Cell Cycle III: Entering and Exiting Mitosis (and the Events Between)

Today is the final part of the cell cycle that I will be writing, though more information about cell division will be posted eventually in the meiosis study notes.  I find that the pathways involved in mitosis are rather easy to understand, as everything fits together - it just takes some effort to realize how everything works with everything else. 

When the cell is ready to begin anaphase, the sister chromatids must be aligned at the metaphase plate, and the proper Cdk/cyclin must remain activated (Cdc2/cyclin B).  When anaphase is to begin, APC/C (anaphase promoting complex / cyclosome) activates via phosphorylation from Cdk/cyclin and association with a cofactor.  APC/C acts as a ubiquitin ligase and is involved in poly-ubiquitinating proteins to target them for degradation.  When phosphorylated APC/C binds Cdc20, it targets securin for degradation.  Securin typically holds a protein called separase.  When securin releases separase, the protein cleaves cohesion complexes and separates sister chromatids.  This prompts the cell to enter anaphase when the sister chromatids migrate away from each other.  This process is regulated by Bub and Mad2, which act to inhibit the action of APC/C^Cdc20.  When a checkpoint is activated by a sister chromatid not being bound to a kinetochore MT, for example, it actively signals to Bub, which activates Mad2 to activate Mad2*.  Mad2* is involved in inhibiting the APC/C^Cdc20 complex.  The end result is that the sister chromatids do not separate. 

During anaphase, the microtubules are undergoing a number of changes.  Initially in anaphase (Anaphase A), the kinetochore microtubules, bound to the kinetochores of the sister chromatids, begin to shorten, pulling the chromatids apart.  During anaphase B, the astral microtubules remain in place, maintaining the spindle pole in place.  The polar microtubules begin to push on each other by elongating, which pushes the spindle poles apart and promotes separation of the nuclei.  When the spindles have moved apart and the chromatids have separated, APC/C targets Ase1p for degradation, which leads to breakdown of the spindle. 

When cohesion is cleaved by separase (see above), Cdc14 phosphatase is released from the nucleolus.  This event is the signal to end mitosis by inducing expression expression of Sic1, which acts to deactivate MPF.  Cdc14 dephosphorylates and activates Cdh1, which complexes with APC/C to target MPF for degradation.

With MPF degraded proteolytically, the nuclear envelope is free to reform.  Constitutive phosphatases dephosphorylate the lamins and nuclear pore complexes.  This results in the formation of karyomeres, which are small vesicles (with nuclear pores in them) that form around the sister chromatids.  The karyomeres then fuse to reform the nuclear envelope. 

In addition to the nuclear lamins and pore complexes, myosin light chain is also dephosphorylated when MPF is degraded.  This results in the activation of the protein and the initiation of cytokinesis when the contractile ring begins to form.

A quick summary…

Phase of the Cell Cycle	Description
Interphase	The cell is preparing to duplicate; centrosomes appear outside the nucleus
Early Prophase	Spindle poles form and sister chromatids condense
Late Prophase	Sister chromatids are condensed and begin to be attached by kinetochore MTs
Metaphase	Sister chromatids align at the metaphase plate
Anaphase	The cell begins to divide the sister chromatids by releasing them to each pole
Telophase	The nuclear envelope reforms and the cells begin to structurally split

The cell cycle isn’t so scary, but it certainly is complex, and not all of the components and regulatory mechanisms have been discussed here (or discovered in the literature either!).  Nonetheless, it is crucial to understand the basics of the cell cycle because it is so intimately related to nearly all biological processes.

Cell Cycle II: Condensins, Cohesins, Microtubules, and All the Fun Stuff

I'm going to try to make this blog post a little different with a short introduction before jumping right into my writings and ramblings about the cell cycle.  This post is the second of three concerning the cell cycle, though more information will be eventually posted that is somewhat related to this topic (notably the meiosis notes).  I haven't drawn a diagram for this particular post, but there are not many mechanisms involved.  Tomorrow's post will include a cool drawing of separase, securin, and a bunch of other cool stuff.  

When I was in college and learning the cell cycle, my professors weren't interesting or very organized.  Therefore, I've tried to organize this in a way that makes sense to me.  Since this organization might not make sense to everyone else, I'll be including a list of links to review articles and other websites that concern the cell cycle when all of the sections have been posted.

Now that we’ve covered the “motor” of the cell cycle, we need to consider regulation in terms of different stages of the cell cycle and the associated checkpoints.  These checkpoints influence the activation of Cdk/cyclin and affect whether the cell arrests or progresses through the cycle.  During S phase, the cell must check that the centrioles are duplicated and MPF has built up.  Before it is able to leave S phase, it must recognize that all DNA has been replicated faithfully and that there has been no damage accumulation. 

One of the important requirements for S phase to proceed is for sister chromatids to be attached to each other.  This cohesion is facilitated by several proteins named cohesins that loop around both chromatids and “tie” them together.  Smc molecules contain a hinge and ATPase domain:  Smc3 and Smc1 together wrap around the sister chromatids like a cord.  Scc1 and Scc3 act as the latches on the ends of the Smc molecules.  In yeast, the cohesions exist on the chromosome until anaphase and the yeast chromosomes do not become highly condensed.  In mammalian cells, however, the chromosomes become condensed via the condensin complex.  Condensins consist of Smc2 and Smc4 loops along with CAP-G, -H, and –D2.  The condensin complex acts to supercoil DNA.  These sister chromatids, which are connected by the cohesin or condensin complexes are held in place by kinetochore microtubules.  These microtubules emanate from the centrosome and connect to the sister chromatids via the kinetochore.  Additionally, the cell contains astral microtubules that connect the centrosomes to the cell periphery.  Polar microtubules connect to each other near the metaphase plate and act to push on each other, thereby separating the two poles of the cell.

When the cell is in metaphase and the sister chromatids must all align on the metaphase plate, the kinetochore microtubules pull on the chromatids.  These chromatids then align by moving back and forth until they have reached a point where the tension is equal in both directions.  Chromatids that are not attached to kinetochore microtubules actively signal (beep) that the sister chromatids are not prepared for separation (in anaphase).

When the cell is ready to split, its DNA content is store in the nucleus, which is surrounded by the nuclear envelope, made of lamins.  MPF phosphorylates these lamins to disrupt the lamina.  When phosphorylated, the lamins vesiculates and dissociate.  Additionally, nuclear pore complexes dissociate as they, too, are phosphorylated.  This allows for the duplicated DNA to separate to the separate poles of the two daughter cells.

As mentioned previously, Cdc25 is the activating phosphatase for Cdk/cyclin.  During interphase, it is phosphorylated on serine 216 and bound to a 14-3-3 protein.  This protein blocks Cdc25’s NLS (nuclear localization sequence) and exposes its NES (nuclear export sequence), causing Cdc25 to be cytoplasmic.  During mitosis, the phosphorylation of Cdc25 changes and 14-3-3 no longer blocks.  This change exposes the NES, and Cdc25 is phosphorylated by Pin1 to upregulate its activity.  This drives the activation of Cdc2/cyclin B, which is active during mitosis only.