scholarly journals An extended γ-tubulin ring functions as a stable platform in microtubule nucleation

2012 ◽  
Vol 197 (1) ◽  
pp. 59-74 ◽  
Author(s):  
Sarah Erlemann ◽  
Annett Neuner ◽  
Linda Gombos ◽  
Romain Gibeaux ◽  
Claude Antony ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Fluorescence Recovery After Photobleaching ◽  
Nucleation Site ◽  
Fluorescence Recovery ◽  
Microtubule Nucleation ◽  
Nucleation Sites ◽  
Small Complex ◽  
Tubulin Binding

γ-Tubulin complexes are essential for microtubule (MT) nucleation. The γ-tubulin small complex (γ-TuSC) consists of two molecules of γ-tubulin and one molecule each of Spc97 and Spc98. In vitro, γ-TuSCs oligomerize into spirals of 13 γ-tubulin molecules per turn. However, the properties and numbers of γ-TuSCs at MT nucleation sites in vivo are unclear. In this paper, we show by fluorescence recovery after photobleaching analysis that γ-tubulin was stably integrated into MT nucleation sites and was further stabilized by tubulin binding. Importantly, tubulin showed a stronger interaction with the nucleation site than with the MT plus end, which probably provides the basis for MT nucleation. Quantitative analysis of γ-TuSCs on single MT minus ends argued for nucleation sites consisting of approximately seven γ-TuSCs with approximately three additional γ-tubulin molecules. Nucleation and anchoring of MTs required the same number of γ-tubulin molecules. We suggest that a spiral of seven γ-TuSCs with a slight surplus of γ-tubulin nucleates MTs in vivo.

scholarly journals Microtubule associated proteins and motors required for ectopic microtubule array formation in S. cerevisiae

2021 ◽  
Author(s):  
Brianna R. King ◽  
Janet B. Meehl ◽  
Tamira Vojnar ◽  
Mark Winey ◽  
Eric G. Muller ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Polymerase Activity ◽  
Nucleation Site ◽  
Microtubule Nucleation ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
A Subunit ◽  
Nucleation Activity

AbstractThe mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules, and the organization of microtubules relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Further, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.

scholarly journals Microtubule associated proteins and motors required for ectopic microtubule array formation in S. cerevisiae

Genetics ◽  
2021 ◽  
Author(s):  
Brianna R King ◽  
Janet B Meehl ◽  
Tamira Vojnar ◽  
Mark Winey ◽  
Eric G Muller ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Polymerase Activity ◽  
Nucleation Site ◽  
Microtubule Nucleation ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
A Subunit ◽  
Nucleation Activity

Abstract The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules, and the organization of microtubules relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Further, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.

scholarly journals Improved Model of Fluorescence Recovery Expands the Application of Multiphoton Fluorescence Recovery after Photobleaching in Vivo

2009 ◽  
Vol 96 (12) ◽  
pp. 5082-5094 ◽  
Author(s):  
Kelley D. Sullivan ◽  
William H. Sipprell ◽  
Edward B. Brown ◽  
Edward B. Brown
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Fluorescence Recovery ◽  
Improved Model

scholarly journals Influence of the centrosome on the structure of nucleated microtubules.

1985 ◽  
Vol 100 (4) ◽  
pp. 1185-1191 ◽  
Author(s):  
L Evans ◽  
T Mitchison ◽  
M Kirschner
Keyword(s):  
Lattice Structure ◽  
Neuroblastoma Cells ◽  
Nucleation Sites ◽  
Self Assembled ◽  
Brain Microtubules

The capacity of the centrosome to influence the lattice structure of nucleated microtubules was studied in vitro. Brain microtubules self-assembled to give predominantly (98%) 14-protofilament microtubules. However, under exactly the same conditions of assembly they grew off of purified centrosomes from neuroblastoma cells to give mostly (82%) 13-protofilament microtubules. Thus, the nucleation sites on the centrosome constrained the microtubule lattice to yield the number of protofilaments usually found in vivo.

Ability to organize microtubules in taxol-treated mitotic PtK2 cells goes with the SPN antigen and not with the centrosome

1992 ◽  
Vol 102 (1) ◽  
pp. 91-102 ◽  
Author(s):  
M. Kallajoki ◽  
K. Weber ◽  
M. Osborn
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Nuclear Matrix ◽  
Essential Role ◽  
Soluble Form ◽  
Microtubule Nucleation ◽  
Brain Microtubules ◽  
Mitotic Cells

The SPN antigen plays an essential role in mitosis, since microinjection of antibodies causes mitotic arrest. Here we show, by examination of the relative locations of SPN antigen, the centrosomal 5051 antigen and tubulin in normal mitotic, and in taxol-treated mitotic cells, that the SPN antigen is involved in organizing the microtubules of the spindle. The 210 kDa protein defined as SPN antigen relocates from the nuclear matrix to the centrosome at prophase, remains associated with the poles at metaphase and anaphase, and dissociates from the centrosomes in telophase. In taxol-treated mitotic cells, SPN staining shows a striking redistribution while 5051 antigen remains associated with centrosomes. SPN antigen is seen at the plasma membrane end of the rearranged microtubules. SPN antigen is always at the center of the multiple microtubule asters (5 to 20 per cell) induced by taxol, whereas 5051 again remains associated with the centrosomal complex (1 to 2 foci per cell). Microtubule nucleation is associated with the SPN antigen rather than with the 5051 antigen. Microinjection of SPN-3 antibody into taxol-treated mitotic PtK2 cells causes disruption of the asters as judged by tubulin staining of the same cells. Finally, SPN antigen extracted in soluble form from synchronized mitotic HeLa cells binds to, and sediments with, pig brain microtubules stabilized by taxol. This association of SPN antigen with microtubules is partially dissociated by 0.5 M NaCl but not by 5 mM ATP. Thus SPN antigen binds to microtubules in vitro and seems to act as a microtubular minus-end organizer in mitotic cells in vivo.

Activity-Related Calcium Dynamics in Motoneurons of the Nucleus Hypoglossus From Mouse

1999 ◽  
Vol 82 (6) ◽  
pp. 2936-2946 ◽  
Author(s):  
Mario B. Lips ◽  
Bernhard U. Keller
Keyword(s):  
Quantitative Analysis ◽  
Action Potential ◽  
Calcium Binding ◽  
Calcium Influx ◽  
Binding Capacity ◽  
Calcium Transients ◽  
Calcium Dynamics ◽  
The Brain

A quantitative analysis of activity-related calcium dynamics was performed in motoneurons of the nucleus hypoglossus in the brain stem slice preparation from mouse by simultaneous patch-clamp and microfluorometric calcium measurements. Motoneurons were analyzed under in vitro conditions that kept them in a functionally intact state represented by rhythmic, inspiratory-related bursts of excitatory postsynaptic currents and associated action potential discharges. Bursts of electrical activity were paralleled by somatic calcium transients resulting from calcium influx through voltage-activated calcium channels, where each action potential accounted for a calcium-mediated charge influx around 2 pC into the somatic compartment. Under in vivo conditions, rhythmic-respiratory activity in young mice occurred at frequencies up to 5 Hz, demonstrating the necessity for rapid calcium elevation and recovery in respiratory-related neurons. The quantitative analysis of hypoglossal calcium homeostasis identified an average extrusion rate, but an exceptionally low endogenous calcium binding capacity as cellular parameters accounting for rapid calcium signaling. Our results suggest that dynamics of somatic calcium transients 1) define an upper limit for the maximum frequency of respiratory-related burst discharges and 2) represent a potentially dangerous determinant of intracellular calcium profiles during pathophysiological and/or excitotoxic conditions.

scholarly journals XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover.

1994 ◽  
Vol 127 (4) ◽  
pp. 985-993 ◽  
Author(s):  
R J Vasquez ◽  
D L Gard ◽  
L Cassimeris
Keyword(s):  
Dynamic Instability ◽  
Fold Increase ◽  
Nucleation Site ◽  
Microtubule Assembly ◽  
Shortening Velocity ◽  
Threefold Increase ◽  
Cell Biol ◽  
Dic Microscopy

We have used video-enhanced DIC microscopy to examine the effects of XMAP, a Mr 215,000 microtubule-associated protein from Xenopus eggs (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), on the dynamic instability of microtubules nucleated from axoneme fragments in vitro. Our results indicate that XMAP substantially alters the parameters of microtubule assembly at plus ends. Specifically, addition of 0.2 microM XMAP resulted in (a) 7-10-fold increase in elongation velocity, (b) approximately threefold increase in shortening velocity, and (c) near elimination of rescue (the switch from rapid shortening to elongation). Thus, addition of XMAP resulted in the assembly of longer, but more dynamic, microtubules from the plus ends of axonemes which upon catastrophe disassembled back to the axoneme nucleation site. In agreement with previous observations (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), the effects of XMAP on the minus end were much less dramatic, with only a 1.5-3-fold increase in elongation velocity. These results indicate that XMAP, unlike brain MAPs, promotes both polymer assembly and turnover, and suggests that the interaction of XMAP with tubulin and the function of XMAP in vivo may differ from previously characterized MAPs.

Inhalable nanostructured lipid particles of 9-bromo-noscapine, a tubulin-binding cytotoxic agent: In vitro and in vivo studies

2015 ◽  
Vol 445 ◽  
pp. 219-230 ◽  
Author(s):  
Kiran Jyoti ◽  
Karanvir Kaur ◽  
Ravi Shankar Pandey ◽  
Upendra Kumar Jain ◽  
Ramesh Chandra ◽  
...  
Keyword(s):  
Cytotoxic Agent ◽  
In Vivo Studies ◽  
Tubulin Binding ◽  
Lipid Particles
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