Biology subjects

Gorbsky, G. J.

Publications and source records attributed to Gorbsky, G. J..

12 recordsLinked to original sources

bioRxiv2026

Functional Interaction of SKA and NDC80 Complexes at Kinetochores Promoting Anaphase Onset in Mitosis

The kinetochore and spindle complex (SKA) and NDC80 complexes are essential kinetochore elements that ensure highly accurate chromosome segregation and successful progression through mitosis. The SKA heterodimer complex consists of SKA1, SKA2, and SKA3 subunits, and the NDC80 complex contains NDC80, NUF2, SPC24, and SPC25 subunits. Through live cell fluorescence timelapse imaging assays and expression of RNAi-resistant SKA3 constructs, we rescue SKA complex function in cells lacking endogenous SKA3. These assays reveal a critical span within SKA3s C-terminus required for successful mitotic progression. Structural protein modeling shows that this span encompasses the majority of a roughly 40 amino acid SKA3 C-terminal structural element that promotes interaction with the coiled-coil NDC80 and NUF2 subunits of the NDC80 complex. Thus, although spindle and kinetochore concentration of the SKA complex is mediated in part by the tubulin and tip-tracking capabilities provided by the SKA1 component of the SKA complex, transition from metaphase to anaphase requires the contribution of SKA3s C-terminal structural interface to mediate interaction between the SKA and NDC80 complexes. Significance StatementAccurate chromosome segregation is essential for genomic stability, and its failure underlies developmental defects, and cancer. The kinetochore-microtubule interface, where the SKA and NDC80 complexes converge, is central, yet the of these two complexes is incompletely defined. We identify a critical segment within the C-terminus of SKA3 required for the metaphase-to-anaphase transition. Using live-cell imaging with RNAi-resistant rescue constructs and structural modeling, we demonstrate that this region mediates SKA engagement with the coiled-coil domains of NDC80 and NUF2. Our findings establish that a small segment of SKA3s C-terminus provides an essential physical bridge between the SKA and NDC80 complexes. This work refines understanding kinetochore-microtubule interaction, functionally identifying a discrete contact whose disruption may be relevant to chromosomal instability in disease.

cell biology↗

bioRxiv2024

Origin of Chromosome 12 Trisomy Surge in Human Induced Pluripotent Stem Cells (iPSCs)

Cultured pluripotent stem cells are unique in being the only fully diploid immortal human cell lines. However, during continued culture, they acquire significant chromosome abnormalities. Chromosome 12 trisomy is the most common whole-chromosome abnormality found during culture of human induced pluripotent stem cells (iPSCs). The conventional paradigm is that trisomy 12 occurs very rarely but provides a proliferative advantage, enabling these cells to outcompete the diploid. Here, we challenge this prevailing model by demonstrating that trisomy 12 arises simultaneously in a very high percentage of diploid cells. Using a single cell line that reproducibly undergoes transition from diploid to trisomy 12, we found that proliferation differences alone do not account for the rapid dominance of trisomic cells. Through careful mapping by fluorescent in-situ hybridization, we identified critical transition passages where trisomic cells first appeared and swiftly gained dominance. Remarkably, single trisomic cells repeatedly emerged de novo from diploid parents. Delving deeper, we discovered an extremely high incidence of chromosome 12 anaphase bridging exclusively during transition passages, along with overrepresentation of chromosome 12 chromatids in micronuclei. These micronuclei fail to replicate during S phase. Subsequently, when these micronucleated cells enter mitosis they contain an unreplicated chromosome 12 chromatids. We also found that nearly 20% of the shorter p arms of chromosome 12 but not the longer q arms exhibited loss of subtelomeric repeats during transition passages. Chromosome 12p arms were exclusively responsible for the bridging observed in anaphase cells. Our findings unveil a novel mechanism of whole-chromosome instability in human stem cells, where chromosome 12p arm-specific segregation errors occur simultaneously in a high percentage of cells. The slight yet significant growth advantage of trisomy 12 cells allows them to persist and eventually dominate the population. Our findings detailing this novel interpretation of the origin of chromosome instability in cultured of human stem cells may have broad implications for understanding the genesis of aneuploidy across diverse biological systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/626470v3_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@dc7cc7org.highwire.dtl.DTLVardef@183fb9eorg.highwire.dtl.DTLVardef@1fc6adaorg.highwire.dtl.DTLVardef@cb4078_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Adeno-Associated Viral Tools to Trace Neural Development and Connectivity Across Amphibians

The development, evolution, and function of the vertebrate central nervous system (CNS) can be best studied using diverse model organisms. Amphibians, with their unique phylogenetic position at the transition between aquatic and terrestrial lifestyles, are valuable for understanding the origin and evolution of the tetrapod brain and spinal cord. Their metamorphic developmental transitions and unique regenerative abilities also facilitate the discovery of mechanisms for neural circuit remodeling and replacement. The genetic toolkit for amphibians, however, remains limited, with only a few species having sequenced genomes and a small number of transgenic lines available. In mammals, recombinant adeno-associated viral vectors (AAVs) have become a powerful alternative to genome modification for visualizing and perturbing the nervous system. AAVs are DNA viruses that enable neuronal transduction in both developing and adult animals with low toxicity and spatial, temporal, and cell-type specificity. However, AAVs have never been shown to transduce amphibian cells efficiently. To bridge this gap, we established a simple, scalable, and robust strategy to screen AAV serotypes in three distantly-related amphibian species: the frogs Xenopus laevis and Pelophylax bedriagae, and the salamander Pleurodeles waltl, in both developing larval tadpoles and post-metamorphic animals. For each species, we successfully identified at least two AAV serotypes capable of infecting the CNS; however, no pan-amphibian serotype was identified, indicating rapid evolution of AAV tropism. In addition, we developed an AAV-based strategy that targets isochronic cohorts of developing neurons - a critical tool for parsing neural circuit assembly. Finally, to enable visualization and manipulation of neural circuits, we identified AAV variants for retrograde tracing of neuronal projections in adult animals. Our findings expand the toolkit for amphibians to include AAVs, establish a generalizable workflow for AAV screening in non-canonical research organisms, generate testable hypotheses for the evolution of AAV tropism, and lay the foundation for modern cross-species comparisons of vertebrate CNS development, function, and evolution.

neuroscience↗

bioRxiv2023

Polyploid yeast are dependent on elevated levels of Mps1 for successful chromosome segregation

Tumor cell lines with elevated chromosome numbers frequently exhibit elevated expression of Mps1. These tumors are also dependent on high Mps1 activity for their survival. Mps1 is a conserved kinase involved in controlling aspects of chromosome segregation in mitosis and meiosis. The mechanistic explanation for the Mps1-addiction of aneuploid cells is unknown. To address this question, we explored Mps1-dependence in yeast cells with increased sets of chromosomes. These experiments revealed that in yeast, increasing ploidy leads to delays and failures in orienting chromosomes on the mitotic spindle. Yeast cells with elevated numbers of chromosomes proved vulnerable to reductions of Mps1 activity. Cells with reduced Mps1 activity exhibit an extended prometaphase with longer spindles and delays in orienting the chromosomes. One known role of Mps1 is in recruiting Bub1 to the kinetochore in meiosis. We found that the Mps1-addiction of polyploid yeast cells is due in part to its role in Bub1 recruitment. Together, the experiments presented here demonstrate that increased ploidy renders cells more dependent on Mps1 for orienting chromosomes on the spindle. The phenomenon described here may be relevant in understanding why high-ploidy cancer cells exhibit elevated reliance on Mps1 expression for successful chromosome segregation. AUTHOR SUMMARYLosing or gaining chromosomes during cell division leads to aneuploidy (an abnormal number of chromosomes) and can contribute to cancer and other diseases. Indeed, most cells in solid tumors carry abnormally elevated numbers of chromosomes. Mps1 is a regulator of the machinery that distributes chromosomes to daughter cells. In tumors with elevated chromosome numbers, the expression of Mps1 is often also elevated. In some aneuploid tumor cell lines these elevated Mps1 levels have been shown to be critical for tumor survival. To determine how cells with higher ploidy become dependent on Mps1, we explored Mps1-dependence in yeast cells with increased numbers of chromosomes. We report that yeast cells with elevated chromosome number are sensitive to reductions Mps1 expression. In cells with high ploidy and reduced levels of Mps1, the progression of the cell cycle is delayed and the ability of the cells to properly orient and segregate their chromosomes on the spindle is greatly reduced.

cell biology↗

bioRxiv2022

Dynamic Features of Chromosomal Instability during Culture of Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) hold great potential for regenerative medicine. By reprogramming a patient"s own cells, immunological rejection can be avoided during transplantation. For expansion and gene editing, iPSCs are grown in artificial culture for extended times. Culture affords potential danger for accumulation of genetic aberrations. To study these, two iPS cell lines were cultured and periodically analyzed using advanced optical mapping to detect and classify chromosome numerical and segmental changes that included deletions, insertions, balanced translocations and inversions. In one of the lines, a population trisomic for chromosome 12 gained dominance over a small number of passages. This appearance and dominance of the culture by chromosome 12 trisomic cells was tracked through intermediate passages by analysis of chromosome spreads. Mathematical modeling suggested that the proliferation rates of diploid versus trisomic cells could not account for the rapid dominance of the trisomic population. In addition, optical mapping revealed hundreds of structural variations distinct from those generally found within the human population. Many of these structural variants were detected in samples taken early in the culturing process and were maintained in late passage samples, while others were acquired over the course of culturing.

cell biology↗

bioRxiv2022

Four novel, immortal cell lines derived from embryos of Xenopus tropicalis

The diploid anuran, Xenopus tropicalis, has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10, and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10, and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30{degrees}C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.

cell biology↗

bioRxiv2021

Generation and diversification of recombinant monoclonal antibodies for studying mitosis

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitotic cell division; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

cell biology↗

bioRxiv2020

Mps1 promotes poleward chromosome movements in meiotic pro-metaphase

In prophase of meiosis I, homologous partner chromosomes pair and become connected by crossovers. Chiasmata, the connections formed between the partners enable the chromosome pair, called a bivalent, to attach as a single unit to the spindle. When the meiosis I spindle forms in prometaphase, most bivalents are associated with a single spindle pole and go through a series of oscillations on the spindle, attaching to and detaching from microtubules until the partners of the bivalent are bi-oriented, that is, attached to microtubules from opposite sides of the spindle, and prepared to be segregated at anaphase I. The conserved, kinetochore-associated kinase, Mps1, is essential for the bivalents to be pulled by microtubules across the spindle in prometaphase. Here we show that MPS1 is not required for kinetochores to attach microtubules but instead is necessary to trigger the migration of microtubule-attached kinetochores towards the poles. Our data support the model that Mps1 triggers depolymerization of microtubule ends once they attach to kinetochores in prometaphase. Thus, Mps1 acts at the kinetochore to co-ordinate the successful attachment of a microtubule and the triggering of microtubule depolymerization to move the chromosome.

cell biology↗

bioRxiv2019

CSAG1 Maintains the Integrity of the Mitotic Centrosome in Cells with Defective p53

Centrosomes focus microtubules to promote mitotic spindle bipolarity, a critical requirement for balanced chromosome segregation. Comprehensive understanding of centrosome function and regulation requires a complete inventory of components. While many centrosome components have been identified, others may yet remain undiscovered. We have used a bioinformatics approach, based on \"guilt by association\" expression to identify novel mitotic components among the large group of predicted human proteins that have yet to be functionally characterized. Here we identify Chondrosarcoma-Associated Gene 1 (CSAG1) in maintaining centrosome integrity during mitosis. Depletion of CSAG1 disrupts centrosomes and leads to multipolar spindles more effectively in cells with compromised p53 function. Thus, CSAG1 may reflect a class of \"mitotic addiction\" genes whose expression is more essential in transformed cells.

cell biology↗

bioRxiv2019

Complexities of Microtubule Population Dynamics within the Mitotic Spindle

The microtubules of the mitotic spindle mediate chromosome alignment to the metaphase plate, then sister chromatid segregation to the spindle poles in anaphase. Previous analyses of spindle microtubule kinetics utilizing fluorescence dissipation after photoactivation described two main populations, a slow and a fast turnover population, and these were ascribed to reflect kinetochore versus non-kinetochore microtubules, respectively. Here, we test this categorization by disrupting kinetochores through depletion of the Ndc80 complex. In the absence of functional kinetochores, microtubule dynamics still exhibit slow and fast turnover populations, though the proportion of each population and the timings of turnover are altered. Importantly, the data obtained following Hec1/Ndc80 depletion suggests other sub-populations, in addition to kinetochore microtubules, contribute to the slow turnover population. Further manipulation of spindle microtubules revealed a complex landscape. Dissection of the dynamics of microtubule populations provides a greater understanding of mitotic spindle kinetics and insight into roles in facilitating chromosome attachment, movement, and segregation during mitosis.

cell biology↗

bioRxiv2017

Multiple Determinants and Consequences of Cohesion Fatigue in Mammalian Cells

Cells delayed in metaphase with intact mitotic spindles undergo cohesion fatigue, where sister chromatids separate asynchronously, while cells remain in M phase. Cohesion fatigue requires release of sister chromatid cohesion. However, the pathways necessary to breach sister chromatid cohesion during cohesion fatigue remain unknown. Using a regulated protein heterodimerization system to lock different cohesin interfaces at specific times in mitosis, we show that the prophase pathway of Cohesin release is not required for cohesion fatigue. By manipulating microtubule stability and Cohesin complex integrity in cell lines with varying sensitivity to cohesion fatigue, we show that rates of cohesion fatigue reflect a dynamic balance between spindle pulling forces and resistance to separation by interchromatid cohesion. Cohesion fatigue that results in complete chromatid separation may be an unrecognized but common source of chromosome instability. Here, we extend the significance of cohesion fatigue by showing that even limited delays at metaphase lead to partial centromere separation and predispose cells to chromosome missegregation.

cell biology↗

bioRxiv2017

PHOSPHATASE-REGULATED RECRUITMENT OF THE SPINDLE AND KINETOCHORE ASSOCIATED (SKA) COMPLEX TO KINETOCHORES

Kinetochores move chromosomes on dynamic spindle microtubules and regulate cell cycle progression by signaling the spindle checkpoint. The Spindle and Kinetochore-Associated (Ska) Complex, a hexamer composed of two copies of Ska1, Ska2 and Ska3, participates in both roles. The mitotic kinases, Cdk1, Aurora B, Plk1, Mps1 and Bub1 play key, overlapping tasks in regulating chromosome movement and checkpoint signaling. However, roles for the phosphatases that oppose these kinases are more poorly defined. Recently, we showed that Ska1 is important for recruiting protein phosphatase 1 (PP1) to kinetochores. Here we show that PP1 and protein phosphatase 2A (PP2A) both promote accumulation of Ska at kinetochores. Depletion of PP1 or PP2A by siRNA reduces Ska binding at kinetochores, impairs alignment of chromosomes to the spindle midplane, and causes metaphase delay or arrest, phenotypes also seen after depletion of Ska. Tethering of PP1 to the kinetochore protein Nuf2 promotes Ska recruitment to kinetochores, and reduces mitotic defects seen after Ska depletion. We propose that kinetochore-associated phosphatases generate a positive feedback cycle to reinforce Ska complex accumulation and function at kinetochores.\n\nSUMMARY STATEMENTPhosphatases reinforce recruitment of the Ska complex at kinetochores to stabilize microtubule attachment and oppose spindle checkpoint signaling.

cell biology↗