scholarly journals Functional Overlap of Microtubule Assembly Factors in Chromatin-Promoted Spindle Assembly

2009 ◽  
Vol 20 (11) ◽  
pp. 2766-2773 ◽  
Author(s):  
Aaron C. Groen ◽  
Thomas J. Maresca ◽  
Jesse C. Gatlin ◽  
Edward D. Salmon ◽  
Timothy J. Mitchison
Keyword(s):  
Mitotic Spindle ◽  
Animal Cell ◽  
Spindle Assembly ◽  
Functional Redundancy ◽  
The Other ◽  
Microtubule Assembly ◽  
Microtubule Polymerization ◽  
Egg Extract ◽  
Assembly Factors ◽  
Mitotic Spindle Assembly

Distinct pathways from centrosomes and chromatin are thought to contribute in parallel to microtubule nucleation and stabilization during animal cell mitotic spindle assembly, but their full mechanisms are not known. We investigated the function of three proposed nucleation/stabilization factors, TPX2, γ-tubulin and XMAP215, in chromatin-promoted assembly of anastral spindles in Xenopus laevis egg extract. In addition to conventional depletion-add back experiments, we tested whether factors could substitute for each other, indicative of functional redundancy. All three factors were required for microtubule polymerization and bipolar spindle assembly around chromatin beads. Depletion of TPX2 was partially rescued by the addition of excess XMAP215 or EB1, or inhibiting MCAK (a Kinesin-13). Depletion of either γ-tubulin or XMAP215 was partially rescued by adding back XMAP215, but not by adding any of the other factors. These data reveal functional redundancy between specific assembly factors in the chromatin pathway, suggesting individual proteins or pathways commonly viewed to be essential may not have entirely unique functions.

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 Coordination of adjacent domains mediates TACC3–ch-TOG–clathrin assembly and mitotic spindle binding

2013 ◽  
Vol 202 (3) ◽  
pp. 463-478 ◽  
Author(s):  
Fiona E. Hood ◽  
Samantha J. Williams ◽  
Selena G. Burgess ◽  
Mark W. Richards ◽  
Daniel Roth ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Coiled Coil ◽  
Spindle Assembly ◽  
The Other ◽  
Cross Linking ◽  
Aurora A ◽  
Dileucine Motif ◽  
Mitotic Spindle Assembly ◽  
Interaction Surface ◽  
Overexpressed Gene

Acomplex of transforming acidic coiled-coil protein 3 (TACC3), colonic and hepatic tumor overexpressed gene (ch-TOG), and clathrin has been implicated in mitotic spindle assembly and in the stabilization of kinetochore fibers by cross-linking microtubules. It is unclear how this complex binds microtubules and how the proteins in the complex interact with one another. TACC3 and clathrin have each been proposed to be the spindle recruitment factor. We have mapped the interactions within the complex and show that TACC3 and clathrin were interdependent for spindle recruitment, having to interact in order for either to be recruited to the spindle. The N-terminal domain of clathrin and the TACC domain of TACC3 in tandem made a microtubule interaction surface, coordinated by TACC3–clathrin binding. A dileucine motif and Aurora A–phosphorylated serine 558 on TACC3 bound to the “ankle” of clathrin. The other interaction within the complex involved a stutter in the TACC3 coiled-coil and a proposed novel sixth TOG domain in ch-TOG, which was required for microtubule localization of ch-TOG but not TACC3–clathrin.

scholarly journals Localized accumulation of tubulin during semi-open mitosis in the Caenorhabditis elegans embryo

2012 ◽  
Vol 23 (9) ◽  
pp. 1688-1699 ◽  
Author(s):  
Hanako Hayashi ◽  
Kenji Kimura ◽  
Akatsuki Kimura
Keyword(s):  
Caenorhabditis Elegans ◽  
Nuclear Envelope ◽  
Mitotic Spindle ◽  
Fluorescence Recovery After Photobleaching ◽  
Spindle Assembly ◽  
Small Gtpase ◽  
Microtubule Assembly ◽  
C Elegans ◽  
Nuclear Envelope Breakdown ◽  
Mitotic Spindle Assembly

The assembly of microtubules inside the cell is controlled both spatially and temporally. During mitosis, microtubule assembly must be activated locally at the nascent spindle region for mitotic spindle assembly to occur efficiently. In this paper, we report that mitotic spindle components, such as free tubulin subunits, accumulated in the nascent spindle region, independent of spindle formation in the Caenorhabditis elegans embryo. This accumulation coincided with nuclear envelope permeabilization, suggesting that permeabilization might trigger the accumulation. When permeabilization was induced earlier by knockdown of lamin, tubulin also accumulated earlier. The boundaries of the region of accumulation coincided with the remnant nuclear envelope, which remains after nuclear envelope breakdown in cells that undergo semi-open mitosis, such as those of C. elegans. Ran, a small GTPase protein, was required for tubulin accumulation. Fluorescence recovery after photobleaching analysis revealed that the accumulation was accompanied by an increase in the immobile fraction of free tubulin inside the remnant nuclear envelope. We propose that this newly identified mechanism of accumulation of free tubulin—and probably of other molecules—at the nascent spindle region contributes to efficient assembly of the mitotic spindle in the C. elegans embryo.

scholarly journals Novel insights into the mechanisms of mitotic spindle assembly by NEK kinases

2016 ◽  
Vol 3 (3) ◽  
pp. e1062952 ◽  
Author(s):  
Suzanna L. Prosser ◽  
Laura O'Regan ◽  
Andrew M. Fry
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Mitotic Spindle Assembly
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