10: Checkpoints, Flagella

Checkpoints: Controls That Ensure the Order of Cell Cycle Events
Hartwell & Weinert Science 1989

This is an older article, but it provides a good framework to think about control of the cell cycle. Here are two initial questions the authors posed:
"What principles does the cell use to establish an ordered pathway of events? Does the existence of such order imply the existence of control mechanisms that enforce order?"

They basically have two models they propose:
One is substrate-product assembly - basically, objects have inherent properties that cause them to come together in the proper way. They cite phage assembly as an example, where the proteins are created simultaneously but require the baseplate to come together first before the outer coating can assemble around it. In this example, the order of the process is controlled by the materials themselves

The other model is subtly different - phage DNA is processed in a matter that depends on a control mechanism. The DNA will not be processed by terminase unless proheads are in place and a special inhibitor is put in place to make sure the system abides by this principle.

So, in the early days of thinking about the cell cycle, I guess scientists were unsure which was true (although it seems so obvious now). But, they were able to use chemical treatment and special mutants to deduce things. They argue that, although you can't prove it for certain, if these types of treatments relieve the dependence relationship, then there's most likely a control mechanism in place. I wonder if they were the first people to coin "checkpoint". Anyway, they go through a few examples of checkpoints, namely how mitosis depends on DNA replication, how replicons depend on each other, how mitosis is dependent on cell growth, how spindle pole replication is dependent on DNA replication, etc. They also mention that they observe embryonic stem cells to be different from somatic cells in these areas of control, and speculate on why that would be - are they sacrificing fidelity for quicker division? They also mention the SOS response, which I will get to probably in the next entry.

So, after class discussion, I no longer felt that I understood the difference between the two models - I thought the presence of an extrinsic sensor/regulator was the key part - yet there are some systems in which the lines start to blur - for example, SlmA binds to nucleoid DNA and prevents FtsZ activity - it's both the sensor and the executioner.

Sensing Structural Intermediates in Bacterial Flagellar Assembly by Export of a Negative Regulator
Hughes, et al. Science 1993

Background: In this paper, they talk about how flagellar assembly is grouped into 3 classes of genes: the early and middle genes are responsible for assembling the basal body and hook, which are attached together and anchored in the membrane. These genes induce the expression of the late genes that build off of the hook by exporting flagellin, hook-associated proteins (HAPs) and the Cap protein. These middle genes affect expression of late genes through encoding an alternate sigma factor (fliA, sigma 28), but if there's a defect in assembly, flgM will negatively modulate this activity.

Results: They come up with a valid model, where if the base of the flagellum is correctly set up, it can export flgM, thus lifting the repressiong of fliA and corresponding late genes that can finish assembling the flagellum. What's interesting is that normally fliA and flgM exist in equilibrium when flagella are not being assembled, but if the flagellum gets sheared, it allows for flgM export and thus rebuilding. They came to this conclusion through a few experiments, namely analyzing spent media for exported flgM, analyzing motility, and determining whether fliC (flagellin) can get exported too. They use some mutants in these experiments, either defective in some of the flagellar components or with a lacZ fusion to the flgM that they hypothesized clogged up the export (though they don't really prove this is what's happening, this paper has yet to be disproven).

A Molecular Clutch Disables Flagella in the Bacillus subtilis Biofilm
Blair, et al. Science 2008

So, bacteria switch between being motile and burying themselves in biofilms, but how is this coordinated? I mean, you wouldn't want to just start lopping off your flagella when you're getting ready to settle down - they're expensive and take a lot of time/effort to make again.

So, these two processes are alternately controlled by the transcription factor SinR in B. subtilis, which represses the eps (Extracellular polysaccharide) operon. Mutants in sinR are nonmotile - but are the flagella just getting stuck in the sticky goo secreted?

No, sinR epsH mutants don't make EPS and are still stuck. But, after searching for suppressors of the nonmotile phenotype, they found mutants in EpsE, a family II glycosyltransferase. And when they continued to look for suppressors of suppressors, they found mutations in FliG, the protein that makes the C-ring at the base of the flagellum. This protein is responsible for transducing the rotational movement generated by MovA/MovB (yes, that's right, flagella rotate because of the proton motive force conducted through these channels, anchored to the peptidoglycan skeleton) to the flagellum. So, they wondered whether EpsE was acting either as a brake, to stop the flagellum from rotating, or if it was acting like a clutch and detaching the flagellum from the motion-generating apparatus. After immobilizing bacteria and inducing expression of EpsE, they measured that flagella were not rotating as normal, but they weren't locked in position - they still underwent Brownian motion. Thus, EpsE serves as a clutch!

Regulated Pole-to-Pole Oscillations of a Bacterial Gliding Motility Protein
Mignot, et al. Science 2005
So, there are all kinds of different ways that bacteria can move, some of which are still not understood. Some bacteria use pili-directed movements, where they shoot out a pilus kind of like a grapling hook, and then try to reel themselves in. One poorly understood way is through gliding.

In this paper, they used FRAP to show that they found another oscillatory protein that goes back and forth, from pole to pole (recall MinD in E. coli), called frzS (they were in Myxococcus xanthus).