Bridging microtubules and generation of forces in mitosis

Na početku stanične diobe stanica konstruira diobeno vreteno, složeni citoskeletni mehanizam sastavljen od mikrotubula i proteina povezanih s mikrotubulima. Dosadašnji model mitotskog diobenog vretena definira dvije odvojene skupine mikrotubula; kinetohorne i ne-kinetohorne mikrotubule. Tijekom metafaze, sile na kinetohorama su uzrokovane kinetohornim mikrotubulima koji čine kinetohorna vlakna. Međutim, nekinetohorni mikrotubuli su uočeni u blizini kinetohora u metafazi. Ovaj rad sumira sva istraživanja temeljena na novoj podskupini antiparalelnih mikrotubula, nazvanih premoščujući mikrotubuli, koji premošćuju regiju ispod sestrinskih kinetohora i ostvaruju lateralne poveznice između odgovarajućih kinetohornih vlakana. Premoščujući mikrotubuli su vizualizirani konfokalnom mikroskopijom u stanicama HeLa i PtK1. Kako bi se premoščujući mikrotubuli mogli razlikovati od ostalih ne-kinetohornih mikrotubula te kako bi se ispitale sile koje premoščujući snop generira, osmišljen je protokol laserske ablacije. Konstruiran je fizikalni model u kojem premoščujući snop osigurava mehaničku otpornost na tenziju i kompresiju unutar kinetohornog vlakna. Rezultati istraživanja indiciraju uključenost nove podskupine mikrotubula u preciznu segregaciju i organizaciju kromosoma uravnotežavanjem sila unutar mitotskog diobenog vretena.At the onset of division, cell constructs a spindle, complex cytoskeletal mechanism formed with microtubules and microtubule-associated proteins. The current model of the mitotic spindle defines two separate populations of microtubules; kinetochore and nonkinetochore microtubules. During metaphase, forces on the kinetochores are exerted by kinetochore fibers consisting of kinetochore microtubules. However, non-kinetochore microtubules have been observed in the vicinity of the kinetochores in metaphase. This paper summarizes all research based on subpopulation of antiparallel microtubules, named bridging microtubules that span the region under sister kinetochores and facilitate a lateral connection between corresponding kinetochore fibers. Bridging microtubules have been visualized by confocal microscopy in HeLa and PtK1 cells. In order to distinct bridging microtubules from other non-kinetochore microtubules and to test forces that bridging bundle generates, laser ablation assay was developed. Physical model in which bridging bundle provides mechanical resistance to the tension and compression within the kinetochore fibers was constructed. Results indicate that the new subpopulation of microtubules is responsible for precise chromosome segregation and organization by balancing forces within mitotic spindle

PRC1‐labeled microtubule bundles and kinetochore pairs show one‐to‐one association in metaphase

In the mitotic spindle, kinetochore microtubules form k‐fibers, whereas overlap or interpolar microtubules form antiparallel arrays containing the cross‐linker protein regulator of cytokinesis 1 (PRC1). We have recently shown that an overlap bundle, termed bridging fiber, links outermost sister k‐fibers. However, the relationship between overlap bundles and k‐fibers throughout the spindle remained unknown. Here, we show that in a metaphase spindle more than 90% of overlap bundles act as a bridge between sister k‐fibers. We found that the number of PRC1‐GFP‐labeled bundles per spindle is nearly the same as the number of kinetochore pairs. Live‐cell imaging revealed that kinetochore movement in the equatorial plane of the spindle is highly correlated with the movement of the coupled PRC1‐GFP‐labeled fiber, whereas the correlation with other fibers decreases with increasing distance. Analysis of endogenous PRC1 localization confirmed the results obtained with PRC1‐GFP. PRC1 knockdown reduced the bridging fiber thickness and interkinetochore distance throughout the spindle, suggesting a function of PRC1 in bridging microtubule organization and force balance in the metaphase spindle

Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment

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 toward the center. We develop speckle microscopy in human spindles and confirm the key prediction that kinetochore fiber flux is length dependent. Kinetochores are better centered when overlaps are shorter and the kinetochore fiber flux 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 support chromosome alignment

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

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

Nuclear chromosome locations dictate segregation error frequencies

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis [1, 2, 3, 4, 5]. Selective pressures then shape distinct aneuploidy and rearrangement patterns—for example, in cancer [6, 7] —but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing [8] after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes [9] and aneuploidies in early development [10] occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development. 1. van Jaarsveld, R. H. & Kops, G. J. P. L. Difference makers: chromosomal instability versus aneuploidy in cancer. Trends Cancer 2, 561–571 (2016). 2. Compton, D. A. Mechanisms of aneuploidy. Curr. Opin. Cell Biol. 23, 109–113 (2011). 3. Zhang, C. Z. et al. Chromothripsis from DNA damage in micronuclei. Nature 522, 179–184 (2015). 4. Ly, P. et al. Chromosome segregation errors generate a diverse spectrum of simple and complex genomic rearrangements. Nat. Genet. 51, 705–715 (2019). 5. Shoshani, O. et al. Chromothripsis drives the evolution of gene amplification in cancer. Nature 591, 137–141 (2021). 6. Davoli, T. et al. Cumulative haploinsufficiency and triplosensitivity drive aneuploidy patterns and shape the cancer genome. Cell 155, 948–962 (2013). 7. Knouse, K. A., Davoli, T., Elledge, S. J. & Amon, A. Aneuploidy in cancer: seq-ing answers to old questions. Annu. Rev. Cancer Biol. 1, 335–354 (2017). 8. Bolhaqueiro, A. C. F. et al. Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids. Nat. Genet. 51, 824–834 (2019). 9. Cortés-Ciriano, I. et al. Comprehensive analysis of chromothripsis in 2, 658 human cancers using whole-genome sequencing. Nat. Genet. 52, 331–341 (2020). 10. McCoy, R. C. et al. Evidence of selection against complex mitotic-origin aneuploidy during preimplantation development. PLoS Genet. 348, 235–238 (2015)

Identification of motor proteins in the bridging fiber of the mitotic spindle

Na početku mitoze, stanica konstruira diobeno vreteno, dinamičnu strukturu odgovornu za preciznu segregaciju repliciranog genoma između stanica kćeri. Ova jedinstvena citoskeletna struktura sastavljena je od kromosoma, mikrotubula i proteina povezanih s mikrotubulima. Nedavna istraživanja su započela s otkrivanjem nove grupe mikrotubula, nazvanih mikrotubuli premosnice, koji premošćuju regiju između sestrinskih kinetohora i pritom tvore premosno vlakno, lateralno povezano sa sestrinskim k-vlaknima. U ovom radu, identificiran je položaj motornih proteina povezanih s mikrotubulima u mitotskom diobenom vretenu stanica HeLa u odnosu na pozicije premosnih vlakana. Protein MKLP1, motor kinezin-6, pronađen je kako obogaćuje centralni dio diobenog vretena na pozicijama premosnih vlakana. Prekomjerna ekspresija proteina PRC1, antiparalelnog veznika koji je potvrđen u premosnom vlaknu, pokazala je korelaciju između lokalizacije proteina MKLP1 i premosnih preklapajućih regija označenih s proteinom PRC1. Nadalje, utišavanje Kif4A, poznatog veznog partnera proteina PRC1, uzrokuje elongaciju preklapajućih regija premosnih vlakana.At the onset of mitosis, cell constructs a spindle, a dynamic structure responsible for precise segregation of replicated genome between daughter cells. This unique cytoskeletal apparatus is made of chromosomes, microtubules and microtubule-associated proteins. Recent studies have begun to uncover a new class of microtubules, termed bridging microtubules, that span the region between sister kinetochores, whilst forming the bridging fiber that laterally connects sister k-fibers. In this thesis, I have identified localization of motor microtubule-associated proteins in the mitotic spindle of HeLa cells with respect to the bridging fiber positions. MKLP1, a kinesin-6 motor, was observed to enrich the central part of the spindle at the positions of the bridging fibers. Overexpression of PRC1, an anti-parallel cross-linker, which is confirmed in the bridging fibers, shows correlation of MKLP1 localization with PRC1-labeled bridging overlaps. Additionally, silencing of Kif4A, a major binding partner of PRC1, elongates overlap regions of the bridging fibers

MEHANIZAM PORAVNANJA KROMOSOMA SILAMA KOJE OVISE O PREKLAPANJU MIKROTUBULA I NJEGOV UTJECAJ NA POGREŠKE U RAZDVAJANJU KROMOSOMA

Chromosome alignment is a hallmark of mitosis and alignment defects, which persist in anaphase, lead to direct aneuploidy. Alignment at the spindle equator depends on pulling forces exerted at kinetochore fiber tips together with polar ejection forces. However, kinetochore fibers are also subjected to forces that drive their poleward flux. Here I introduce a microtubule flux-driven centering mechanism 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. Kinetochores are better centered when overlaps are shorter and the kinetochore fiber flux is markedly slower than the bridging fiber flux. I identified Kif18A and Kif4A proteins as overlap and flux regulators and NuMA protein as a fiber coupler. Thus, I propose that length-dependent sliding forces exerted by the bridging fiber onto kinetochore fibers support chromosome alignment.Poravnanje kromosoma je jedno od obilježja mitoze i problemi u poravnavanju, koji opstaju u anafazi, vode izravnoj aneuploidiji. Poravnanje na ekvatoru vretena ovisi o silama vučenja koje djeluju na vrhove kinetohornih vlakana zajedno sa silama izbacivanja s pola. Međutim, kinetohorna vlakna također su podvrgnuta silama koje pokreću njihov tok prema polu. U ovom radu predstavljam mehanizam za centriranje vođen tokom mikrotubula koji se oslanja na tok generiran silama unutar preklopa između premošćujućih i kinetohornih vlakana. Ovaj mehanizam centriranja radi tako da duže kinetohorno vlakno teče brže od kraćeg, što pomiče kinetohore prema centru. Kinetohore su bolje centrirane kada su preklapanja kraća i tok kinetohornih vlakana izrazito sporiji od toka premošćujućih vlakana. Identificirao sam proteine Kif18A i Kif4A kao regulatore preklapanja i toka mikrotubula i protein NuMA kao spojnicu vlakana. Stoga predlažem da sile klizanja koje su ovisne o duljini, a koje premošćujuće vlakno prenosi na kinetohorna vlakna, podržavaju poravnanje kromosoma