scholarly journals A liquid +TIP-network drives microtubule dynamics through tubulin condensation

2021 ◽  
Author(s):  
Julie Miesch ◽  
Robert T. Wimbish ◽  
Marie-Claire Velluz ◽  
Charlotte Aumeier
Keyword(s):  
Phase Separation ◽  
Binding Proteins ◽  
Tip Growth ◽  
Regulatory Mechanism ◽  
Microtubule Dynamics ◽  
Microtubule Network ◽  
Cell Behaviors ◽  
Dynamic Cell ◽  
Microtubule Growth ◽  
Dynamic Microtubule

Tubulin dimers assemble into a dynamic microtubule network throughout the cell. Microtubule dynamics and network organization must be precisely tuned for the microtubule cytoskeleton to regulate a dazzling array of dynamic cell behaviors. Since tubulin concentration determines microtubule growth, we studied here a novel regulatory mechanism of microtubule dynamics: local tubulin condensation. We discovered that two microtubule tip-binding proteins, CLIP-170 and EB3, undergo phase separation and form an EB3/CLIP-170 droplet at the growing microtubule tip. We prove that this +TIP-droplet has the capacity to locally condense tubulin. This process of tubulin co-condensation is spatially initiated at the microtubule tip and temporally regulated to occur only when there is tip growth. Tubulin condensation at the growing microtubule tip increases growth speeds three-fold and strongly reduces depolymerization events. With this work we establish a new mechanism to regulate microtubule dynamics by enrichment of tubulin at strategically important locations: the growing microtubule tips.

scholarly journals Mammalian end binding proteins control persistent microtubule growth

2009 ◽  
Vol 184 (5) ◽  
pp. 691-706 ◽  
Author(s):  
Yulia Komarova ◽  
Christian O. De Groot ◽  
Ilya Grigoriev ◽  
Susana Montenegro Gouveia ◽  
E. Laura Munteanu ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Microtubule Dynamics ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dimerization Domain ◽  
Negative Effect ◽  
Microtubule Growth ◽  
Tracking Behavior ◽  
In Cells

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.

scholarly journals An ELMO2-RhoG-ILK network modulates microtubule dynamics

2015 ◽  
Vol 26 (14) ◽  
pp. 2712-2725 ◽  
Author(s):  
Bradley C. Jackson ◽  
Iordanka A. Ivanova ◽  
Lina Dagnino
Keyword(s):  
Focal Adhesions ◽  
Cellular Level ◽  
Microtubule Dynamics ◽  
Epidermal Keratinocytes ◽  
Microtubule Network ◽  
Microtubule Stability ◽  
Integrin Linked Kinase ◽  
Exogenous Expression ◽  
Microtubule Growth ◽  
Gsk 3Β

ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting β1 integrin–dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3β, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca2+-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes.

scholarly journals Desmin intermediate filaments and tubulin detyrosination stabilize growing microtubules in the cardiomyocyte

2021 ◽  
Author(s):  
Alexander Salomon ◽  
Naima Okami ◽  
Julie Heffler ◽  
Jia-Jye Lee ◽  
Patrick Robison ◽  
...  
Keyword(s):  
Intermediate Filaments ◽  
Muscle Mechanics ◽  
High Energy ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Microtubule Network ◽  
Biochemical Assays ◽  
Molecular Determinants ◽  
Microtubule Growth ◽  
Dynamic Microtubule

The microtubule network of the cardiomyocyte exhibits specialized architecture, stability and mechanical behavior that accommodate the demands of working muscle cells. Stable, post-translationally detyrosinated microtubules are physical coupled to the sarcomere, the contractile apparatus of muscle, and resist sarcomere motion to regulate muscle mechanics and mechanosignaling. Control of microtubule growth and shrinkage dynamics represents a potential intermediate in the formation of a stable, physically coupled microtubule network, yet the molecular determinants that govern dynamics are unknown. Here we test the hypothesis that desmin intermediate filaments may stabilize growing microtubules at the sarcomere Z-disk in a detyrosination-dependent manner. Using a combination of biochemical assays and direct observation of microtubule plus-end dynamics in primary adult cardiomyocytes, we determine that: 1) tyrosination increases the frequency of microtubule depolymerization and reduces the pausing of microtubules at the Z-disk, leading to a more dynamic microtubule; and 2) desmin intermediate filaments stabilize both growing and shrinking microtubules specifically at the Z-disk and protect them from depolymerization. This stabilizes iteratively growing, detyrosinated microtubules between adjacent sarcomeres, which promotes the formation of high-energy microtubules that buckle between sarcomeres and elevates myocyte viscoelasticity. Our findings inform on how the tubulin code and intermediate filaments regulate microtubule dynamics, and provide mechanism to the establishment of a spatially organized, physically coupled, and long-lived microtubule network in the cardiomyocyte.

scholarly journals Arabidopsis SPIRAL2 promotes uninterrupted microtubule growth by suppressing the pause state of microtubule dynamics

2008 ◽  
Vol 121 (14) ◽  
pp. 2372-2381 ◽  
Author(s):  
M. Yao ◽  
Y. Wakamatsu ◽  
T. J. Itoh ◽  
T. Shoji ◽  
T. Hashimoto
Keyword(s):  
Microtubule Dynamics ◽  
Microtubule Growth

scholarly journals Yeast Kinetochores Do Not Stabilize Stu2p-dependent Spindle Microtubule Dynamics

2003 ◽  
Vol 14 (10) ◽  
pp. 4181-4195 ◽  
Author(s):  
Chad G. Pearson ◽  
Paul S. Maddox ◽  
Ted R. Zarzar ◽  
E.D. Salmon ◽  
Kerry Bloom
Keyword(s):  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Regulatory Mechanism ◽  
Metaphase Plate ◽  
Microtubule Dynamics ◽  
Spindle Microtubule ◽  
Microtubule Binding ◽  
Microtubule Stabilization ◽  
Kinetochore Function

The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule–kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.

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.

The Kar3p and Kip2p motors function antagonistically at the spindle poles to influence cytoplasmic microtubule numbers

1998 ◽  
Vol 111 (3) ◽  
pp. 295-301 ◽  
Author(s):  
A. Huyett ◽  
J. Kahana ◽  
P. Silver ◽  
X. Zeng ◽  
W.S. Saunders
Keyword(s):  
Molecular Motors ◽  
Motor Proteins ◽  
Single Gene ◽  
Intermediate Phenotype ◽  
Microtubule Network ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cytoplasmic Microtubule ◽  
Spindle Poles ◽  
Microtubule Motors ◽  
Microtubule Growth

Microtubules provide the substrate for intracellular trafficking by association with molecular motors of the kinesin and dynein superfamilies. Motor proteins are generally thought to function as force generating units for transport of various cargoes along the microtubule polymer. Recent work suggests additional roles for motor proteins in changing the structure of the microtubule network itself. We report here that in the budding yeast Saccharomyces cerevisiae microtubule motors have antagonistic effects on microtubule numbers and lengths. As shown previously, loss of the Kar3p motor stimulates cytoplasmic microtubule growth while loss of Kip2p leads to a sharp reduction in cytoplasmic microtubule numbers. Loss of both the Kip2p and Kar3p motors together in the same cell produces an intermediate phenotype, suggesting that these two motors act in opposition to control cytoplasmic microtubule density. A Kip2p-GFP fusion from single gene expression is most concentrated at the spindle poles, as shown previously for an epitope tagged Kar3p-HA, suggesting both of these motors act from the minus ends of the microtubules to influence microtubule numbers.

scholarly journals Dynein efficiently navigates the dendritic cytoskeleton to drive the retrograde trafficking of BDNF/TrkB signaling endosomes

2017 ◽  
Vol 28 (19) ◽  
pp. 2543-2554 ◽  
Author(s):  
Swathi Ayloo ◽  
Pedro Guedes-Dias ◽  
Amy E. Ghiretti ◽  
Erika L. F. Holzbaur
Keyword(s):  
Real Time ◽  
Axonal Transport ◽  
Hippocampal Neurons ◽  
Cytoplasmic Dynein ◽  
Microtubule Dynamics ◽  
Neuronal Function ◽  
Retrograde Trafficking ◽  
Basic Understanding ◽  
Dependent Transport ◽  
Dynamic Microtubule

The efficient transport of cargoes within axons and dendrites is critical for neuronal function. Although we have a basic understanding of axonal transport, much less is known about transport in dendrites. We used an optogenetic approach to recruit motor proteins to cargo in real time within axons or dendrites in hippocampal neurons. Kinesin-1, a robust axonal motor, moves cargo less efficiently in dendrites. In contrast, cytoplasmic dynein efficiently navigates both axons and dendrites; in both compartments, dynamic microtubule plus ends enhance dynein-dependent transport. To test the predictions of the optogenetic assay, we examined the contribution of dynein to the motility of an endogenous dendritic cargo and found that dynein inhibition eliminates the retrograde bias of BDNF/TrkB trafficking. However, inhibition of microtubule dynamics has no effect on BDNF/TrkB motility, suggesting that dendritic kinesin motors may cooperate with dynein to drive the transport of signaling endosomes into the soma. Collectively our data highlight compartment-specific differences in kinesin activity that likely reflect specialized tuning for localized cytoskeletal determinants, whereas dynein activity is less compartment specific but is more responsive to changes in microtubule dynamics.

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