Biology subjects

Sarkar, A.

Publications and source records attributed to Sarkar, A..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

Genome-wide DNA methylation and multi-omics study of human chondrocyte ontogeny and an epigenetic clock analysis of adult chondrocytes

Articular chondrocytes undergo functional changes and their regenerative potential declines with age. Although the molecular mechanisms guiding articular cartilage aging is poorly understood, DNA methylation is known to play a mechanistic role in aging. However, our understanding of DNA methylation in chondrocyte development across human ontogeny is limited. To better understand DNA methylome changes, methylation profiling was performed in human chondrocytes. This study reveals association between methylation of specific CpG sites and chondrocyte age. We also determined the putative binding targets of STAT3, a key age-patterned transcription factor in fetal chondrocytes and genetic ablation of STAT3 induced a global genomic hypermethylation. Moreover, an epigenetic clock built for adult human chondrocytes revealed that exposure of aged adult human chondrocytes to STAT3 agonist, decreased epigenetic age. Taken together, this work will serve as a foundation to understand development and aging of chondrocytes with a new perspective for development of rejuvenation agents for synovial joints.

molecular biology↗

bioRxiv2021

NAD+ repletion by the PARP inhibitor PJ34 prevents Sarm1 activation and rotenone-induced cell death

Sarm1 is an evolutionary conserved innate immune adaptor protein that has emerged as a primary regulator of programmed axonal degeneration over the past decade. In vitro structural insights have revealed that although Sarm1 induces energy depletion by breaking down NAD+, it is also allosterically inhibited by NAD+. However, how NAD+ levels modulate the activation of intracellular Sarm1 has not been elucidated so far. This study focuses on understanding the events leading to Sarm1 activation in both neuronal and non-neuronal cells using the mitochondrial complex I inhibitor rotenone. Here we report the regulation of rotenone-induced cell death by loss of NAD+ that may act as a "biological trigger" of Sarm1 activation. Our study revealed that early loss of endogenous NAD+ levels arising due to PARP1 hyperactivation preceded Sarm1 induction following rotenone treatment. Interestingly, replenishing NAD+ levels by the PARP1 inhibitor, PJ34 restored mitochondrial homeostasis and prevented subsequent Sarm1 activation in rotenone treated cells. These cellular data were further validated in Drosophila melanogaster where a significant reduction in rotenone mediated loss of locomotor abilities and reduced dSarm expression was observed in the flies following PARP inhibition. Taken together, these observations not only uncovers a novel regulation of Sarm1 induction by endogenous NAD+ levels but also point towards an important understanding on how PARP inhibitors could be repurposed in the treatment of mitochondrial complex I deficiency disorders mediated by Sarm1.

cell biology↗

bioRxiv2021

Long-term repair of porcine articular cartilage using cryopreservable, clinically compatible human embryonic stem cell-derived chondrocytes

Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injuries such as focal defects to the articular surface are a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Here we describe the development, transcriptomic ontogenetic characterization and quality assessment at the single cell level, as well as the scaled manufacturing of an allogeneic human pluripotent stem cell-derived articular chondrocyte formulation that exhibits long-term functional repair of porcine articular cartilage. These results define a new potential clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA.

bioengineering↗

bioRxiv2021

Signaling through FcγRIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the Fc{gamma}RIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection. Cover illustration O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY One-sentence summaryThe Fc{gamma}RIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19

immunology↗

bioRxiv2021

A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elements, and higher-order chromatin structures. Expectedly, these chromatin marks are indispensable for mammalian development and alterations often lead to diseases such as cancer. Molecularly, epigenetic mechanisms rely on factors to establish patterns, interpret them into a transcriptional output, and maintain them across cell divisions. A global picture of these phenomena has started to emerge over the years, yet many of the molecular actors remain to be discovered. In this context, we have developed a reporter system sensitive to epigenetic perturbations to report on repressive pathways based on Dazl, which is normally repressed in mouse ES cells. We used this system for a genome-wide CRISPR knock-out screen, which yielded expected hits (DNMT1, UHRF1, MGA), as well as novel candidates. We prioritized the candidates by secondary screens, and led further experiments on 6 of them: ZBTB14, KDM5C, SPOP, MCM3AP, BEND3, and KMT2D. Our results show that all 6 candidates regulate the expression of germline genes. In addition, we find that removal of ZBTB14, KDM5C, SPOP and MCM3AP led to similar transcriptional responses, including a reactivation of the 2-cell like cell (2CLC) signature. Therefore, our genetic screen has identified new regulators of key cellular states.

molecular biology↗

bioRxiv2020

miRNA-mediated loss of m6A increases nascent translation in glioblastoma

Within the glioblastoma cellular niche, glioma stem cells (GSCs) can give rise to differentiated glioma cells (DGCs) and, when necessary, DGCs can reciprocally give rise to GSCs to maintain the cellular equilibrium necessary for optimal tumor growth. Here, using ribosome profiling, transcriptome and m6A RNA sequencing, we show that GSCs from patients with different subtypes of glioblastoma share a set of transcripts, which exhibit a pattern of m6A loss and increased protein translation during differentiation. The target sequences of a group of miRNAs overlap the canonical RRACH m6A motifs of these transcripts, many of which confer a survival advantage in glioblastoma. Ectopic expression of the RRACH-binding miR-145 induces loss of m6A, formation of FTO/AGO1/ILF3/miR-145 complexes on a clinically relevant tumor suppressor gene (CLIP3) and significant increase in its nascent translation. Inhibition of miR-145 maintains RRACH m6A levels of CLIP3 and inhibits its nascent translation. This study highlights a critical role of miRNAs in assembling complexes for m6A demethylation and induction of protein translation during GSC state transition. Author SummaryCellular plasticity and epigenetic adaptation of human glioblastoma stem cells to the tumor microenvironment is a hallmark of this devastating disease. With our present work, we discover the relationship between miRNAs and the RNA methylation machinery in human glioblastoma and show how miRNA-induced loss of m6A results in increase in protein translation of clinically important transcripts during glioblastoma stem cell differentiation. Leveraging the dynamic functions of these miRNAs can be important in the design of optimal therapeutics targeted at cancer cell plasticity.

cancer biology↗

bioRxiv2020

Studies on C. elegans probed with estradiol, elucidate the critical role of Na+/H+ exchanger, nhx-2 in reproductive senescence and neuronal health

Setting in of reproductive senescence (RS) gives rise to several changes, making aged individuals susceptible to multiple disorders including neurodegenerative diseases, cardiovascular ailments and bone disorders amongst others. The present study, employing transgenic C. elegans that expresses human alpha synuclein, endeavors to decipher the association of reproductive senescence with age-associated neurodegenerative diseases and behavioral ageing, under normal conditions and after being probed with estradiol. We carried out RNAi induced silencing of a subset of 22 genes that are known to delay RS, followed by studies on alpha-Synuclein aggregation and associated effects. These studies led us to functional characterization of the Na+/H+ exchanger; nhx-2, expressed exclusively in gut. We found that RNAi of nhx-2 not only ameliorates the effects associated with alpha-Synuclein aggregation, but it also attunes effects related to behavioral aging including that of reproductive health-span and neuroprotection via mimicking dietary restriction, as it alters food absorption from the gut. We further elucidated that these effects are Sir-2.1 driven as nhx-2 knock out did not delay reproductive senescence in knock down condition of sir-2.1. To substantiate our findings, we performed whole transcriptome analysis in nhx-2 mutant strain. Our data revealed differential expression of 61 out of 62 hallmark genes of CR described by GenDR, in knock out condition of nhx-2. As estradiol plays a central role in both reproductive health as well as neuronal health, we subjected worms to exogenous estradiol treatment and observed that it led to elevated levels of nhx-2. Studies on structural binding analysis demonstrated significant binding potential of estradiol receptor NHR-14 with nhx-2 gene and ChIP analysis revealed that estradiol treatment gives rise to enhanced NHX-2 levels through inducing the promoter specific histone H3 acetylation (H3K9) and lysine methylation (H3K4me3). These studies identify nhx-2 as an important modulator that extends reproductive longevity and ameliorates effects associated with alpha synuclein aggregation in C elegans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/229344v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@917377org.highwire.dtl.DTLVardef@133a07dorg.highwire.dtl.DTLVardef@6d6f28org.highwire.dtl.DTLVardef@9cde9a_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LISilencing of sodium proton antiporter nhx-2 affords neuroprotection and ameliorates effects associated with alpha-Synuclein aggregation via mimicking dietary restriction in C. elegans. C_LIO_LIExogenous 17-{beta}-Estradiol treatment induces the expression of nhx-2, through inducing the promoter specific histone H3 acetylation (H3K9) and lysine methylation (H3K4me3). C_LIO_LIEffects associated with nhx-2, including prolonged reproductive span and neuroprotective effects, are SIR-2.1 driven. C_LIO_LInhx-2 silencing decreases alpha-Synuclein aggregation, however; estradiol mediated overexpression above the endogenous levels, does not amend the aggregation any further. C_LI

neuroscience↗

bioRxiv2020

MAGIC: Mosaic Analysis by gRNA-Induced Crossing-over

Mosaic animals have provided the platform for many fundamental discoveries in developmental biology, cell biology, and other fields. Techniques to produce mosaic animals by mitotic recombination have been extensively developed in Drosophila melanogaster but are less common for other laboratory organisms. Here, we report mosaic analysis by gRNA-induced crossing-over (MAGIC), a new technique for generating mosaic animals based on DNA double-strand breaks produced by CRISPR/Cas9. MAGIC efficiently produces mosaic clones in both somatic tissues and the germline of Drosophila. Further, by developing a MAGIC toolkit for one chromosome arm, we demonstrate the method’s application in characterizing gene function in neural development and in generating fluorescently marked clones in wild-derived Drosophila strains. Eliminating the need to introduce recombinase-recognition sites in the genome, this simple and versatile system simplifies mosaic analysis in Drosophila and can be applied in any organism that is compatible with CRISPR/Cas9.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genetics↗

bioRxiv2020

Phagocytosis drives NAD+ reduction-induced dendrite degeneration in Drosophila

After injury, severed dendrites and axons expose the "eat-me" signal phosphatidylserine (PS) on their surface and degenerate by disassembly. While axon degeneration is controlled by a conserved "axon-death" pathway that is thought to activate self-destruction, how PS exposure is regulated by this pathway and whether PS-induced phagocytosis contributes to neurite breakdown in vivo remain unknown. Here we show that in Drosophila sensory dendrites, PS exposure and self-destruction are triggered by two distinct levels of NAD+ reduction downstream of Sarm activation. Surprisingly, phagocytosis is the main driver of dendrite degeneration induced by both genetic NAD+ disruptions and injury. Furthermore, the axon-death factor Axed is only partially required for self-destruction of injured dendrites, acting in parallel with PS-induced phagocytosis. Lastly, injured dendrites exhibit a unique rhythmic calcium flashing that correlates with self-destruction. Therefore, a special genetic program coordinates PS exposure and self-destruction in injury-induced dendrite degeneration in vivo.

cell biology↗

bioRxiv2020

Ferric Heme as a CO/NO Sensor in the Nuclear Receptor Reverbβ by Coupling Gas binding to Electron Transfer

Rev-Erb{beta} is a nuclear receptor that couples circadian rhythm, metabolism, and inflammation.1-7 Heme binding to the protein modulates its function as a repressor, its stability, its ability to bind other proteins, and its activity in gas sensing.8-11 Rev-Erb{beta} binds Fe3+-heme tighter than Fe2+-heme, suggesting its activities may be regulated by the heme redox state.9 Yet, this critical role of heme redox chemistry in defining the proteins resting state and function is unknown. We demonstrate by electrochemical and whole-cell electron paramagnetic resonance experiments that Rev-Erb{beta} exists in the Fe3+ form within the cell essentially allowing the protein to be heme-replete even at low concentrations of labile heme in the nucleus. However, being in the Fe3+ redox state contradicts Rev-Erbs known function as a gas sensor, which dogma asserts must be a Fe2+ protein This paper explains why the resting Fe3+-state is congruent both with heme-binding and cellular gas sensing. We show that the binding of CO/NO elicits a striking increase in the redox potential of the Fe3+/Fe2+ couple, characteristic of an EC mechanism in which the unfavorable Electrochemical reduction of heme is coupled to the highly favorable Chemical reaction of gas binding, making the reduction spontaneous. Thus, Fe3+-Rev-Erb{beta} remains heme-loaded, crucial for its repressor activity, and only undergoes reduction when diatomic gases are present. This work has broad implications for hemoproteins where ligand-triggered redox changes cause conformational changes influencing proteins function or inter-protein interactions, like NCoR1 for Rev-Erb{beta}. This study opens up the possibility of CO/NO-mediated regulation of the circadian rhythm through redox changes in Rev-Erb{beta}.

biochemistry↗

bioRxiv2020

Gene expression variability in human and chimpanzee populations share common determinants

Inter-individual variation in gene expression has been shown to be heritable and it is often associated with differences in disease susceptibility between individuals. Many studies focused on mapping associations between genetic and gene regulatory variation, yet much less attention has been paid to the evolutionary processes that shape the observed differences in gene regulation between individuals in humans or any other primate. To begin addressing this gap, we performed a comparative analysis of gene expression variability and expression quantitative trait loci (eQTLs) in humans and chimpanzees, using gene expression data from primary heart samples. We found that expression variability in both species is often determined by non-genetic sources, such as cell-type heterogeneity. However, we also provide evidence that inter-individual variation in gene regulation can be genetically controlled, and that the degree of such variability is generally conserved in humans and chimpanzees. In particular, we found a significant overlap of orthologous genes associated with eQTLs in both species. We conclude that gene expression variability in humans and chimpanzees often evolves under similar evolutionary pressures.

genomics↗

bioRxiv2020

Study of cell to cell transmission of SARS CoV 2 virus particle using gene network from micro array data

Microarray data from SARS CoV2 patients helps to construct a gene network relating to this disease. Analysis of these genes, present in network, highlights their biological functions and related cellular pathways. With the assistance of these information, a drug(s) can be identified to treat COVID-19. A detailed analysis of the human host response to SARS CoV 2 with expression profiling by high throughput sequencing has executed with primary human lung epithelium cell lines. Clustered genes annotation and gene network construction are performed with the help of String database. Among four cluster of genes from microarray data, cluster 1 is identified as basal cells with p= 1.37e-05 with 44 genes. Functional enrichment analysis of genes present in gene network has completed using String database, ToppFun online tool and NetworkAnalyst tool. For SARS CoV2 virus particles, keratin proteins, which are part of cytoskeleton structure of host cell, play a major role in cell to cell virus particle transmission. Among three types of cell- cell communication, only anchoring junction between basal cell membrane and basal lamina, is involved in this virus transmission. In this junction point, hemidesmosome structure play a vital role in virus spread from one cell to basal lamina in respiratory tract. In this protein complex structure, keratin, integrin and laminin proteins of host cell is used to promote the spread of virus infection into extracellular matrix. So, small molecular blockers of different anchoring junction proteins i.e. ITGA3, ITGA2 can provide efficient protection against this deadly virus disease. Understanding the human host response against this virus is very important to develop novel therapeutics for the treatment of SARS-CoV 2.

bioinformatics↗

bioRxiv2020

A draft genome of grass pea (Lathyrus sativus), a resilient diploid legume

We have sequenced the genome of grass pea (Lathyrus sativus), a resilient diploid (2n=14) legume closely related to pea (Pisum sativum). We determined the genome size of the sequenced European accession (LS007) as 6.3 Gbp. We generated two assemblies of this genome, i) EIv1 using Illumina PCR-free paired-end sequencing and assembly followed by long-mate-pair scaffolding and ii) Rbp using Oxford Nanopore Technologies long-read sequencing and assembly followed by polishing with Illumina paired-end data. EIv1 has a total length of 8.12 Gbp (including 1.9 billion Ns) and scaffold N50 59,7 kbp. Annotation has identified 33,819 high confidence genes in the assembly. Rbp has a total length of 6.2 Gbp (with no Ns) and a contig N50 of 155.7 kbp. Gene space assessment using the eukaryote BUSCO database showed completeness scores of 82.8 % and 89.8%, respectively.

genomics↗

bioRxiv2020

Dynamic effects of genetic variation on gene expression revealed following hypoxic stress in cardiomyocytes

One life-threatening outcome of cardiovascular disease is myocardial infarction, where cardiomyocytes are deprived of oxygen. To study inter-individual differences in response to hypoxia, we established an in vitro model of induced pluripotent stem cell-derived cardiomyocytes from 15 individuals. We measured gene expression levels, chromatin accessibility, and methylation levels in four culturing conditions that correspond to normoxia, hypoxia and short or long-term re-oxygenation. We characterized thousands of gene regulatory changes as the cells transition between conditions. Using available genotypes, we identified 1,573 genes with a cis expression quantitative locus (eQTL) in at least one condition, as well as 367 dynamic eQTLs, which are classified as eQTLs in at least one, but not in all conditions. A subset of genes with dynamic eQTLs is associated with complex traits and disease. Our data demonstrate how dynamic genetic effects on gene expression, which are likely relevant for disease, can be uncovered under stress.

genomics↗

bioRxiv2020

Strategies for vaccine design for corona virus using Immunoinformatics techniques

The cutting-edge technology vaccinomics is the combination of two topics immunogenetics and immunogenomics with the knowledge of systems biology and immune profiling for designing vaccine against infectious disease. In our present study, an epitope-based peptide vaccine against nonstructural protein 4 of beta coronavirus, using a combination of B cell and T cell epitope predictions, followed by molecular docking methods are performed. Here, protein sequences of homologous nonstructural protein 4 of beta coronavirus are collected and conserved regions present in them are investigated via phylogenetic study to determine the most immunogenic part of protein. From the identified region of the target protein, the peptide sequence IRNTTNPSAR from the region ranging from 38-47 and the sequence PTDTYTSVYLGKFRG from the positions of 76-90 are considered as the most potential B cell and T cell epitopes respectively. Furthermore, this predicted T cell epitopes PTDTYTSVY and PTDTYTSVYLGKFRG interacted with MHC allelic proteins HLA-A*01:01 and HLA-DRB5*01:01 respectively with the low IC50 values. These epitopes are perfectly fitted into the epitope binding grooves of alpha helix of MHC I molecule and MHC II molecule with binding energy scores -725.0 Kcal/mole and -786.0 Kcal/mole respectively, showing stability in MHC molecules binding. This MHC restricted epitope PTDTYTSVY also showed a good conservancy of 50.16% in world population coverage. This MHC I HLA-A*01:01 allele is present among 58.87% of Chinese population also. Therefore, the epitopes IRNTTNPSAR and PTDTYTSVYLGKFRG may be considered as potential peptides for peptide-based vaccine for coronavirus after further experimental study.

immunology↗

bioRxiv2020

Correlated motions of conserved polar motifs lay out a plausible mechanism of G protein-coupled receptor activation.

Recent advancements in the field of experimental structural biology have provided high-resolution structures of active and inactive state G protein-coupled receptors (GPCRs), a highly important pharmaceutical target family, but the process of transition between these states is poorly understood. According to the current theory, GPCRs exist in structurally distinct, dynamically interconverting functional states of which populations are shifted upon binding of ligands and intracellular signaling proteins. However, explanation of the activation mechanism on an entirely structural basis gets complicated when multiple activation pathways and active receptor states are considered. Our unbiased, atomistic molecular dynamics simulations of the mu-opioid receptor in a physiological environment revealed that external stimulus is propagated to the intracellular surface of the receptor through subtle, concerted movements of highly conserved polar amino acid side chains along the 7th transmembrane helix. To amend the widely accepted theory we suggest that the initiation event of GPCR activation is the shift of macroscopic polarization between the ortho- and allosteric binding pockets and the intracellular G protein-binding interface. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/920769v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@102f33org.highwire.dtl.DTLVardef@8af6bforg.highwire.dtl.DTLVardef@14d9192org.highwire.dtl.DTLVardef@1903819_HPS_FORMAT_FIGEXP M_FIG Table of Contents Graphic C_FIG

molecular biology↗

bioRxiv2020

Tandem tension sensor reveals substrate rigidity-dependence of integrin molecular tensions in live cells

Response of integrin tensions to substrate rigidity is important in cell rigidity sensing but has not been confirmed. Current fluorescent tension sensors produce cellular force signals collectively resulted from integrin tension magnitude, tension dwell time, integrin density and activity, ligand density and accessibility, etc., making it challenging to monitor the absolute molecular force level of integrin tensions in live cells. Here we developed a tandem tension sensor (TTS) consisting of two coupled tension sensing units which are subject to the same tension and respond with different activation probabilities to the tension. Reported by fluorescence, the activation probability ratio of these two units solely responds to the force level of local integrin tensions, excluding the bias from other non-force factors. We verified the feasibility of TTS in detecting integrin tensions and applied it to study cells on elastic substrates. TTS unambiguously reported that integrin tensions in platelets decrease monotonically with the substrate rigidity, verifying the rigidity-dependence of integrin tensions in live cells.

biophysics↗

bioRxiv2019

Mechanics of multi-centrosomal clustering in bipolar mitotic spindles

To segregate chromosomes in mitosis, cells assemble mitotic spindle, a molecular machine with centrosomes at two opposing cell poles and chromosomes at the equator. Microtubules and molecular motors connect the poles to kinetochores, specialized protein assemblies on the centromere regions of the chromosomes. Bipolarity of the spindle is crucial for the proper cell division, and two centrosomes in animal cells naturally become two spindle poles. Cancer cells are often multi-centrosomal, yet they are able to assemble bipolar spindles by clustering centrosomes into two spindle poles. Mechanisms of this clustering are debated. In this study, we computationally screen effective forces between a) centrosomes, b) centrosomes and kineto-chores, c) centrosomes and chromosome arms, d) centrosomes and cell cortex, to understand mechanics that determines three-dimensional spindle architecture. To do this, we use stochastic Monte Carlo search for stable mechanical equilibria in effective energy landscape of the spindle. We find that the following conditions have to be met to robustly assemble the bipolar spindle in a multi-centrosomal cell: 1) strengths of centrosomes attraction to each other and to the cell cortex have to be proportional to each other; 2) strengths of centrosomes attraction to kinetochores and repulsion from the chromosome arms have to be proportional to each other. We also find that three other spindle configurations emerge if these conditions are not met: a) collapsed, b) monopolar, c) multipolar spindles, and the computational screen reveal mechanical conditions for these abnormal spindles. Significance statementTo segregate chromosomes, cells assemble bipolar mitotic spindle. Multiple mechanical forces generated by microtubules and molecular motors in the spindle govern the spindle architecture, but it is unclear what force balances support the bipolarity of the spindle. This problem is especially difficult and important in cancer cells, which often have multiple centrosomes that somehow are able to cluster into two spindle poles. By using stochastic energy minimization in an effective energy landscape of the spindle and computationally screening forces, we find mechanical conditions for mono-, multi- and bi-polar spindles. We predict how microtubule and motor parameters have to be regulated in mitosis in multi-centrosomal cells.

cell biology↗