Annual Biophysical Society Meeting,
2 to 6 March 1997
New Orlean, Louisiana

Knowles D.W., Gimm J.A., Oddone G., Mohandas N.
Lawrence Berkeley National Laboratory, University of California, Berkeley, CA.

Micropipette aspiration is a well established technique for measuring elastic shear rigidity of the red cell membrane. Consideration of the compressibility of the membrane skeleton has added a new level of complexity to the analysis of these experiments. The recently developed technique of Fluorescence Imaged Microdeformation (FIMD) shows that aspiration of a single cell into a glass micropipette creates a local deformation of the underlying skeleton which must condense to enter the pipette and then dilate down the aspirated portion. This local membrane skeletal deformation drives molecular redistribution of the associated membrane components. A complete understanding of these processes will depend on our understanding membrane skeletal deformation at the molecular level. Our studies of Rhodamine Phalodin labelled skeletal actin in human red blood cells show that the normalized skeletal density at the pipette entrance (Pe) and the aspiradted cap (Pc) exhibit a linear increase and decrease, respectively, with increasing aspiration length scaled by the pipette radius (L/Rp), up to a moderate value of L/Rp which is measured from the aspirated cap. Analysis of the entrance and cap densities show that the skeletal density gradient is dependent on Rp and increases monotonically as Rp decreases. Most importantly, when the aspiration length exceeds a specific value, L2/Rp, we measure a discontinuity in the actin density gradient. Analysis of Pe and Pc showed that this discontinuity results from a discoutinuity in the slope of Pe, at L2/Rp, whereas the slope of Pc remained constant. For normal cells this second deformation phase occured at L2/Rp=5.8+0.6 which was Rp independent. Since deformed membrane elements at the pipette entrance have a maximal (1) skeletal density; (2) skeletal asmuthial compression and (3) skeletal axial dilitation, it is plausable that the second phase of deformation be explained  by a discontinuity in one or both of the asmuthial compression or axial dilitation of the skeleton. To obtain some insights into the mechanism(s) we calculate the principle stretch ratios from experimental density profiles. This shows that the second phase of deformation is accompanied by a discontinuity in the slope of the axial (dilitation) stretch ratio with almost no change in the asmuthial (compressive) stretch ratio. The analysis indicates that in the first phase of the deformation, skeletal elements at the pipette entrance reach a maximum limiting axial stretch which is then abruptly exceeded in the second deformation phase. This could result if molecular skeletal components are maximially stretched (first phase) and then fail (second phase). These data reenforce the complexities of membrane skeletal re-organization during micropipette aspiration and raise interesting issues regarding the behaviour of complex networks during mechanical deformation.