lab running for ~6 months now.
- bacterial cell organization (in rod like cells): ends, middle, equally spaced
- ParM finds the ends by pushing — polymerizing until it starts bending.
How bacteria grow
- cell wall holds shape. Digest this, get spherical bacteria.
- proteins acting at a local scale cause organization at 5 um scale
- MreB – structural similarity to Actin, polymerizes
- Ftz – structurally similar to Tubulin, polymerizes (core sequence not recognizable)
- chromosome organization / shape looks different in Bacillus than E coli.
- might be imaging condition difference.
Back to Bacteria growth
- helical structure seen. Believed to be helical cytoskeleton
- Motion still happens in absence of polymer dynamics — not treadmilling
- maybe cell wall synthesis drives the motion
- antibiotics and genetics that prevent cell wall synthesis prevent this motion.
- all components move at a similar velocity.
- All components are oriented perpendicular to the long axis of the cell.
- clockwise/non-clockwise motion is uncorrelated at all length scales.
- right before everything freezes (under drug / removal of cell wall) get random motion.
How do disconnected units organize in a coherent motion?
- Tag0, have cell walls but still round. components move in all directions.
- can induce rod formation in by activating the native gene — quickly start forming rods.
what is the function of MreB?
- Can polyermerize and depolymerize reversibly with a drug
- Class A PBPs (involved in inserting new cell wall), either diffusive or stationary, does not show coherent motion like the rest of the complex.
How is this growth regulated?
- more enzymatic sites or faster synthesis?
- Rate of motion doesn’t change. Must add more components
- New fluorescent amino-acids. incorporate into cell wall, label newly synthesized cell wall.
- Functional fusion proteins substantial challenge: MreB fusion protein makes nice helices. — These don’t compliment and these don’t move.
- In ecoli, fusions do compliment and move, but different constructs behave differently.
- some fusion proteins polymerize and never leave the polymer. Natural MreB makes small
- long linkers 30+ and non-dimerization domains behave more natively.
- lipid involved in flipping glycoprotein chains from inside to outside, when inhibited, leads to filament polymerization.
- amount of precursor determines amount of mREB determines amount of cell wall synthesis.
- not clear if it is sugar polymerization driving the motion or something else.
Q -> can you target the incorporation of the labeled amino-acids into particular proteins / what’s the chance of doing this with clever genetics in the future? A -> currently not sure how they incorporate in the first place. Concievable to make labeled proteins with the technique (but probably a ways off).