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Stanica tijekom mitoze stvara diobeno vreteno: dinamičnu i kompleksnu strukturu sastavljenu od mikrotubula, motornih i nemotornih proteina. Nemotorni protein NuMA (eng. nuclear and mitotic apparatus) tijekom mitoze je uglavnom prisutan na polovima vretena gdje sudjeluje u njihovom održavanju i u manjoj mjeri na staničnom korteksu u kompleksu s proteinom LGN gdje sudjeluje u pozicioniranju diobenog vretena unutar stanice. U ovome radu promatrana je njegova uloga u mitozi kada je utišana ekspresija proteina LGN pomoću siRNA u stanicama linije HeLa. Prilikom utišavanja došlo je do promjene arhitekture diobenog vretena i smanjenja heliciteta njegovih mikrotubula, međutim, lokalizacija proteina NuMA i polovi diobenog vretena su ostali očuvani. Također, konstruiran je optogenetički sustav za protein NuMA koji, iako se pokazao funkcionalnim, nije bio dovoljno efikasan da bi se iskoristio za detaljno određivanje njegove funkcije u metafazi mitoze. Ipak, moguće je da bi ovaj sustav bio iznimno moćan alat u drugoj staničnoj liniji ili drugoj fazi mitoze. Na temelju dobivenih rezultata može se zaključiti da protein LGN najvjerojatnije ima dodatnu funkciju tijekom mitoze vezanu uz diobeno vreteno koja vjerojatno ne ovisi o njegovoj interakciji s proteinom NuMA. During mitosis, the cells create a dynamic and complex structure called mitotic spindle made of microtubules, motor and crosslinking proteins. NuMA (Nuclear and Mitotic Apparatus) is a crosslinking protein which is, during mitosis, mostly present on spindle poles maintaining them and in a smaller amount on the cell cortex in complex with LGN protein which is responsible for spindle orientation. In this thesis the role of NuMA in mitosis was investigated in cells in which LGN protein expression had been silenced by siRNA in HeLa cells. The result of silencing was change in spindle architecture and decrease in the helicity of microtubule bundles, but NuMA localization and spindle poles were preserved. Furthermore, an optogenetic system for NuMA was constructed. It was shown to be functional, but not efficient enough to investigate NuMA’s function in the metaphase of mitosis. Nevertheless, the constructed system could be a very powerful tool in a different cell line or in another phase of mitosis. It could be concluded that LGN protein has, beside NuMA regulation, an additional function in mitosis that is connected to the spindle and probably does not depend on interaction with NuMA. |
