A Division Inhibitor and a Topological Specificity Factor Coded for by the Minicell Locus Determine Proper Placement of the Division Septum in E. coli
de Boer, et al. Cell 1989
They identified this MinCDE locus that's connected to cell division in E. coli.
Highlights:
Without MinCDE, E. coli forms mini-cells (divides in wrong places and pinches off parts lacking DNA and other material)
MinC+D without E inhibits cell division, leading to long, long cells.
Overexpressing MinC+D with E around inhibits cell division.
And finally, overexpressing E leads to minicells.
Something's going on, where E directs cell division, but C and D prevent E from gathering at places other than the middle of the cell.
Well, that's weird, but maybe the next paper can help clarify things:
Rapid pole-to-pole oscillation of a protein required for directing division to the middle of Escherichia coli
Raskin, PNAS 1999
This paper is pretty cool. They visualized GFP-tagged MinD.....and saw that it very rapidly moves in clumps back and forth from one pole of the cell to the other! Imagine seeing that in your microscope...
MinE is necessary for this oscillation, but not MinC. This cool technique basically verified the previous model.
Spatial Regulators for Bacterial Cell Division Self-Organize into Surface Waves in Vitro
Loose, et al. Science 2008
So, even with this model, the specific interactions between these proteins still isn't well understood. But this recent paper showed something cool:
If you just isolate fluorescently-labelled pure MinD+E and put them on a pseudomembrane, they will spontaneously organize themselves into waves (and they made movies!), where MinE basically sweeps MinD along, plucking it out of the membrane. This helps to explain MinD oscillations back and forth.
Amazingly, after class, this type of system helps to explain why successive cell divisions always occur orthogonal to each other - I don't know the eukaryotic division machinery that well, but : )
Prokaryotic DNA segregation by an actin-like filament
Mùller-Jensen, et al. EMBO J 2002
So, in one of my previous entries, I talked about a bacterial protein MreB that acts a lot like actin.
Just to review, that's what actin polymerization looks like. Actin monomers with ATP bound (Actin G for globular) bind to the barbed end of an actin filament (Actin F for filament), and the ATP is hydrolyzed to ADP. Sometimes there are nucleation factors that promote filamentation at a given site. These actin proteins have conserved domains for where the ATP binds.
Ok, so first, they identified this actin homolog, ParM, that exists on sequenced plasmids. When they express the plasmid in E. coli with fluorescently-labelled polyclonal antibodies for ParM, they see filaments from pole-to-pole (40% of the time), 20% have them at foci in the center, and the remaining 40% have fluorescence nonspecifically everywhere. They quantified the filament at roughly 15,000-18,000 molecules/cell. They found that parC and ParR are required for the filaments. This is strange, because parC is a promoter. This suggests that ParR bound to parC is a nucleation site for ParM filamentation. Interestingly, a previous group that used a GFP-ParM fusion didn't see filaments, and this was probably an artifact of the construct!
Using ParM mutants, they showed that ATPase activity wasn't required for forming filaments. D170 mutants formed inflexible, rigid filaments that extended t
he entire length of the cell (this residue is in the phosphate-chelating domain). And then, using ultracentrifugation, they demonstrated that Mg2+ and ATP were required for filament formation, which they also analyzed under an EM. They subsequently used light scattering to analyze filamentation kinetics in vitro, and showed using a non-hydrolyzable ATP analog that depolymerization was dependent on ATP hydrolysis. Similarly, they threw in the complex, and observed differential light scattering with signficant added ParR (but still <1000 style="font-weight: bold;">Reconstitution of DNA Segregation Driven by Assembly of a Prokaryotic Actin HomologGarner, et al. Science 2007
ParM is similar to actin based on filament structure, but they differ in that ParM nucleation is rapid, polymerization is symmetrical and bidirectional (as opposed to polarized actin growth), and finally, that it's dynamically unstable.
So, this group purified the machinery and watched it go in vitro using fluorescently-labelled ParM, and parC DNA attached to microbeads. They saw that filaments formed aster-looking arrays to a maximum length of 3um, unless they added nonhydrolyzable ATP, in which case they grew much longer. They also observed some beads that became connected to each other by ParM bundles that interact with parC at both ends, and these beads were pushed apart from each other over time. The end that attaches to parR/parC becomes resistant to depolymerization, and when they cut a filament with a laser, the frayed ends start to depolymerize.
Oh wow, this is pretty cool. Then they put the machinery in microtube channels they created, and watched as the filaments oriented themselves along the long axis of the channels, and pushed beads apart, until they hit the ends of the channel. They then used "speckle microscopy" and photobleaching to show that nascent polymerization occurs at the parR/parC interface (I think speckle microscopy is where they rapidly mix in the protein with a newly colored conjugated fluorophore)
Cool stuff!
Today, I led a paper discussion on:
An Expanded Eukaryotic Genetic Code
Chin, et al. Science 2003
In this paper, they evolved E. coli Tyr-tRNA synthetase to accept unnatural amino acids and incorporate them into stop codons in yeast via E. coli tRNAcua. They had this complicated series of screens where they used positive and negative selection to screen over 10^8 library members, and miraculously isolated ones for 5 different unnatural amino acids. They then demonstrated that the system works by expressing SOD with the unnatural incorporations, though they could only get 20% of the yield of native protein.
The second paper we discussed was:
Improving Nature's Enzyme Active Site with Genetically Encoded Unnatural Amino Acids
Jackson, et al. JACS 2006
In this paper, they mutated various active site residues to unnatural amino acids in the enzyme nitroreductase from E. coli (which converts the prodrugs CB1954 and LH7 into active DNA alkylating agents poised to kill cancer cells) in an attempt to improve enzymatic activity. While they found ones that improved activity, apparently they didn't do a good job justifying why (they came up with ideas, but didn't show evidence). It was interesting though to hear the reaction from my TF - basically, he didn't take them seriously because they came from a small place. He recognized that their resources limited the experiments they could do, but didn't consider it real science, because it didn't come from some place like Harvard. I spoke up and said I know exactly where they come from!
Finally, I was reading about pharmacogenetics/pharmacogenomics (perhaps I'll save that for another time), and they mentioned Torsades de pointes, which is basically a cardiac arrhythmia (specifically a tachycardia), in this class' case, potentially induced by a large number of drugs depending on patient genotype.
So, of course, I discovered that on wikipedia, and I found something about long QT syndrome, which I remember hearing about during some discussion of genetics. So, I looked up what a QT interval is, and through wikipedia, learned about both the polarizaion/depolarization of heart cells, as well as the path of electricity that induces cardiac contraction (and produces bloodflow!). The heart is SO amazing. Obviously, I won't remember everything, but here's a bit (I hope this gif works):
The P wave illustrates the generation of the action potential at the sinoatrial (SA) node and its conduction to the atrioventricular (AV) node. This enables spatial resolution of atrial and ventricular contractions, otherwise blood could have backflow. The signal then propagates through the bundle of His (PR interval) and through the Purkinje fibers (QRS complex). Finally the ventricles repolarize during the T wave. Thus, the QT interval is the distance between the initiation of the QRS complex and the end of the T wave (though there is some disagreement about how to calculate this accurately from ECGs).
Wow, that was long. I have more to talk about, but I'll save it for next time!
gif file doesn't work, boo.
ReplyDeletebut this is what we were tested on most recently - lots of ecg reading (ugh!)
random transition from all that bacterial stuff to cardiac electrophysiology ^^