scholarly journals Emergent properties of a mitotic Kif18b-MCAK-EB network

2020 ◽  
Author(s):  
Toni McHugh ◽  
Julie P.I. Welburn
Keyword(s):  
Single Molecule ◽  
Mitotic Spindle ◽  
Microtubule Dynamics ◽  
Emergent Properties ◽  
Single Molecule Imaging ◽  
Prior Work ◽  
Microtubule Depolymerization ◽  
Mechanistic Basis ◽  
The Individual

AbstractThe precise regulation of microtubule length during mitosis is essential to assemble and position the mitotic spindle and segregate chromosomes. Prior work has identified key molecular players in this process, including the kinesin-18 Kif18b and the kinesin-13 Kif2C/MCAK, which both promote microtubule depolymerization. MCAK acts as a potent microtubule depolymerase diffusing short distances on microtubules, while Kif18b is a mitotic processive motor with weak depolymerase activity. However the individual activities of these factors cannot explain the dramatic increase in microtubule dynamics in mitosis. Using in vitro reconstitution and single molecule imaging, we demonstrate that Kif18b, MCAK and the plus-end tracking protein EB3 act in an integrated manner to potently promote microtubule depolymerization. We find Kif18b acts as a microtubule plus end delivery factor for its cargo MCAK, and that Kif18b also promotes EB accumulation to plus ends independently of lattice nucleotide state. Together, our work defines the mechanistic basis for a cooperative Kif18b-EB-MCAK network with emergent properties, that acts to efficiently shorten microtubules in mitosis.

scholarly journals The mitotic spindle protein CKAP2 potently increases formation and stability of microtubules

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Thomas S McAlear ◽  
Susanne Bechstedt
Keyword(s):  
Cell Division ◽  
Growth Factor ◽  
Mitotic Spindle ◽  
Apparent Rate Constant ◽  
Microtubule Dynamics ◽  
Proliferation Marker ◽  
Microtubule Growth ◽  
Large Rearrangements ◽  
And Function

Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2's role as an important spindle protein, proliferation marker, and oncogene.

scholarly journals Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

1998 ◽  
Vol 142 (6) ◽  
pp. 1519-1532 ◽  
Author(s):  
Yasmina Saoudi ◽  
Rati Fotedar ◽  
Ariane Abrieu ◽  
Marcel Dorée ◽  
Jürgen Wehland ◽  
...  
Keyword(s):  
Model System ◽  
Microtubule Dynamics ◽  
In Vitro Assays ◽  
Microtubule Depolymerization ◽  
Dynamic Activity ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Cell Extracts

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.

scholarly journals Intrinsically disordered domain of kinesin-3 Kif14 enables unique functional diversity

2020 ◽  
Author(s):  
Ilia Zhernov ◽  
Stefan Diez ◽  
Marcus Braun ◽  
Zdenek Lansky
Keyword(s):  
Functional Diversity ◽  
Single Molecule ◽  
Mitotic Spindle ◽  
Cellular Functions ◽  
Protein Tau ◽  
Intrinsically Disordered ◽  
Order Of Magnitude ◽  
Pass Through ◽  
Domain I

ABSTRACTIn addition to their force-generating motor domains, kinesin motor proteins feature various accessory domains enabling them to fulfil a variety of functions in the cell. Human kinesin-3, Kif14, localizes to the midbody of the mitotic spindle and is involved in the progression of cytokinesis. The specific motor properties enabling Kif14’s cellular functions, however, remain unknown. Here, we show in vitro that it is the intrinsically disordered N-terminal domain of Kif14 that enables unique functional diversity of the motor. Using single molecule TIRF microscopy we observed that the presence of the disordered domain i) increased the Kif14 run-length by an order of magnitude, rendering the motor super-processive and enabling the motor to pass through highly crowded microtubule areas shielded by cohesive layers of microtubule-associated protein tau, which blocks less processive motors ii) enabled robust, autonomous Kif14 tracking of growing microtubule tips, independent of microtubule end-binding (EB) proteins and iii) enabled Kif14 to crosslink parallel microtubules and to drive the relative sliding of antiparallel ones. We explain these features of Kif14 by the observed increased affinity of the disordered domain for GTP-like tubulin and the observed diffusible interaction of the disordered domain with the microtubule lattice. We hypothesize that the disordered domain tethers the motor domain to the microtubule forming a diffusible foothold. We suggest that the intrinsically disordered N-terminal anchoring domain of Kif14 is a regulatory hub supporting the various cellular functions of Kif14 by tuning the motor’s interaction with microtubules.

scholarly journals Single-molecule imaging reveals the interplay between transcription factors, nucleosomes, and transcriptional bursting

2018 ◽  
Author(s):  
Benjamin T. Donovan ◽  
Anh Huynh ◽  
David A. Ball ◽  
Michael G. Poirier ◽  
Daniel R. Larson ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Single Molecule ◽  
Target Genes ◽  
Burst Size ◽  
Yeast Cells ◽  
Single Molecule Imaging ◽  
Transcriptional Bursting

SummaryTranscription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the transcription factor Gal4 with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell times sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model where multiple polymerases initiate during a burst as long as the transcription factor is bound to DNA, and a burst terminates upon transcription factor dissociation.

Mitotic Spindle Dysfunction Promotes Genomic Instability In Marrow Failure

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 880-880 ◽  
Author(s):  
Katie Lombardo ◽  
Jason Stumpff ◽  
Susan Parkhurst ◽  
Linda Wordeman ◽  
Akiko Shimamura
Keyword(s):  
Genomic Instability ◽  
Mitotic Spindle ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Microtubule Dynamics ◽  
Published Data ◽  
Hematopoietic Progenitor ◽  
Microtubule Stability ◽  
Cd34 Cells

Abstract Abstract 880 Shwachman-Diamond syndrome (SDS) is an autosomal recessively inherited disorder associated with bone marrow failure and leukemia predisposition. The majority of patients harbor biallelic mutations in the SBDS gene. The SBDS protein has been implicated in several cellular functions including ribosome biogenesis and microtubule stabilization during mitosis. We have previously found that SBDS deficiency results in multipolar spindles, centrosome amplification and aneuploidy, implicating a role for SBDS in cell division. The mechanism by which SBDS functions to ensure proper spindle assembly and DNA segregation during mitosis remains unknown. Here we present evidence that SBDS functions to promote mitotic spindle stability both by directly modifying microtubule dynamics and through a microtubule crosslinking activity. Importantly, the microtubule stabilizing effects of SBDS appear to be essential for the growth and differentiation of hematopoietic progenitor cells. Specifically, we found that SBDS deficiency resulted in shortened mitotic spindle length and decreased spindle acetylation, a marker of microtubule stability. The loss of microtubule stability in the absence of SBDS function may be due to changes in microtubule dynamics or reduction in microtubule crosslinking activity, as we found that addition of recombinant purified wild-type SBDS to polymerized microtubules in vitro increases their polymerization rate and strongly promotes microtubule bundling. Interestingly, recombinant patient-derived missense mutant SBDS proteins showed a marked decrease in their microtubule bundling ability. To assess whether spindle destabilization contributes to marrow failure, we modeled hematopoiesis in the absence of SBDS in vitro. When SBDS expression was knocked down in human CD34+ cells, proliferation, differentiation, and hematopoietic progenitor colony formation were impaired, consistent with published data on primary marrow from SDS patients. The addition of taxol at concentrations that significantly impaired hematopoiesis in control CD34+ cells resulted in stable to improved hematopoiesis in the SBDS-deficient CD34+ cells. Based on these data, we hypothesize that spindle destabilization by SBDS loss promotes genomic instability, which in turn, contributes to marrow failure and leukemia predisposition. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Benjamin A Smith ◽  
Shae B Padrick ◽  
Lynda K Doolittle ◽  
Karen Daugherty-Clarke ◽  
Ivan R Corrêa ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Network Growth ◽  
Cell Locomotion ◽  
Branch Formation ◽  
Membrane Bound

During cell locomotion and endocytosis, membrane-tethered WASP proteins stimulate actin filament nucleation by the Arp2/3 complex. This process generates highly branched arrays of filaments that grow toward the membrane to which they are tethered, a conflict that seemingly would restrict filament growth. Using three-color single-molecule imaging in vitro we revealed how the dynamic associations of Arp2/3 complex with mother filament and WASP are temporally coordinated with initiation of daughter filament growth. We found that WASP proteins dissociated from filament-bound Arp2/3 complex prior to new filament growth. Further, mutations that accelerated release of WASP from filament-bound Arp2/3 complex proportionally accelerated branch formation. These data suggest that while WASP promotes formation of pre-nucleation complexes, filament growth cannot occur until it is triggered by WASP release. This provides a mechanism by which membrane-bound WASP proteins can stimulate network growth without restraining it.

scholarly journals The Microtubule Plus-End Proteins EB1 and Dynactin Have Differential Effects on Microtubule Polymerization

2003 ◽  
Vol 14 (4) ◽  
pp. 1405-1417 ◽  
Author(s):  
Lee A. Ligon ◽  
Spencer S. Shelly ◽  
Mariko Tokito ◽  
Erika L.F. Holzbaur
Keyword(s):  
Cultured Cells ◽  
Cell Types ◽  
Microtubule Dynamics ◽  
In Vitro Assays ◽  
Nucleation Effect ◽  
Microtubule Polymerization ◽  
The Individual ◽  
Different Cell Types ◽  
Dynactin Complex

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150Glued subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150Glued in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150Glued on microtubule polymerization, and they show that p150Gluedhas a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics.

Sign in / Sign up

Export Citation Format

Share Document