In Vivo Assembly and Arrangement of the DNA Translocase SpoIIIE During Chromosome Segregation and Membrane Fission in B. Subtilis

Escherichia coli FtsK is a septum-located DNA translocase that co-ordinates the late stages of cytokinesis and chromosome segregation. Relatives of FtsK are present in most bacteria; in Bacillus subtilis, the FtsK orthologue, SpoIIIE, transfers the majority of a chromosome into the forespore during sporulation. DNA translocase activity is contained within a ~ 512-amino-acid C-terminal domain, which is divided into three subdomains: α, β and γ. α and β comprise the translocation motor, and γ is a regulatory domain that interacts with DNA and with the XerD recombinase. In vitro rates of translocation of ~ 5 kb·s−1 have been measured for both FtsK and SpoIIIE, whereas, in vivo, SpoIIIE has a comparable rate of translocation. Translocation by both of these proteins is not only rapid, but also directed by DNA sequence. This directionality requires interaction of the γ subdomain with specific 8 bp DNA asymmetric sequences that are oriented co-directionally with replication direction of the bacterial chromosome. The γ subdomain also interacts with the XerCD site-specific recombinase to activate chromosome unlinking by recombination at the chromosomal dif site. In the present paper, the properties in vivo and in vitro of FtsK and its relatives are discussed in relation to the biological functions of these remarkable enzymes.

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Visualization and functional dissection of coaxial paired SpoIIIE channels across the sporulation septum

eLife ◽

10.7554/elife.06474 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 25

Author(s):

Jae Yen Shin ◽

Javier Lopez-Garrido ◽

Sang-Hyuk Lee ◽

Cesar Diaz-Celis ◽

Tinya Fleming ◽

...

Keyword(s):

Protein Degradation ◽

Mother Cell ◽

Chromosome Translocation ◽

Specific Protein ◽

Dna Translocation ◽

Membrane Fission ◽

Photoactivated Localization Microscopy ◽

And Function ◽

Dna Translocase

SpoIIIE is a membrane-anchored DNA translocase that localizes to the septal midpoint to mediate chromosome translocation and membrane fission during Bacillus subtilis sporulation. Here we use cell-specific protein degradation and quantitative photoactivated localization microscopy in strains with a thick sporulation septum to investigate the architecture and function of the SpoIIIE DNA translocation complex in vivo. We were able to visualize SpoIIIE complexes with approximately equal numbers of molecules in the mother cell and the forespore. Cell-specific protein degradation showed that only the mother cell complex is required to translocate DNA into the forespore, whereas degradation in either cell reverses membrane fission. Our data suggest that SpoIIIE assembles a coaxially paired channel for each chromosome arm comprised of one hexamer in each cell to maintain membrane fission during DNA translocation. We show that SpoIIIE can operate, in principle, as a bi-directional motor that exports DNA.

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Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

Essays in Biochemistry ◽

10.1042/ebc20190080 ◽

2020 ◽

Vol 64 (2) ◽

pp. 251-261

Author(s):

Jessica E. Fellmeth ◽

Kim S. McKim

Keyword(s):

Chromosome Segregation ◽

New Technologies ◽

Early Prophase ◽

Unique Role ◽

Kinetochore Assembly ◽

M Phase ◽

In Vivo Analysis ◽

Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

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Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

Molecular and Cellular Biology ◽

10.1128/mcb.25.12.4977-4992.2005 ◽

2005 ◽

Vol 25 (12) ◽

pp. 4977-4992 ◽

Cited By ~ 100

Author(s):

Hao G. Nguyen ◽

Dharmaraj Chinnappan ◽

Takeshi Urano ◽

Katya Ravid

Keyword(s):

Chromosome Segregation ◽

Fluorescent Protein ◽

Point Mutations ◽

Degradation Pathway ◽

Aurora B ◽

Stable Form ◽

Wild Type ◽

Green Fluorescent ◽

Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

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Pericentric chromatin loops function as a nonlinear spring in mitotic force balance

The Journal of Cell Biology ◽

10.1083/jcb.201208163 ◽

2013 ◽

Vol 200 (6) ◽

pp. 757-772 ◽

Cited By ~ 48

Author(s):

Andrew D. Stephens ◽

Rachel A. Haggerty ◽

Paula A. Vasquez ◽

Leandra Vicci ◽

Chloe E. Snider ◽

...

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Force Balance ◽

Nonlinear Spring ◽

Sister Chromatids ◽

Spindle Dynamics ◽

Restoring Forces ◽

Cross Links ◽

Mean And Variance

The mechanisms by which sister chromatids maintain biorientation on the metaphase spindle are critical to the fidelity of chromosome segregation. Active force interplay exists between predominantly extensional microtubule-based spindle forces and restoring forces from chromatin. These forces regulate tension at the kinetochore that silences the spindle assembly checkpoint to ensure faithful chromosome segregation. Depletion of pericentric cohesin or condensin has been shown to increase the mean and variance of spindle length, which have been attributed to a softening of the linear chromatin spring. Models of the spindle apparatus with linear chromatin springs that match spindle dynamics fail to predict the behavior of pericentromeric chromatin in wild-type and mutant spindles. We demonstrate that a nonlinear spring with a threshold extension to switch between spring states predicts asymmetric chromatin stretching observed in vivo. The addition of cross-links between adjacent springs recapitulates coordination between pericentromeres of neighboring chromosomes.

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In vivo analysis of the Saccharomyces cerevisiae centromere CDEIII sequence: requirements for mitotic chromosome segregation

Molecular and Cellular Biology ◽

10.1128/mcb.11.10.5212-5221.1991 ◽

1991 ◽

Vol 11 (10) ◽

pp. 5212-5221

Author(s):

B Jehn ◽

R Niedenthal ◽

J H Hegemann

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Segregation ◽

Artificial Chromosome ◽

Complete Information ◽

Saturation Mutagenesis ◽

Chromosome Loss ◽

Single Mutation ◽

Yeast Saccharomyces Cerevisiae ◽

Double Base

In the yeast Saccharomyces cerevisiae, the complete information needed in cis to specify a fully functional mitotic and meiotic centromere is contained within 120 bp arranged in the three conserved centromeric (CEN) DNA elements CDEI, -II, and -III. The 25-bp CDEIII is most important for faithful chromosome segregation. We have constructed single- and double-base substitutions in all highly conserved residues and one nonconserved residue of this element and analyzed the mitotic in vivo function of the mutated CEN DNAs, using an artificial chromosome. The effects of the mutations on chromosome segregation vary between wild-type-like activity (chromosome loss rate of 4.8 x 10(-4)) and a complete loss of CEN function. Data obtained by saturation mutagenesis of the palindromic core sequence suggest asymmetric involvement of the palindromic half-sites in mitotic CEN function. The poor CEN activity of certain single mutations could be improved by introducing an additional single mutation. These second-site suppressors can be found at conserved and nonconserved positions in CDEIII. Our suppression data are discussed in the context of natural CDEIII sequence variations found in the CEN sequences of different yeast chromosomes.

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Overexpressed Pituitary Tumor-Transforming Gene Causes Aneuploidy in Live Human Cells

Endocrinology ◽

10.1210/en.2003-0305 ◽

2003 ◽

Vol 144 (11) ◽

pp. 4991-4998 ◽

Cited By ~ 76

Author(s):

Run Yu ◽

Wenge Lu ◽

Jiandong Chen ◽

Chris J. McCabe ◽

Shlomo Melmed

Keyword(s):

Cancer Cells ◽

Chromosome Segregation ◽

Pituitary Tumor ◽

Fluorescent Protein ◽

Human Cancer ◽

Live Cells ◽

Pituitary Tumor Transforming Gene ◽

Transforming Gene

Abstract The mammalian securin, pituitary tumor-transforming gene (PTTG), is overexpressed in several tumors and transforms cells in vitro and in vivo. To test the hypothesis that PTTG overexpression causes aneuploidy, enhanced green fluorescent protein (EGFP)-tagged PTTG (PTTG-EGFP) was expressed in human H1299 cancer cells (with undetectable endogenous PTTG expression) and mitosis of individual live cells observed. Untransfected cells and cells expressing EGFP alone exhibited appropriate mitosis. PTTG-EGFP markedly prolonged prophase and metaphase, indicating that PTTG blocks progression of mitosis to anaphase. In cells that underwent apparently normal mitosis (35 of 65 cells), PTTG-EGFP was degraded about 1 min before anaphase onset. Cells that failed to degrade PTTG-EGFP exhibited asymmetrical cytokinesis without chromosome segregation (18 of 65 cells) or chromosome decondensation without cytokinesis (9 of 65 cells), resulting in appearance of a macronucleus. Fifty-one of 55 cells expressing a nondegradable mutant PTTG exhibited asymmetrical cytokinesis without chromosome segregation, and some (4 of 55) decondensed chromosomes, both resulting in macronuclear formation. During this abnormal cytokinesis, all chromosomes and spindles and both centrosomes moved to one daughter cell, suggesting potential chaos in the subsequent mitosis. In conclusion, failure of PTTG degradation or enhanced PTTG accumulation, as a consequence of overexpression, inhibits mitosis progression and chromosome segregation but does not directly affect cytokinesis, resulting in aneuploidy. These results demonstrate that PTTG induces aneuploidy in single, live, human cancer cells.

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Virulence protein VirD5 ofAgrobacterium tumefaciensbinds to kinetochores in host cells via an interaction with Spt4

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706166114 ◽

2017 ◽

Vol 114 (38) ◽

pp. 10238-10243 ◽

Cited By ~ 9

Author(s):

Xiaorong Zhang ◽

G. Paul H. van Heusden ◽

Paul J. J. Hooykaas

Keyword(s):

Chromosome Segregation ◽

High Frequency ◽

Chromosomal Instability ◽

Plant Cells ◽

Tumor Formation ◽

Host Cells ◽

Infection Site ◽

Virulence Proteins ◽

Virulence Protein

The bacteriumAgrobacterium tumefacienscauses crown gall tumor formation in plants. During infection the bacteria translocate an oncogenic piece of DNA (transferred DNA, T-DNA) into plant cells at the infection site. A number of virulence proteins are cotransported into host cells concomitantly with the T-DNA to effectuate transformation. Using yeast as a model host, we find that one of these proteins, VirD5, localizes to the centromeres/kinetochores in the nucleus of the host cells by its interaction with the conserved protein Spt4. VirD5 promotes chromosomal instability as seen by the high-frequency loss of a minichromosome in yeast. By using both yeast and plant cells with a chromosome that was specifically marked by alacOrepeat, chromosome segregation errors and the appearance of aneuploid cells due to the presence of VirD5 could be visualized in vivo. Thus, VirD5 is a prokaryotic virulence protein that interferes with mitosis.

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The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip–coupling protein

The Journal of Cell Biology ◽

10.1083/jcb.201711181 ◽

2018 ◽

Vol 217 (11) ◽

pp. 3886-3900 ◽

Cited By ~ 12

Author(s):

Aida Llauró ◽

Hanako Hayashi ◽

Megan E. Bailey ◽

Alex Wilson ◽

Patryk Ludzia ◽

...

Keyword(s):

Chromosome Segregation ◽

Binding Activity ◽

Significant Similarity ◽

Load Bearing ◽

Kinetochore Protein ◽

Microtubule Binding ◽

Binding Domains ◽

Microtubule Binding Domain ◽

Coupling Protein

Kinetochores are multiprotein machines that drive chromosome segregation by maintaining persistent, load-bearing linkages between chromosomes and dynamic microtubule tips. Kinetochores in commonly studied eukaryotes bind microtubules through widely conserved components like the Ndc80 complex. However, in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here, we show that the T. brucei kinetochore protein KKT4 binds directly to microtubules and maintains load-bearing attachments to both growing and shortening microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Thus, despite its lack of significant similarity to other known microtubule-binding proteins, KKT4 has key functions required for driving chromosome segregation. We propose that it represents a primary element of the kinetochore–microtubule interface in kinetoplastids.

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Molecular requirements for the formation of a kinetochore–microtubule interface by Dam1 and Ndc80 complexes

The Journal of Cell Biology ◽

10.1083/jcb.201210091 ◽

2012 ◽

Vol 200 (1) ◽

pp. 21-30 ◽

Cited By ~ 49

Author(s):

Fabienne Lampert ◽

Christine Mieck ◽

Gregory M. Alushin ◽

Eva Nogales ◽

Stefan Westermann

Keyword(s):

Chromosome Segregation ◽

Protein Complexes ◽

Structural Elements ◽

Large Protein ◽

Kinetochore Assembly ◽

Sister Chromatids ◽

Dam1 Complex ◽

Shed Light ◽

Kinetochore Function

Kinetochores are large protein complexes that link sister chromatids to the spindle and transduce microtubule dynamics into chromosome movement. In budding yeast, the kinetochore–microtubule interface is formed by the plus end–associated Dam1 complex and the kinetochore-resident Ndc80 complex, but how they work in combination and whether a physical association between them is critical for chromosome segregation is poorly understood. Here, we define structural elements required for the Ndc80–Dam1 interaction and probe their function in vivo. A novel ndc80 allele, selectively impaired in Dam1 binding, displayed growth and chromosome segregation defects. Its combination with an N-terminal truncation resulted in lethality, demonstrating essential but partially redundant roles for the Ndc80 N-tail and Ndc80–Dam1 interface. In contrast, mutations in the calponin homology domain of Ndc80 abrogated kinetochore function and were not compensated by the presence of Dam1. Our experiments shed light on how microtubule couplers cooperate and impose important constraints on structural models for outer kinetochore assembly.

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