scholarly journals KIF18A's neck linker permits navigation of microtubule-bound obstacles within the mitotic spindle

2019 ◽  
Vol 2 (1) ◽  
pp. e201800169 ◽  
Author(s):  
Heidi LH Malaby ◽  
Dominique V Lessard ◽  
Christopher L Berger ◽  
Jason Stumpff
Keyword(s):  
Single Molecule ◽  
Mitotic Spindle ◽  
Binding Proteins ◽  
Mitotic Chromosome ◽  
Chromosome Movement ◽  
Wild Type ◽  
Microtubule Associated Proteins ◽  
Chromosome Alignment ◽  
Associated Proteins ◽  
Fiber Dynamics

KIF18A (kinesin-8) is required for mammalian mitotic chromosome alignment. KIF18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that KIF18A's relatively long neck linker is required for the motor's accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of KIF18A display a deficiency in accumulation at the ends of K-fibers at the center of the spindle. Depletion of K-fiber–binding proteins reduces the KIF18A sNL localization defect, whereas their overexpression reduces wild-type KIF18A's ability to accumulate on this same K-fiber subset. Furthermore, single-molecule assays indicate that KIF18A sNL motors are less proficient in navigating microtubules coated with microtubule-associated proteins. Taken together, these results support a model in which KIF18A's neck linker length permits efficient navigation of obstacles to reach K-fiber ends during mitosis.

scholarly journals The Human Kinesin Kif18A’s Neck Linker Permits Navigation of Microtubule Bound Obstacles within the Mitotic Spindle

2018 ◽  
Author(s):  
Heidi L. H. Malaby ◽  
Dominique V. Lessard ◽  
Christopher L. Berger ◽  
Jason Stumpff
Keyword(s):  
Single Molecule ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Mitotic Chromosome ◽  
Genetic Material ◽  
Daughter Cells ◽  
Chromosome Alignment ◽  
A Cell ◽  
Fiber Dynamics ◽  
Robust Separation

AbstractMitotic chromosome alignment is essential for the robust separation of genetic material into daughter cells. In mammalian cells, this process requires the function of Kif18A, a kinesin-8 motor protein. Kif18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that Kif18A’s relatively long (17 amino acid) neck linker is required for the motor’s accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of Kif18A display a deficiency in K-fiber accumulation, especially on K-fibers near the center of the spindle. This pattern correlates with the more uniform concentration of the microtubule bundling protein HURP on central K-fibers compared to peripheral K-fibers. Depletion of HURP permits Kif18A sNL to accumulate on central K-fibers, while HURP overexpression reduces wild-type Kif18A’s ability to accumulate on this same K-fiber subset. Furthermore, single molecule assays indicate that Kif18A sNL motors are less proficient at navigating microtubules coated with the microtubule associated protein tau. Taken together, these results support a model in which Kif18A’s neck linker length permits efficient navigation of obstacles such as HURP to reach K-fiber ends during mitosis.Signficiance StatementKinesin motor proteins play key roles in controlling chromosome alignment and segregation during cell division. The kinesin Kif18A confines chromosomes to the middle of the spindle by accumulating at the ends of microtubules attached to chromosomes. We show here that Kif18A’s ability to accumulate at the end of these microtubules requires navigation of microtubule-associated protein obstacles, and that this activity is imparted by a relatively long neck linker region. These findings demonstrate a molecular mechanism for navigation of densely populated microtubules inside a cell.

Filaments and membranes in the mitotic apparatus

1983 ◽  
Vol 41 ◽  
pp. 544-547
Author(s):  
Kent McDonald
Keyword(s):  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mitotic Apparatus ◽  
Light Microscope Level ◽  
Microtubule Associated Proteins ◽  
Recent Developments ◽  
Associated Proteins ◽  
Force Generator ◽  
Spindle Function ◽  
Antibody Technology

At the light microscope level the recent developments and interest in antibody technology have permitted the localization of certain non-microtubule proteins within the mitotic spindle, e.g., calmodulin, actin, intermediate filaments, protein kinases and various microtubule associated proteins. Also, the use of fluorescent probes like chlorotetracycline suggest the presence of membranes in the spindle. Localization of non-microtubule structures in the spindle at the EM level has been less rewarding. Some mitosis researchers, e.g., Rarer, have maintained that actin is involved in mitosis movements though the bulk of evidence argues against this interpretation. Others suggest that a microtrabecular network such as found in chromatophore granule movement might be a possible force generator but there is little evidence for or against this view. At the level of regulation of spindle function, Harris and more recently Hepler have argued for the importance of studying spindle membranes. Hepler also believes that membranes might play a structural or mechanical role in moving chromosomes.

Regulation of Microtubule Assembly: The View From The End

2000 ◽  
Vol 6 (S2) ◽  
pp. 80-81
Author(s):  
L. Cassimeris ◽  
C. Spittle ◽  
M. Kratzer
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Dynamic Instability ◽  
Intrinsic Property ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
Chromosome Movement ◽  
Spindle Formation ◽  
Associated Proteins

The mitotic spindle is responsible for chromosome movement during mitosis. It is composed of a dynamic array of microtubules and associated proteins whose assembly and constant turnover are required for both spindle formation and chromosome movement. Because microtubule assembly and turnover are necessary for chromosome segregation, we are studying how cells regulate microtubule dynamics. Microtubules are polarized polymers composed of tubulin subunits; they assemble by a process of dynamic instability where individual microtubules exist in persistent phases of elongation or rapid shortening with abrupt transitions between these two states. The switch from elongation to shortening is termed catastrophe, and the switch from shortening to elongation, rescue. Although dynamic instability is an intrinsic property of the tubulin subunits, cells use associated proteins to both speed elongation (∼ 10 fold) and regulate transitions.The only protein isolated to date capable of promoting fast polymerization consistent with rates in vivo is XMAP215, a 215 kD protein from Xenopus eggs.

scholarly journals The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA

2020 ◽  
Vol 48 (19) ◽  
pp. 10820-10831
Author(s):  
Kiyoto Kamagata ◽  
Kana Ouchi ◽  
Cheng Tan ◽  
Eriko Mano ◽  
Sridhar Mandali ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Binding Modes ◽  
Dna Structures ◽  
Bacterial Proteins ◽  
Associated Proteins ◽  
Diffusion Mechanisms ◽  
Dynamics Simulations

Abstract DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.

scholarly journals Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo.

1989 ◽  
Vol 109 (6) ◽  
pp. 2977-2991 ◽  
Author(s):  
D R Kellogg ◽  
C M Field ◽  
B M Alberts
Keyword(s):  
Affinity Chromatography ◽  
Mitotic Spindle ◽  
Polyclonal Antibodies ◽  
Drosophila Embryo ◽  
Microtubule Cytoskeleton ◽  
Microtubule Associated Proteins ◽  
Early Drosophila Embryo ◽  
Individual Mouse ◽  
Associated Proteins

We have developed affinity chromatography methods for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts and have used them to analyze the cytoskeleton of the early Drosophila embryo. More than 50 Drosophila embryo proteins bind to microtubule affinity columns. To begin to characterize these proteins, we have generated individual mouse polyclonal antibodies that specifically recognize 24 of them. As judged by immunofluorescence, some of the antigens localize to the mitotic spindle in the early Drosophila embryo, while others are present in centrosomes, kinetochores, subsets of microtubules, or a combination of these structures. Since 20 of the 24 antibodies stain microtubule structures, it is likely that most of the proteins that bind to our columns are associated with microtubules in vivo. Very few MAPS seem to be identically localized in the cell, indicating that the microtubule cytoskeleton is remarkably complex.

scholarly journals Contribution of hCAP-D2, a Non-SMC Subunit of Condensin I, to Chromosome and Chromosomal Protein Dynamics during Mitosis

2005 ◽  
Vol 25 (2) ◽  
pp. 740-750 ◽  
Author(s):  
Erwan Watrin ◽  
Vincent Legagneux
Keyword(s):  
Rna Interference ◽  
Mitotic Spindle ◽  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosomal Protein ◽  
Structural Components ◽  
Mitotic Chromosomes ◽  
Sister Chromatids ◽  
Chromosome Alignment ◽  
Structural Maintenance Of Chromosomes

ABSTRACT Condensins are heteropentameric complexes that were first identified as structural components of mitotic chromosomes. They are composed of two SMC (structural maintenance of chromosomes) and three non-SMC subunits. Condensins play a role in the resolution and segregation of sister chromatids during mitosis, as well as in some aspects of mitotic chromosome assembly. Two distinct condensin complexes, condensin I and condensin II, which differ only in their non-SMC subunits, exist. Here, we used an RNA interference approach to deplete hCAP-D2, a non-SMC subunit of condensin I, in HeLa cells. We found that the association of hCAP-H, another non-SMC subunit of condensin I, with mitotic chromosomes depends on the presence of hCAP-D2. Moreover, chromatid axes, as defined by topoisomerase II and hCAP-E localization, are disorganized in the absence of hCAP-D2, and the resolution and segregation of sister chromatids are impaired. In addition, hCAP-D2 depletion affects chromosome alignment in metaphase and delays entry into anaphase. This suggests that condensin I is involved in the correct attachment between chromosome kinetochores and microtubules of the mitotic spindle. These results are discussed relative to the effects of depleting both condensin complexes.

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.

Proteins of the mammalian mitotic spindle: phosphorylation/dephosphorylation of MAP-4 during mitosis

1991 ◽  
Vol 98 (4) ◽  
pp. 577-588 ◽  
Author(s):  
D.D. Vandre ◽  
V.E. Centonze ◽  
J. Peloquin ◽  
R.M. Tombes ◽  
G.G. Borisy
Keyword(s):  
Mitotic Spindle ◽  
Immunoblot Analysis ◽  
Entry And Exit ◽  
Immunofluorescence Analysis ◽  
Temporal And Spatial Distribution ◽  
Microtubule Associated Proteins ◽  
Before And After ◽  
Associated Proteins ◽  
Temporal And Spatial ◽  
Monospecific Antibodies

The phosphoprotein composition of isolated CHO spindles was analyzed using the MPM-1 and MPM-2 antibodies, which are reactive with a phosphorylated epitope enriched in mitotic cells and present on the centrosome, kinetochores, midbody and fibers of the mitotic spindle. Several high molecular weight phosphorylated spindle proteins were detected on immunoblots, including species of 410 × 10(3) Mr, 350 × 10(3) Mr, a 230–240 X 10(3) Mr doublet, 210 × 10(3) Mr and 120 × 10(3) Mr. The temporal and spatial distribution of the MPM-reactive phosphoproteins was determined by examining spindle structures isolated from cells at various stages of mitosis. The susceptibility of the staining pattern to extraction with salt, a procedure known to remove most microtubule-associated proteins (MAPs), was also examined. The phosphorylated 210 × 10(3) Mr species was identified as MAP-4 and localized to the spindle fibers using (1) a polyclonal antibody raised against this species, that reacted with known MAPs, and (2) established MAP-4 antibodies that reacted with the spindle 210 × 10(3) Mr MPM-reactive proteins. The comparative immunoblot and immunofluorescence analysis establishes a cycle of phosphorylation/dephosphorylation of MAP-4 upon entry and exit from mitosis. Regarding the other MPM-reactive proteins, comparative immunofluorescence staining and immunoblot analysis of isolated spindle samples before and after salt extraction indicate that they may be constituents of the centrosome, kinetochores or midbody, but their definitive identification awaits the production of monospecific antibodies.

Sign in / Sign up

Export Citation Format

Share Document