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Current Biology : CB Mar 2022Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to...
Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex. In mammalian cells, end-binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for diverse +TIPs that control microtubule dynamics and interactions with other intracellular structures. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation, the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light-sensitive EB1 variant, π-EB1, that allows local, acute, and reversible inactivation of +TIP association with growing microtubule ends in live cells. We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase but instead increases astral microtubule length and number. Yet in the absence of EB1 activity, astral microtubules fail to engage the cortical dynein/dynactin machinery, and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments, likely due to compensatory molecular systems regulating vertebrate spindle dynamics.
Topics: Animals; Mammals; Metaphase; Microtubule-Associated Proteins; Microtubules; Optogenetics; Spindle Apparatus
PubMed: 35090591
DOI: 10.1016/j.cub.2022.01.017 -
Blood Jul 2020B-cell acute lymphoblastic leukemia (ALL; B-ALL) is the most common pediatric cancer, and high hyperdiploidy (HyperD) identifies the most common subtype of pediatric... (Clinical Trial)
Clinical Trial
B-cell acute lymphoblastic leukemia (ALL; B-ALL) is the most common pediatric cancer, and high hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD being an initiating oncogenic event affiliated with childhood B-ALL, the mitotic and chromosomal defects associated with HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated with chromosome-alignment defects at the metaphase plate leading to robust chromosome-segregation defects and nonmodal karyotypes. Mechanistically, biochemical, functional, and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness, and mislocalization of the chromosome passenger complex proteins Aurora B kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and an impaired spindle assembly checkpoint (SAC), thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated with a defective condensin complex, AURKB, and SAC.
Topics: Adenosine Triphosphatases; Aurora Kinase B; Chromosome Aberrations; Chromosomes, Human; DNA-Binding Proteins; Humans; Metaphase; Multiprotein Complexes; Neoplasm Proteins; Ploidies; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 32321174
DOI: 10.1182/blood.2019002538 -
PLoS Genetics Jan 2021Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases...
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.
Topics: Animals; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; Chromosome Segregation; DNA Topoisomerases, Type II; Drosophila melanogaster; Kinetochores; Larva; Metaphase; Microtubules; Mitosis; Spindle Apparatus; Cohesins
PubMed: 33513180
DOI: 10.1371/journal.pgen.1009304 -
EMBO Reports Apr 2024Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear....
Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear. Here, we show that CKAP5, a conserved microtubule plus tip protein, regulates CENP-E at kinetochores in human cells. Depletion of CKAP5 impairs CENP-E localization at kinetochores at the metaphase plate and results in increased kinetochore-microtubule stability and attachment errors. Erroneous attachments are also supported by computational modeling. Analysis of CKAP5 knockout cancer cells of multiple tissue origins shows that CKAP5 is preferentially essential in aneuploid, chromosomally unstable cells, and the sensitivity to CKAP5 depletion is correlated to that of CENP-E depletion. CKAP5 depletion leads to reduction in CENP-E-BubR1 interaction and the interaction is rescued by TOG4-TOG5 domain of CKAP5. The same domain can rescue CKAP5 depletion-induced CENP-E removal from the kinetochores. Interestingly, CKAP5 depletion facilitates recruitment of PP1 to the kinetochores and furthermore, a PP1 target site-specific CENP-E phospho-mimicking mutant gets stabilized at kinetochores in the CKAP5-depleted cells. Together, the results support a model in which CKAP5 controls mitotic chromosome attachment errors by stabilizing CENP-E at kinetochores and by regulating stability of the kinetochore-attached microtubules.
Topics: Humans; Kinetochores; Chromosomal Proteins, Non-Histone; Microtubules; Metaphase; Kinesins; HeLa Cells; Mitosis; Chromosome Segregation; Microtubule-Associated Proteins
PubMed: 38424231
DOI: 10.1038/s44319-024-00106-9 -
Developmental Cell Aug 2013Reporting in Developmental Cell, Schlaitz et al. (2013) show that endoplasmic reticulum (ER) membrane exclusion from the mitotic spindle is an active process requiring...
Reporting in Developmental Cell, Schlaitz et al. (2013) show that endoplasmic reticulum (ER) membrane exclusion from the mitotic spindle is an active process requiring REEP membrane proteins. REEP protein depletion results in ER membrane retention on the spindle and chromosomes, leading to defects in chromosome segregation and nuclear envelope assembly.
Topics: Chromatin; Endoplasmic Reticulum; Humans; Membrane Transport Proteins; Metaphase; Nuclear Envelope
PubMed: 23948250
DOI: 10.1016/j.devcel.2013.07.018 -
The Journal of Cell Biology Jun 2022Errors in mitosis that cause chromosome missegregation lead to aneuploidy and micronucleus formation, which are associated with cancer. Accurate segregation requires the...
Errors in mitosis that cause chromosome missegregation lead to aneuploidy and micronucleus formation, which are associated with cancer. Accurate segregation requires the alignment of all chromosomes by the mitotic spindle at the metaphase plate, and any misalignment must be corrected before anaphase is triggered. The spindle is situated in a membrane-free "exclusion zone"; beyond this zone, endomembranes (mainly endoplasmic reticulum) are densely packed. We investigated what happens to misaligned chromosomes localized beyond the exclusion zone. Here we show that such chromosomes become ensheathed in multiple layers of endomembranes. Chromosome ensheathing delays mitosis and increases the frequency of chromosome missegregation and micronucleus formation. We use an induced organelle relocalization strategy in live cells to show that clearance of endomembranes allows for the rescue of chromosomes that were destined for missegregation. Our findings indicate that endomembranes promote the missegregation of misaligned chromosomes that are outside the exclusion zone and therefore constitute a risk factor for aneuploidy.
Topics: Anaphase; Aneuploidy; Cell Membrane; Chromosome Segregation; Chromosomes; Endoplasmic Reticulum; Humans; Metaphase; Mitosis; Spindle Apparatus
PubMed: 35486148
DOI: 10.1083/jcb.202203021 -
The Plant Journal : For Cell and... Jul 2013The microtubular cytoskeleton plays a major role in cellular organization and proliferation. The first step in construction of a microtubule is microtubule nucleation.... (Review)
Review
The microtubular cytoskeleton plays a major role in cellular organization and proliferation. The first step in construction of a microtubule is microtubule nucleation. Individual microtubules then participate in organization of more complex microtubule arrays. A strong body of evidence suggests that the underlying molecular mechanisms involve protein complexes that are conserved among eukaryotes. However, plant cell specificities, mainly characterized by the presence of a cell wall and the absence of centrosomes, must be taken into account to understand their mitotic processes. The goal of this review is to summarize and discuss current knowledge regarding the mechanisms involved in plant spindle assembly during early mitotic events. The functions of the proteins currently characterized at microtubule nucleation sites and involved in spindle assembly are considered during cell-cycle progression from G2 phase to metaphase.
Topics: Chromosomes, Plant; Cytoskeleton; Metaphase; Microtubule-Associated Proteins; Microtubules; Mitosis; Nuclear Envelope; Plant Cells; Spindle Apparatus
PubMed: 23521421
DOI: 10.1111/tpj.12179 -
Seminars in Cell & Developmental Biology May 2010Kinetochores have been proposed to play multiple roles in mitotic chromosome alignment, including initial microtubule (MT) capture, monitoring MT attachments,... (Review)
Review
Kinetochores have been proposed to play multiple roles in mitotic chromosome alignment, including initial microtubule (MT) capture, monitoring MT attachments, prometaphase and anaphase chromosome movement and tension generation at metaphase. In addition, kinetochores are essential components of the spindle assembly checkpoint (SAC), and couple chromosome alignment with SAC silencing at metaphase. Although the molecular details of these activities remain under investigation, cytoplasmic dynein has been implicated in several aspects of MT and SAC regulation. Recent work clarifies the contribution of dynein to MT interactions and to events that drive anaphase onset. This review summarizes these studies and provides new models for dynein function.
Topics: Anaphase; Animals; Aspergillus; Cytoplasm; Dyneins; Gene Silencing; Humans; Kinetochores; Metaphase; Microtubules; Mitosis; Models, Biological; Phosphorylation
PubMed: 20045078
DOI: 10.1016/j.semcdb.2009.12.015 -
International Review of Cell and... 2013During mitosis, duplicated sister chromatids are properly aligned at the metaphase plate of the mitotic spindle before being segregated into two daughter cells. This... (Review)
Review
During mitosis, duplicated sister chromatids are properly aligned at the metaphase plate of the mitotic spindle before being segregated into two daughter cells. This requires a complex process to ensure proper interactions between chromosomes and spindle microtubules. The kinetochore, the proteinaceous complex assembled at the centromere region on each chromosome, serves as the microtubule attachment site and powers chromosome movement in mitosis. Numerous proteins/protein complexes have been implicated in the connection between kinetochores and dynamic microtubules. Recent studies have advanced our understanding on the nature of the interface between kinetochores and microtubule plus ends in promoting and maintaining their stable attachment. These efforts have demonstrated the importance of this process to ensure accurate chromosome segregation, an issue which has great significance for understanding and controlling abnormal chromosome segregation (aneuploidy) in human genetic diseases and in cancer progression.
Topics: Chromosomes; Humans; Kinetochores; Metaphase; Microtubules; Models, Biological; Spindle Apparatus
PubMed: 23445812
DOI: 10.1016/B978-0-12-407697-6.00006-4 -
Molecular Cell Sep 2020A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of...
A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.
Topics: Adenosine Triphosphatases; Anaphase; Animals; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; Chromosomes; DNA Topoisomerases, Type II; DNA-Binding Proteins; Imaging, Three-Dimensional; Mammals; Metaphase; Mitosis; Prophase
PubMed: 32768407
DOI: 10.1016/j.molcel.2020.07.002