scholarly journals The mitotic spindle protein CKAP2 potently increases formation and stability of microtubules

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Thomas S McAlear ◽  
Susanne Bechstedt
Keyword(s):  
Cell Division ◽  
Growth Factor ◽  
Mitotic Spindle ◽  
Apparent Rate Constant ◽  
Microtubule Dynamics ◽  
Proliferation Marker ◽  
Microtubule Growth ◽  
Large Rearrangements ◽  
And Function

Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2's role as an important spindle protein, proliferation marker, and oncogene.

scholarly journals The contribution of αβ-tubulin curvature to microtubule dynamics

2014 ◽  
Vol 207 (3) ◽  
pp. 323-334 ◽  
Author(s):  
Gary J. Brouhard ◽  
Luke M. Rice
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Fundamental Property ◽  
Microtubule Dynamics ◽  
Cellular Structures ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Microtubule Networks ◽  
And Control

Microtubules are dynamic polymers of αβ-tubulin that form diverse cellular structures, such as the mitotic spindle for cell division, the backbone of neurons, and axonemes. To control the architecture of microtubule networks, microtubule-associated proteins (MAPs) and motor proteins regulate microtubule growth, shrinkage, and the transitions between these states. Recent evidence shows that many MAPs exert their effects by selectively binding to distinct conformations of polymerized or unpolymerized αβ-tubulin. The ability of αβ-tubulin to adopt distinct conformations contributes to the intrinsic polymerization dynamics of microtubules. αβ-Tubulin conformation is a fundamental property that MAPs monitor and control to build proper microtubule networks.

scholarly journals Systems cell biology of the mitotic spindle

2010 ◽  
Vol 188 (1) ◽  
pp. 7-9
Author(s):  
Ramsey A. Saleem ◽  
John D. Aitchison
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Cell Biology ◽  
Systems Genetics ◽  
Mitotic Spindles ◽  
High Content Imaging ◽  
Controlled Assembly ◽  
Daughter Cells ◽  
And Function ◽  
Insight Into

Cell division depends critically on the temporally controlled assembly of mitotic spindles, which are responsible for the distribution of duplicated chromosomes to each of the two daughter cells. To gain insight into the process, Vizeacoumar et al., in this issue (Vizeacoumar et al. 2010. J. Cell Biol. doi:10.1083/jcb.200909013), have combined systems genetics with high-throughput and high-content imaging to comprehensively identify and classify novel components that contribute to the morphology and function of the mitotic spindle.

scholarly journals Class V β-tubulin alters dynamic instability and stimulates microtubule detachment from centrosomes

2011 ◽  
Vol 22 (7) ◽  
pp. 1025-1034 ◽  
Author(s):  
Rajat Bhattacharya ◽  
Hailing Yang ◽  
Fernando Cabral
Keyword(s):  
Cell Division ◽  
Dynamic Instability ◽  
Microtubule Assembly ◽  
Class V ◽  
Tubulin Isotypes ◽  
Specific Manner ◽  
Microtubule Growth ◽  
A Minor ◽  
And Function

A multigene family produces tubulin isotypes that are expressed in a tissue-specific manner, but the role of these isotypes in microtubule assembly and function is unclear. Recently we showed that overexpression or depletion of β5-tubulin, a minor isotype with wide tissue distribution, inhibits cell division. We now report that elevated β5-tubulin causes uninterrupted episodes of microtubule shortening and increased shortening rates. Conversely, depletion of β5-tubulin reduces shortening rates and causes very short excursions of growth and shortening. A tubulin conformation-sensitive antibody indicated that the uninterrupted shortening can be explained by a relative absence of stabilized patches along the microtubules that contain tubulin in an assembly-competent conformation and normally act to restore microtubule growth. In addition to these changes in dynamic instability, overexpression of β5-tubulin causes fragmentation that results from microtubule detachment from centrosomes, and it is this activity that best explains the effects of β5 on cell division. Paclitaxel inhibits microtubule detachment, increases the number of assembly-competent tubulin patches, and inhibits microtubule shortening, thus providing an explanation for why the drug can counteract the phenotypic effects of β5 overexpression. On the basis of these observations, we propose that cells can use β5-tubulin expression to adjust the behavior of the microtubule cytoskeleton.

scholarly journals Effect of nerve growth factor on the proliferation in newborn bovine testicular Sertoli cells

Reproduction ◽  
2020 ◽  
Vol 160 (3) ◽  
pp. 405-415
Author(s):  
Qiaoge Niu ◽  
Maosheng Cao ◽  
Chenfeng Yuan ◽  
Yuwen Huang ◽  
Zijiao Zhao ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Male Reproduction ◽  
Nerve Growth ◽  
And Function

Nerve growth factor (NGF) has been proved to play important roles in male reproductive physiology, but the molecular mechanisms of NGF action remain unclear. In this study, the effects of NGF on the growth of newborn bovine testicular Sertoli (NBS) cells and the related signaling pathways were investigated. The NBS cells were treated in vitro with NGF (100 ng/mL) for 18 h. The expression levels of cell proliferation related genes, INHBB, and cytoplasmic specialization related gene were determined using real-time PCR and Western blot. The roles of PI3K/AKT and MAPK/ERK pathways in NGF-induced cell proliferation were investigated. It was found that NGF regulates proliferation and function of NBS cells via its receptor NTRK1 by activating the PI3K/ATK and MAPK/ERK signaling pathways. The study will help to further understand the role of NGF in male reproduction and provide new therapeutic targets for reproductive dysfunctions in male animals.

scholarly journals Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

2014 ◽  
Vol 204 (7) ◽  
pp. 1111-1121 ◽  
Author(s):  
Emmanuel Gallaud ◽  
Renaud Caous ◽  
Aude Pascal ◽  
Franck Bazile ◽  
Jean-Philippe Gagné ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Microtubule Associated Proteins ◽  
Microtubule Polymerization ◽  
Sister Chromatids ◽  
Centrosome Separation ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Mitotic Spindle Assembly

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

Effect of Epidermal Growth Factor on Adult Rat Hepatocyte Monolayer Cultures

1980 ◽  
Vol 38 ◽  
pp. 778-779
Author(s):  
W. A. Samsonoff ◽  
R. Jansing
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Function ◽  
Parenchymal Cell ◽  
Male Rats ◽  
Monolayer Cultures ◽  
Epidermal Growth ◽  
And Function ◽  
Hepatocyte Monolayer

Primary hepatocyte monolayer cultures have proven useful for in vitro study of parenchymal cell function. Unfortunately these cultures lose liver function rapidly and are often short-lived. Modified subtrates (1,2) can overcome some of these problems, but they produce fewer viable cells. Specific media supplements can also prolong cellular function. Epidermal growth factor (EGF) for example, significantly affects the structure and function of cells in culture (3). In this study we determined the effect of EGF on the ultrastructure and function of hepatocytes from Fisher 344 adult male rats. They were maintained as primary monolayer cultures in Leibovitz L-15 medium with supplements.

Mitotic Spindle Dysfunction Promotes Genomic Instability In Marrow Failure

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 880-880 ◽  
Author(s):  
Katie Lombardo ◽  
Jason Stumpff ◽  
Susan Parkhurst ◽  
Linda Wordeman ◽  
Akiko Shimamura
Keyword(s):  
Genomic Instability ◽  
Mitotic Spindle ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Microtubule Dynamics ◽  
Published Data ◽  
Hematopoietic Progenitor ◽  
Microtubule Stability ◽  
Cd34 Cells

Abstract Abstract 880 Shwachman-Diamond syndrome (SDS) is an autosomal recessively inherited disorder associated with bone marrow failure and leukemia predisposition. The majority of patients harbor biallelic mutations in the SBDS gene. The SBDS protein has been implicated in several cellular functions including ribosome biogenesis and microtubule stabilization during mitosis. We have previously found that SBDS deficiency results in multipolar spindles, centrosome amplification and aneuploidy, implicating a role for SBDS in cell division. The mechanism by which SBDS functions to ensure proper spindle assembly and DNA segregation during mitosis remains unknown. Here we present evidence that SBDS functions to promote mitotic spindle stability both by directly modifying microtubule dynamics and through a microtubule crosslinking activity. Importantly, the microtubule stabilizing effects of SBDS appear to be essential for the growth and differentiation of hematopoietic progenitor cells. Specifically, we found that SBDS deficiency resulted in shortened mitotic spindle length and decreased spindle acetylation, a marker of microtubule stability. The loss of microtubule stability in the absence of SBDS function may be due to changes in microtubule dynamics or reduction in microtubule crosslinking activity, as we found that addition of recombinant purified wild-type SBDS to polymerized microtubules in vitro increases their polymerization rate and strongly promotes microtubule bundling. Interestingly, recombinant patient-derived missense mutant SBDS proteins showed a marked decrease in their microtubule bundling ability. To assess whether spindle destabilization contributes to marrow failure, we modeled hematopoiesis in the absence of SBDS in vitro. When SBDS expression was knocked down in human CD34+ cells, proliferation, differentiation, and hematopoietic progenitor colony formation were impaired, consistent with published data on primary marrow from SDS patients. The addition of taxol at concentrations that significantly impaired hematopoiesis in control CD34+ cells resulted in stable to improved hematopoiesis in the SBDS-deficient CD34+ cells. Based on these data, we hypothesize that spindle destabilization by SBDS loss promotes genomic instability, which in turn, contributes to marrow failure and leukemia predisposition. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mammalian end binding proteins control persistent microtubule growth

2009 ◽  
Vol 184 (5) ◽  
pp. 691-706 ◽  
Author(s):  
Yulia Komarova ◽  
Christian O. De Groot ◽  
Ilya Grigoriev ◽  
Susana Montenegro Gouveia ◽  
E. Laura Munteanu ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Microtubule Dynamics ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dimerization Domain ◽  
Negative Effect ◽  
Microtubule Growth ◽  
Tracking Behavior ◽  
In Cells

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.

Regulation of thymic epithelium by keratinocyte growth factor

Blood ◽  
2002 ◽  
Vol 100 (9) ◽  
pp. 3269-3278 ◽  
Author(s):  
Matthew Erickson ◽  
Stanislaw Morkowski ◽  
Sophie Lehar ◽  
Geoffrey Gillard ◽  
Courtney Beers ◽  
...  
Keyword(s):  
Growth Factor ◽  
Keratinocyte Growth Factor ◽  
Cathepsin L ◽  
Developmentally Regulated ◽  
Thymic Epithelium ◽  
Histocompatibility Complex ◽  
Fetal Thymic Organ Culture ◽  
And Function

Abstract Here we demonstrate that keratinocyte growth factor (KGF) and FGFR2IIIb signaling can affect development and function of thymic epithelium (TE) and that αβ-lineage thymocytes contribute to intrathymic levels of KGF. Thymocyte expression of KGF is developmentally regulated, being undetectable in CD3−4−8− thymocytes and expressed at highest levels by mature CD4 or CD8 thymocytes. Exposure of thymocyte-depleted fetal thymic lobes to KGF resulted in reduced thymic epithelial expression of class II major histocompatibility complex (MHC), invariant chain (Ii), and cathepsin L (CatL) molecules involved in thymocyte-positive selection and also stimulated expression of the cytokines interleukin 6 (IL-6) and thymic stromal-derived lymphopoietin (TSLP), while having little effect on IL-7 or stem cell factor expression. Within intact fetal thymic organ culture (FTOC), exogenous KGF impairs the generation of CD4 thymocytes. Two lines of evidence point to responsiveness of the medullary TE compartment to KGF and FGFR2IIIb signaling. First, the medullary compartment is expanded in intact FTOC exposed to KGF in vitro. Second, in the RAG-deficient thymus, where the thymocytes do not express detectable levels of KGF message, the hypoplastic medullary TE compartment can be expanded by administration of recombinant KGF in vivo. This expansion is accompanied by restoration of the normal profile of medullary TE–associated chemokine expression in the RAG2−/−thymus. Collectively, these findings point to a role for KGF and FGFR signaling in the development and function of thymic epithelium.

scholarly journals Thirty years of search and capture: The complex simplicity of mitotic spindle assembly

2015 ◽  
Vol 211 (6) ◽  
pp. 1103-1111 ◽  
Author(s):  
Rebecca Heald ◽  
Alexey Khodjakov
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Microtubule Dynamics ◽  
Adaptive Changes ◽  
Bipolar Spindle ◽  
Chromosome Architecture ◽  
Self Assembling ◽  
Motor Activities ◽  
Mitotic Spindle Assembly

Cell division is enacted by a microtubule-based, self-assembling macromolecular machine known as the mitotic spindle. In 1986, Kirschner and Mitchison proposed that by undergoing dynamic cycles of growth and disassembly, microtubules search for chromosomes. Capture of microtubules by the kinetochores progressively connects chromosomes to the bipolar spindle. 30 years later, “search and capture” remains the cornerstone of spindle assembly. However, a variety of facilitating mechanisms such as regulation of microtubule dynamics by diffusible gradients, spatially selective motor activities, and adaptive changes in chromosome architecture have been discovered. We discuss how these mechanisms ensure that the spindle assembles rapidly and with a minimal number of errors.

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