scholarly journals A proteomic survey of microtubule-associated proteins in a R402H TUBA1A mutant mouse

PLoS Genetics ◽  
2020 ◽  
Vol 16 (11) ◽  
pp. e1009104
Author(s):  
Ines Leca ◽  
Alexander William Phillips ◽  
Iris Hofer ◽  
Lukas Landler ◽  
Lyubov Ushakova ◽  
...  
Keyword(s):  
Neuronal Migration ◽  
Molecular Mechanisms ◽  
Mutant Mouse ◽  
Critical Role ◽  
Recurrent Mutation ◽  
Intrinsic Defect ◽  
Quantitative Mass Spectrometry ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
A Cell

Microtubules play a critical role in multiple aspects of neurodevelopment, including the generation, migration and differentiation of neurons. A recurrent mutation (R402H) in the α-tubulin gene TUBA1A is known to cause lissencephaly with cerebellar and striatal phenotypes. Previous work has shown that this mutation does not perturb the chaperone-mediated folding of tubulin heterodimers, which are able to assemble and incorporate into the microtubule lattice. To explore the molecular mechanisms that cause the disease state we generated a new conditional mouse line that recapitulates the R402H variant. We show that heterozygous mutants present with laminar phenotypes in the cortex and hippocampus, as well as a reduction in striatal size and cerebellar abnormalities. We demonstrate that homozygous expression of the R402H allele causes neuronal death and exacerbates a cell intrinsic defect in cortical neuronal migration. Microtubule sedimentation assays coupled with quantitative mass spectrometry demonstrated that the binding and/or levels of multiple microtubule associated proteins (MAPs) are perturbed by the R402H mutation including VAPB, REEP1, EZRIN, PRNP and DYNC1l1/2. Consistent with these data we show that the R402H mutation impairs dynein-mediated transport which is associated with a decoupling of the nucleus to the microtubule organising center. Our data support a model whereby the R402H variant is able to fold and incorporate into microtubules, but acts as a gain of function by perturbing the binding of MAPs.

scholarly journals New Insights Into Functions of IQ67-Domain Proteins

2021 ◽  
Vol 11 ◽  
Author(s):  
Chunyue Guo ◽  
Jun Zhou ◽  
Dengwen Li
Keyword(s):  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Critical Role ◽  
Biological Functions ◽  
Microtubule Associated Proteins ◽  
Calmodulin Binding ◽  
Associated Proteins ◽  
Review Current ◽  
Iq67 Domain

IQ67-domain (IQD) proteins, first identified in Arabidopsis and rice, are plant-specific calmodulin-binding proteins containing highly conserved motifs. They play a critical role in plant defenses, organ development and shape, and drought tolerance. Driven by comprehensive genome identification and analysis efforts, IQDs have now been characterized in several species and have been shown to act as microtubule-associated proteins, participating in microtubule-related signaling pathways. However, the precise molecular mechanisms underpinning their biological functions remain incompletely understood. Here we review current knowledge on how IQD family members are thought to regulate plant growth and development by affecting microtubule dynamics or participating in microtubule-related signaling pathways in different plant species and propose some new insights.

529. PROPROTEIN CONVERTASE 6 PLAYS A CRITICAL ROLE IN MODULATING THE HUMAN ENDOMETRIAL EPITHELIUM FOR RECEPTIVITY AND IMPLANTATION

2009 ◽  
Vol 21 (9) ◽  
pp. 128
Author(s):  
G. Nie ◽  
Y. Li ◽  
L. A. Salamonsen ◽  
C. Simon ◽  
A. Quiñonero ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Cell Model ◽  
Embryo Implantation ◽  
Critical Role ◽  
Proprotein Convertase ◽  
Gynecological Disorders ◽  
Proteomics Technology ◽  
A Cell ◽  
Endometrial Epithelium

Successful embryo implantation is an important step in establishing pregnancy, requiring a healthy embryo and a receptive endometrium. Establishment of endometrial receptivity involves morphological and physiological changes initially in the endometrial epithelium, but the underlying molecular mechanisms are not fully understood. We have previously demonstrated that proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is up-regulated in the endometrium specifically at implantation in association with epithelial differentiation, in the human and monkey. PCs convert a range of precursor proteins of important functions into their bioactive forms; they are thus regarded as critical “master switch” molecules. The present study aimed to determine whether PC6 is a critical regulator in the endometrial epithelium for receptivity and implantation. We examined whether endometrial epithelial PC6 dys-regulation is associated with implantation failure in women and whether knockdown of PC6 by siRNA in human endometrial epithelial cells affects embryo adhesion in a cell culture model. Endometrial PC6 expression was assessed by immunohistochemistry in the mid-secretory phase of the menstrual cycle (receptive phase) in two unique clinical cohorts comprising women of known fertility and infertility (with no obvious gynecological disorders, and with fertile males). Endometrial epithelial PC6 levels were significantly lower in infertile vs fertile women in both cohorts. To further establish that PC6 is important for receptivity, a cell model relevant to human implantation was used involving co-culture of uterine epithelial cells with mouse embryos. The epithelial cells were stably transfected with PC6 siRNA and PC6 knock down was confirmed at the levels of mRNA, protein, and activity by real-time RT-PCR, Western blotting and activity assay respectively. Embryos readily adhered to normal epithelial cells, but the adhesion was significantly reduced in the PC6 knockdown epithelial cells. We are currently using proteomics technology to identify the pathways affected by PC6 knockdown. These results strongly suggest that PC6 plays a critical role in modulating the human endometrial epithelium for receptivity and implantation.

scholarly journals Defects in Axonal Elongation and Neuronal Migration in Mice with Disrupted tau and map1b Genes

2000 ◽  
Vol 150 (5) ◽  
pp. 989-1000 ◽  
Author(s):  
Yosuke Takei ◽  
Junlin Teng ◽  
Akihiro Harada ◽  
Nobutaka Hirokawa
Keyword(s):  
Knockout Mice ◽  
Neuronal Migration ◽  
Hippocampal Neurons ◽  
Primary Cultures ◽  
Double Knockout ◽  
Microtubule Organization ◽  
Axonal Elongation ◽  
Microtubule Associated Proteins ◽  
Associated Proteins

Tau and MAP1B are the main members of neuronal microtubule-associated proteins (MAPs), the functions of which have remained obscure because of a putative functional redundancy (Harada, A., K. Oguchi, S. Okabe, J. Kuno, S. Terada, T. Ohshima, R. Sato-Yoshitake, Y. Takei, T. Noda, and N. Hirokawa. 1994. Nature. 369:488–491; Takei, Y., S. Kondo, A. Harada, S. Inomata, T. Noda, and N. Hirokawa. 1997. J. Cell Biol. 137:1615–1626). To unmask the role of these proteins, we generated double-knockout mice with disrupted tau and map1b genes and compared their phenotypes with those of single-knockout mice. In the analysis of mice with a genetic background of predominantly C57Bl/6J, a hypoplastic commissural axon tract and disorganized neuronal layering were observed in the brains of the tau+/+map1b−/− mice. These phenotypes are markedly more severe in tau−/−map1b−/− double mutants, indicating that tau and MAP1B act in a synergistic fashion. Primary cultures of hippocampal neurons from tau−/−map1b−/− mice showed inhibited axonal elongation. In these cells, a generation of new axons via bundling of microtubules at the neck of the growth cones appeared to be disturbed. Cultured cerebellar neurons from tau−/−map1b−/− mice showed delayed neuronal migration concomitant with suppressed neurite elongation. These findings indicate the cooperative functions of tau and MAP1B in vivo in axonal elongation and neuronal migration as regulators of microtubule organization.

scholarly journals c-Myc–mediated control of cell fate in megakaryocyte-erythrocyte progenitors

Blood ◽  
2009 ◽  
Vol 114 (10) ◽  
pp. 2097-2106 ◽  
Author(s):  
Yinshi Guo ◽  
Chao Niu ◽  
Peter Breslin ◽  
Minghui Tang ◽  
Shubin Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cell Fate ◽  
Critical Role ◽  
Fold Increase ◽  
Intrinsic Defect ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
A Cell ◽  
B And T Lymphocytes

Abstract It has been found that c-Myc protein plays a critical role in controlling self-renewal versus differentiation in hematopoietic stem cells. We report that c-Myc also controls the fate of megakaryocyte-erythrocyte progenitors through regulating the differentiation of erythroid and megakaryocytic progenitors. In addition to the significant reduction of granulocytes/macrophages and B and T lymphocytes because of the reduction of their corresponding progenitors, we found significantly increased numbers of megakaryocytic progenitors and mature megakaryocytes in bone marrow and spleens of c-Myc-knockout (c-Myc−/−) mice. Differentiation of erythrocytes was blocked at the erythroid progenitor stage. This increased megakaryocytopoiesis is a cell-intrinsic defect of c-Myc-mutant hematopoietic stem cells, as shown by transplantation studies. Furthermore, we found that c-Myc is required for polyploidy formation but not for cytoplasmic maturation of megakaryocytes. Megakaryocytes from c-Myc−/− mice are significantly smaller in size and lower in ploidy than those of control mice; however, because of the dramatic increase in megakaryocyte number, although fewer platelets are produced by each megakaryocyte, a greater than 3-fold increase in platelet number was consistently observed in c-Myc−/− mice. Thus, c-Myc−/− mice develop a syndrome of severe thrombocytosis-anemia-leukopenia because of significant increases in megakaryocytopoiesis and concomitant blockage of erythrocyte differentiation and reductions in myelolymphopoiesis.

Tubulin polyglutamylase: isozymic variants and regulation during the cell cycle in HeLa cells

1999 ◽  
Vol 112 (23) ◽  
pp. 4281-4289 ◽  
Author(s):  
C. Regnard ◽  
E. Desbruyeres ◽  
P. Denoulet ◽  
B. Edde
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Molecular Motors ◽  
Dependent Manner ◽  
Proliferating Cells ◽  
Beta Tubulin ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
A Cell ◽  
Cycle Dependent

Polyglutamylation is a posttranslational modification of tubulin that is very common in neurons and ciliated or flagellated cells. It was proposed to regulate the binding of microtubule associated proteins (MAPs) and molecular motors as a function of the length of the polyglutamyl side-chain. Though much less common, this modification of tubulin also occurs in proliferating cells like HeLa cells where it is associated with centrioles and with the mitotic spindle. Recently, we partially purified tubulin polyglutamylase from mouse brain and described its enzymatic properties. In this work, we focused on tubulin polyglutamylase activity from HeLa cells. Our results support the existence of a tubulin polyglutamylase family composed of several isozymic variants specific for alpha- or beta-tubulin subunits. In the latter case, the specificity probably also concerns the different beta-tubulin isotypes. Interestingly, we found that tubulin polyglutamylase activity is regulated in a cell cycle dependent manner and peaks in G(2)-phase while the level of glutamylated tubulin peaks in mitosis. Consistent results were obtained by treating the cells with hydroxyurea, nocodazole or taxotere. In particular, in mitotic cells, tubulin polyglutamylase activity was always low while glutamylation level was high. Finally, tubulin polyglutamylase activity and the level of glutamylated tubulin appeared to be inversely related. This paradox suggests a complex regulation of both tubulin polyglutamylase and the reverse deglutamylase activity.

scholarly journals The catenin/cadherin adhesion system is localized in synaptic junctions bordering transmitter release zones.

1996 ◽  
Vol 135 (3) ◽  
pp. 767-779 ◽  
Author(s):  
N Uchida ◽  
Y Honjo ◽  
K R Johnson ◽  
M J Wheelock ◽  
M Takeichi
Keyword(s):  
Transmitter Release ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Adherens Junction ◽  
Ultrastructural Level ◽  
Beta Catenin ◽  
Adhesion System ◽  
Associated Proteins ◽  
Synaptic Junctions ◽  
Similar Site

Molecular mechanisms linking pre- and postsynaptic membranes at the interneuronal synapses are little known. We tested the cadherin adhesion system for its localization in synapses of mouse and chick brains. We found that two classes of cadherin-associated proteins, alpha N- and beta-catenin, are broadly distributed in adult brains, colocalizing with a synaptic marker, synaptophysin. At the ultrastructural level, these proteins were localized in synaptic junctions of various types, forming a symmetrical adhesion structure. These structures sharply bordered the transmitter release sites associated with synaptic vesicles, although their segregation was less clear in certain types of synapses. N-cadherin was also localized at a similar site of synaptic junctions but in restricted brain nuclei. In developing synapses, the catenin-bearing contacts dominated their junctional structures. These findings demonstrate that interneuronal synaptic junctions comprise two subdomains, transmitter release zone and catenin-based adherens junction. The catenins localized in these junctions are likely associated with certain cadherin molecules including N-cadherin, and the cadherin/ catenin complex may play a critical role in the formation or maintenance of synaptic junctions.

scholarly journals LIS1 and DCX: Implications for Brain Development and Human Disease in Relation to Microtubules

Scientifica ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-17 ◽  
Author(s):  
Orly Reiner
Keyword(s):  
Brain Development ◽  
Neuronal Migration ◽  
Place Of Birth ◽  
Microtubule Associated Proteins ◽  
Final Destination ◽  
Brain Malformations ◽  
Wide Range ◽  
Associated Functions ◽  
Associated Proteins ◽  
Neuronal Polarization

Proper lamination of the cerebral cortex requires the orchestrated motility of neurons from their place of birth to their final destination. Improper neuronal migration may result in a wide range of diseases, including brain malformations, such as lissencephaly, mental retardation, schizophrenia, and autism. Ours and other studies have implicated that microtubules and microtubule-associated proteins play an important role in the regulation of neuronal polarization and neuronal migration. Here, we will review normal processes of brain development and neuronal migration, describe neuronal migration diseases, and will focus on the microtubule-associated functions of LIS1 and DCX, which participate in the regulation of neuronal migration and are involved in the human developmental brain disease, lissencephaly.

scholarly journals Human CLASP2 specifically regulates microtubule catastrophe and rescue

2018 ◽  
Vol 29 (10) ◽  
pp. 1168-1177 ◽  
Author(s):  
Elizabeth J. Lawrence ◽  
Göker Arpag˘ ◽  
Stephen R. Norris ◽  
Marija Zanic
Keyword(s):  
Growth Rates ◽  
Molecular Mechanisms ◽  
Direct Interaction ◽  
Microtubule Dynamics ◽  
Microtubule Associated Proteins ◽  
Cellular Processes ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Protein Components

Cytoplasmic linker-associated proteins (CLASPs) are microtubule-associated proteins essential for microtubule regulation in many cellular processes. However, the molecular mechanisms underlying CLASP activity are not understood. Here, we use purified protein components and total internal reflection fluorescence microscopy to investigate the effects of human CLASP2 on microtubule dynamics in vitro. We demonstrate that CLASP2 suppresses microtubule catastrophe and promotes rescue without affecting the rates of microtubule growth or shrinkage. Strikingly, when CLASP2 is combined with EB1, a known binding partner, the effects on microtubule dynamics are strongly enhanced. We show that synergy between CLASP2 and EB1 is dependent on a direct interaction, since a truncated EB1 protein that lacks the CLASP2-binding domain does not enhance CLASP2 activity. Further, we find that EB1 targets CLASP2 to microtubules and increases the dwell time of CLASP2 at microtubule tips. Although the temporally averaged microtubule growth rates are unaffected by CLASP2, we find that microtubules grown with CLASP2 display greater variability in growth rates. Our results provide insight into the regulation of microtubule dynamics by CLASP proteins and highlight the importance of the functional interplay between regulatory proteins at dynamic microtubule ends.

Dis1/TOG universal microtubule adaptors - one MAP for all?

2001 ◽  
Vol 114 (21) ◽  
pp. 3805-3812 ◽  
Author(s):  
Hiroyuki Ohkura ◽  
Miguel A. Garcia ◽  
Takashi Toda
Keyword(s):  
Molecular Mechanisms ◽  
Binding Activity ◽  
Mitotic Spindles ◽  
Microtubule Associated Proteins ◽  
Cellular Processes ◽  
Microtubule Motors ◽  
Associated Proteins ◽  
On Line ◽  
And Function ◽  
Centrosomal Proteins

Microtubules play central roles in various cellular processes in eukaryotes. The dynamics and organisation of interphase microtubules and mitotic spindles are dramatically altered during the cell cycle and development. However, the molecular mechanisms underlying this dynamic behaviour remain to be understood. In recent years, a novel family of microtubule-associated proteins (MAPs), the Dis1/TOG family, has emerged as a versatile regulator of microtubule function. These MAPs are highly conserved in eukaryotes from yeasts and plants to humans. The localisation and function of these MAPs are not determined simply by their intrinsic microtubule-binding activity. Instead this family executes its diverse roles by interacting with other regulatory molecules, including microtubule motors and centrosomal proteins. The modular structure of these MAPs may allow them to interact with multiple proteins and thereby be involved in a wide variety of microtubule and spindle functions. Movies available on-line

scholarly journals The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin

2021 ◽  
Author(s):  
Charlotte Repton ◽  
C Fiona Cullen ◽  
Mariana FA Costa ◽  
Christos Spanos ◽  
Juri Rappsilber ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Regulatory Mechanism ◽  
Mass Spectrometry Data ◽  
Quantitative Mass Spectrometry ◽  
Microtubule Associated Proteins ◽  
Chromosomal Passenger ◽  
Associated Proteins ◽  
And Function ◽  
Systematic Identification ◽  
Docking Protein

Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. We previously uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. Here we report systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, we identified proteins or complexes whose ability to binding microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. We showed that 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function. The mass spectrometry data are available from ProteomeXchange, identifier ID to be provided when available, PXD000xxx.

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