Pallav Kosuri (Zhuang Lab)

Intro

• 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

Intro: VWF

• 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
• Questions
  • 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.

Approach:

• fluorescent photo-convertable proteins.
• take a collection of proteins fused to photo-convertable dendra (DDR). Study in ~7 different conditions.

observations

• 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)

Background

• 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.