Kruno Vukušić

Rudjer Boskovic Institute

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Iva M. Tolić

Rudjer Boskovic Institute

Patrik Risteski

Rudjer Boskovic Institute

Renata Buđa

Rudjer Boskovic Institute

Ivana Ponjavić

Rudjer Boskovic Institute

Steven Abel

University of California, Riverside

Snježana Ramić

Sisters of Charity Hospital

Krešo Bendelja

University of Zagreb

Nenad Pavin

University of Zagreb

Declan Brady

University of Nottingham

Edward Rea

Oxford Brookes University

Cooperative Institutions

University of Edinburgh

Oncode Institute

Rudjer Boskovic Institute

University Medical Center Utrecht

University of Zagreb

Royal Netherlands Academy of Arts and Sciences

Sisters of Charity Hospital

University Hospital Centre Zagreb

Max Planck Institute of Molecular Cell Biology and Genetics

Rutgers, The State University of New Jersey

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Torques and Forces in the Mitotic Spindle

Biophysical Journal (2018)

Kruno Vukušić Renata Buđa Juraj Simunić Bruno Polak Maja Novak

Spindle pole body

Astral microtubules

10.1016/j.bpj.2017.11.2147

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New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

Stem Cell Research & Therapy (2023)

Marija Zekušić Marina Bujić Mihica Marija Skoko Kruno Vukušić Patrik Risteski

Abstract Background Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications. Methods After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined. Results Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype. Conclusion This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.

10.1186/s13287-023-03586-z

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Expansion microscopy of the mitotic spindle

Methods in cell biology (2020)

Ivana Ponjavić Kruno Vukušić Iva M. Tolić

10.1016/bs.mcb.2020.04.014

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New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

Research Square (Research Square) (2023)

Marija Zekušić Marina Bujić Mihica Marija Skoko Kruno Vukušić Patrik Risteski

Abstract Background Limbal stem cells(LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications. Methods After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Through a clinically applicable protocol,the collected data on LSCs at passage 1 were summarized: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency were analyzed by trypan blue staining); expression of putative stem cell markers by flow cytometry, immunofluorescence and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined. Results Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n=873) was performed, and only a small fraction <2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype. Conclusion This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.

10.21203/rs.3.rs-3239976/v1

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Two Functionally Redundant Sliding Modules Drive Chromosome Segregation

SSRN Electronic Journal (2020)

Kruno Vukušić Renata Buđa Ivana Ponjavić Patrik Risteski Iva M. Tolić

Proper chromosome segregation into two future daughter cells requires the mitotic spindle to elongate in anaphase. Yet, the molecular mechanism that drives spindle elongation in human cells is unknown. Using combined depletion and inactivation assays together with CRISPR technology to explore redundancy between multiple targets, we discovered that the force-generating mechanism of spindle elongation consists of the PRC1-dependent motor KIF4A/kinesin-4 together with EG5/kinesin-5. Disruption of both motors leads to total failure of chromosome segregation due to blocked spindle elongation, despite poleward chromosome motion. Tubulin photoactivation and super-resolution microscopy show that perturbation of both proteins impairs midzone microtubule sliding without affecting microtubule stability. Thus, two mechanistically distinct sliding modules, one based on a self-sustained and the other on a crosslinker-assisted motor, power the mechanism that drives spindle elongation in human cells.

10.2139/ssrn.3671936

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Plasmodium ARK2 and EB1 drive unconventional spindle dynamics, during chromosome segregation in sexual transmission stages

Nature Communications (2023)

Mohammad Zeeshan Edward Rea Steven Abel Kruno Vukušić Róbert Márkus

Abstract The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities by distinct protein scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes with three unique and highly divergent aurora-related kinases (ARK1-3) that are essential for asexual cellular proliferation but lack most canonical scaffolds/activators. Here we investigate the role of ARK2 during sexual proliferation of the rodent malaria Plasmodium berghei , using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging. We find that ARK2 is primarily located at spindle microtubules in the vicinity of kinetochores during both mitosis and meiosis. Interactomic and co-localisation studies reveal several putative ARK2-associated interactors including the microtubule-interacting protein EB1, together with MISFIT and Myosin-K, but no conserved eukaryotic scaffold proteins. Gene function studies indicate that ARK2 and EB1 are complementary in driving endomitotic division and thereby parasite transmission through the mosquito. This discovery underlines the flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite.

Plasmodium (life cycle)

Plasmodium berghei

Spindle pole body

10.1038/s41467-023-41395-3

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PlasmodiumARK2-EB1 axis drives the unconventional spindle dynamics, scaffold formation and chromosome segregation of sexual transmission stages

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Mohammad Zeeshan Edward Rea Steven Abel Kruno Vukušić Róbert Márkus

Abstract 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 .

Spindle pole body

Plasmodium (life cycle)

10.1101/2023.01.29.526106

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Citations (3)

Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores

Nature Communications (2016)

Janko Kajtez Anastasia I. Solomatina Maja Novak Bruno Polak Kruno Vukušić

Abstract During metaphase, forces on kinetochores are exerted by k-fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser-cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non-kinetochore microtubules, which we term ‘bridging fibre’, bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape.

10.1038/ncomms10298

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The mechanisms of microtubule associated proteins in chromosome segregation

Kruno Vukušić

Successful cell division requires chromosomes to be separated on opposite sides of the cell. This is achieved by depolymerisation of kinetochore fibers and spindle elongation. Forces required for spindle elongation 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 during anaphase in the spindles of human cells remains unknown. Here I show, by using combined depletion and inactivation assays to explore redundancy between multiple targets together with CRISPR technology, that PRC1-dependent motor KIF4A/kinesin-4, together with EG5/kinesin-5 motor is essential for spindle elongation in human cells. Depletion of KIF4A, or its partner PRC1, together with EG5 inhibition, completely arrests cell division in early anaphase while depletion of other PRC1-interacting partners had minor effect on anaphase progression. On the other hand, decreased stability of midzone microtubules measured by photoactivation assay or their structural changes studied by expansion microscopy cannot explain the observed anaphase arrest. Conclusively, my work demonstrates that two independent sliding modules power sliding mechanism that drives anaphase spindle elongation in human cells thus resolving longstanding question in the field and bridging the gap between extensive in vitro work on sliding motors and their concrete actions within the cell.

Kinesin

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Mehanizmi razvoja B-staničnih limfoma

Kruno Vukušić

Mechanisms that control normal B-cell differentiation and activation are frequently disrupted in lymphoma cells which in the end leads to their unlimited growth and survival. B cells are particulary prone to malignant transformation because processes that normally create imunoglobulin diversity, which is necessary for normal imunnologic response, often can cause chromosomal translocations and oncogenic mutations which include strong promoters of Ig genes, tumor–supressor genes, and oncogenes. Such aberations often lead to block in differentiation, prevention of apotosis and/or promotion of proliferation which can represent first oncogenic hit in B-cell transformation. In addition, most of B-cell lymphoma depend on expression of a BCR for their survival. In some lymphomas antigen activation of lyphoma cells through BCR signalling snd specific factors from the microenviroment are equally important in tumorogenesis. Reserach and development of funtional and structural genomics greatly improved understanding of mechanisms involved in B-cell lymphoma pathogenesis. Discovery of many signalling pathways which are aberrant in B cell lymphomas, leading to constitutive activation of many prosurvival pathways are now known. In addition, such research showed that B-cell lymhoma can be classified by their cell of origin because transformed cells often resemble normal stages of B-cell differentiation by immunophenotypic and genetic characteristics. In conclusion, development od field of genomics could lead to better understanding of mechanisms that drive B-cell lymphoma pathogenesis, all for one cause - efficient lymphoma therapies.

breakpoint cluster region

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