Physicists of Biology hangout 03/20/14
Pallav Kosuri (Zhuang Lab)
- passive elacticity — stored energy in (in-elastic) muscle.
- contrast running to yoga.
- muscle structure z-bands spanned by titin. Titin stretches elastically.
Atomic Force Microscope and Titin
- Force clamp setup: fixed force (through feedback, measure force based on deflection of cantilever.
- Titin unfolds is fixed stochastic steps under constant force.
- remove the force protein collapses back to unstretched length in a single step. (entropic recoil, as opposed to instantaneous refolding).
- Titin made of Ig domains (very common structural motif, also found in antibodies).
- each unfolding step is a ~10 fold increase in length.
Mechanism for mechanical memory
- stems from oxidative stress (during exercise)
- mediated largely by glutathione (absorbs reactive oxygen species to protect cells) Oxidized gultathoine attaches to protein cysteines.
- most cysteines in titin are buried inside the folds. But are still gultathioni-ated during excercise stress.
- leave exposed to glutathione for a long time (40s instead of 2 seconds), domains don’t refold. (pull again and they instantly reach full extension).
- mutate the cysteines, remove this effect (happily refolds as well as it used to).
- cells have an enzyme GRX to remove glutathione from proteins — allows regulation.
test on heart cells
- step length, measure tension/force. Increase in force followed by relaxation.
- add glutathione muscle becomes much more pliant
- add DTT (remove all gulathionalition) become stiffer than usual.
Darren Yang (W. Wong Lab): Investigation of von Willebrand Factor in Hydrodynamic Flow
- multimeric blood glycoprotein, serves as a ligand for platelet adhesion and aggregation in vascular injury.
- Domain structure: A1 — binds to platelet cells. A3 domain binds to collagen. A2 domain – has a cleavage site.
- Hydrodynamic stress in blood flow. Shear flow (even volume), elongation flow (narrowing volume), tethered flow.
- molecule extends under velocity / shear flow.
- experimental setup — DNA stretches under elongational flow.
### vWF under hydro stress
- when secreted, protein is cleaved by proteolytic enzyme. Cleavage requires protein unfolding.
- Q -> difference in A2 domain vs. rest of the protein to unfold under stress.
- built microscope compatible with conventional desktop centrifuge.
Rotem (Jeremy England lab)
Intro: how things move in cells
- standard model — thermally driven diffusion.
- dense environment, lots of motors, ATP hydrolysis, far from equilibrium
- do proteins diffuse?
- do thermal fluctuations drive the motion?
- FRAP – motion of some proteins fits a diffusion model (e.g. Lippincott Schwatrz Science 2003). Better predicition for GFP than ‘functional proteins’
- recent single molecule tracking Parry et al Cell 2014. Treat cells with DNP / (no ATP) or even stationary growth phase, motion of protein becomes much more localized.
- fluorescent photo-convertable proteins.
- take a collection of proteins fused to photo-convertable dendra (DDR). Study in ~7 different conditions.
- HSP70-dendra propigates much slower than free dendra.
- fit to exponentional the decay rate of fluorescence in the photo-converted spot.
- diffusion rate not related to size of molecules.
- ATP depletion might lead to immobilization of a sub-population of dendra.
Sudha Kumari (Irvine MIT and Dustin Labs, NYU)
- cell biologist interested in immune cells
- basis of specific recognition of ‘antigen’
- intra and inter-cellular signal amplification of antigen recognition
- physical contact? — a ’tissue’ lacking physical contact.
- Wiskott-Aldrich syndrome protein (WASP) mutant — T cells fail to amplify signal.
- WASP aids in polymerization of actin
- observe small patches of actin at Tcell contact interface in WT cells, lost in WASP mutants
- Actin patches correlate with TCR (signaling) micro-clusters.
- pattern antigen on the surface. (keeps clusters from moving / aggregating which was intefering with tracking).
- FRET polarization / anisotropy imaging in the patch. FRET pair have different alignment and different anisotropy. Patches have low anisotropy. Patches are ordered structures. Compartmental amplification?
- down regulation of WASP observed at time of aggregation of cells (cell-cell amplification part of the process).
- remove WASP, more cell-cell contact,
- less affinity to antigen presenting surface? (competition model)
- changes existing affinity between cells? (independent model)
- remove wasp, cells stick to eachother more (WASP prevents cell-cell sticking).
- mix WT cells and WASP negative cells, the null cells still stick to the positive cells.
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