Search

Za promatranje procesa unutar žive stanice, većinom se koristi fluorescencijska mikroskopija. Glavni zahtjev fluorescencijske mikroskopije je to da željeni predmet proučavanja u stanici mora fluorescirati. Jedan od načina za promatranje stanice pod fluorescencijskim mikroskopom je da se stanični proteini obilježe fluorescentnim proteinima. Međutim, egzogeno unešene sekvence za fuzionirane proteine generiraju proteine s neprirodnom razinom ekspresije, a endogeni protein ostaje neobilježen. Endogeno obilježavanje staničnih proteina s fluorescentnim proteinima jedan je od načina za njihovu vizualizaciju bez utjecaja na jačinu ekspresije. Tehnologija CRISPR-Cas (engl. Clustered Regularly Interspaced Short Palindromic Repeats) često je korištena u genetičkom inženjerstvu za jednostavno i precizno uređivanje genoma. U ovom radu je korištena novo razvijena metoda CRISPaint (engl. CRISPR-assisted insertion tagging). To je sustav koji uz pomoć tehnologije CRISPR-Cas omogućava ugradnju gena za fluorescentni protein u genom tj. u ciljanu sekvencu gena, da bi se proizveo endogeni protein obilježeni fluorescentnim biljegom. Optimizirala sam protokol za transfekciju uz pomoć plazmida sustava CRISPaint-a i uspostavila sam dvije ljudske stanične linije, HeLa i RPE1, s endogeno obilježenim proteinom tubulinom sa zelenim fluorescentnim proteinom. Ove stanične linije se dalje mogu koristiti za promatranje stanične diobe pod fluorescencijskim mikroskopom, a metoda CRISPaint se može primijeniti za endogeno obilježavanje i drugih proteina od interesa. Fluorescence microscopy is used to observe processes inside a living cell and its main requirement is that the desired object of study in the cell must fluoresce. One way to observe a cell under a fluorescence microscope is to label cellular proteins with fluorescent proteins (FP). However, exogenously introduced sequences for fusion proteins produce proteins with unnatural expression levels while the endogenous protein remains unlabeled. Endogenous labeling of cellular proteins with FP is one way to visualize them without affecting the strength of expression. CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats) technology is today often used in genetic engineering to simply and precisely edit the genome. In this work, the newly developed method CRISPaint (CRISPR-assisted insertion tagging) was used. It is a system that, using CRISPR-Cas technology, enables the incorporation of a gene for FP into the genome in order to produce an endogenous fluorescently tagged protein. I optimized the transfection protocol using CRISPaint system plasmids and established two human cell lines, HeLa and RPE1, with endogenously labeled protein tubulin with green FP. These cell lines can be used to observe cell division under a fluorescence microscope and the CRISPaint method can be applied for endogenous labeling of other proteins.

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 reveals 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. Steric interactions between the bundle-associated chromosomes at the spindle midplane drive bundle separation and spindle widening. In agreement with experiments, theoretical modeling suggests that bundles arise through competing attractive and repulsive mechanisms. Finally, perturbation of overlap bundles leads 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.

Stanična dioba je efikasan je i pouzdan proces koji omogućuje raspodjelu genetičkog i staničnog materijala. Brojni proteini koji omogućuju i reguliraju staničnu diobu vezani su na diobeno vreteno. Jedan od njih je i protein regulator citokineze 1, PRC1. U ovom radu proučavana su biofizička svojstva PRC1 fuzioniranog na zeleni fluorescentni protein, GFP, metodom oporavka fluorescencije nakon fotoizblijeđenja (FRAP) u ljudskim stanicama. Od početka do kraja diobe afinitet vezanja PRC1-GFP na vreteno se izrazito mijenja. Prilagođavanjem kinetičkog modela na eksperimentalne podatke, pokazano je da je PRC1- GFP na početku diobe mobilan protein koji se prema kraju veže na vreteno sve jačim afinitetom. Na ovaj način dobivene su pouzdane informacije o kinetičkim svojstvima PRC1- GFP u nativnim uvjetima žive stanice. Cell division is efficient and precise process which enables the distribution of genetic and cellular material. Numerous proteins that enable and regulate cell division are associated with the microtubule structure called mitotic spindle. One of them is the protein regulator of cytokinesis 1, PRC1. Biophysical properties of PRC1 fused to green fluorescent protein, GFP, were investigated by recovery of fluorescence after photobleaching (FRAP) in human cells. From the beginning to the end of the division, the binding affinity of PRC1-GFP to the spindle changes markedly. By adjusting the kinetical model on the experimental data, it was shown that PRC1-GFP at the beginning of division is a mobile protein loosely bound to the spindle. As the division progresses, binding affinity increases. In this way, reliable information on the kinetic properties of PRC1-GFP was gained in real time in live cells.

Tijekom stanične diobe, diobeno vreteno dijeli genetski materijal u dvije identične stanice kćeri. Kinetohorni mikrotubuli u diobenom vretenu povezuju kromosome s polovima vretena i generiraju sile na kromosome putem proteinskih kompleksa zvanih kinetohore. Eksperimentalnim mjerenjima uočeni su premosni mikrotubuli koji su stabilno vezani za kinetohorne mikrotubule te su u ovom radu razvijeni fizikalni modeli koji razjašnjavaju njihovu ulogu tijekom stanične diobe. Uz kinetohorne i premosne mikrotubule i kinetohore, pri izradi modela uključena je interakcija mikrotubula s polovima diobenog vretena te pasivni i aktivni molekularni motori koji lateralno povezuju kinetohorne i premosne mikrotubule i omogućuju njihovo klizanje te aktivni motori koji reguliraju dinamiku mikrotubula. Za slučaj staničnog sustava koji se nalazi u metafazi, rješenja modela pokazuju da aktivni motori na plus krajevima mikrotubula, reguliraju prijelaz mikrotubula iz faze rasta u fazu skraćivanja te time ograničavaju kretanje kinetohora na središnju trećinu diobenog vretena. Model predviđa da stohastički član u modelu osigurava robustnost pri kretanju kinetohora za veliki raspon parametara. Također, za slučaj metafaznog diobeog vretena koje sadrži premosne mikrotubule, predviđanje je modela da će kinetohore izmaknute od središta diobenog vretena postići novi stabilni položaj koji korelira s pozicijom svežnja premosnih mikrotubula. Za slučaj sustava koji opisuje diobeno vreteno u anafazi pokazali smo da lateralno klizanje između premosnih i kinetohornih mikrotubula generirano aktivnim motorima koji se nalaze u antiparalelnim preklopima svežnjeva mikrotubula omogućuje robusnu separaciju kromosoma koja je neovisna od sila generiranih na polovu diobenog vretena. During mitosis, mitotic spindle segregates genetic material into two identical daughter cells. Kinetochore microtubules in the mitotic spindle connect the chromosomes to the spindle poles and generate forces on the chromosomes via protein complexes called kinetochores. Measurements have shown that bridging microtubules are stably bound to kinetochore microtubules and in this thesis we developed physical models that clarify their role during cell division. In addition to kinetochore microtubules, bridging microtubules and kinetochores, the model includes the interaction of microtubules with the poles of the mitotic spindle and passive and active molecular motors that laterally connect kinetochore and bridging microtubules and enable their sliding and active motors that regulate dynamics of the microtubule plus ends. For the case of a system that is in metaphase, the model solutions show that active motors at the plus ends of microtubules regulate the transition of microtubules from the growing phase to the shrinking phase and thus limit the movement of kinetochores to the central third of the mitotic spindle. The model predicts that the stochastic contribution in the model provides robustness in the motion of the kinetochore for a wide range of parameters. Also, for the case of a metaphase spindle, that contains bridging microtubules, the model predicts that the kinetochores away from the center of the spindle will achieve a new stable position that correlates with the position of the bridging microtubules overlap. For the case of a system describing the mitotic spindle in anaphase, we have shown that lateral sliding between bridging and kinetochore microtubules generated by active motors located in the antiparallel microtubule bundles allows robust chromosome separation independent of forces generated at the spindle pole.

Ekspanzijska mikroskopija je nova metoda mikroskopije u kojoj se kemijskim postupcima fizički povećava fiksirani uzorak te dozvoljava da strukture bliže od granice difrakcije svjetlosti (250 nm) postanu razlučive. To je tehnika u kojoj su fluorofori na fiksiranom uzorku vezani za polimer koji ima mogućnost izotropnog ekspandiranja te kada se polimer ekspandira, omogućava super-rezolucijsku mikroskopiju sa standardnim mikroskopom. Cilj ovog rada bio je uz pomoć razvijene metode ekspanzijske mikroskopije, proučiti arhitekturu diobenog vretena, molekularne strukture odgovorne za jednaku raspodjelu kromosoma među stanicama kćerima. Vizualizirane i analizirane su RPE1 i U2OS stanice. U ovom radu razvijen je protokol za ekspanzijsku mikroskopiju, te je dobivena slika cjelovitog diobenog vretena u ljudskim stanicama uz upotrebu ekspanzijske mikroskopije. Pokazano je da snimke ekspandiranih diobenih vretena nude mnoštvo informacija o arhitekturi vretena te je u budućnosti potrebno optimizirati protokol za bilo koji protein, koji se želi vizualizirati u diobenom vretenu. Expansion microscopy is a newly discovered microscopy technique that uses physical expansion of fixed specimens to allow features closer than the diffraction limit of light (250 nm) to become resolvable in the expanded specimen. It is a technique in which fluorophores on fixed specimens are linked to a swellable polymer that is physically expanded to enable super-resolution microscopy with ordinary microscopes. The goal of this thesis was, with the help od the developed expansion microscopy technique, to study the architecture od the mitotic spindle, a molecular machine responsible for equal distribution of chromosomes between the daughter cells. RPE1 and U2OS cells were visualized and analyzed. In this thesis the protocol for expansion micoscropy was developed and used for imaging the whole mitotic spindle in human cells. It was shown that the images of the expanded mitotic spindles offer plenty information about the architecture of the spindle and in the future it would be interesting to optimize the protocol for visualising any protein in the mitotic spindle with the expansion microscopy.

Diobeno vreteno je stanična struktura koja je odgovorna za raspodjelu genetskog materijala medu stanicama kćeri, a sastoji se od mikrotubula i pridruženih proteina. Sile i momenti sila igraju ključnu ulogu u funkcioniranju vretena te su odgovorni za pomicanje kromosoma i održavanje njegove grade, a važnu ulogu igraju elastična svojstva mikrotubula. Međutim, sile i momente nije lako odrediti današnjim eksperimentalnim tehnikama. U ovom teorijskom istraživanju uvodimo model za ravnotežu sila i momenata sila u diobenom vretenu. Korištenjem aproksimacije srednjeg polja opisujemo sve mikrotubule u diobenom vretenu, kao i sile i momente sila koje djeluju na njih. Model daje predvidanja za oblik diobenog vretena. Dobivene rezultate planiramo usporediti s eksperimentalno određenim oblicima koje su dobili naši suradnici. Teorijska predviđanja dobivena ovim modelom, zajedno s eksperimentima, trebala bi odgovoriti na pitanje kolike sile djeluju u diobenom vretenu. The mitotic spindle is a cellular structure which is responsible for the partition of genetic material when a cell divides into two daughter cells. It is comprised of microtubules and accompanying proteins. Forces and torques play a key role in the functioning of the spindle, for they are chiefly responsible for the movement of chromosomes and maintaining the overall shape of the spindle. This is facilitated by the elastic properties of microtubules. The forces and torques are, however, hard to determine with contemporary experimental techniques. In this theoretical research we develop a model for the balance of forces and torques in a mitotic spindle. Using a mean-field approximation we describe all microtubules in a mitotic spindle and the forces acting upon them. The model gives a prediction for the shape of the spindle. The results are to be matched to experimental imaging done by our associates, with the aim of answering what magnitude of forces and torques act in mitotic spindles.

Na početku mitoze mikrotubuli i pripadajući proteini formiraju diobeno vreteno, strukturu koja omogućuje točnu raspodjelu kromosoma između dvije stanice kćeri. U ovom radu razvijena je optogenetička metoda koja omogućuje brzu i reverzibilnu translokaciju proteina iz diobenog vretena na mitohondrije korištenjem plavog svjetla. Metoda je primijenjena za proučavanje proteina TACC3 i PRC1 koji sudjeluju u formiranju paralelnih, odnosno antiparalelnih svežnjeva mikrotubula. Translokacija oba proteina događala se na vremenskim skalama od nekoliko minuta. Mijenjanjem koncentracije proteina PRC1 na diobenom vretenu bilo je moguće inducirati raspadanje i ponovno formiranje antiparalelnih svežnjeva mikrotubula što je rezultiralo promjenom oblika diobenog vretena. Dakle, ova metoda predstavlja novi moćni alat za proučavanje proteina diobenog vretena kontrolom njihove lokalizacije u pojedinim fazama mitoze. At the onset of mitosis microtubules and microtubule associated proteins form a spindle, which is responsible for proper segregation of chromosomes between two daughter cells. In this thesis, I have developed optogenetic method for rapid and reversible translocation of proteins from the mitotic spindle to mitochondria using blue light. This method was used for studying TACC3 and PRC1, proteins which are required for formation of parallel and antiparallel microtubule bundles, respectively. Translocation of both proteins occurred at time scales of several minutes. By changing concentration of PRC1 in the mitotic spindle, I was able to induce disassembly and formation of antiparallel microtubule bundles, which resulted in changes in the spindle shape. Thus, this method represents powerful new tool for studying spindle proteins by controlling their localization at specific stages of mitosis.

Diobeno vreteno je stanična struktura koja se formira za vrijeme stanične diobe kako bi se genetski materijal jednoliko podijelio medu stanicama kćeri. Sile igraju ključnu ulogu u funkcioniranju vretena, sudjelujući u pomicanju njegovih dijelova i održavanju oblika. Unatoč tome, te sile nije lako odrediti korištenjem dosadašnjih eksperimentalnih metoda pa se stoga o njima jako malo zna. Kako bismo riješili ovaj problem, proučavamo oblik vretena i izvlačimo zaključke o silama na temelju poznavanja elastičnih svojstava mikrotubula. Promatranjem diobenih vretena ljudskih stanica STED superrezolucijskom mikroskopijom, vidjeli smo da svežnjevi mikrotubula nalikuju oblicima slova C i S. Uz pomoć konfokalne mikroskopije, snimana su vretena orijentirana približno okomito na ravninu snimanja te je otkriveno da posjeduju kiralnost. Pokazano je da je kiralnost lijeva sa srednjim helicitetom svežnjeva od oko -1 stupanj/μm. Kako bi se perturbirao iznos heliciteta, mijenjana je aktivnost motornog proteina kinezina 5. Razvijen je i teorijski model u kojemu je svežanj mikrotubula opisan kao tanki elastični štap. Kako bi se objasnila eksperimentalna opažanja, model predviđa postojanje momenata sila uz same sile. Zaključujemo kako uz sile, oblik diobenog vretena određuju i momenti sila generirani od strane motornih proteina. The mitotic spindle is a micromachine formed during cell division to ensure even distribution of genetic material amongst daughter cells. Forces in the spindle play a key role in its functioning, by driving movement of its constituents and maintaining its shape. However, these forces are not easily accessible by current experimental techniques and, therefore, remain to a large extent unknown. To tackle this problem, we study spindle shape and infer forces from elastic properties of microtubules. We used STED super-resolution microscopy to image human cell spindles and found that microtubule bundles resemble C and S letter shapes. By using confocal imaging of spindles oriented roughly perpendicular to the imaging plane, we show that they possess chirality. We found that chirality is left-handed with an average bundle helicity around -1 deg/μm. To perturb the twisting moment in our spindles, we altered the activity of motor protein kinesin-5 (Eg5/Kif11). A theoretical model is developed with a microtubule bundle described as an elastic slender rod, which suggests that a torque is required to generate the observed helical shapes. We conclude that torques generated by motor proteins, in addition to forces, exist in the spindle and determine its shape.

Tijekom stanične diobe, genetski materijal stanice se precizno dijeli u dvije stanice kćeri, cime mehanički i biološki upravlja mreža proteinskih filamenata koju nazivamo diobeno vreteno. Poznato je da ti proteinski filamenti, mikrotubuli, imaju elastična svojstva. Skupina mikrotubula, koju nazivamo kinetohorni mikrotubuli, djeluje silom tenzije na kromosome. Kinetohore su proteinski kompleksi koji su vezani na kromosome. Premda su sile koje djeluju u diobenom vretenu iscrpno istraživane, i dalje je nepoznato kako su one uravnotežene. Poznato je da se između sestrinskog para kinetohora nalazi još jedna skupina mikrotubula, koju mi ovdje nazivamo premosni mikrotubuli. Razvili smo model koji uključuje kinetohorne i premosne mikrotubule, te sile i momente sila koji djeluju me.u njima. Mikrotubuli su u modelu opisani kao tanki elastični štapovi koji se svijaju i uvijaju pod djelovanjem sila i momenata sila na njihovim krajevima. Iz oblika snopova mikrotubula, pomoću modela možemo odrediti kolike sile i momenti sila djeluju unutar diobenog vretena. Usporedbom sa izmjerenim oblicima predvidjeli smo da je sila na kinetohori Fk ~ 300 pN, a u polu F0 ~ 30 pN. Pokazali smo da premosni mikrotubuli omogućuju koegzistenciju sile kompresije u blizini polova te sile tenzije u blizini kinetohora, što je već niz godina otvoreno pitanje. Pokazali smo da je diobeno vreteno kiralno, te da su za kiralni oblik snopova mikrotubula odgovorni momenti sila u sustavu dva sestrinska kinetohorna vlakna i premosnog vlakna. Zaključujemo da uz sile u diobenom postoje i momenti sila, koje generiraju molekularni motori, te da oni zajedno sa silama određuju njegovu arhitekturu. At the onset of division, the cell forms a network of protein filaments, mitotic spindle, which divides genetic material between the two nascent daughter cells. It is known that protein filaments, microtubules, behave as elastic objects. Forces that govern the division are exerted by k-fibres, bundles of microtubules which end at the kinetochores, protein complexes which mechanically couple microtubules to the chromosomes. Although forces that exist in mitotic spindle have been studied thoroughly, how they are balanced still remains elusive. It is known that nonkinetochore microtubules exist between the sister pairs of kinetochores, which we term bridging microtubules. We have developed a model that includes k-fibres and bridging fibre, as well as forces and torques acting between them. We have modelled the microtubule bundles as slender elastic rods that undergo bending and torsion due to the forces and torques exerted at its ends. By using our model and known shape of microtubule bundles, we can infer the forces and torques that are present in mitotic spindle. We have compared shapes obtained from theory and experiment, and predicted that the force at kinetochore is Fk ~ 300 pN and the force at pole is F0 ~ 30 pN. By using a theoretical model that includes the bridging fibre, we explained the coexistence of the tension at kinetochores and compression at poles, which has been an open question for a few years now. In addition, we show that mitotic spindle is chiral, and that torques acting on the bridging and k- fibres spindle are responsible for chiral shapes of microtubule bundles. We conclude that, in addition to forces, torques that are generated by the motor proteins exist in the spindle and determine its architecture.

The main task of the mitotic spindle is to generate forces that position the chromosomes at the metaphase plate and subsequently pull them apart toward the opposite spindle poles. These forces in living cells are, unfortunately, not easily accessible by current experimental techniques. However, much about the forces can be inferred from the shape of the spindle because the shape is an outcome of forces. K-fibers, which are bundles of microtubules ending at the kinetochore, are typically curved, suggesting that they are under compression. By severing a k-fiber with a laser we have shown that the bridging fiber moves together with sister k-fibers, revealing that these 3 fibers are strongly linked and thus able to survive large physical perturbations such as cutting of k-fibers. Severing of a k-fiber at different locations also revealed that the bridging fiber is linked laterally to the k-fibers in the region away from the kinetochore, while these fibers separate from each other close to the kinetochore.

Na početku diobe, stanica formira diobeno vreteno, molekularnu strukturu odgovornu za jednaku raspodjelu kromosoma među stanicama kćerima. To je vrlo kompleksna struktura koju čine mikrotubuli i pripadajući proteini. Tijekom prometafaze, kinetohorni mikrotubuli rastu iz polova diobenog vretena prema drugom kraju stanice i povezuju se s kinetohorama, multi-proteinskim strukturama na centromerama kromosoma. U ovom radu, fluorescencijska mikroskopija je korištena za vizualizaciju ljudskih stanica. Vizualizirane su i analizirane U2OS (CENP-A-GFP, mCherry--alpha-tubulin) stanice. Pokazano je da se duljina diobenog vretena smanjuje, a širina povećava tijekom prometafaze. Unatoč promjenama duljine i širine diobenog vretena, kontura ove stanične strukture ostaje nepromijenjena. At the onset of division, the cell forms a spindle, a molecular machine responsible for equal distribution of chromosomes between the daughter cells. It is a highly complex structure made of microtubules and associated proteins. During prometaphase, kinetochore microtubules extend from poles on either end of the cell and attach to the kinetochores, multiprotein structures on the centromeres of the chromosomes. In this thesis, fluorescence microscopy was used for live-cell imaging of human cells. U2OS (CENP-A-GFP, mCherry-- alpha tubulin) cells were visualized and analyzed. It was shown that the spindle length decreases and the width increases during the prometaphase. Despite the changes in spindle length and width, the contour of this cell structure remains unchanged.

Tijekom stanične diobe, sestrinske kromatide razdvajaju se formiranjem metafaznog vretena, bipolarne molekularne makrostrukture samosastavljene od mikrotubula i pridruženih proteina. Trenutačno su u modelima metafaznog vretena opisane tri vrste mikrotubula: kinetohorni, interpolarni i astralni. U ovom radu analizirana su antiparalelna nekinetohorna vlakna mikrotubula koja se nalaze odmah ispod sestrinskih kinetohornih parova, a nazvana su premošćujuća vlakna. Primjenom fluorescentne mikroskopije živih stanica, pokazao sam da je ova struktura prisutna u različito obilježenim HeLa i Ptk1 stanicama, i to s različitim brojem mikrotubula u svakoj liniji. Također, analiziran je odgovor vanjskoga kinetohornog vlakna na lasersku ablaciju u staničnoj liniji Ptk1. Uočio sam zajedničku putanju kinetohornih vlakana, kinetohora i premoščujućih vlakana prema van što ukazuje na lateralnu povezanost između kinetohornih i premošćujućih vlakana. Također, pokazao sam da se udaljenost između kinetohora smanjuje nakon ablacije i da razina smanjivanja obrnuto korelira s udaljenošću mjesta ablacije od kinetohore. Dodatno, pokazao sam da se vanjski element vretena izravnava nakon ablacije, i to najbrže i najviše u liniji s najdebljim premošćujućim vlaknom, što ukazuje da su ta dva parametra korelirana. Konačno, usporedio sam neke od rezultata s predikcijama teorijskog modela, kako bih pokazao njegovu robusnost koristeći neke eksperimentalne podatke kao ulazne parametre. Dobiveni rezultati govore da su premošćujuća vlakna važna strukturalna komponenta koja ima ulogu u ravnoteži sila kompresije i tenzije u metafaznom vretenu. During cell division, sister chromatids are segregated by mitotic spindle, a bipolar self-assembly of microtubules and associated proteins. Current models of mitotic spindle recognize three distinct subpopulations of microtubules: kinetochore, interpolar and astral microtubules. The role of antiparallel non-kinetochore microtubule bundles, termed bridging fibers, positioned under sister kinetochores in HeLa and Ptk1 cell lines, was analysed in this study. Using live-fluorescent imaging analysis it was shown that these bundles are present in differently labelled HeLa and Ptk1 cell lines with distinct thicknesses. The response of outermost spindle element to laser ablation in Ptk1 cell line was also analysed. Joint outward movement of bridging and k-fibers was observed suggesting connection into single mechanical unit. Decrease in inter-kinetochore distance was also observed after ablation. That decrease is inversely correlated with distance of the cut from the kinetochore. In addition, spindle element straightening after ablation was observed in all cell lines but with strongest straightening in HeLa cell line with thickest bridging fiber, demonstrating that response to ablation and thickness of bridging fiber are correlated. Finally, some of obtained results were compared with theoretical model of the HeLa metaphase spindle confirming robustness of the model by using some of our experimental data as inputs. In conclusion, obtained results demonstrate that bridging fiber is important structural component of metaphase spindle involved in balancing compressive and tensile forces in spindle.