Sunday, July 3, 2016

SPOC vs MEN: Mechanics of Cell Division

Some of the self-regulating mechanisms underlying the cell division process, including the spindle position checkpoint.

Cell division looks elegantly choreographed, and indeed, "alive". Yet we know in principle that it is also a mechanical contrivance, composed entirely of chemical reactions that through their slowly evolved complexity have achieved a highly reliable, self-checking mechanism of DNA and cytoplasmic segregation. Figuring out just what that mechanism is continues to fascinate many researchers.

Yeast cell spindle, combined fluorescence and DIC image. Microtubules (alpha tubulin) are green, pushing the respective DNA/nuclei to opposite ends of the incipient mother (large) and daughter (small) cells. Gamma tubulin, which is a special component of the core of the spindle pole body, is red, and the DNA is blue.
A couple of recent papers studied one of these homeostatic mechanisms- also called checkpoints- by which each side of the mitotic spindle knows that it has gotten to the right place in the cell, and can initiate disassembly. The spindle is the complex of microtubules in all eukaryotic cells that is nucleated from the centrioles/basal bodies/astral center/MTOC/spindle pole body and extends to the individual chromosomes, holding them in an organized array (the metaphase plate) before pulling each divided half apart into the two nascent cells, at which point it disassembles again, allowing nuclei to re-form around the DNA of each new cell. (Though unlike in other eukaryotes, yeast nuclei never break down, but are divided and dragged to their destination intact.) Several points of this process have checkpoints to prevent further steps from taking place before the current one is complete. No mitosis can be going on, and the cell must have reached some new threshold of size, before a new round of DNA synthesis can be kicked off for a new cell division. All the DNA has to be replicated before microtubules can engage at the centromeres. All the chromosomes have to be captured before separation between the homologs can be initiated. And so forth.

The particular checkpoint dealt with here is called the SPOC, or spindle position checkpoint. Each side of the spindle, centered at its respective spindle pole body, needs to know somehow that it is at a site within the new cell, rather than just floating around in the old cell. In yeast, where this work was done, the new cell starts off as a little bud that fills with cytoplasm for a while before the DNA replication and segregation process happens. So there is a pre-prepared site for the new cell spindle pole body to go, and that site is marked by a special molecule, called Lte1.

How one end of the spindle knows to get into the bud, and the other end to remain in the mother, is a different story we won't get into here. At any rate, as long as neither end has made it into the daughter bud, a complex of molecules enforce the SPOC. How this works is that protein kinase Kin4 is also asymmetric, located in the mother cell, and inhibits a key function at the centriole. The protein Spc72 is a dock for the core tubulin (gamma) at the centriole, which in turn attracts the major alpha tubulin. Spc72 also is the docking point for Kin4, allowing it to encourage (by preventing their inactivation by CDC5, one of the classic cell division cycle kinases) the activity of Bfa1/Bub2, two proteins that in combination are key inhibitors of Tem1, a GTPase that begins the molecular cascade of the mitotic escape network, or MEN.

Model of SPOC to MEN transition, where the spindle pole (gold) that gets into the daughter bud (green) triggers / undergoes the molecular steps that license entry into anaphase, or exit from mitosis. 

But as soon as one of the spindle ends has made it into the daugher bud, it escapes the influence of Kin4, and enters the zone of Lte1 activity. Lte1 inhibits the kinase activity of Kin4 directly, and also apparently activates Tem1 since it has the exact opposite activity (guanine exchange factor, or GEF) from the Bfa1/Bub2 pair, which constitute a GTPase-activating complex (GAP). Tem1 then activates the escape from mitosis, (MEN), which includes disassembly of the spindle, decondensation of the DNA,  as well as the closing and abscission of the bud neck. Thus yeast cells have taken advantage of their unusual shape characteristics to create a clean, if in our terms still complicated, system to enforce the correct placement of the daughter's genetic material.

While one of the recent papers was a better analysis of the system, doing some very intricate ablation of select microtubules in tiny dividing yeast cells to conclude that the SPOC is not so much a measure of spindle mis-alignment as it is a brake while both spindle ends are still in the mother, the other paper looked at the molecular structure at the centriole with a particularly interesting method.

It is difficult to get structural details about systems like this, unless one is willing to do a great deal of protein crystalization. But a few workarounds have been developed, one of which is fluorescence energy transfer, or FRET. If you engineer a pair of molecular sites, such as two proteins, with emitting and absorbing fluorophores, such that the one is close to the other, (in the 10 to 100 ångstrom range), and such that the one emits at wavelengths that the other absorbs, then you can roughly measure the distance between them at angstrom scales simply by exciting the emitter, and measuring the degree of local quenching by the absorber. And this can be done in live cells and in real time.

Model derived from FRET and other data, suggesting that Bfa1 under SPOC conditions is prevented from interacting with Spc72 by a Kin4 phosphorylation that allows interaction with Bmh1 and allows continued inhibitory activity of Bfa1/Bub2 over Tem1 and the MEN.

Using the fluorophores positioned on Bfa1 and either Spc72, Cnm67 or Nud1 (all stably associated centrosomal proteins), these authors find that Bfa1 is clearly closest to the Spc72 protein, and also near the Nud1 protein, and not detectably close to the Cnm67, which serves here as a control. In addition, they found that while the association of Bfa1 with Nud1 is more or less stable through the SPOC and MEN process, its proximity to Spc72, which as noted above is the docking site for the SPOC-activating Kin4, is reduced when in the active presence of Kin4, presumably due to the arrival of yet another protein, Bmh1, which can bind to Bfa1 once Bfa1 has been phosphorylated by Kin4.

In addition, they deploy yet another fluorescence technique, photo-bleaching, to show that in the absence of Kin4, the Bfa1 association with the whole centrosome, including Spc72 and Nud1, is loosened substantially. They bleached the region close to one spindle pole, and waited for natural exchange and diffusion to restore fluorescence and especially FRET from the spindle pole site. In settings where Kin4 is not active, they see six times the speed of exchange, indicating that phosphorylation of Bfa1 by Cdc5, even though it correlates with closer proximity to Spc72, also correlates with an overall loosening of Bfa1 attachment, which makes sense given that its presence is key to promoting SPOC and inhibiting MEN via its inhibition of Tem1.

The molecular system of the cell cycle was first worked out in the yeast model system, and it is gratifying to see continued, if slow, progress in this system on a variety of fronts to work out its details.

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