Biology subjects

Vukusic, K.

Publications and source records attributed to Vukusic, K..

10 recordsLinked to original sources

bioRxiv2026

Mitotic adaptations shape acquired resistance and vulnerabilities to KIF18A inhibition in cancer

KIF18A inhibition selectively kills cancer cells by inducing chromosome alignment defects that activate a spindle assembly checkpoint (SAC)-dependent mitotic arrest. The mechanisms by which initially sensitive cancer cells acquire resistance to KIF18A inhibitors (KIF18Ai), the vulnerabilities of resistant cells, and the initial genetic determinants of KIF18Ai sensitivity remain poorly understood. Using orthogonal CRISPR-Cas9 screening and long-term drug adaptation approaches, we identify two convergent resistance mechanisms. Resistant cells either partially override the SAC, permitting mitotic exit despite chromosome misalignment, or adapt spindle microtubule dynamics to restore chromosome alignment in the absence of KIF18A. Both strategies sustain mitotic progression without inducing KIF18Ai dependence or increasing sensitivity to other mitotic perturbations. In contrast, reduced activity of the mitotic exit regulators PP2A or APC/C enhances KIF18Ai sensitivity. Accordingly, Mps1 inhibitor-driven APC/C mutations enhance responsiveness to KIF18A inhibition. In conclusion, resistance to KIF18A inhibition emerges rapidly, yet distinct genetic contexts create exploitable vulnerabilities to KIF18Ai.

cancer biology↗

bioRxiv2026

The SKA complex is a mitotic vulnerability of chromosomally unstable cancers

Aneuploidy and chromosomal instability are common characteristics of cancer but remain underexploited therapeutically. Because chromosomally unstable cells exhibit altered mitotic control, they are selectively vulnerable to inhibition of the kinesin KIF18A. Here, we identify the spindle and kinetochore-associated (SKA) complex as a mitotic regulator that establishes a similar dependency. SKA loss is tolerated in cells with a defective mitotic checkpoint, but when mitosis is prolonged, it leads to mitotic errors and loss of viability. Genetic analysis reveals that SKA and KIF18A are epistatic, although they function through mechanistically distinct mitotic pathways. SKA depletion selectively impacts chromosomally unstable cancer cells and mirrors the effects of KIF18A inhibition. We thus identify a cellular vulnerability associated with prolonged mitosis in chromosomally unstable cancers.

cell biology↗

bioRxiv2026

Mitotic errors drive rapid clearance of polyploidy during intestinal regeneration despite robust centrosome clustering

Polyploid cells are increasingly recognized not only as hallmarks of cancer but also as features of regenerating tissues. During intestinal regeneration, polyploid cells are transient, yet the mechanisms underlying their clearance remain unknown. Using mouse intestinal organoids as a regeneration model, we show that, unlike in many cancer cell-line models, this clearance occurs without immediate cell-cycle arrest and is not driven by failure to establish spindle bipolarity. Instead, polyploid intestinal cells efficiently cluster supernumerary centrosomes to form bipolar spindles in an HSET-dependent manner, facilitated by delayed centrosome separation at mitotic onset. Despite this, polyploid divisions frequently produce chromosome segregation errors, including catastrophic chronocrisis. Lineage tracing reveals that progeny of such divisions is rapidly lost over subsequent generations. Increasing polyploidy during early regeneration disrupts organoid maturation, indicating that timely polyploidy clearance is required for successful regeneration. Polyploid cells are also detected in regenerating human colonic organoids, suggesting that transient polyploidy is a conserved feature of intestinal regeneration.

cell biology↗

bioRxiv2025

Mitotic BLM functions are required to maintain genomic stability

The BLM helicase is a critical genome maintenance protein involved in diverse cellular processes including DNA replication, repair, transcription, and chromosome segregation. During mitosis, it cooperates with the PICH helicase and topoisomerases to resolve ultrafine DNA bridges (UFBs) - non-chromatinized DNA structures that link sister chromatids - through a mechanism that is not yet fully understood. Here we tagged endogenous BLM and PICH with fluorescent proteins and BLM with an auxin-inducible degron to generate a cell model system that enables temporal tracking of UFB dynamics in the presence or absence of BLM. Time-resolved lattice light sheet microscopy established the dynamic localization patterns of BLM and PICH throughout the cell cycle. While BLM cycles between PML bodies and DNA repair foci in interphase, it dissociates from chromatin at the mitotic entry, and re-associates during anaphase to UFBs as well as to CENP-B-positive mitotic foci. Acute BLM depletion during mitosis increased the fraction of unresolved UFBs, micronuclei containing acentric fragments, binucleation, and resulted in subtle genomic abnormalities detected by single-cell whole genome sequencing. These findings highlight a mitosis-specific role for BLM in UFB resolution and underscore its function in preserving genomic stability. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/659902v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@92d8b2org.highwire.dtl.DTLVardef@d0a915org.highwire.dtl.DTLVardef@10cac60org.highwire.dtl.DTLVardef@108da58_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

A Universal Scaling Law for Mitotic Spindles Driven by Chromosome Crowding

Cells regulate the size of their internal structures to maintain function in diverse biological settings1. The mitotic spindle, a molecular micro-machine responsible for chromosome segregation2, must scale to accommodate genomes varying in size by over 10,000-fold across eukaryotes3. Yet, how spindle biomechanics adapts to vastly different genome sizes remains unknown. Here, we uncover a universal spindle scaling law, where metaphase plate width scales with genome size following a power law with an exponent of [~]1/3. We hypothesize that chromosome crowding within the metaphase plate generates compressive forces as chromosomes push against each other, thereby determining spindle size and shape. Our experiments with altered chromosome number and mechanical properties in healthy and cancerous human and mouse cells, together with a theoretical model based on inter-chromosome pushing forces and mechanical manipulations of cells, confirm this hypothesis. Extending these insights across eukaryotes, we demonstrate that chromosome crowding predicts the observed power-law scaling. The biophysical constraint of chromosome crowding offers a mechanistic explanation for the evolution of open mitosis and mitotic cell rounding, enabling the division of larger genomes. Spindle adaptability to larger genomes may promote the proliferation of polyploid cells, driving not only tumor progression but also speciation during evolution.

biophysics↗

bioRxiv2024

Kinetochore-centrosome feedback linking CENP-E and Aurora kinases controls chromosome congression

Chromosome congression is crucial for accurate cell division, with key roles played by kinetochore components CENP-E/kinesin-7 and Aurora B kinase. However, Aurora kinases both inhibit and promote congression, suggesting the presence of a larger signaling network. Our study demonstrates that centrosomes inhibit congression initiation when CENP-E is inactive by regulating the activity of kinetochore components. Depleting centrioles via Plk4 inhibition allows chromosomes near acentriolar poles to initiate congression independently of CENP-E. At centriolar poles high Aurora A enhances Aurora B activity, increasing phosphorylation of microtubule-binding proteins at kinetochores and preventing stable microtubule attachments in the absence of CENP-E. Conversely, inhibiting Aurora A or expressing a dephosphorylatable Hec1 mutant enables congression initiation without CENP-E. We propose a self-limiting feedback mechanism involving Aurora kinases and CENP-E that regulates the timing of chromosome movement by modulating kinetochore-microtubule attachments and fibrous corona expansion, with Aurora A activity gradient providing critical spatial cues for the networks function.

cell biology↗

bioRxiv2024

Cardiomyocyte antisense transcription regulates exon usage in the elastic spring region of Titin to modulate sarcomere function

BackgroundThe spring-like sarcomere protein Titin (TTN) is a key determinant of cardiac passive stiffness and diastolic function. Alternative splicing of TTN I-band exons produce protein isoforms with variable size and elasticity, but the mechanisms regulating TTN exon skipping and isoform composition in the human heart are not well studied. Non-coding RNA transcripts from the antisense strand of protein-coding genes have been shown to regulate alternative splicing of the sense gene. The TTN gene locus harbours >80 antisense transcripts with unknown function in the human heart. The aim of this study was to determine if TTN antisense transcripts play a role in alternative splicing of TTN. MethodsRNA-sequencing and RNA in situ hybridization (ISH) of cardiac tissue from unused organ donor hearts (n=7) and human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) were used to determine the expression and localization of TTN antisense transcripts. The effect of siRNA-mediated knock down of TTN antisense transcripts on TTN exon usage in iPS-CMs was determined using RNA-sequencing. Live cell imaging with sarcomere tracking was used to analyze the effect of antisense transcript knock down on sarcomere length, organization and contraction dynamics. RNA ISH, immunofluorescence and high content microscopy was performed in iPS-CMs to study the interaction between antisense transcripts, TTN mRNA and splice factor protein RBM20. ResultsIn mapping TTN antisense transcription, we found that TTN-AS1-276 was the predominant transcript in the human heart and that it was mainly localized in cardiomyocyte nuclear chromatin. Knock down of TTN-AS1-276 in human iPS-CMs resulted in decreased interaction between the splicing factor RBM20 and TTN pre-mRNA, decreased TTN I-band exon skipping, and markedly lower expression of the less elastic TTN isoform N2B. The effect on TTN exon usage was independent of sense-antisense exon overlap and polymerase II elongation rate. Furthermore, knockdown resulted in longer sarcomeres with preserved alignment, improved fractional shortening and relaxation times. ConclusionsWe demonstrate a role for the cardiac TTN antisense transcript TTN-AS1-276 in facilitating alternative splicing of TTN and regulating sarcomere properties. This transcript could constitute a target for improving cardiac passive stiffness and diastolic function in conditions such as heart failure with preserved ejection fraction.

molecular biology↗

bioRxiv2023

CENP-E initiates chromosome congression by opposing Aurora kinases to promote end-on microtubule attachments close to centrosomes

Accurate cell division relies on rapid chromosome congression. The kinetochore motor protein CENP-E/kinesin-7 is uniquely required for congression of polar chromosomes. It is currently assumed that CENP-E drives congression by gliding kinetochores along microtubules independently of their biorientation. Here, by studying chromosome movement under different levels of CENP-E activity, we favor an alternative model in which CENP-E initiates congression by promoting stabilization of end-on attachments. In this way, CENP-E accelerates congression initiation without significantly contributing to subsequent movement. Stabilization of end-on attachments on polar chromosomes without CENP-E is delayed due to Aurora kinase-mediated hyperphosphorylation of microtubule-binding proteins and expansion of the fibrous corona. CENP-E counters this by reducing Aurora B-mediated phosphorylation in a BubR1-dependent manner, thereby stabilizing initial end-on attachments, facilitating removal of the fibrous corona, and triggering biorientation-dependent chromosome movement. These findings support a unified model of chromosome movement in which congression is intrinsically coupled to biorientation.

cell biology↗

bioRxiv2023

Plasmodium ARK2-EB1 axis drives the unconventional spindle dynamics, scaffold formation, and chromosome segregation of sexual transmission stages

Mechanisms of cell division are remarkably diverse, suggesting the underlying molecular networks among eukaryotes differ extensively. The Aurora family of kinases orchestrates the process of chromosome segregation and cytokinesis during cell division through precise spatiotemporal regulation of their catalytic activities by distinct scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes that have three divergent aurora-related kinases (ARKs) and lack most canonical scaffolds/activators. The parasite uses unconventional modes of chromosome segregation during endomitosis and meiosis in sexual transmission stages within mosquito host. This includes a rapid threefold genome replication from 1N to 8N with successive cycles of closed mitosis, spindle formation and chromosome segregation within eight minutes (termed male gametogony). Kinome studies had previously suggested likely essential functions for all three Plasmodium ARKs during asexual mitotic cycles; however, little is known about their location, function, or their scaffolding molecules during unconventional sexual proliferative stages. Using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging, we set out to investigate the role of the atypical Aurora paralog ARK2 to proliferative sexual stages using rodent malaria model Plasmodium berghei. We find that ARK2 primarily localises to the spindle apparatus in the vicinity of kinetochores during both mitosis and meiosis. Interactomics and co-localisation studies reveal a unique ARK2 scaffold at the spindle including the microtubule plus end-binding protein EB1, lacking conserved Aurora scaffold proteins. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora kinase spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite Plasmodium.

cell biology↗

bioRxiv2019

Mitotic spindle elongation is driven by joint microtubule sliding action of kinesins KIF4A and EG5

Successful cell division requires proper chromosome segregation during anaphase. Forces required for chromosome segregation in human cells are linked to sliding of antiparallel microtubules and sliding capacity has been demonstrated in vitro for multiple motor proteins, but the molecular mechanism of sliding in the spindle of human cells remains unknown. Using combined depletion and inactivation assays to explore redundancy between multiple targets together with CRISPR technology, we found that PRC1-dependent motor KIF4A/kinesin-4, together with EG5/kinesin-5 motor is essential for spindle elongation in human cells. Photoactivation of tubulin and super-resolution microscopy show that perturbation of both proteins impairs sliding, while decreased midzone microtubule stability cannot explain the observed anaphase arrest. Thus, two independent sliding modules power sliding mechanism that drives spindle elongation in human cells.

cell biology↗