scholarly journals Kinetochore-independent mechanisms of sister chromosome separation

2020 ◽  
Author(s):  
Hannah Vicars ◽  
Travis Karg ◽  
Brandt Warecki ◽  
Ian Bast ◽  
William Sullivan
Keyword(s):  
Topoisomerase Ii ◽  
Sliding Mode ◽  
Synthetic Lethality ◽  
Daughter Cells ◽  
Sister Chromosome ◽  
Associated Proteins ◽  
Chromosome Fragments ◽  
Chromosome Separation ◽  
Additional Image ◽  
Oriented Parallel

ABSTRACTAlthough kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore-independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.AUTHOR SUMMARYKinetochores, the site on the chromosomes to which microtubules attach driving the separation and segregation of replicated sister chromosomes, have been viewed as essential for proper cell division and accurate transmission of chromosomes into daughter cells. However previous studies demonstrated that sister chromosomes lacking kinetochores (acentrics) often undergo separation, segregation and transmission. Here we demonstrate that sister acentrics are held together through DNA intertwining. We show that during anaphase, acentric sister separation is achieved through Topoisomerase activity, an enzyme that resolves these DNA linkages, as well as forces generated on the acentrics by the growing ends of highly dynamic microtubule polymers. We found that acentric sister chromatids display unique patterns of separation using mechanisms independent of the kinetochore. Additionally, we identified the specific microtubule-associated proteins required for the successful mitotic transmission of acentric chromosomes to daughter cells. These studies reveal unsuspected, distinct forces that likely act on all chromosomes during mitosis independent of kinetochore-microtubule attachments.

scholarly journals Kinetochore-independent mechanisms of sister chromosome separation

PLoS Genetics ◽  
2021 ◽  
Vol 17 (1) ◽  
pp. e1009304
Author(s):  
Hannah Vicars ◽  
Travis Karg ◽  
Brandt Warecki ◽  
Ian Bast ◽  
William Sullivan
Keyword(s):  
Mitotic Spindle ◽  
Topoisomerase Ii ◽  
Sliding Mode ◽  
Synthetic Lethality ◽  
Sister Chromosome ◽  
Chromosome Fragments ◽  
Chromosome Separation ◽  
Additional Image ◽  
Oriented Parallel ◽  
Cell Cycle Kinase

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.

scholarly journals THE TIME OF SYNTHESIS AND THE CONSERVATION OF MITOSIS-RELATED PROTEINS IN CULTURED HUMAN AMNION CELLS

1967 ◽  
Vol 34 (1) ◽  
pp. 97-110 ◽  
Author(s):  
Jesse E. Sisken ◽  
Elaina Wilkes
Keyword(s):  
Time Lapse ◽  
Major Effect ◽  
Human Amnion ◽  
Daughter Cells ◽  
Low Concentrations ◽  
Associated Proteins ◽  
A Cell ◽  
Quantitative Analyses ◽  
Related Proteins ◽  
Kinetics Of

p-Fluorophenylalanine (PFPA), an analogue of phenylalanine which may be incorporated into proteins, increases the duration of mitosis. In the present experiments, based upon quantitative analyses of time-lapse cinemicrographic films, brief treatments of cells with PFPA are shown to affect the duration of metaphase in only those cells which enter division during or shortly after treatment. The offspring of cells with prolonged metaphases also tend to have prolonged metaphases. Analyses of the kinetics of the appearance of prolonged metaphases indicate that some protein specifically associated with mitosis is synthesized primarily during a period which corresponds closely to G2. The manner in which the defect is passed on to daughter cells indicates that the protein involved is conserved and reutilized by daughter cells for their subsequent divisions. Comparable experiments performed with low concentrations of puromycin indicate that the major effect of PFPA is due to its incorporation into protein rather than its ability to inhibit protein synthesis. The fact that puromycin-induced effects can also be passed on to daughter cells is interpreted to mean that cells make only specific amounts of some mitosis-associated proteins and that if a cell "inherits" a deficiency in such protein it is not able to compensate for the deficiency.

scholarly journals Inhibition of Chromosomal Separation Provides Insights into Cleavage Furrow Stimulation in Cultured Epithelial Cells

1998 ◽  
Vol 9 (8) ◽  
pp. 2173-2184 ◽  
Author(s):  
Sally P. Wheatley ◽  
Christopher B. O’Connell ◽  
Yu-li Wang
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Topoisomerase Ii ◽  
Rat Kidney ◽  
Myosin Ii ◽  
Daughter Cells ◽  
Irregular Shapes ◽  
Anaphase Onset ◽  
Cultured Epithelial Cells ◽  
Normal Rat

While astral microtubules are believed to be primarily responsible for the stimulation of cytokinesis in Echinodermembryos, it has been suggested that a signal emanating from the chromosomal region and mediated by the interzonal microtubules stimulates cytokinesis in cultured mammalian cells. To test this hypothesis, we examined cytokinesis in normal rat kidney cells treated with an inhibitor of topoisomerase II, (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane, which prevents the separation of sister chromatids and the formation of a spindle interzone. The majority of treated cells showed various degrees of abnormality in cytokinesis. Furrows frequently deviated from the equatorial plane, twisting daughter cells into irregular shapes. Some cells developed furrows in regions outside the equator or far away from the spindle. In addition, F-actin and myosin II accumulated at the lateral ingressing margins but did not form a continuous band along the equator as in control cells. Imaging of microinjected 5- (and 6-) carboxymtetramethylrhodamine-tubulin revealed that a unique set of microtubules projected out from the chromosomal vicinity upon anaphase onset. These microtubules emanated toward the lateral cortex, where they delineated sites of microtubule bundle formation, cortical ingression, and F-actin and myosin II accumulation. As centrosome integrity and astral microtubules appeared unperturbed by (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane treatment, the present observations cannot be easily explained by the conventional model involving astral microtubules. We suggest that in cultured epithelial cells the organization of the chromosomes dictates the organization of midzone microtubules, which in turn determines and maintains the cleavage activity.

scholarly journals Nuclear Pore Complex Acetylation Regulates mRNA Export and Cell Cycle Commitment in Budding Yeast

2021 ◽  
Author(s):  
Mercè Gomar-Alba ◽  
Vasilisa Pozharskaia ◽  
Celia Schaal ◽  
Arun Kumar ◽  
Basile Jacquel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Mrna Export ◽  
Cell Types ◽  
Nuclear Pore ◽  
Specific Cell ◽  
Nuclear Pore Complexes ◽  
Daughter Cells ◽  
Associated Proteins ◽  
Pore Complexes

AbstractNuclear pore complexes (NPCs) mediate communication between the nucleus and the cytoplasm and regulate gene expression by interacting with transcription and mRNA export factors. Lysine acetyl-transferases (KATs) promote transcription through acetylation of chromatin-associated proteins. We find that Esa1, the KAT subunit of the yeast NuA4 complex, also acetylates the nuclear pore basket component Nup60 to promote mRNA export. Acetylation of Nup60 recruits mRNA export factors to the nuclear basket, including the scaffolding subunit of the Transcription and Export 2 (TREX-2) complex, Sac3. Esa1-dependent nuclear export of mRNAs promotes entry into S phase, and is inhibited by the Hos3 deacetylase in G1 daughter cells to restrain their premature commitment to a new cell division cycle. This mechanism also inhibits expression of the nutrient-regulated GAL1 gene specifically in daughter cells. These results reveal how acetylation contributes to the functional plasticity of NPCs in specific cell types, and demonstrate how the evolutionarily conserved NuA4 complex regulates gene expression dually at the level of transcription and mRNA export, by modifying the nucleoplasmic entrance to nuclear pores.

Immunolocalization of microfibril and microfibril-associated proteins in the subendothelial matrix of the developing mouse aorta

1994 ◽  
Vol 107 (3) ◽  
pp. 727-736 ◽  
Author(s):  
E.C. Davis
Keyword(s):  
Endothelial Cell ◽  
Structural Integrity ◽  
Cell Layer ◽  
Endothelial Cell Layer ◽  
Associated Proteins ◽  
Subendothelial Matrix ◽  
Oriented Parallel ◽  
Mouse Aorta ◽  
Structural Entity ◽  
Elastic Lamina

In the developing aorta, endothelial cell connecting filaments extend from the abluminal surface of the endothelial cell to the subjacent elastic lamina. The connecting filaments are in alignment with intracellular stress fibers and are oriented parallel to the direction of blood flow. In the present study, the composition of the endothelial cell connecting filaments was investigated by indirect immunogold labeling with antibodies to the microfibril proteins, MP340 (fibrillin) and MAGP, and to fibronectin and heparan sulfate proteoglycan (HSPG). In the subendothelial matrix of both 15-day gestational and 5-day post-natal mouse aortae, the connecting filaments showed moderate immunoreactivity with anti-MP340; however, no significant immunoreaction was seen with anti-MAGP. Anti-fibronectin strongly labeled the connecting filaments and a weak immunoreaction was seen with anti-HSPG. In contrast, the adjacent ‘elastin-associated microfibrils’ showed a very strong immunoreaction with anti-MP340 and a moderate reaction with anti-MAGP. Little or no reaction was seen with anti-fibronectin or anti-HSPG. The filaments that connect endothelial cells to the subjacent elastic lamina during aortic development are thus microfibrillar in nature and related to elastin-associated microfibrils as evidenced by their positive immunoreaction with anti-MP340. The absence of labeling with anti-MAGP, however, suggests that either these fibrillin-containing filaments do not contain MAGP or that the immunoreactive epitopes are blocked by the proteins that coat the connecting filaments such as fibronectin. These results suggest that microfibrils not in association with elastin may play a role in cell anchorage and, more specifically, in the aorta may be involved in maintaining the structural integrity of the endothelial cell layer during early development of the vessel wall. Furthermore, the absence of immunoreactivity with anti-MAGP on the fibrillin-containing endothelial cell connecting filaments raises the possibility that microfibrils may consist of a family of related filaments rather than a single structural entity.

An essential yeast protein, CBF5p, binds in vitro to centromeres and microtubules

1993 ◽  
Vol 13 (8) ◽  
pp. 4884-4893
Author(s):  
W Jiang ◽  
K Middleton ◽  
H J Yoon ◽  
C Fouquet ◽  
J Carbon
Keyword(s):  
Topoisomerase Ii ◽  
Yeast Cells ◽  
Affinity Column ◽  
Bacterial Cells ◽  
Binding Domains ◽  
C Terminus ◽  
Binding Factor ◽  
Associated Proteins

Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability of Saccharomyces cerevisiae. CBF5 specifies a 55-kDa highly charged protein that contains a repeating KKD/E sequence domain near the C terminus, similar to known microtubule-binding domains in microtubule-associated proteins 1A and 1B, CBF5p, obtained by overexpression in bacterial cells, binds microtubules in vitro, whereas C-terminal deleted proteins lacking the (KKD/E)n domain do not. Dividing yeast cells containing a C-terminal truncated CBF5 gene, producing CBF5p containing only three copies of the KKD/E repeat, delay with replicated genomes at the G2/M phase of the cell cycle, while depletion of CBF5p arrests most cells in G1/S. Overproduction of CBF5p in S. cerevisiae complements a temperature sensitivity mutation in the gene (CBF2) specifying the 110-kDa subunit of the high-affinity CEN DNA-binding factor CBF3, suggesting in vivo interaction of CBF5p and CBF3. A second low-affinity centromere-binding factor has been identified as topoisomerase II.

scholarly journals Recombination and chromosome segregation

2004 ◽  
Vol 359 (1441) ◽  
pp. 61-69 ◽  
Author(s):  
David J. Sherratt ◽  
Britta Søballe ◽  
François–Xavier Barre ◽  
Sergio Filipe ◽  
Ivy Lau ◽  
...  
Keyword(s):  
Cell Division ◽  
Homologous Recombination ◽  
Chromosome Segregation ◽  
Atp Hydrolysis ◽  
Daughter Cells ◽  
Recombination Proteins ◽  
Recombination System ◽  
Associated Proteins ◽  
Stalled Replication Forks ◽  
Circular Chromosomes

The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes. The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins. In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the conserved Xer site–specific recombination system. Dimer resolution also requires FtsK, a division septum–located protein, which coordinates chromosome segregation with cell division, and uses the energy of ATP hydrolysis to activate the dimer resolution reaction. FtsK can also translocate DNA, facilitate synapsis of sister chromosomes and minimize entanglement and catenation of newly replicated sister chromosomes. The visualization of the replication/recombination–associated proteins, RecQ and RarA, and specific genes within living Escherichia coli cells, reveals further aspects of the processes that link replication with recombination, chromosome segregation and cell division, and provides new insight into how these may be coordinated.

scholarly journals Cdc20 hypomorphic mice fail to counteract de novo synthesis of cyclin B1 in mitosis

2010 ◽  
Vol 191 (2) ◽  
pp. 313-329 ◽  
Author(s):  
Liviu Malureanu ◽  
Karthik B. Jeganathan ◽  
Fang Jin ◽  
Darren J. Baker ◽  
Janine H. van Ree ◽  
...  
Keyword(s):  
De Novo ◽  
Cyclin B1 ◽  
Anaphase Promoting Complex ◽  
De Novo Synthesis ◽  
Cytoplasmic Polyadenylation ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Element Binding Protein ◽  
Sister Chromosome ◽  
Chromosome Separation

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome–microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element–binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.

scholarly journals Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase

1992 ◽  
Vol 117 (5) ◽  
pp. 921-934 ◽  
Author(s):  
CE Shamu ◽  
AW Murray
Keyword(s):  
Sister Chromatid ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Egg Extracts ◽  
Chromosome Separation ◽  
Sister Chromatid Separation ◽  
Sperm Nuclei ◽  
Cytostatic Factor

We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.

scholarly journals Opto-Katanin: An Optogenetic Tool for Localized Microtubule Disassembly

2021 ◽  
Author(s):  
Joyce C.M. Meiring ◽  
Ilya Grigoriev ◽  
Wilco Nijenhuis ◽  
Lukas C. Kapitein ◽  
Anna Akhmanova
Keyword(s):  
Intracellular Transport ◽  
Experimental Manipulation ◽  
Microtubule Depolymerization ◽  
Microtubule Associated Proteins ◽  
Microtubule Severing ◽  
Associated Proteins ◽  
Dividing Cells ◽  
Chromosome Separation ◽  
Organelle Morphology ◽  
Spatiotemporal Control

Microtubules are major cytoskeletal filaments that drive chromosome separation during cell division, serve as rails for intracellular transport and as a scaffold for organelle positioning. Experimental manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubule integrity are currently lacking. Here, we exploit the dependence of the mammalian microtubule-severing protein katanin on microtubule-targeting co-factors to generate a light-activated system for localized microtubule disassembly that we named opto-katanin. Targeted illumination with blue light induces rapid and localized opto-katanin recruitment and local microtubule depolymerization, which is quickly reversible after stopping light-induced activation. Opto-katanin can be employed to locally perturb microtubule-based transport and organelle morphology in dividing cells and differentiated neurons with high spatiotemporal precision. We show that different microtubule-associated proteins can be used to recruit opto-katanin to microtubules and induce severing, paving the way for spatiotemporally precise manipulation of specific microtubule subpopulations.

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