The Dynamic Cell Surface
Presentation by Prof. George Barisas, recipient of the CO-ACS Chemistry Award
Monday, December 18, 2017
11:30 – 2:00
Room 382, Lory Student Center/CSU
Reserved Lunches Only – (**Deadline to RSVP: Wednesday, December 13**)
ACS Members: $20 / Students: $10
Contact: Janet.Moder@colostate.edu (or call 970-491-5409)
That molecules in living cell surfaces are in constant motion and that this motion is physiologically significant is a relatively recent concept. Semi-quantitative imaging studies by Mike Edidin in 1972 showed that proteins diffuse laterally in the plane of the cell membrane at a rate of approximately 1×10-10 cm2sec-1. Quantitating such motions was first attempted in 1974 by Reiner Peters and this approach is today called FRAP or fluorescence recovery after photobleaching. In 1975 we began developing various implementations of this technique to examine diffusion of molecules in solution and on cell surfaces. These measurements allow estimating degrees of receptor aggregation by ligands such as antigens and hormones. A common feature of several studies has been that modest degrees of receptor clustering lead to cell activation while more extensive crosslinking places cells in a refractory state.
However, lateral motions of molecules in two-dimensional environments such as cell surfaces are only weak functions of molecular size. Recognition of this led to interest in examining membrane protein rotation as a more sensitive probe of receptor clustering. Protein rotation measurements are challenging since typical receptors rotate over times of perhaps 20-200 microseconds and techniques useful on this timescale such as transient absorption and time-resolved phosphorescence anisotropy dahave limited sensitivity. We developed fluorescence depletion anisotropy or FDA methods to extend that sensitivity and, for example, showed on individual cells that activation of Type I Fc¬ε receptors responsible for allergy symptoms can arise from simple receptor dimer formation by allergen.
Many aspects of cell surface dynamics cannot be studied by ensemble measurements such as FRAP or FDA but rather require observation of single molecules. Lateral motions of individual molecules were first evaluated by differential interference contrast microscopy of cells with gold nanoparticle-labeled receptors and subsequently by fluorescence microscopy of cells where receptor labels are fluorescent quantum dots. Such methods show that particular cell surface proteins are confined to cytoskeletally-defined 100 nanometer-scale regions within which they diffuse hydrodynamically at rates of 1×10-9 cm2sec-1. Thus, what is actually observed, in most FRAP measurements is the “hopping” of receptors between such regions at rates between about 1×10 10 and 1×10 12 cm2sec-1.
We are currently exploring rotation of single cell surface molecules. Asymmetric quantum dots emit polarized fluorescence and the time-autocorrelation function of fluctuations in fluorescence anisotropy reflects molecular rotation or reorientation. We find that various receptors exhibit slow orientational fluctuations on the 100-millisecond timescale and that these fluctuations are independent of treatments affecting receptor aggregation or the overall membrane environment. This suggests that small, sub-micrometer membrane regions may liberate with respect to the overall cell surface. This would represent a new aspect of membrane dynamics not considered previously. Our long-term goal is to extend single-molecule rotation measurements into the microsecond timescale of membrane protein hydrodynamic motion.
Event Date: 2017-12-18
Event Start Time: 11:30 am
Event End Time: 2:00 pm
Event Location: Room 382, Lory Student Center-CSU