MITOSIS

This is the process from which one cell divides in two daughter cells, genome information is duplicated and segregated. There are different stages in mitosis: - chromosome condensation in profase, nuclear envelope breakdown which brings you into pro-metaphase at which point the chromosomes are starting to align on the mitotic spindle that is alreading forming on the outside of the nucleous. - In metaphase there is an align state; once the cell has conformed that indeed al the chromosomes are properly attached to microtubules emanating from the two spindle poles, anaphase is on set, chromosomes segregate. - In telophase the chromosomes start to decondense beginning to assemble the cleavage fur that will physically separate the two cells. in 1980s people did know that mitosis was an important process and noticed that in cancer cells this was going wrong, with mitotic mitotic figures. Theodor Boveri came up with what is now know as the aneuploidy hypothesis  the origin of cancer is one cell with an incorrectly combined karyotype and this aneuploid state is the source of this abnormal growth tendency that constitutes cancer.  Anatomy of the mitotic spindle - Microtubules base spindle that has at its poles the replicated centrosomes; you have condensed chromosomes and site of attachment of those chromosomes to the spindle in the form of kinetochores. Mitotic chromosome segregation is a very complicated process; yet there are three main elements: condensed chromosomes, the centrosomes and kinetochores; they can give an overview of mitosis. o Chromosome condensation This involves taking the decontained interphase dna and further compacting it 20x fold and then also resolving those individual chromosomes into individual units that can then be align on the metaphase plate. The proteins that are essential for chromosome organization protein interphase and in mitosis are those stabilio maintence of chromosomes SMC proteins  the cohesins and the condensisns.  Cohesin SMC proteins are involved in loop extrusion, and thereby in the organization of the interphase chromatine. Through this process you are developing the topologically associated domain TADs, seen in the map.  Condensin Depetion of cohesion release factor called wapl would induce a hyper compaction of the interphase dna the made a nucleus turn into something called Vermicello.  depletion of cohesion release factor results in DNA hyper-compaction in interphase (vermicelli chromosomes). What condensin does is very similar, forms dna loops in an atp dependent manner. This was shown using fna curtain assay (you attach dna, add recombinant complex, add atp, apply flow and you can see dna looping occurring in vitro). From experiments like this was concluded that condensing does indeed loop dna on one side only off this ring around the dna. There is condensin I and II that share the same SMC subunits. Condensin 2 is involved in chromosome compaction and prophase, and Condensin 1 is involved on further looping on top of the loops that you got with the condensing 2. These condensins will inset in the chromosome axis and this is why in the early experiments it was seen that chromosomes scuffles of proteins that seemed to remain in the shape of a x shaped mitotic chromosome and those proteins were those condensins as well as one other proteins which is topoisomerase II. Topoisomerase II decatenate dna, it set off one dsDNA through the other. As dna is replicating in s phase, you’re entangling this two stands but once you try to segregate them in mitosis you need to be ab le to have this nice them apart with a nice X shape where only at the centromere the sister chromatids are held together.  Keeping mitotic chromosomes apart – the role of Ki-67 After we nicely condenced chromosomes, we need to make sure they stay apart. One protein involved in this is Ki-67 that sits in the nucleolus in the interphase and then re-localizes to the chromosome periphery. If this protein is depleted the consequences are dramatic: - In the image is found on the left the wt and on the right the protein repleted rnai; you have the condensed chromosomes in red and the nuclear envelope marker. It can be seen that dna will have a hard time being properly segregated in anaphase. The model that is proposed is that Ki67 coats the mitotic chromosomes and creats a charged enviroments that will repel other chromosomes.  A role for histone modifications in mitotic chromosome condensation An other thing that is important for chromosome condensation is histone modification, in particular de-acetylation. It was known that interphase chromosomes are hyper-acetylated and as you go into mitosis this acetylation is lost. If you perturb this mitotic de-acetylation using TSA, something goes wrong with chromosomes condensation, everything looked a little bit fuzzy. The consequencies don’t seem very dramatinc because you will have the condensins; but what’s really quite striking is what happens if you get rid of both condensins and de-acetilation  no real process of condensing mitosis is completed. This tells us that we need both histone de-acetilation and the action of condensing to condense mitotic chromosome.  What is de-acetylation good for? This is where liquid-liquid phase separation comes into play; it was found that if you take nucleosomes you can, under physiologically salt conditions, see liquid-liquid phase separation behavior in vitro. This behavior was actually dependent on histone-tail; if removed (histone tails) you are left with no condensation. The same could be seen if you work to acetylate these histones. So, this is important for chromosome compaction, but also or establishing a different kind of environment around the condensed chromosomes. This is extremely important because in mitosis there are spindle microtubules that are supposed to be interacting with the kinetochores, but they’re basically coming at the chromosomes from all angles. You want to avoid that you damage this fragile dna as the microtubule hits them. It was found that if you prevent de-acetilation, tubuline permeates the chromatine.  in the presence of the de-acetylation inhibitor TSA tubuline is all over the place, even where the dna is.  HISTONE DE-ACETYLATION IS CRITICAL TO PREVENT MITOTIC CHROMATIN FROM BEING PERFORATED BY SPINDLE MICROTIBULES In normal conditions microtubules get close to chromatin but then stops, yet this does not happens if de-acetylation is inhibit, and they would go through physically damaging the dna. In this case this phase separation behavior is important to exclude tubuline, which means that as a microtubuline polymerase is towards the chromatine, it runs out of tubulines subunits for further incorporation and that holds further growth.