scholarly journals Kinetochore–microtubule coupling mechanisms mediated by the Ska1 complex and Cdt1

2020 ◽  
Vol 64 (2) ◽  
pp. 337-347
Author(s):  
Amit Rahi ◽  
Manas Chakraborty ◽  
Kristen Vosberg ◽  
Dileep Varma
Keyword(s):  
Outer Layer ◽  
Binding Protein ◽  
Load Bearing ◽  
Microtubule Associated Proteins ◽  
Sister Chromatids ◽  
Recent Advances ◽  
Ndc80 Complex ◽  
Associated Proteins ◽  
Essential Function ◽  
Coupling Mechanisms

Abstract The faithful segregation of duplicated sister chromatids rely on the remarkable ability of kinetochores to sustain stable load bearing attachments with the dynamic plus ends of kinetochore–microtubules (kMTs). The outer layer of the kinetochore recruits several motor and non-motor microtubule-associated proteins (MAPs) that help the kinetochores establish and maintain a load bearing dynamic attachment with kMTs. The primary kMT-binding protein, the Ndc80 complex (Ndc80c), which is highly conserved among diverse organisms from yeast to humans, performs this essential function with assistance from other MAPs. These MAPs are not an integral part of the kinetochore, but they localize to the kinetochore periodically throughout mitosis and regulate the strength of the kinetochore microtubule attachments. Here, we attempt to summarize the recent advances that have been made toward furthering our understanding of this co-operation between the Ndc80c and these MAPs, focusing on the spindle and kinetochore-associated 1 (Ska1) complex (Ska1c) and Cdc10-dependent transcript 1 (Cdt1) in humans.

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.

scholarly journals KIF14 and citron kinase act together to promote efficient cytokinesis

2006 ◽  
Vol 172 (3) ◽  
pp. 363-372 ◽  
Author(s):  
Ulrike Gruneberg ◽  
Rüdiger Neef ◽  
Xiuling Li ◽  
Eunice H.Y. Chan ◽  
Ravindra B. Chalamalasetty ◽  
...  
Keyword(s):  
Cell Division ◽  
Human Cell ◽  
Cleavage Furrow ◽  
General Requirement ◽  
Central Spindle ◽  
Microtubule Associated Proteins ◽  
Cell Biol ◽  
Associated Proteins ◽  
Essential Function ◽  
Citron Kinase

Multiple mitotic kinesins and microtubule-associated proteins (MAPs) act in concert to direct cytokinesis (Glotzer, M. 2005. Science. 307:1735–1739). In anaphase cells, many of these proteins associate with an antiparallel array of microtubules termed the central spindle. The MAP and microtubule-bundling protein PRC1 (protein-regulating cytokinesis 1) is one of the key molecules required for the integrity of this structure (Jiang, W., G. Jimenez, N.J. Wells, T.J. Hope, G.M. Wahl, T. Hunter, and R. Fukunaga. 1998. Mol. Cell. 2:877–885; Mollinari, C., J.P. Kleman, W. Jiang, G. Schoehn, T. Hunter, and R.L. Margolis. 2002. J. Cell Biol. 157:1175–1186). In this study, we identify an interaction between endogenous PRC1 and the previously uncharacterized kinesin KIF14 as well as other mitotic kinesins (MKlp1/CHO1, MKlp2, and KIF4) with known functions in cytokinesis (Hill, E., M. Clarke, and F.A. Barr. 2000. EMBO J. 19:5711–5719; Matuliene, J., and R. Kuriyama. 2002. Mol. Biol. Cell. 13:1832–1845; Kurasawa, Y., W.C. Earnshaw, Y. Mochizuki, N. Dohmae, and K. Todokoro. 2004. EMBO J. 23:3237–3248). We find that KIF14 targets to the central spindle via its interaction with PRC1 and has an essential function in cytokinesis. In KIF14-depleted cells, citron kinase but not other components of the central spindle and cleavage furrow fail to localize. Furthermore, the localization of KIF14 and citron kinase to the central spindle and midbody is codependent, and they form a complex depending on the activation state of citron kinase. Contrary to a previous study (Di Cunto, F., S. Imarisio, E. Hirsch, V. Broccoli, A. Bulfone, A. Migheli, C. Atzori, E. Turco, R. Triolo, G.P. Dotto, et al. 2000. Neuron. 28:115–127), we find a general requirement for citron kinase in human cell division. Together, these findings identify a novel pathway required for efficient cytokinesis.

scholarly journals Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules

2004 ◽  
Vol 384 (2) ◽  
pp. 327-336 ◽  
Author(s):  
Josefa ANDRADE ◽  
Sandy Timm PEARCE ◽  
Hu ZHAO ◽  
Margarida BARROSO
Keyword(s):  
Binding Protein ◽  
Direct Interaction ◽  
Independent Manner ◽  
Microtubule Associated Proteins ◽  
Binding Partners ◽  
Ef Hand ◽  
Associated Proteins ◽  
Microsomal Membranes ◽  
Terminal Amino

Previously, we have shown that p22, an EF-hand Ca2+-binding protein, interacts indirectly with microtubules in an N-myristoylation-dependent and Ca2+-independent manner. In the present study, we report that N-myristoylated p22 interacts with several microtubule-associated proteins within the 30–100 kDa range using overlay blots of microtubule pellets containing cytosolic proteins. One of those p22-binding partners, a 35–40 kDa microtubule-binding protein, has been identified by MS as GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Several lines of evidence suggest a functional relationship between GAPDH and p22. First, endogenous p22 interacts with GAPDH by immunoprecipitation. Secondly, p22 and GAPDH align along microtubule tracks in analogous punctate structures in BHK cells. Thirdly, GAPDH facilitates the p22-dependent interactions between microtubules and microsomal membranes, by increasing the ability of p22 to bind microtubules but not membranes. We have also shown a direct interaction between N-myristoylated p22 and GAPDH in vitro with a KD of ∼0.5 μM. The removal of either the N-myristoyl group or the last six C-terminal amino acids abolishes the binding of p22 to GAPDH and reduces the ability of p22 to associate with microtubules. In summary, we report that GAPDH is involved in the ability of p22 to facilitate microtubule–membrane interactions by affecting the p22–microtubule, but not the p22–membrane, association.

scholarly journals Doublecortin facilitates the elongation of the somatic Golgi apparatus into proximal dendrites

2021 ◽  
pp. mbc.E19-09-0530
Author(s):  
Peijun Li ◽  
Luyao Li ◽  
Binyuan Yu ◽  
Xinye Wang ◽  
Qi Wang ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Binding Protein ◽  
Underlying Mechanism ◽  
Neurite Length ◽  
Microtubule Binding ◽  
Microtubule Associated Proteins ◽  
Cortical Malformations ◽  
Subcortical Band Heterotopia ◽  
Associated Proteins ◽  
Band Heterotopia

Mutations in the doublecortin ( DCX) gene, which encodes a microtubule-binding protein, cause human cortical malformations, including lissencephaly and subcortical band heterotopia. A deficiency in DCX and doublecortin-like kinase 1 (DCLK1), a functionally redundant and structurally similar cognate of DCX, decreases neurite length and increases the number of primary neurites directly arising from the soma. The underlying mechanism is not completely understood. In this study, the elongation of the somatic Golgi apparatus into proximal dendrites, which have been implicated in dendrite patterning, was significantly decreased in the absence of DCX/DCLK1. Phosphorylation of DCX at S47 or S327 was involved in this process. DCX deficiency shifted the distribution of CLASP2 proteins to the soma from the dendrites. In addition to CLASP2, dynein and its co-factor JIP3 were abnormally distributed in DCX-deficient neurons. The association between JIP3 and dynein was significantly increased in the absence of DCX. Downregulation of CLASP2 or JIP3 expression with specific shRNAs rescued the Golgi phenotype observed in DCX-deficient neurons. We conclude that DCX regulates the elongation of the Golgi apparatus into proximal dendrites through microtubule-associated proteins and motors.

The Physics of the Metaphase Spindle

2018 ◽  
Vol 47 (1) ◽  
pp. 655-673 ◽  
Author(s):  
David Oriola ◽  
Daniel J. Needleman ◽  
Jan Brugués
Keyword(s):  
Mitotic Spindle ◽  
The Self ◽  
Current Understanding ◽  
Bipolar Structure ◽  
Collective Behaviors ◽  
Microtubule Associated Proteins ◽  
Self Organized ◽  
Sister Chromatids ◽  
Associated Proteins ◽  
Organized Action

The assembly of the mitotic spindle and the subsequent segregation of sister chromatids are based on the self-organized action of microtubule filaments, motor proteins, and other microtubule-associated proteins, which constitute the fundamental force-generating elements in the system. Many of the components in the spindle have been identified, but until recently it remained unclear how their collective behaviors resulted in such a robust bipolar structure. Here, we review the current understanding of the physics of the metaphase spindle that is only now starting to emerge.

Characterization of microtubules by dark-field STEM and replication

1991 ◽  
Vol 49 ◽  
pp. 152-153
Author(s):  
S.B. Andrews ◽  
R.D. Leapman ◽  
P.E. Gallant ◽  
T.S. Reese
Keyword(s):  
Protein Interactions ◽  
Low Dose ◽  
Unit Length ◽  
Dark Field ◽  
Carbon Films ◽  
Microtubule Associated Proteins ◽  
Molecular Weights ◽  
Freeze Dried ◽  
Protein Motors ◽  
Associated Proteins

As part of a study on protein interactions involved in microtubule (MT)-based transport, we used the VG HB501 field-emission STEM to obtain low-dose dark-field mass maps of isolated, taxol-stabilized MTs and correlated these micrographs with detailed stereo images from replicas of the same MTs. This approach promises to be useful for determining how protein motors interact with MTs. MTs prepared from bovine and squid brain tubulin were purified and free from microtubule-associated proteins (MAPs). These MTs (0.1-1 mg/ml tubulin) were adsorbed to 3-nm evaporated carbon films supported over Formvar nets on 600-m copper grids. Following adsorption, the grids were washed twice in buffer and then in either distilled water or in isotonic or hypotonic ammonium acetate, blotted, and plunge-frozen in ethane/propane cryogen (ca. -185 C). After cryotransfer into the STEM, specimens were freeze-dried and recooled to ca.-160 C for low-dose (<3000 e/nm2) dark-field mapping. The molecular weights per unit length of MT were determined relative to tobacco mosaic virus standards from elastic scattering intensities. Parallel grids were freeze-dried and rotary shadowed with Pt/C at 14°.

Filaments and membranes in the mitotic apparatus

1983 ◽  
Vol 41 ◽  
pp. 544-547
Author(s):  
Kent McDonald
Keyword(s):  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mitotic Apparatus ◽  
Light Microscope Level ◽  
Microtubule Associated Proteins ◽  
Recent Developments ◽  
Associated Proteins ◽  
Force Generator ◽  
Spindle Function ◽  
Antibody Technology

At the light microscope level the recent developments and interest in antibody technology have permitted the localization of certain non-microtubule proteins within the mitotic spindle, e.g., calmodulin, actin, intermediate filaments, protein kinases and various microtubule associated proteins. Also, the use of fluorescent probes like chlorotetracycline suggest the presence of membranes in the spindle. Localization of non-microtubule structures in the spindle at the EM level has been less rewarding. Some mitosis researchers, e.g., Rarer, have maintained that actin is involved in mitosis movements though the bulk of evidence argues against this interpretation. Others suggest that a microtrabecular network such as found in chromatophore granule movement might be a possible force generator but there is little evidence for or against this view. At the level of regulation of spindle function, Harris and more recently Hepler have argued for the importance of studying spindle membranes. Hepler also believes that membranes might play a structural or mechanical role in moving chromosomes.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

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