Microtubule Sliding within the Bridging Fiber Pushes Kinetochore Fibers Apart to Segregate Chromosomes

During cell division, mitotic spindle microtubules segregate chromosomes by exerting forces on kinetochores. What forces drive chromosome segregation in anaphase remains a central question. The current model for anaphase in human cells includes shortening of kinetochore fibers and separation of spindle poles. Both processes require kinetochores to be linked with the poles. Here we show, by combining laser ablation, photoactivation, and theoretical modeling, that kinetochores can separate without any attachment to one spindle pole. This separation requires the bridging fiber, a microtubule bundle that connects sister kinetochore fibers. Bridging fiber microtubules in intact spindles slide apart with kinetochore fibers, indicating strong crosslinks between them. We conclude that sliding of microtubules within the bridging fibers drives pole separation and pushes kinetochore fibers poleward by the friction of passive crosslinks between these fibers. Thus, sliding within the bridging fiber works together with the shortening of kinetochore fibers to segregate chromosomes

Uloga premošćujućih mikrotubula u anafazi stanične diobe

During cell division, microtubules of the mitotic spindle segregate chromosomes by exerting forces on kinetochores, protein complexes on the chromosomes. The central question is which forces drive chromosome segregation. The current model for anaphase in human cells includes shortening of kinetochore fibers and separation of spindle poles, processes that require kinetochore-pole connections. By combining laser ablation, photoactivation and protein depletion, we show that kinetochores can separate without the attachment to the pole. This separation requires the bridging fiber, which connects sister k-fibers whereby astral microtubules do not contribute to the separation. Bridging microtubules in intact spindles slide apart together with k-fibers, indicating strong crosslinks between them. Kinetochore segregation and pole separation is slower after depletion of MKLP1/KIF23 (kinesin-6), faster after depletion of KIF4a (kinesin-4), and unaffected by reduction of Eg5/KIF11 (kinesin-5) or KIF15/Hklp2 (kinesin-12). We assume that motor-generated sliding in the bridging fibers drives pole separation and pushes k-fibers poleward, thereby working together with k-fiber shortening to segregate chromosomes.Tijekom stanične diobe, mikrotubuli u diobenom vretenu razdvajaju kromosome stvarajući sile na kinetohore, proteinske komplekse na kromosomima. Unatoč mnogim istraživanjima u ovom području, ostalo je upitno koje sile upravljaju segregacijom kromosoma. Trenutno prihvaćen model za anafazu u ljudskim stanicama obuhvaća skraćivanje kinetohornih mikrotubula i razdvajanje polova diobenog vretena, procese koji zahtijevaju vezu između kinetohore i pola. Primjenom laserske ablacije, fotoaktivacije i deplecije proteina, u ovom radu je pokazano da se kinetohore mogu razdvajati i kada nema veze sa polom diobenog vretena. Takvo razdvajanje zahtjeva antiparalelna premošćujuća vlakna, koja povezuju sestrinska kinetohorna vlakna i kližu jedan preko drugog, dok astralni mikrotubuli ne doprinose navedenom procesu razdvajanja. Također, premošćujuća vlakna u netaknutom diobenom vretenu kližu zajedno sa kinetohornim vlaknima, a to upućuje na snažnu povezanost između te dvije populacije vlakana. Brzine razdvajanje kinetohora i polova su manje nakon utišavanja MKLP1/KIF23 (kinezin-6), više nakon utišavanja KIF4a (kinezin-4) i nepromijenjene nakon inhibicije/utišavanja Eg5/KIF11 (kinezin-5) i/ili KIF15/Hklp2 (kinezin-12) proteina. Zaključak ovog rada je da premošćujući mikrotubuli kližu, vjerojatno uz pomoć motornih proteina, odgurujući tako kinetohorna vlakna sa kinetohorama, kao i polove, jedne od drugih. Takav mehanizam djeluje skupa sa skraćivanjem kinetohornih vlakana i posljedica njihovog zajedničkog djelovanja je razdvajanje kromosoma u ljudskim stanicama

Uloga premošćujućih mikrotubula u anafazi stanične diobe

During cell division, microtubules of the mitotic spindle segregate chromosomes by exerting forces on kinetochores, protein complexes on the chromosomes. The central question is which forces drive chromosome segregation. The current model for anaphase in human cells includes shortening of kinetochore fibers and separation of spindle poles, processes that require kinetochore-pole connections. By combining laser ablation, photoactivation and protein depletion, we show that kinetochores can separate without the attachment to the pole. This separation requires the bridging fiber, which connects sister k-fibers whereby astral microtubules do not contribute to the separation. Bridging microtubules in intact spindles slide apart together with k-fibers, indicating strong crosslinks between them. Kinetochore segregation and pole separation is slower after depletion of MKLP1/KIF23 (kinesin-6), faster after depletion of KIF4a (kinesin-4), and unaffected by reduction of Eg5/KIF11 (kinesin-5) or KIF15/Hklp2 (kinesin-12). We assume that motor-generated sliding in the bridging fibers drives pole separation and pushes k-fibers poleward, thereby working together with k-fiber shortening to segregate chromosomes.Tijekom stanične diobe, mikrotubuli u diobenom vretenu razdvajaju kromosome stvarajući sile na kinetohore, proteinske komplekse na kromosomima. Unatoč mnogim istraživanjima u ovom području, ostalo je upitno koje sile upravljaju segregacijom kromosoma. Trenutno prihvaćen model za anafazu u ljudskim stanicama obuhvaća skraćivanje kinetohornih mikrotubula i razdvajanje polova diobenog vretena, procese koji zahtijevaju vezu između kinetohore i pola. Primjenom laserske ablacije, fotoaktivacije i deplecije proteina, u ovom radu je pokazano da se kinetohore mogu razdvajati i kada nema veze sa polom diobenog vretena. Takvo razdvajanje zahtjeva antiparalelna premošćujuća vlakna, koja povezuju sestrinska kinetohorna vlakna i kližu jedan preko drugog, dok astralni mikrotubuli ne doprinose navedenom procesu razdvajanja. Također, premošćujuća vlakna u netaknutom diobenom vretenu kližu zajedno sa kinetohornim vlaknima, a to upućuje na snažnu povezanost između te dvije populacije vlakana. Brzine razdvajanje kinetohora i polova su manje nakon utišavanja MKLP1/KIF23 (kinezin-6), više nakon utišavanja KIF4a (kinezin-4) i nepromijenjene nakon inhibicije/utišavanja Eg5/KIF11 (kinezin-5) i/ili KIF15/Hklp2 (kinezin-12) proteina. Zaključak ovog rada je da premošćujući mikrotubuli kližu, vjerojatno uz pomoć motornih proteina, odgurujući tako kinetohorna vlakna sa kinetohorama, kao i polove, jedne od drugih. Takav mehanizam djeluje skupa sa skraćivanjem kinetohornih vlakana i posljedica njihovog zajedničkog djelovanja je razdvajanje kromosoma u ljudskim stanicama

Construction of mutant SLX4 yeast Saccharomyces cerevisiae

Palindromske sekvencije prisutne su u genetičkom materijalu svih živih organizama te su vrlo često uključene u regulaciju fundamentalnih staničnih procesa. Međutim, dulji palindromi mogu u DNA formirati rekombinogene sekundarne strukture koje mogu uzrokovati pucanje kromosoma, genetičke prerasporede i genetičke bolesti kod ljudi. Dosadašnji rezultati sugeriraju da bi za nastanak loma u palindromu u uvjetima in vivo mogao biti odgovoran gen SLX4. Upravo zbog toga je cilj ovoga rada bio provesti inaktivaciju navedenog gena u kvaščevom soju BYUHA-8 koji se otprije koristi za istraživanje rekombinogenosti palindroma. U tu je svrhu najprije lančanom reakcijom polimeraze sintetiziran linearni fragment DNA za inaktivaciju gena SLX4 i tim je fragmentom zatim transformiran kvaščev soj BYUHA-8. Molekularna analiza dobivenih transformanata pokazala je da je u svim transformantima došlo do ilegitimne integracije transformirajuće DNA, a uzrok tome bi mogla biti nedovoljna duljina homologija na krajevima korištenog linearnog fragmenta koja je u ovom radu iznosila 45 parova baza.Palindromic sequences are present in the genetic material of all living organisms and are often involved in the regulation of fundamental cellular processes. However, longer palindromes can form recombinogenic secondary structures that can induce chromosomal breaks, genome rearrangements and cause genetic diseases in humans. Previous results suggest that the SLX4 gene product could be responsible for the introduction of breaks in palindromic sequences in vivo. Therefore, the goal of this paper was to inactivate the SLX4 gene in the yeast strain BYUHA-8, which is used to study palindrome recombinogenicity. To achieve this goal, we synthesized the SLX4 disruption cassette by PCR, and used it to transform yeast strain BYUHA-8. Molecular analysis of the transformants demonstrated that in all cases the disruption cassette integrated at random sites in the genome, which could be due to insufficent length of homology on the ends of the disruption cassette used for transformation

Stress response of E. coli to the in vivo hypoosmotic shock

U ovom istraživanju proučavan je odgovor bakterije E. coli na stres izazvan hipoosmotskim šokom in vivo. E. coli je uzgajana u medijima visoke osmolarnosti (125 – 650 mM NaCl), a potom naglo prebačena u medij niske osmolarnosti (0 mM NaCl). U tu svrhu korišteni si različiti sojevi: divlji tip, dvostruki mutant (ΔmscSΔmscL) te ΔaqpZ mutant. Na osnovi rezultata fluorescentne mikroskopije i analize podataka vidljivo je da se stanicama divljeg tipa i mutanata povećava volumen proporcionalno intenzitetu šoka za 7 – 18 %. Divlji tip se uspijeva oporaviti nakon svakog hipoosmotskog šoka te nastaviti s rastom. Međutim, kod dvostrukog mutanta E. coli vidljiv je prestanak oporavka kod 450 mM NaCl kao i povećan broj odumrlih stanica kod 650 mM NaCl. Nadalje, opaženo je da se sve stanice nakon povećanja volumena, osim divljeg tipa kod hipoosmotskog šoka od 650 mM NaCl, vraćaju na početni volumen nakon određenog vremena. Hipoosmotski šok od 650 mM NaCl kod divljeg tipa izaziva prekomjerni gubitak vode (ispod početne vrijednosti), a primjenom šoka iste takve magnitude na ΔaqpZ mutanta utvrđeno je da protein Aquaporin Z nema ulogu u prekomjernom gubitku vode. Također, takve stanice postepeno regeneriraju svoj volumen uzimanjem kalija iz okoline.In this research, the E. coli stress response on the in vivo hypoosmotic shock has been studied. E. coli was grown in a high osmolarity medium (125 mM – 650 mM sodium chloride) and then rapidly transferred into the low osmolarity medium (0 mM sodium chloride). For this purpose different strains were used: wild type, double mutant (ΔmscSΔmscL) and the ΔaqpZ mutant. Based on the single cell results of the fluorescence microscopy and the data analysis, it has been observed that wild type and the mutant cells volume was increased for 7 – 18%, proportionally to the shock magnitude. The wild type was able to recover and continue to grow after each hypoosmotic shock. However, for the E. coli double mutant the termination of the recovery was observed at 450 mM NaCl as well as the increased number of the dead cells at 650 mM NaCl. Furthermore, it was observed that all of the cells after the volume increase, except the wild type at 650 mM NaCl, were recovering their initial volume after a specific time. The hypoosmotic shock from 650 mM NaCl causes the excessive water loss in the wild type (below the initial value). By applying the shock of such magnitude on the ΔaqpZ mutant , it was established that the protein Aquaporin Z doesn’t partake in the excessive water loss. Likewise, such cells recover their volume gradually by potassium uptake

Uloga premošćujućih mikrotubula u anafazi stanične diobe

During cell division, microtubules of the mitotic spindle segregate chromosomes by exerting forces on kinetochores, protein complexes on the chromosomes. The central question is which forces drive chromosome segregation. The current model for anaphase in human cells includes shortening of kinetochore fibers and separation of spindle poles, processes that require kinetochore-pole connections. By combining laser ablation, photoactivation and protein depletion, we show that kinetochores can separate without the attachment to the pole. This separation requires the bridging fiber, which connects sister k-fibers whereby astral microtubules do not contribute to the separation. Bridging microtubules in intact spindles slide apart together with k-fibers, indicating strong crosslinks between them. Kinetochore segregation and pole separation is slower after depletion of MKLP1/KIF23 (kinesin-6), faster after depletion of KIF4a (kinesin-4), and unaffected by reduction of Eg5/KIF11 (kinesin-5) or KIF15/Hklp2 (kinesin-12). We assume that motor-generated sliding in the bridging fibers drives pole separation and pushes k-fibers poleward, thereby working together with k-fiber shortening to segregate chromosomes.Tijekom stanične diobe, mikrotubuli u diobenom vretenu razdvajaju kromosome stvarajući sile na kinetohore, proteinske komplekse na kromosomima. Unatoč mnogim istraživanjima u ovom području, ostalo je upitno koje sile upravljaju segregacijom kromosoma. Trenutno prihvaćen model za anafazu u ljudskim stanicama obuhvaća skraćivanje kinetohornih mikrotubula i razdvajanje polova diobenog vretena, procese koji zahtijevaju vezu između kinetohore i pola. Primjenom laserske ablacije, fotoaktivacije i deplecije proteina, u ovom radu je pokazano da se kinetohore mogu razdvajati i kada nema veze sa polom diobenog vretena. Takvo razdvajanje zahtjeva antiparalelna premošćujuća vlakna, koja povezuju sestrinska kinetohorna vlakna i kližu jedan preko drugog, dok astralni mikrotubuli ne doprinose navedenom procesu razdvajanja. Također, premošćujuća vlakna u netaknutom diobenom vretenu kližu zajedno sa kinetohornim vlaknima, a to upućuje na snažnu povezanost između te dvije populacije vlakana. Brzine razdvajanje kinetohora i polova su manje nakon utišavanja MKLP1/KIF23 (kinezin-6), više nakon utišavanja KIF4a (kinezin-4) i nepromijenjene nakon inhibicije/utišavanja Eg5/KIF11 (kinezin-5) i/ili KIF15/Hklp2 (kinezin-12) proteina. Zaključak ovog rada je da premošćujući mikrotubuli kližu, vjerojatno uz pomoć motornih proteina, odgurujući tako kinetohorna vlakna sa kinetohorama, kao i polove, jedne od drugih. Takav mehanizam djeluje skupa sa skraćivanjem kinetohornih vlakana i posljedica njihovog zajedničkog djelovanja je razdvajanje kromosoma u ljudskim stanicama

Stress response of E. coli to the in vivo hypoosmotic shock

U ovom istraživanju proučavan je odgovor bakterije E. coli na stres izazvan hipoosmotskim šokom in vivo. E. coli je uzgajana u medijima visoke osmolarnosti (125 – 650 mM NaCl), a potom naglo prebačena u medij niske osmolarnosti (0 mM NaCl). U tu svrhu korišteni si različiti sojevi: divlji tip, dvostruki mutant (ΔmscSΔmscL) te ΔaqpZ mutant. Na osnovi rezultata fluorescentne mikroskopije i analize podataka vidljivo je da se stanicama divljeg tipa i mutanata povećava volumen proporcionalno intenzitetu šoka za 7 – 18 %. Divlji tip se uspijeva oporaviti nakon svakog hipoosmotskog šoka te nastaviti s rastom. Međutim, kod dvostrukog mutanta E. coli vidljiv je prestanak oporavka kod 450 mM NaCl kao i povećan broj odumrlih stanica kod 650 mM NaCl. Nadalje, opaženo je da se sve stanice nakon povećanja volumena, osim divljeg tipa kod hipoosmotskog šoka od 650 mM NaCl, vraćaju na početni volumen nakon određenog vremena. Hipoosmotski šok od 650 mM NaCl kod divljeg tipa izaziva prekomjerni gubitak vode (ispod početne vrijednosti), a primjenom šoka iste takve magnitude na ΔaqpZ mutanta utvrđeno je da protein Aquaporin Z nema ulogu u prekomjernom gubitku vode. Također, takve stanice postepeno regeneriraju svoj volumen uzimanjem kalija iz okoline.In this research, the E. coli stress response on the in vivo hypoosmotic shock has been studied. E. coli was grown in a high osmolarity medium (125 mM – 650 mM sodium chloride) and then rapidly transferred into the low osmolarity medium (0 mM sodium chloride). For this purpose different strains were used: wild type, double mutant (ΔmscSΔmscL) and the ΔaqpZ mutant. Based on the single cell results of the fluorescence microscopy and the data analysis, it has been observed that wild type and the mutant cells volume was increased for 7 – 18%, proportionally to the shock magnitude. The wild type was able to recover and continue to grow after each hypoosmotic shock. However, for the E. coli double mutant the termination of the recovery was observed at 450 mM NaCl as well as the increased number of the dead cells at 650 mM NaCl. Furthermore, it was observed that all of the cells after the volume increase, except the wild type at 650 mM NaCl, were recovering their initial volume after a specific time. The hypoosmotic shock from 650 mM NaCl causes the excessive water loss in the wild type (below the initial value). By applying the shock of such magnitude on the ΔaqpZ mutant , it was established that the protein Aquaporin Z doesn’t partake in the excessive water loss. Likewise, such cells recover their volume gradually by potassium uptake

Construction of mutant SLX4 yeast Saccharomyces cerevisiae

Palindromske sekvencije prisutne su u genetičkom materijalu svih živih organizama te su vrlo često uključene u regulaciju fundamentalnih staničnih procesa. Međutim, dulji palindromi mogu u DNA formirati rekombinogene sekundarne strukture koje mogu uzrokovati pucanje kromosoma, genetičke prerasporede i genetičke bolesti kod ljudi. Dosadašnji rezultati sugeriraju da bi za nastanak loma u palindromu u uvjetima in vivo mogao biti odgovoran gen SLX4. Upravo zbog toga je cilj ovoga rada bio provesti inaktivaciju navedenog gena u kvaščevom soju BYUHA-8 koji se otprije koristi za istraživanje rekombinogenosti palindroma. U tu je svrhu najprije lančanom reakcijom polimeraze sintetiziran linearni fragment DNA za inaktivaciju gena SLX4 i tim je fragmentom zatim transformiran kvaščev soj BYUHA-8. Molekularna analiza dobivenih transformanata pokazala je da je u svim transformantima došlo do ilegitimne integracije transformirajuće DNA, a uzrok tome bi mogla biti nedovoljna duljina homologija na krajevima korištenog linearnog fragmenta koja je u ovom radu iznosila 45 parova baza.Palindromic sequences are present in the genetic material of all living organisms and are often involved in the regulation of fundamental cellular processes. However, longer palindromes can form recombinogenic secondary structures that can induce chromosomal breaks, genome rearrangements and cause genetic diseases in humans. Previous results suggest that the SLX4 gene product could be responsible for the introduction of breaks in palindromic sequences in vivo. Therefore, the goal of this paper was to inactivate the SLX4 gene in the yeast strain BYUHA-8, which is used to study palindrome recombinogenicity. To achieve this goal, we synthesized the SLX4 disruption cassette by PCR, and used it to transform yeast strain BYUHA-8. Molecular analysis of the transformants demonstrated that in all cases the disruption cassette integrated at random sites in the genome, which could be due to insufficent length of homology on the ends of the disruption cassette used for transformation