scholarly journals NDC80 clustering modulates microtubule dynamics under force

2017 ◽  
Author(s):  
Vladimir A. Volkov ◽  
Pim J. Huis in't Veld ◽  
Marileen Dogterom ◽  
Andrea Musacchio
Keyword(s):  
Residence Time ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Microtubule Binding ◽  
Multiple Interfaces ◽  
Biological Phenomena ◽  
Ndc80 Complex ◽  
Multiple Copies ◽  
Spindle Microtubules ◽  
Incremental Addition

AbstractMultivalency, the presence of multiple interfaces for intermolecular interactions, underlies many biological phenomena, including receptor clustering and cytosolic condensation. One of its ultimate purposes is to increase binding affinity, but systematic analyses of its role in complex biological assemblies have been rare. Presence of multiple copies of the microtubule-binding NDC80 complex is an evolutionary conserved but poorly characterized feature of kinetochores, the points of attachment of chromosomes to spindle microtubules. To address its significance, we engineered modules allowing incremental addition of NDC80 complexes. The modules’ residence time on microtubules increased exponentially with the number of NDC80 complexes. While modules containing a single NDC80 complex were unable to track depolymerizing microtubules, modules with two or more complexes tracked depolymerizing microtubules and stiffened the connection with microtubules under force. Cargo-conjugated modules of divalent or trivalent NDC80 stalled and rescued microtubule depolymerization in a force-dependent manner. Thus, multivalent microtubule binding through NDC80 clustering is crucial for force-induced modulation of kinetochore-microtubule attachments.

scholarly journals Multivalency of NDC80 in the outer kinetochore is essential to track shortening microtubules and generate forces

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Vladimir A Volkov ◽  
Pim J Huis in 't Veld ◽  
Marileen Dogterom ◽  
Andrea Musacchio
Keyword(s):  
Residence Time ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Microtubule Binding ◽  
Ndc80 Complex ◽  
Multiple Copies ◽  
Biased Diffusion ◽  
Spindle Microtubules ◽  
Incremental Addition ◽  
Do So

Presence of multiple copies of the microtubule-binding NDC80 complex is an evolutionary conserved feature of kinetochores, points of attachment of chromosomes to spindle microtubules. This may enable multivalent attachments to microtubules, with implications that remain unexplored. Using recombinant human kinetochore components, we show that while single NDC80 complexes do not track depolymerizing microtubules, reconstituted particles containing the NDC80 receptor CENP-T bound to three or more NDC80 complexes do so effectively, as expected for a kinetochore force coupler. To study multivalency systematically, we engineered modules allowing incremental addition of NDC80 complexes. The modules’ residence time on microtubules increased exponentially with the number of NDC80 complexes. Modules with two or more complexes tracked depolymerizing microtubules with increasing efficiencies, and stalled and rescued microtubule depolymerization in a force-dependent manner when conjugated to cargo. Our observations indicate that NDC80, rather than through biased diffusion, tracks depolymerizing microtubules by harnessing force generated during microtubule disassembly.

scholarly journals The kinesin-8 Kip3 depolymerizes microtubules with a collective force-dependent mechanism

2019 ◽  
Author(s):  
Michael Bugiel ◽  
Mayank Chugh ◽  
Tobias Jörg Jachowski ◽  
Erik Schäffer ◽  
Anita Jannasch
Keyword(s):  
Cell Division ◽  
Optical Tweezers ◽  
High Precision ◽  
Residence Time ◽  
Binding Activity ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Cellular Processes ◽  
Length Changes ◽  
Stall Force

ABSTRACTMicrotubules are highly dynamic filaments with dramatic structural rearrangements and length changes during the cell cycle. An accurate control of the microtubule length is essential for many cellular processes in particular, during cell division. Motor proteins from the kinesin-8 family depolymerize microtubules by interacting with their ends in a collective and length-dependent manner. However, it is still unclear how kinesin-8 depolymerizes microtubules. Here, we tracked the microtubule end-binding activity of yeast kinesin-8, Kip3, under varying loads and nucleotide conditions using high-precision optical tweezers. We found that single Kip3 motors spent up to 200 s at the microtubule end and were not stationary there but took several 8-nm forward and backward steps that were suppressed by loads. Interestingly, increased loads, similar to increased motor concentrations, also exponentially decreased the motors’ residence time at the microtubule end. On the microtubule lattice, loads also exponentially decreased the run length and time. However, for the same load, lattice run times were significantly longer compared to end residence times suggesting the presence of a distinct force-dependent detachment mechanism at the microtubule end. The force dependence of the end residence time enabled us to estimate what force must act on a single motor to achieve the microtubule depolymerization speed of a motor ensemble. This force is higher than the stall force of a single Kip3 motor, supporting a collective force-dependent depolymerization mechanism that unifies the so-called “bump-off” and “switching” models. Understanding the mechanics of kinesin-8’s microtubule end activity will provide important insights into cell division with implications for cancer research.STATEMENT OF SIGNIFICANCEKinesin-8 motors are important for microtubule length regulation and are over-expressed in different types of cancer. Yet, on the molecular level, it is unclear how these motors depolymerize microtubules. Using high-precision optical tweezers, we measured how single yeast kinesin-8 motors, Kip3, interacted with the microtubule end. Interestingly, we found that single Kip3 motors were still motile at the microtubule end. The force dependence of how long single motors were associated with the microtubule end enabled us to estimate what force motors must exert onto each other to achieve the collective microtubule depolymerization speed of many motors. Our data support a collective force-dependent depolymerization mechanism. A better understanding of Kip3’s microtubule end activity has implications for cell division and associated diseases.

scholarly journals Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores

2015 ◽  
Vol 208 (2) ◽  
pp. 181-196 ◽  
Author(s):  
Soonjoung Kim ◽  
Hongtao Yu
Keyword(s):  
Spindle Checkpoint ◽  
Aurora B ◽  
Dependent Manner ◽  
Checkpoint Proteins ◽  
Multiple Strategies ◽  
Complete Inactivation ◽  
Microtubule Attachment ◽  
Ndc80 Complex ◽  
Spindle Microtubules ◽  
Multiple Assembly

During mitosis, the spindle checkpoint senses kinetochores not properly attached to spindle microtubules and prevents precocious sister-chromatid separation and aneuploidy. The constitutive centromere-associated network (CCAN) at inner kinetochores anchors the KMN network consisting of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C) at outer kinetochores. KMN is a critical kinetochore receptor for both microtubules and checkpoint proteins. Here, we show that nearly complete inactivation of KMN in human cells through multiple strategies produced strong checkpoint defects even when all kinetochores lacked microtubule attachment. These KMN-inactivating strategies reveal multiple KMN assembly mechanisms at human mitotic kinetochores. In one mechanism, the centromeric kinase Aurora B phosphorylates Mis12C and strengthens its binding to the CCAN subunit CENP-C. In another, CENP-T contributes to KMN attachment in a CENP-H-I-K–dependent manner. Our study provides insights into the mechanisms of mitosis-specific assembly of the checkpoint platform KMN at human kinetochores.

scholarly journals The EF-Hand Ca2+-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca2+-binding Requirements

2004 ◽  
Vol 15 (2) ◽  
pp. 481-496 ◽  
Author(s):  
Josefa Andrade ◽  
Hu Zhao ◽  
Brian Titus ◽  
Sandra Timm Pearce ◽  
Margarida Barroso
Keyword(s):  
Endoplasmic Reticulum ◽  
Conformational Changes ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Network Formation ◽  
Binding Protein ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Independent Manner ◽  
Ef Hand

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

scholarly journals Impact of evolutionary selection on dynamic behavior of MCAK protein

2020 ◽  
Author(s):  
Shiwani Limbu
Keyword(s):  
Amino Acid ◽  
Amino Acid Position ◽  
Coding Region ◽  
Microtubule Depolymerization ◽  
Protein Coding ◽  
Microtubule Binding ◽  
Evolutionary Selection ◽  
Family Protein ◽  
Α Helix

AbstractKinesins of class 13 (kinesin-13s), also known as KinI family proteins, are non-motile microtubule binding kinesin proteins. Mitotic centromere-associated kinesin (MCAK), a member of KinI family protein, diffuses along the microtubule and plays a key role in microtubule depolymerization. Here we have demonstrated the role of evolutionary selection in MCAK protein coding region in regulating its dynamics associated with microtubule binding and stability. Our results indicate that evolutionary selection within MCAK motor domain at amino acid position 440 in carnivora and artiodactyla order results in significant change in the dynamics of α – helix and loop 11, indicating its likely impact on changing the microtubule binding and depolymerization process. Furthermore, evolutionary selections at amino acid position 600, 617 and 698 are likely to affect MCAK stability. A deeper understanding of evolutionary selections in MCAK can reveal the mechanism associated with change in microtubule dynamics within eutherian mammals.

Inscuteable-dependent apical localization of the microtubule-binding protein Cornetto suggests a role in asymmetric cell division

2001 ◽  
Vol 114 (20) ◽  
pp. 3655-3662 ◽  
Author(s):  
Silvia Bulgheresi ◽  
Elke Kleiner ◽  
Juergen A. Knoblich
Keyword(s):  
Cell Fate ◽  
Mitotic Spindle ◽  
Binding Protein ◽  
Protein Localization ◽  
Apical Cell ◽  
Coiled Coil ◽  
Cell Cortex ◽  
Dependent Manner ◽  
Genetic Redundancy ◽  
Microtubule Binding

Drosophila neuroblasts divide asymmetrically along the apical-basal axis. The Inscuteable protein localizes to the apical cell cortex in neuroblasts from interphase to metaphase, but disappears in anaphase. Inscuteable is required for correct spindle orientation and for asymmetric localization of cell fate determinants to the opposite (basal) cell cortex. Here, we show that Inscuteable also directs asymmetric protein localization to the apical cell cortex during later stages of mitosis. In a two-hybrid screen for Inscuteable-binding proteins, we have identified the coiled-coil protein Cornetto, which shows a highly unusual subcellular distribution in neuroblasts. Although the protein is uniformly distributed in the cytoplasm during metaphase, it concentrates apically in anaphase and forms an apical crescent during telophase in an inscuteable-dependent manner. Upon overexpression, Cornetto localizes to astral microtubules and microtubule spin-down experiments demonstrate that Cornetto is a microtubule-binding protein. After disruption of the actin cytoskeleton, Cornetto localizes with microtubules throughout the cell cycle and decorates the mitotic spindle during metaphase. Our results reveal a novel pattern of asymmetric protein localization in Drosophila neuroblasts and are consistent with a function of Cornetto in anchoring the mitotic spindle during late phases of mitosis, even though our cornetto mutant analysis suggests that this function might be obscured by genetic redundancy.

Spatiotemporal control of functional specification and distribution of spindle microtubules with 13, 14 and 15 protofilaments during mitosis in the ciliate Nyctotherus

1985 ◽  
Vol 76 (1) ◽  
pp. 337-355
Author(s):  
U. Eichenlaub-Ritter
Keyword(s):  
Differential Sensitivity ◽  
Microtubule Depolymerization ◽  
Functional Specification ◽  
Microtubule Stability ◽  
Early Anaphase ◽  
Late Telophase ◽  
Spindle Microtubules ◽  
Elongation Phase ◽  
Spatiotemporal Control ◽  
Macronuclear Division

The formation of microtubules with more than 13 protofilaments in the ciliate Nyctotherus ovalis Leidy seems to be a highly ordered process. Such microtubules are restricted to the nucleoplasm and, moreover, to certain stages of nuclear division. They assemble during anaphase of micronuclear mitosis and during the elongation phase of macronuclear division. The number of microtubules with more than 13 protofilaments in the micronuclear nucleoplasm increases as anaphase progresses. Furthermore, assembly of microtubules with 14 and 15 protofilaments seems to proceed concomitantly with net disassembly of 13-protofilament microtubules, because the total amount of polymerized tubulin in the interpolar spindle region remains approximately constant between mid anaphase and late telophase. In addition, evidence for spatial control of the distribution of microtubules with different protofilament numbers in the micronuclear stembody has been found. The percentage of microtubules with 13 protofilaments per stembody cross-section is highest at the ends of the stembody, while the percentage of microtubules with either 14 or 15 protofilaments increases as the middle of the stembody is approached. Temporal control of polymerization of microtubules with high protofilament numbers seems to be exerted independently in the two types of nuclei. For example, when the macronucleus starts to elongate it contains microtubules with more than 13 protofilaments but the metaphase micronucleus still possesses only microtubules with 13 protofilaments at this stage. Control of fidelity of protofilament numbers is not lost in the early stages of micronuclear or macronuclear division when cells are exposed to 2H2O or media containing taxol. Even microtubules that reassemble during recovery of metaphase micronuclei from nocodazole-induced microtubule depolymerization, in either the absence or presence of 2H2O and taxol, possess 13 protofilaments. Similarly, if the introduction of microtubules with 14 and 15 protofilaments is inhibited during early micronuclear anaphase and delayed for 60 min by exposure to nocodazole, such microtubules still assemble during telophase when recovery is permitted. Microtubules that have been assembled under normal conditions show differential sensitivity to nocodazole. During metaphase, nocodazole induces disassembly of most microtubules. There is an increase in microtubule stability that coincides with the appearance of microtubules with high protofilament numbers during early anaphase. However, considerable numbers of 13-protofilament microtubules, as well as microtubules with 14 and 15 protofilaments, exhibit such stability during anaphase.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau

1994 ◽  
Vol 124 (5) ◽  
pp. 769-782 ◽  
Author(s):  
BL Goode ◽  
SC Feinstein
Keyword(s):  
Amino Acid ◽  
Electrostatic Interactions ◽  
Weak Interactions ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Microtubule Organization ◽  
Developmentally Regulated ◽  
Microtubule Binding ◽  
Amino Acid Repeats

Tau is a developmentally regulated microtubule-associated protein that influences microtubule behavior by directly associating with tubulin. The carboxyl terminus of tau contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter-repeat (IR) regions previously thought to function as "linkers." Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site-directed mutagenesis reveal that the binding activity of this site is derived primarily from lys265 and lys272, with a lesser contribution from lys271. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed array of weak interactions thought to underlie the association between 18-amino acid repeats and microtubules (Butner, K. A., and M. W. Kirschner. J. Cell Biol. 115:717-730). Moreover, competition assays suggest that the R1-R2 IR associates with microtubules at tubulin site(s) distinct from those bound by the repeats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence-dependent manner. Since the R1-R2 IR is specifically expressed in adult tau, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult-specific, high affinity anchor that tethers the otherwise mobile tau molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons.

scholarly journals Adult teleost heart expresses two distinct troponin C paralogs: cardiac TnC and a novel and teleost-specific ssTnC in a chamber- and temperature-dependent manner

2013 ◽  
Vol 45 (18) ◽  
pp. 866-875 ◽  
Author(s):  
Christine E. Genge ◽  
William S. Davidson ◽  
Glen F. Tibbits
Keyword(s):  
Rainbow Trout ◽  
Single Gene ◽  
Temperature Acclimation ◽  
Troponin C ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Adult Zebrafish ◽  
Temperature Dependent ◽  
Tandem Gene Duplication ◽  
Multiple Copies

The teleost-specific whole genome duplication created multiple copies of genes allowing for subfunctionalization of isoforms. In this study, we show that the teleost cardiac Ca2+-binding troponin C (TnC) is the product of two distinct genes: cardiac TnC (cTnC, TnnC1a) and a fish-specific slow skeletal TnC (ssTnC, TnnC1b). The ssTnC gene is novel to teleosts as mammals have a single gene commonly referred as cTnC but which is also expressed in slow skeletal muscle. In teleosts, the data strongly indicate that these are two TnC genes are different paralogs. Because we determined that ssTnC exists across many teleosts but not in basal ray-finned fish (e.g., bichir), we propose that these paralogs are the result of an ancestral tandem gene duplication persisting only in teleosts. Quantification of mRNA levels was used to demonstrate distinct expression localization patterns of the paralogs within the chambers of the heart. In the adult zebrafish acclimated at 28°C, ssTnC mRNA levels are twofold greater than cTnC mRNA levels in the atrium, whereas cTnC mRNA was almost exclusively expressed in the ventricle. Meanwhile, rainbow trout acclimated at 5°C showed cTnC mRNA levels in both chambers significantly greater than ssTnC. Distinct responses to temperature acclimation were also quantified in both adult zebrafish and rainbow trout, with mRNA in both chambers shifting to express higher levels of cTnC in 18°C acclimated zebrafish and 5°C acclimated trout. Possible subfunctionalization of TnC isoforms may provide insight into how teleosts achieve physiological versatility in chamber-specific contractile properties.

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