scholarly journals Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end

2021 ◽  
Author(s):  
Joseph M Cleary ◽  
Tae Kim ◽  
Annan SI Cook ◽  
William O Hancock ◽  
Luke M Rice
Keyword(s):  
Dissociation Rate ◽  
Regulatory Proteins ◽  
Early Event ◽  
Gtp Hydrolysis ◽  
Microtubule Polymerization ◽  
Assembly Kinetics ◽  
Quantitative Understanding ◽  
Microtubule Growth ◽  
Association Kinetics ◽  
Do So

Microtubule polymerization dynamics result from the biochemical interactions of αβ-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αβ-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently "poisoned" growth, yielding a wider range of growth rate compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP tubulin at the growing microtubule end may be an important early event determining catastrophe.

scholarly journals Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner

2015 ◽  
Vol 26 (11) ◽  
pp. 2020-2029 ◽  
Author(s):  
Wallis Nahaboo ◽  
Melissa Zouak ◽  
Peter Askjaer ◽  
Marie Delattre
Keyword(s):  
Caenorhabditis Elegans ◽  
Dependent Manner ◽  
Microtubule Polymerization ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Chromatid Segregation ◽  
Nucleation Agents ◽  
Interesting Possibility ◽  
Spindle Midzone ◽  
Microtubule Growth

During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.

scholarly journals Purification of functional Plasmodium falciparum tubulin allows for the identification of parasite-specific microtubule inhibitors

2021 ◽  
Author(s):  
William Graham Hirst ◽  
Dominik Fachet ◽  
Benno Kuropka ◽  
Christoph Weise ◽  
Kevin J Saliba ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Drug Targets ◽  
Cytoskeletal Proteins ◽  
Microtubule Inhibitors ◽  
Microtubule Polymerization ◽  
Molecular Building Block ◽  
Structural Divergence ◽  
Microtubule Growth ◽  
Exciting Possibility

Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore represent promising drug targets. While αβ-tubulin, the molecular building block of microtubules, is an established drug target in a variety of cancers, we still lack substantial knowledge of the biochemistry of parasite tubulins, which would allow us to exploit the structural divergence between parasite and human tubulins. Indeed, mechanistic insights have been limited by the lack of purified, functional parasite tubulin. In this study, we isolated Plasmodium falciparum tubulin that is assembly-competent and shows specific microtubule dynamics in vitro. We further present mechanistic evidence that two compounds selectively interact with parasite over host microtubules and inhibit Plasmodium microtubule polymerization at substoichiometric compound concentrations. The ability of compounds to selectively disrupt protozoan microtubule growth without affecting human microtubules provides the exciting possibility for the targeted development of novel antimalarials.

scholarly journals Mechanisms of Aggresome Biogenesis: Ubiquitination, Transport, & Maintenance

2019 ◽  
Author(s):  
AJ Keefe
Keyword(s):  
Intermediate Filament ◽  
Retrograde Transport ◽  
Regulatory Proteins ◽  
Misfolded Proteins ◽  
Misfolded Protein ◽  
Proteotoxic Stress ◽  
Organizing Center ◽  
Critical Insight ◽  
Insight Into ◽  
Do So

Neurodegenerative diseases are universally marked by the accumulation of misfolded protein. Neurons respond to these proteostatic disturbances by sequestering, and thus inactivating, toxic misfolded proteins into a perinuclear organelle called the aggresome. The aggresome can be subsequently degraded in bulk by autophagy, a process termed aggrephagy. The formation of protein aggregates has historically been considered a spontaneous and unregulated process, but emerging research has instead discovered a diverse cohort of regulatory proteins that mediate protein aggregation. Chaperones are the first proteins to respond to misfolded proteins, and do so by recognizing the aberrant exposure of hydrophobic domains. When chaperones are unable to correctly refold proteins, their substrates are  transferred to ubiquitin ligating machinery to catalyze polyubiquitination. Although ubiquitin chains typically direct proteins towards proteasomes, severe proteotoxic stress can overwhelm, or even directly inhibit, proteasomes. As an alternative to proteasomal degradation, misfolded proteins are redirected towards the mitotic organizing center (MTOC) and, following retrograde transport by dynein, are packaged and sequestered within an intermediate filament (IF) cage to form the aggresome. The biogenesis of the aggresome is thus a highly regulated event, and a better understanding of the mechanisms facilitating this process will provide critical insight into neurodegenerative disease.

scholarly journals Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions

2021 ◽  
Author(s):  
Ali Imran ◽  
Brandon S. Moyer ◽  
Ashley J. Canning ◽  
Dan Kalina ◽  
Thomas M Duncan ◽  
...  
Keyword(s):  
Binding Site ◽  
Primary Role ◽  
Dissociation Kinetics ◽  
High Affinity ◽  
Histone 3 ◽  
Quantitative Understanding ◽  
Slow Dissociation ◽  
Kinetics Of ◽  
Association Kinetics

Recent advances in quantitative proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their physical associations with other proteins are ambiguous. A quantitative understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of  transient associations between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow association kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissociation kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnology.

scholarly journals Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

2014 ◽  
Vol 204 (7) ◽  
pp. 1111-1121 ◽  
Author(s):  
Emmanuel Gallaud ◽  
Renaud Caous ◽  
Aude Pascal ◽  
Franck Bazile ◽  
Jean-Philippe Gagné ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Microtubule Associated Proteins ◽  
Microtubule Polymerization ◽  
Sister Chromatids ◽  
Centrosome Separation ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Mitotic Spindle Assembly

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

scholarly journals Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate.

1991 ◽  
Vol 114 (1) ◽  
pp. 73-81 ◽  
Author(s):  
R A Walker ◽  
N K Pryer ◽  
E D Salmon
Keyword(s):  
Dynamic Instability ◽  
Elongation Rate ◽  
Gtp Hydrolysis ◽  
Microtubule Stability ◽  
Assembly Mechanism ◽  
High Elongation ◽  
Microtubule Growth ◽  
Hydrolysis Of ◽  
Brain Tubulin

Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build-up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP-tubulin association.

scholarly journals Tracking individual secretory vesicles during exocytosis reveals an ordered and regulated process

2015 ◽  
Vol 210 (2) ◽  
pp. 181-189 ◽  
Author(s):  
Kirk W. Donovan ◽  
Anthony Bretscher
Keyword(s):  
Plasma Membrane ◽  
Secretory Vesicle ◽  
Regulatory Proteins ◽  
Regulatory Mechanisms ◽  
Secretory Vesicles ◽  
Myosin V ◽  
Gtp Hydrolysis ◽  
Associated Proteins ◽  
Hydrolysis Of

Post-Golgi secretory vesicle trafficking is a coordinated process, with transport and regulatory mechanisms to ensure appropriate exocytosis. While the contributions of many individual regulatory proteins to this process are well studied, the timing and dependencies of events have not been defined. Here we track individual secretory vesicles and associated proteins in vivo during tethering and fusion in budding yeast. Secretory vesicles tether to the plasma membrane very reproducibly for ∼18 s, which is extended in cells defective for membrane fusion and significantly lengthened and more variable when GTP hydrolysis of the exocytic Rab is delayed. Further, the myosin-V Myo2p regulates the tethering time in a mechanism unrelated to its interaction with exocyst component Sec15p. Two-color imaging of tethered vesicles with Myo2p, the GEF Sec2p, and several exocyst components allowed us to document a timeline for yeast exocytosis in which Myo2p leaves 4 s before fusion, whereas Sec2p and all the components of the exocyst disperse coincident with fusion.

scholarly journals Mechanisms of Aggresome Biogenesis Ubiquitination, Transport, & Maintenance

2018 ◽  
Author(s):  
AJ Keefe
Keyword(s):  
Intermediate Filament ◽  
Retrograde Transport ◽  
Regulatory Proteins ◽  
Misfolded Proteins ◽  
Misfolded Protein ◽  
Proteotoxic Stress ◽  
Organizing Center ◽  
Critical Insight ◽  
Insight Into ◽  
Do So

Neurodegenerative diseases are universally marked by the accumulation of misfolded protein. Neurons respond to these proteostatic disturbances by sequestering, and thus inactivating, toxic misfolded proteins into a perinuclear organelle called the aggresome. The aggresome can be subsequently degraded in bulk by autophagy, a process termed aggrephagy. The formation of protein aggregates has historically been considered a spontaneous and unregulated process, but emerging research has instead discovered a diverse cohort of regulatory proteins that mediate protein aggregation. Chaperones are the first proteins to respond to misfolded proteins, and do so by recognizing the aberrant exposure of hydrophobic domains. When chaperones are unable to correctly refold proteins, their substrates are  transferred to ubiquitin ligating machinery to catalyze polyubiquitination. Although ubiquitin chains typically direct proteins towards proteasomes, severe proteotoxic stress can overwhelm, or even directly inhibit, proteasomes. As an alternative to proteasomal degradation, misfolded proteins are redirected towards the mitotic organizing center (MTOC) and, following retrograde transport by dynein, are packaged and sequestered within an intermediate filament (IF) cage to form the aggresome. The biogenesis of the aggresome is thus a highly regulated event, and a better understanding of the mechanisms facilitating this process will provide critical insight into neurodegenerative disease.

scholarly journals Kinesin-5 promotes microtubule nucleation and assembly by stabilizing a lattice-competent conformation of tubulin

2019 ◽  
Author(s):  
Geng-Yuan Chen ◽  
Ana B. Asenjo ◽  
Yalei Chen ◽  
Jake Mascaro ◽  
David F. J. Arginteanu ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Polymerase Activity ◽  
Microtubule Nucleation ◽  
Domain Swap ◽  
Microtubule Polymerization ◽  
The Family ◽  
Microtubule Growth ◽  
Spindle Elongation ◽  
Necessary And Sufficient ◽  
Induced Changes

SummaryBesides sliding apart antiparallel microtubules during spindle elongation, the mitotic kinesin-5, Eg5 promotes microtubule polymerization, emphasizing its importance in mitotic spindle length control. Here, we characterize the Eg5 microtubule polymerase mechanism by assessing motor-induced changes in the longitudinal and lateral tubulin-tubulin bonds that form the microtubule lattice. Isolated Eg5 motor domains promote microtubule nucleation, growth and stability. Eg5 binds preferentially to microtubules over free tubulin, and colchicine-like inhibitors that stabilize the bent conformation of tubulin allosterically inhibit Eg5 binding, consistent with a model in which Eg5 induces a curved-to-straight transition in tubulin. Domain swap experiments establish that the family-specific Loop11, which resides near the nucleotide-sensing Switch-II domain, is necessary and sufficient for the polymerase activity of Eg5. Thus, we propose a microtubule polymerase mechanism in which Eg5 at the plus-end promotes a curved-to-straight transition in tubulin that enhances lateral bond formation and thereby promotes microtubule growth and stability.

scholarly journals Stable centrosomal roots disentangle to allow interphase centriole independence

2017 ◽  
Author(s):  
Robert Mahen
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Lipid Membrane ◽  
Cellular Compartment ◽  
Ciliary Function ◽  
Assembly Kinetics ◽  
Pericentriolar Material ◽  
Membrane Bound ◽  
Kinetics Of ◽  
Do So

AbstractThe centrosome is a non-membrane bound cellular compartment consisting of two centrioles surrounded by a protein coat termed the pericentriolar material (PCM). Centrioles must remain physically associated together (a phenomenon called centrosome cohesion) for cell migration, ciliary function and mitosis, yet how this occurs in the absence of a bounding lipid membrane is unclear. One model posits that pericentriolar fibres formed from rootletin protein directly link centrioles, yet little is known about the structure, biophysical properties or assembly kinetics of such fibres. Here, I combine live cell imaging of endogenously tagged rootletin with cell fusion, and find previously unrecognised plasticity in centrosome cohesion. Rootletin forms large, diffusionally stable, bifurcating fibres, which amass slowly on mature centrioles over many hours from anaphase. Nascent centrioles (procentrioles) in contrast do not form roots, and must be licensed to do so through polo-like kinase 1 (PLK1) activity. Transient separation of roots accompanies centriolar repositioning during the interphase, suggesting that centrioles organize as independent units, each containing a discrete root. Indeed, forced induction of duplicate centriole pairs allows independent re-shuffling of individual centrioles between the pairs. Thus, collectively, these findings suggest that progressively nucleated polymers mediate the dynamic association of centrioles as either one or two interphase centrosomes, with implications for our understanding of how non-membrane bound organelles self-organise.

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