Biology subjects

Tolic, I. M.

Publications and source records attributed to Tolic, I. M..

At least 19 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↗

bioRxiv2026

Spheroid culture remodels mitosis and the proteome in tumor cells

Mitosis depends on precise spindle assembly and positioning, processes influenced by cell shape, size, and microenvironment. Most mechanistic insights into mitosis come from two-dimensional (2D) monolayer cultures, which lack the spatial constraints and extracellular matrix found in tissues, leaving the influence of the tissue environment on mitosis poorly understood. Here, we combine high-resolution imaging and quantitative proteomics to compare mitosis in three-dimensional (3D) multicellular spheroids, generated by magnetic levitation, with that in 2D monolayers. Using a non-transformed cell line and three cancer cell lines from breast, bone, and ovary, we show that 3D culture reshapes mitotic cells and their spindles. Tumor spheroids exhibited a prometaphase delay together with minor chromosome alignment defects, yet chromosome segregation remained largely accurate. Cells in spheroids were rounder, and their spindles were smaller, with increased multipolarity and defects in orientation and position, which varied by cell line. Proteomic profiling revealed broad downregulation of mitotic regulators in spheroids, including kinesins (KIF11, KIF4A), spindle checkpoint proteins, and APC/C components, accompanied by enrichment of metabolic and mitochondrial pathways. Together, our results reveal both shared and cell line-specific modes of mitotic restructuring and establish a framework that connects proteome state to mitotic architecture in 3D environments.

cell biology↗

bioRxiv2026

Coordinated control of both microtubule ends regulates mitotic spindle length

The mitotic spindle is a biomechanical structure whose length must be precisely controlled to ensure faithful chromosome segregation. The treadmilling of microtubules towards centrosomes, termed poleward flux, is involved in spindle length control. However, poleward flux has been shown to be both inversely and directly proportional to spindle length, a contradiction that remains unexplained by current mechanisms. Here we introduce a model which demonstrates that length-dependent regulation at both microtubule ends allows plus- and minus-end dynamics to synchronize with one another, enabling pathways of poleward flux-based spindle length control which can rectify previous results. Moreover, our model predicts that spindle length and poleward flux can vary independently via simultaneous perturbations at both microtubule ends, which we experimentally validate with combinations of KIF18A, KIF2A, and KATNB1 depletions in human cells. Our results thus resolve a longstanding paradox and provide mechanistic insight into the reciprocal control of spindle length and poleward flux.

biophysics↗

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

Microtubule pivoting driven by spindle elongation rescues polar chromosomes to ensure faithful mitosis

Polar chromosomes, which initially attach to the mitotic spindle behind the pole, are prone to missegregation and micronuclear entrapment, contributing to chromosomal instability in cancer. Yet, the mechanisms ensuring their faithful segregation remain unclear. Here, we show that polar chromosomes require a unique step involving spindle elongation, which repositions chromosome-bound astral microtubules by pivoting them around the centrosome toward the spindle surface. By modulating Eg5/KIF11 activity, we demonstrate that spindle elongation determines the direction and extent of pivoting, with microtubules from the opposite spindle half facilitating final movement. Kinetochores on polar chromosomes form mixed lateral and immature monotelic attachments, recruiting corona components and partially Mad2, but lacking Astrin. In several cancer cell lines, limited spindle elongation delays polar chromosome resolution, whereas enhanced elongation accelerates it. These findings highlight the role of spindle elongation in the timely rescue of polar chromosomes from the danger zone behind the pole, and provide mechanistic insight into how chromosome congression errors can arise in cancer.

cell biology↗

bioRxiv2024

Microtubule poleward flux as a target for modifying chromosome segregation errors

Cancer cells often display errors in chromosome segregation, some of which result from improper chromosome alignment at the spindle midplane. Chromosome alignment is facilitated by different rates of microtubule poleward flux between sister kinetochore fibers. However, the role of the poleward flux in supporting mitotic fidelity remains unknown. Here, we introduce the hypothesis that the finely tuned poleward flux safeguards against lagging chromosomes and micronuclei at mitotic exit by promoting chromosome alignment in metaphase. We used human untransformed RPE-1 cells depleted of KIF18A/kinesin-8 as a system with reduced mitotic fidelity, which we rescued by three mechanistically independent treatments, comprising low-dose taxol or co-depletion of the spindle proteins HAUS8 or NuMA. The rescue of mitotic errors was due to shortening of the excessively long overlaps of antiparallel microtubules, serving as a platform for motor proteins that drive the flux, which in turn slowed down the overly fast flux and improved chromosome alignment. In contrast to the prevailing view, the rescue was not accompanied by reduction of overall microtubules growth rates. Speckle microscopy revealed that the improved chromosome alignment in the rescue treatments was associated with slower growth and flux of kinetochore microtubules. In a similar manner, a low-dose taxol treatment rescued mitotic errors in a high-grade serous ovarian carcinoma cell line OVKATE. Collectively, our results highlight the potential of targeting microtubule poleward flux to modify chromosome instability and provide insight into the mechanism through which low doses of taxol rescue mitotic errors in cancer cells.

cell 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

Life cycle-coupled evolution of mitosis in close relatives of animals

Eukaryotes have evolved towards one of two extremes along a spectrum of strategies for remodelling the nuclear envelope (NE) during cell division: disassembling the NE in an open mitosis or constructing an intranuclear spindle in a closed mitosis. Both classes of mitotic remodelling involve key differences in the core division machinery, but the evolutionary reasons for adopting a specific mechanism are unclear. Here, we use an integrated comparative genomics and ultrastructural imaging approach to investigate mitotic strategies in Ichthyosporea, close relatives of animals and fungi. We show that species within this clade have diverged towards either a fungal-like closed or an animal-like open mitosis, most likely to support distinct multi- or uninucleated states. Our results suggest that multinucleated life cycles favour the evolution of closed mitosis. One-Sentence SummaryMitotic specialization in animal relatives reveal that multinucleated life cycles favor the evolution of closed mitosis

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↗

bioRxiv2022

Proliferative advantage of specific aneuploid cells drives evolution of tumor karyotypes

Most tumors have abnormal karyotypes, which arise from mistakes during mitotic division of healthy euploid cells and evolve through numerous complex mechanisms. In a recent mouse model with high levels of chromosome missegregation, chromosome gains dominate over losses both in pretumor and tumor tissues, whereas tumors are characterized by gains of chromosomes 14 and 15. However, the mechanisms driving clonal selection leading to tumor karyotype evolution remain unclear. Here we show, by introducing a mathematical model based on a concept of a macro-karyotype, that tumor karyotypes can be explained by proliferation-driven evolution of aneuploid cells. In pretumor cells, increased apoptosis and slower proliferation of cells with monosomies lead to predominant chromosome gains over losses. Tumor karyotypes with gain of one chromosome can be explained by karyotype-dependent proliferation, while for those with two chromosomes an interplay with karyotype-dependent apoptosis is an additional possible pathway. Thus, evolution of tumor-specific karyotypes requires proliferative advantage of specific aneuploid karyotypes. SignificanceMost tumors have an erroneous number of chromosomes, which arise from mistakes during division of healthy cells and evolve through numerous complex mechanisms, including chromosome missegregation, cell proliferation and cell death. However, understanding the mechanisms leading to tumor evolution from healthy cells is a hot topic. Here we show, by introducing a "macro-karyotype model", that perturbed number of chromosomes in tumor cells arises predominantly from faster division of cells characterized by a specific combination of chromosomes, or together with irregular cell death. This finding, strengthened by comparison of our theory with experimentally observed combination of chromosomes in different stages of tumor development, gives a direction for future experiments in identifying the key processes underlying tumor development.

biophysics↗

bioRxiv2022

Kinetochore- and chromosome-driven transition of microtubules into bundles promotes spindle assembly

Mitotic spindle assembly is crucial for chromosome segregation and relies on bundles of microtubules that extend from the poles and overlap in the middle. However, how these structures form remains poorly understood. Here we show that overlap bundles arise through a network-to-bundles transition driven by kinetochores and chromosomes. STED super-resolution microscopy revealed that PRC1-crosslinked microtubules initially form loose arrays, which become rearranged into bundles. Kinetochores promote microtubule bundling by lateral binding via CENP-E/kinesin-7 in an Aurora B-regulated manner. Bundle separation is driven by steric interactions of the bundle-associated chromosomes at the spindle midplane since spindles with uncondensed or uncongressed chromosomes are narrower. In agreement with experiments, theoretical modeling suggests that bundles arise through competing attractive and repulsive mechanisms. Finally, perturbation of overlap bundles led to inefficient correction of erroneous kinetochore-microtubule attachments. Thus, kinetochores and chromosomes drive coarsening of a uniform microtubule array into overlap bundles, which promote not only spindle formation but also chromosome segregation fidelity.

cell biology↗

bioRxiv2021

Naegleria's mitotic spindles are built from unique tubulins and highlight core spindle features

Naegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis, and thus provides a unique system to study the evolution and functional specificity of mitotic tubulins and the resulting spindle. Previous studies showed that Naegleria amoebae express a divergent -tubulin during mitosis and we now show that Naegleria amoebae express a second mitotic - and two mitotic {beta}-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical - and {beta}- tubulins, contain residues that suggest distinct microtubule properties, and may represent drug targets for the "brain-eating amoeba" Naegleria fowleri. Using quantitative light microscopy, we find that Naeglerias mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naeglerias spindle is twisted and its length increases during mitosis suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers, and secondary bundles function as bridging fibers.

cell biology↗

bioRxiv2021

Measurement of curvature and twist of microtubule bundles in the mitotic spindle

The highly ordered spatial organization of microtubule bundles in the mitotic spindle is crucial for its proper functioning. The recent discovery of twisted shapes of microtubule bundles and spindle chirality suggests that the bundles extend along curved paths in three dimensions, rather than being confined to a plane. This in turn implies that rotational forces exist in the spindle in addition to the widely studied linear forces. However, studies of spindle architecture and forces are impeded by a lack of a robust method for the geometric quantification of microtubule bundles in the spindle. In this paper, we describe a simple method for measuring and evaluating the shapes of microtubule bundles, by characterizing them in terms of their curvature and twist. By using confocal microscopy, we obtain three-dimensional images of spindles, which allow us to trace the entire microtubule bundles. For each traced bundle, we first fit a plane, and then fit a circle lying in that plane. With this easily reproducible method, we extract the curvature and twist, which represent the geometric information characteristic for each bundle. As the bundle shapes reflect the forces within them, this method is valuable for the understanding of forces that act on chromosomes during mitosis.

biophysics↗

bioRxiv2021

Sliding of kinetochore fibers along bridging fibers helps center the chromosomes on the spindle

Chromosome alignment at the spindle equator promotes proper chromosome segregation and depends on pulling forces exerted at kinetochore fiber tips together with polar ejection forces. However, kinetochore fibers are also subjected to forces driving their poleward flux. Here we introduce a flux-driven centering model that relies on flux generated by forces within the overlaps of bridging and kinetochore fibers. This centering mechanism works so that the longer kinetochore fiber fluxes faster than the shorter one, moving the kinetochores towards the center. We developed speckle microscopy in human spindles and confirmed the key prediction that kinetochore fiber flux is length-dependent. Kinetochores are better centered when overlaps are shorter and the kinetochore fiber flux markedly slower than the bridging fiber flux. We identify Kif18A and Kif4A as overlap and flux regulators and NuMA as a fiber coupler. Thus, length-dependent sliding forces exerted by the bridging fiber onto kinetochore fibers promote chromosome alignment.

cell biology↗