ◽  
2010 ◽  
Vol 116 (10) ◽  
pp. 1767-1775 ◽  
Author(s):  
Markus Bender ◽  
Anita Eckly ◽  
John H. Hartwig ◽  
Margitta Elvers ◽  
Irina Pleines ◽  
...  
Keyword(s):  
Actin Filament ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Actin Dynamics ◽  
Complex Process ◽  
First Time ◽  
Late Stages ◽  
Platelet Formation

Abstract The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/n-cofilin–mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton.

Sign in / Sign up

Export Citation Format

Share Document