scholarly journals Profilin—A master coordinator of actin and microtubule organization in mammalian cells

2021 ◽  
Author(s):  
Roger Karlsson ◽  
Pavel Dráber
Keyword(s):  
Mammalian Cells ◽  
Microtubule Organization

scholarly journals Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells

2002 ◽  
Vol 156 (1) ◽  
pp. 87-100 ◽  
Author(s):  
Toshiro Ohta ◽  
Russell Essner ◽  
Jung-Hwa Ryu ◽  
Robert E. Palazzo ◽  
Yumi Uetake ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Amino Acid Residues ◽  
Protein Overexpression ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Centrosomal Protein ◽  
Wide Range ◽  
Spindle Microtubules

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.

scholarly journals Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

2003 ◽  
Vol 162 (5) ◽  
pp. 757-764 ◽  
Author(s):  
Yasuhiko Terada ◽  
Yumi Uetake ◽  
Ryoko Kuriyama
Keyword(s):  
Mammalian Cells ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Centrosomal Protein ◽  
Spindle Poles

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

scholarly journals 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells

2020 ◽  
Vol 220 (2) ◽  
Author(s):  
Andreas Müller ◽  
Deborah Schmidt ◽  
C. Shan Xu ◽  
Song Pang ◽  
Joyson Verner D’Costa ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Function ◽  
Secretory Granules ◽  
Detailed Knowledge ◽  
Β Cells ◽  
Microtubule Organization ◽  
Supportive Role ◽  
Primary Mouse ◽  
Microtubule Networks ◽  
Insulin Secretory Granule

Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is under debate. Here, we use FIB-SEM to image islet β cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules, and microtubules of seven β cells, and generate a comprehensive spatial map of microtubule–organelle interactions. We find that microtubules form nonradial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane, where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus their supportive role in insulin secretion.

scholarly journals The Golgi Protein GM130 Regulates Centrosome Morphology and Function

2008 ◽  
Vol 19 (2) ◽  
pp. 745-753 ◽  
Author(s):  
Andrew Kodani ◽  
Christine Sütterlin
Keyword(s):  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Human Cell Lines ◽  
Microtubule Organizing Center ◽  
Microtubule Organization ◽  
Cycle Progression ◽  
Multipolar Spindles ◽  
Organizing Center ◽  
Golgi Protein ◽  
And Function

The Golgi apparatus (GA) of mammalian cells is positioned in the vicinity of the centrosome, the major microtubule organizing center of the cell. The significance of this physical proximity for organelle function and cell cycle progression is only beginning to being understood. We have identified a novel function for the GA protein, GM130, in the regulation of centrosome morphology, position and function during interphase. RNA interference–mediated depletion of GM130 from five human cell lines revealed abnormal interphase centrosomes that were mispositioned and defective with respect to microtubule organization and cell migration. When GM130-depleted cells entered mitosis, they formed multipolar spindles, arrested in metaphase, and died. We also detected aberrant centrosomes during interphase and multipolar spindles during mitosis in ldlG cells, which do not contain detectable GM130. Although GA proteins have been described to regulate mitotic centrosomes and spindle formation, this is the first report of a role for a GA protein in the regulation of centrosomes during interphase.

scholarly journals NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization

2003 ◽  
Vol 162 (6) ◽  
pp. 1017-1029 ◽  
Author(s):  
Tim Raemaekers ◽  
Katharina Ribbeck ◽  
Joël Beaudouin ◽  
Wim Annaert ◽  
Mark Van Camp ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Direct Interaction ◽  
Microscopic Analysis ◽  
Mitotic Spindles ◽  
Proliferating Cells ◽  
Microtubule Organization ◽  
Protein Levels ◽  
Microtubule Binding Domain ◽  
Interphase Cells

Here, we report on the identification of nucleolar spindle–associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.

scholarly journals A Ubiquitous β-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

2004 ◽  
Vol 15 (7) ◽  
pp. 3123-3131 ◽  
Author(s):  
Rajat Bhattacharya ◽  
Fernando Cabral
Keyword(s):  
Cell Proliferation ◽  
Mammalian Cells ◽  
Microtubule Assembly ◽  
Microtubule Organization ◽  
Class V ◽  
Tubulin Isotypes ◽  
Concomitant Reduction ◽  
Mitotic Spindle Assembly ◽  
Dose Dependent ◽  
Β Tubulin

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the α- and β-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 β-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 β-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V β-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V β-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.

scholarly journals Disruption of Microtubule Organization and Centrosome Function by Expression of Tobacco Mosaic Virus Movement Protein

2006 ◽  
Vol 80 (12) ◽  
pp. 5807-5821 ◽  
Author(s):  
Jacqueline Ferralli ◽  
Jamie Ashby ◽  
Monika Fasler ◽  
Vitaly Boyko ◽  
Manfred Heinlein
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Movement Protein ◽  
Infected Plant ◽  
In Vivo Studies ◽  
Microtubule Organization

ABSTRACT The movement protein (MP) of Tobacco mosaic virus mediates the cell-to-cell transport of viral RNA through plasmodesmata, cytoplasmic cell wall channels for direct cell-to-cell communication between adjacent cells. Previous in vivo studies demonstrated that the RNA transport function of the protein correlates with its association with microtubules, although the exact role of microtubules in the movement process remains unknown. Since the binding of MP to microtubules is conserved in transfected mammalian cells, we took advantage of available mammalian cell biology reagents and tools to further address the interaction in flat-growing and transparent COS-7 cells. We demonstrate that neither actin, nor endoplasmic reticulum (ER), nor dynein motor complexes are involved in the apparent alignment of MP with microtubules. Together with results of in vitro coprecipitation experiments, these findings indicate that MP binds microtubules directly. Unlike microtubules associated with neuronal MAP2c, MP-associated microtubules are resistant to disruption by microtubule-disrupting agents or cold, suggesting that MP is a specialized microtubule binding protein that forms unusually stable complexes with microtubules. MP-associated microtubules accumulate ER membranes, which is consistent with a proposed role for MP in the recruitment of membranes in infected plant cells and may suggest that microtubules are involved in this process. The ability of MP to interfere with centrosomal γ-tubulin is independent of microtubule association with MP, does not involve the removal of other tested centrosomal markers, and correlates with inhibition of centrosomal microtubule nucleation activity. These observations suggest that the function of MP in viral movement may involve interaction with the microtubule-nucleating machinery.

Overexpression of normal and mutant Arp1alpha (centractin) differentially affects microtubule organization during mitosis and interphase

1999 ◽  
Vol 112 (20) ◽  
pp. 3507-3518 ◽  
Author(s):  
I.B. Clark ◽  
D.I. Meyer
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Mitotic Apparatus ◽  
Mutant Proteins ◽  
Cell Extracts ◽  
Dynactin Complex

Dynactin is a large multisubunit complex that regulates cytoplasmic dynein-mediated functions. To gain insight into the role of dynactin's most abundant component, Arp1alpha was transiently overexpressed in mammalian cells. Arp1alpha overexpression resulted in a cell cycle delay at prometaphase. Intracellular dynactin, dynein and nuclear/mitotic apparatus (NuMA) protein were recruited to multiple foci associated with ectopic cytoplasmic aggregates of Arp1alpha in transfected cells. These ectopic aggregates nucleated supernumerary microtubule asters at prometaphase. Point mutations were generated in Arp1alpha that identified specific amino acids required for the prometaphase delay and for the formation of supernumerary microtubule asters. The mutant Arp1alpha proteins formed aggregates in cells that colocalized with dynactin and dynein peptides, but in contrast to wild-type Arp1alpha, NuMA localization remained unaffected. Although expression of mutant Arp1alpha proteins had no effect on mitotic cells, in interphase cells expression of the mutants resulted in disruption of the microtubule network. Immunoprecipitation studies demonstrated that overexpressed Arp1alpha interacts with dynactin and NuMA proteins in cell extracts, and that these interactions are destabilized in the Arp1alpha mutants. We conclude that the amino acids altered in the Arp1alpha mutant proteins participate in stabilizing interactions between overexpressed Arp1alpha and components of the endogenous dynactin complex as well as the NuMA protein.

scholarly journals CCDC74A/B are K-fiber crosslinkers required for chromosomal alignment

BMC Biology ◽  
2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Haining Zhou ◽  
Tao Zheng ◽  
Tianning Wang ◽  
Qi Li ◽  
Fulin Wang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genetic Material ◽  
Fiber Formation ◽  
Microtubule Organization ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Fiber Organization

Abstract Background Spindle microtubule organization, regulated by microtubule-associated proteins, is critical for cell division. Proper organization of kinetochore fiber (K-fiber), connecting spindle poles and kinetochores, is a prerequisite for precise chromosomal alignment and faithful genetic material transmission. However, the mechanisms of K-fiber organization and dynamic maintenance are still not fully understood. Results We reveal that two previously uncharacterized coiled-coil domain proteins CCDC74A and CCDC74B (CCDC74A/B) are spindle-localized proteins in mammalian cells. They bind directly to microtubules through two separate domains and bundle microtubules both in vivo and in vitro. These functions are required for K-fiber organization, bipolar spindle formation, and chromosomal alignment. Moreover, CCDC74A/B form homodimers in vivo, and their self-association activity is necessary for microtubule bundling and K-fiber formation. Conclusions We characterize CCDC74A and CCDC74B as microtubule-associated proteins that localize to spindles and are important K-fiber crosslinkers required for bipolar spindle formation and chromosome alignment.

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