scholarly journals The Spindle Midzone Microtubule-Associated Proteins Ase1p and Cin8p Affect the Number and Orientation of Astral Microtubules in Saccharomyces cerevisiae

Cell Cycle ◽  
2007 ◽  
Vol 6 (10) ◽  
pp. 1231-1241 ◽  
Author(s):  
Armand de Gramont ◽  
Leslie Barbour ◽  
Karen E. Ross ◽  
Orna Cohen-Fix
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Spindle Midzone

scholarly journals A TOGL domain specifically targets yeast CLASP to kinetochores to stabilize kinetochore microtubules

2014 ◽  
Vol 205 (4) ◽  
pp. 555-571 ◽  
Author(s):  
Caroline Funk ◽  
Verena Schmeiser ◽  
Jennifer Ortiz ◽  
Johannes Lechner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Associated Proteins ◽  
Spindle Midzone ◽  
Spindle Microtubules ◽  
The Stability ◽  
Overexpressed Gene

Cytoplasmic linker–associated proteins (CLASPs) are proposed to function in cell division based on their ability to bind tubulin via arrayed tumor overexpressed gene (TOG)–like (TOGL) domains. Structure predictions suggest that CLASPs have at least two TOGL domains. We show that only TOGL2 of Saccharomyces cerevisiae CLASP Stu1 binds to tubulin and is required for polymerization of spindle microtubules (MTs) in vivo. In contrast, TOGL1 recruits Stu1 to kinetochores (KTs), where it is essential for the stability and tension-dependent regulation of KT MTs. Stu1 is also recruited to spindle MTs by different mechanisms depending on the mitotic phase: in metaphase, Stu1 binds directly to the MT lattice, whereas in anaphase, it is localized indirectly to the spindle midzone. In both phases, the activity of TOGL2 is essential for interpolar MT stability, whereas TOGL1 is not involved. Thus, the two TOGL domains of yeast CLASP have different activities and execute distinct mitotic functions.

scholarly journals Two Microtubule-associated Proteins of Arabidopsis MAP65s Promote Antiparallel Microtubule Bundling

2008 ◽  
Vol 19 (10) ◽  
pp. 4534-4544 ◽  
Author(s):  
Jérémie Gaillard ◽  
Emmanuelle Neumann ◽  
Daniel Van Damme ◽  
Virginie Stoppin-Mellet ◽  
Christine Ebel ◽  
...  
Keyword(s):  
Preprophase Band ◽  
Spindle Microtubule ◽  
Microtubule Associated Proteins ◽  
Late Anaphase ◽  
Associated Proteins ◽  
Planar Network ◽  
Spindle Midzone ◽  
Spindle Microtubules

The Arabidopsis MAP65s are a protein family with similarity to the microtubule-associated proteins PRC1/Ase1p that accumulate in the spindle midzone during late anaphase in mammals and yeast, respectively. Here we investigate the molecular and functional properties of AtMAP65-5 and improve our understanding of AtMAP65-1 properties. We demonstrate that, in vitro, both proteins promote the formation of a planar network of antiparallel microtubules. In vivo, we show that AtMAP65-5 selectively binds the preprophase band and the prophase spindle microtubule during prophase, whereas AtMAP65-1-GFP selectively binds the preprophase band but does not accumulate at the prophase spindle microtubules that coexists within the same cell. At later stages of mitosis, AtMAP65-1 and AtMAP65-5 differentially label the late spindle and phragmoplast. We present evidence for a mode of action for both proteins that involves the binding of monomeric units to microtubules that “zipper up” antiparallel arranged microtubules through the homodimerization of the N-terminal halves when adjacent microtubules encounter.

scholarly journals Two modes of PRC1-mediated mechanical resistance to kinesin-driven microtubule network disruption

2020 ◽  
Author(s):  
April Alfieri ◽  
Ignas Gaska ◽  
Scott Forth
Keyword(s):  
Mechanical Load ◽  
Mechanical Resistance ◽  
Microtubule Network ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Spindle Midzone ◽  
Network Disruption ◽  
Molecular Brake ◽  
Insight Into ◽  
Do So

AbstractThe proper structural organization of the microtubule-based spindle during cell division requires the collective activity of many different types of proteins. These include non-motor microtubule-associated proteins (MAPs) whose functions include crosslinking microtubules to regulate filament sliding rates and assembling microtubule arrays. One such protein is PRC1, an essential MAP that has been shown to preferentially crosslink overlapping antiparallel microtubules at the spindle midzone. PRC1 has been proposed to act as a molecular brake, but insight into the mechanism of how PRC1 molecules function cooperatively to resist motor-driven microtubule sliding and to allow for the formation of stable midzone overlaps has been lacking. Here we employ a modified microtubule gliding assay to rupture PRC1-mediated microtubule pairs using surface-bound kinesins. We discovered that PRC1 crosslinks always reduce bundled filament sliding velocities relative to single microtubule gliding rates, and do so via two distinct emergent modes of mechanical resistance to motor-driven sliding. We term these behaviors braking and coasting, where braking events exhibit substantially slowed microtubule sliding compared to coasting events. Strikingly, braking behavior requires the formation of two distinct high-density clusters of PRC1 molecules near microtubule tips. Our results suggest a cooperative mechanism for PRC1 accumulation when under mechanical load that leads to a unique state of enhanced resistance to filament sliding and provides insight into collective protein ensemble behavior in regulating the mechanics of spindle assembly.

scholarly journals One-step purification of assembly-competent tubulin from diverse eukaryotic sources

2012 ◽  
Vol 23 (22) ◽  
pp. 4393-4401 ◽  
Author(s):  
Per O. Widlund ◽  
Marija Podolski ◽  
Simone Reber ◽  
Joshua Alper ◽  
Marko Storch ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Protein ◽  
High Capacity ◽  
Affinity Matrix ◽  
Related Research ◽  
Microtubule Associated Proteins ◽  
Mild Conditions ◽  
Cell Extracts ◽  
Associated Proteins ◽  
One Step

We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research.

scholarly journals Stu2p, the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end–binding microtubule destabilizer

2003 ◽  
Vol 161 (2) ◽  
pp. 359-369 ◽  
Author(s):  
Mark van Breugel ◽  
David Drechsel ◽  
Anthony Hyman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Quantitative Analysis ◽  
Family Member ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Biochemical Activity ◽  
Microtubule Associated Proteins ◽  
Associated Proteins

The Dis1/XMAP215 family of microtubule-associated proteins conserved from yeast to mammals is essential for cell division. XMAP215, the Xenopus member of this family, has been shown to stabilize microtubules in vitro, but other members of this family have not been biochemically characterized. Here we investigate the properties of the Saccharomyces cerevisiae homologue Stu2p in vitro. Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends. Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends. These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.

scholarly journals The multifunctional spindle midzone in vertebrate cells at a glance

2021 ◽  
Vol 134 (10) ◽  
Author(s):  
Patricia Wadsworth
Keyword(s):  
Chromosome Segregation ◽  
Future Research ◽  
Contractile Ring ◽  
Microtubule Associated Proteins ◽  
Ring Assembly ◽  
Open Questions ◽  
Associated Proteins ◽  
Spindle Midzone ◽  
Spindle Elongation ◽  
And Function

ABSTRACT During anaphase, a microtubule-containing structure called the midzone forms between the segregating chromosomes. The midzone is composed of an antiparallel array of microtubules and numerous microtubule-associated proteins that contribute to midzone formation and function. In many cells, the midzone is an important source of signals that specify the location of contractile ring assembly and constriction. The midzone also contributes to the events of anaphase by generating forces that impact chromosome segregation and spindle elongation; some midzone components contribute to both processes. The results of recent experiments have increased our understanding of the importance of the midzone, a microtubule array that has often been overlooked. This Journal of Cell Science at a Glance article will review, and illustrate on the accompanying poster, the organization, formation and dynamics of the midzone, and discuss open questions for future research.

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”).

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