scholarly journals Roles of Polymerization Dynamics, Opposed Motors, and a Tensile Element in Governing the Length of Xenopus Extract Meiotic Spindles

2005 ◽  
Vol 16 (6) ◽  
pp. 3064-3076 ◽  
Author(s):  
T. J. Mitchison ◽  
P. Maddox ◽  
J. Gaetz ◽  
A. Groen ◽  
M. Shirasu ◽  
...  
Keyword(s):  
Steady State ◽  
Relative Stability ◽  
Motor Proteins ◽  
Microtubule Dynamics ◽  
Microtubule Polymerization ◽  
Hexylene Glycol ◽  
Meiotic Spindles

Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.

scholarly journals TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux

2015 ◽  
Vol 210 (3) ◽  
pp. 373-383 ◽  
Author(s):  
Jingyan Fu ◽  
Minglei Bian ◽  
Guangwei Xin ◽  
Zhaoxuan Deng ◽  
Jia Luo ◽  
...  
Keyword(s):  
Steady State ◽  
Mitotic Spindle ◽  
Microtubule Dynamics ◽  
Flux Rate ◽  
Aurora A ◽  
Null Mutant ◽  
Constant Length ◽  
Microtubule Polymerization ◽  
Spindle Microtubules

A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A–dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux.

scholarly journals Theory of microtubule length regulation in antiparallel overlaps

2019 ◽  
Author(s):  
Hui-Shun Kuan ◽  
Meredith D. Betterton
Keyword(s):  
Steady State ◽  
Cell Division ◽  
Mitotic Spindle ◽  
Motor Proteins ◽  
Critical Concentration ◽  
Binding Kinetics ◽  
Kinesin Motor ◽  
Microtubule Polymerization ◽  
Length Regulation ◽  
Theory And Experiment

AbstractDuring cell division, microtubules in the mitotic spindle form antiparallel overlaps near the center of the spindle. Kinesin motor proteins alter microtubule polymerization dynamics to regulate the length of these overlaps to maintain spindle integrity. Length regulation of antiparallel overlaps has been reconstituted with purified microtubules, crosslinkers, and motors. Here we develop a theory of steady-state overlap length which depends on the filament plus-end motor concentration, determined by a balance between motor arrival (motor binding and stepping in the overlap) and motor departure (motor unbinding from filament tips during depolymerization) in the absence of motor-driven sliding. Assuming that motors processively depolymerize and exhibit altered binding kinetics near MT plus-ends improves the agreement between theory and experiment. Our theory explains the origin of the experimentally observed critical concentration, a minimum motor concentration to observe a steady-state overlap length.

scholarly journals Regulation of Microtubule Dynamics by Bim1 and Bik1, the Budding Yeast Members of the EB1 and CLIP-170 Families of Plus-End Tracking Proteins

2010 ◽  
Vol 21 (12) ◽  
pp. 2013-2023 ◽  
Author(s):  
Kristina A. Blake-Hodek ◽  
Lynne Cassimeris ◽  
Tim C. Huffaker
Keyword(s):  
Budding Yeast ◽  
Family Members ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
Microtubule Polymerization

Microtubule dynamics are regulated by plus-end tracking proteins (+TIPs), which bind microtubule ends and influence their polymerization properties. In addition to binding microtubules, most +TIPs physically associate with other +TIPs, creating a complex web of interactions. To fully understand how +TIPs regulate microtubule dynamics, it is essential to know the intrinsic biochemical activities of each +TIP and how +TIP interactions affect these activities. Here, we describe the activities of Bim1 and Bik1, two +TIP proteins from budding yeast and members of the EB1 and CLIP-170 families, respectively. We find that purified Bim1 and Bik1 form homodimers that interact with each other to form a tetramer. Bim1 binds along the microtubule lattice but with highest affinity for the microtubule end; however, Bik1 requires Bim1 for localization to the microtubule lattice and end. In vitro microtubule polymerization assays show that Bim1 promotes microtubule assembly, primarily by decreasing the frequency of catastrophes. In contrast, Bik1 inhibits microtubule assembly by slowing growth and, consequently, promoting catastrophes. Interestingly, the Bim1-Bik1 complex affects microtubule dynamics in much the same way as Bim1 alone. These studies reveal new activities for EB1 and CLIP-170 family members and demonstrate how interactions between two +TIP proteins influence their activities.

BEYOND THE STEADY STATE: MANAGING DISCONTINUOUS PRODUCT AND PROCESS INNOVATION

2006 ◽  
Vol 10 (02) ◽  
pp. 175-196 ◽  
Author(s):  
WENDY PHILLIPS ◽  
HANNAH NOKE ◽  
JOHN BESSANT ◽  
RICHARD LAMMING
Keyword(s):  
Steady State ◽  
Relative Stability ◽  
Good Practice ◽  
Innovation Process ◽  
Effective Management ◽  
Product And Process Innovation ◽  
Good Guide ◽  
Product And Process ◽  
Discontinuous Innovation ◽  
Selection Of

Research on the innovation process and its effective management has consistently highlighted a set of themes constituting "good practice". The limitation of such "good practice" is that it relates to what might be termed "steady state" innovation — essentially innovative activity in product and process terms which is about "doing what we do, but better". The prescription works well under these conditions of (relative) stability in terms of products and markets but is not a good guide when elements of discontinuity come into the equation. Discontinuity arises from shifts along technological, market, political and other frontiers and requires new or at least significantly adapted approaches to their effective management. This paper highlights empirical findings from a selection of companies involved in a project sponsored by the U.K. Department of Trade and Industry. The results indicate a number of key routines that organisations could implement to enable discontinuous innovation.

scholarly journals EB1 Targets to Kinetochores with Attached, Polymerizing Microtubules

2002 ◽  
Vol 13 (12) ◽  
pp. 4308-4316 ◽  
Author(s):  
Jennifer S. Tirnauer ◽  
Julie C. Canman ◽  
E.D. Salmon ◽  
Timothy J. Mitchison
Keyword(s):  
Spindle Checkpoint ◽  
Time Lapse ◽  
Microtubule Dynamics ◽  
Microtubule Polymerization ◽  
Spindle Microtubules ◽  
Chromosome Movements

Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end–kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.

scholarly journals Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts.

1992 ◽  
Vol 118 (5) ◽  
pp. 1097-1108 ◽  
Author(s):  
F Verde ◽  
M Dogterom ◽  
E Stelzer ◽  
E Karsenti ◽  
S Leibler
Keyword(s):  
Steady State ◽  
Fold Increase ◽  
Cyclin A ◽  
Microtubule Dynamics ◽  
Cyclin B ◽  
Free System ◽  
Egg Extracts ◽  
Dynamical Parameters ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

In eukaryotic cells, the onset of mitosis involves cyclin molecules which interact with proteins of the cdc2 family to produce active kinases. In vertebrate cells, cyclin A dependent kinases become active in S- and pro-phases, whereas a cyclin B-dependent kinase is mostly active in metaphase. It has recently been shown that, when added to Xenopus egg extracts, bacterially produced A- and B-type cyclins associate predominantly with the same kinase catalytic subunit, namely p34cdc2, and induce its histone H1 kinase activity with different kinetics. Here, we show that in the same cell free system, both the addition of cyclin A and cyclin B changes microtubule behavior. However, the cyclin A-dependent kinase does not induce a dramatic shortening of centrosome-nucleated microtubules whereas the cyclin B-dependent kinase does, as previously reported. Analysis of the parameters of microtubule dynamics by fluorescence video microscopy shows that the dramatic shortening induced by the cyclin B-dependent kinase is correlated with a several fold increase in catastrophe frequency, an effect not observed with the cyclin A-dependent kinase. Using a simple mathematical model, we show how the length distributions of centrosome-nucleated microtubules relate to the four parameters that describe microtubule dynamics. These four parameters define a threshold between unlimited microtubule growth and the establishment of steady-state dynamics, which implies that well defined steady-state length distributions can be produced by regulating precisely the respective values of the dynamical parameters. Moreover, the dynamical model predicts that increasing catastrophe frequency is more efficient than decreasing the rescue frequency to reduce the average steady state length of microtubules. These theoretical results are quantitatively confirmed by the experimental data.

scholarly journals cdc2 kinase-induced destabilization of MAP2-coated microtubules in Xenopus egg extracts

1992 ◽  
Vol 101 (1) ◽  
pp. 69-78 ◽  
Author(s):  
S. Faruki ◽  
M. Doree ◽  
E. Karsenti
Keyword(s):  
Steady State ◽  
Bovine Brain ◽  
Microtubule Dynamics ◽  
Cdc2 Kinase ◽  
Stabilizing Effect ◽  
Egg Extracts ◽  
Phosphorylation Event ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Stabilizing Factors

During the interphase to metaphase transition, microtubules are destabilized by a cdc2 kinase-dependent phosphorylation event. This destabilization is due to a dramatic increase in the rate at which each growing microtubule starts to shrink (catastrophe rate). In principle, this could be brought about by lowering the affinity of stabilizing MAPs for the microtubule wall, by activating a factor that would actively increase the catastrophe rate or by an alteration of both parameters. Here we examine the stabilizing effect of bovine brain MAP2 on microtubules assembled in interphase Xenopus egg extracts. We show that this MAP strongly stabilizes microtubules assembled in the extracts against nocodazole-induced depolymerization. However, it does not protect them from the cdc2 kinase-induced shortening and destabilization. Moreover, the steady-state length of centrosome-nucleated microtubules in cdc2-treated extracts containing MAP2 is similar to that found in extracts lacking exogenous MAP2. We also show that although exogenous MAP2 is phosphorylated by cdc2 kinase in the extract, this is not the cause of microtubule destabilization. These results indicate that increased microtubule dynamics during mitosis is due to the activation of a factor that can function independently of the presence of active, stabilizing factors.

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