Studying Cell Biology
Preface | Introduction to Problem Solving | Problem Sets | Acknowledgments

Just how mobile are integral membrane proteins and phospholipid in the plasma membrane? One fundamental tenet of our modern view of membrane organization is its fluid nature of membrane organization: that is, membrane lipid and integral protein components are relatively free to diffuse randomly within the plane of the membrane (see www.middlebury.edu/membranes for dynamic simulations of this behavior). In this problem set, you are asked to interpret the results of more recent, higher resolution experiments designed to examine the behavior of individual membrane constituents (rather than large populations of such components that were the subjects of earlier FRAP experiments).

In a series of experiments, Tomishige and associates (cf., Tomishige and Kusumi, 1999, Mol Biol Cell 10: 2475-2479 PMID:10436005) treated human erythrocyte “ghosts” (plasma membranes washed free of cytoplasm) with antibodies specific for various membrane components (Band 3 protein, spectrin, or phospholipid). The ghosts had been attached to coverslips to keep them from moving and the antibodies had been modified for microscopic observation by linking them to either colloidal gold spheres or to much larger latex beads. The behavior of these particles (and presumably, the underlying membrane components) at 37 degrees C was then monitored by contrast-enhanced bright-field microscopy, with images being captured with a high-speed video camera. The images in the following video movies are blurred and grainy due to technical limitations unrelated to the experiment. The beads appear very bright while the gold spheres are black and, due to their great differences in size, they are never simultaneously in focus.

The first 3 experiments entail simple observations of 40 nm gold spheres linked to membrane components:

A. Does a gold sphere attached to a single Band 3 IMP move in the plane of the membrane?

B. Does Band 3 movement change over a longer period?

C. Does removing the RBC cytoskeleton (spectrin) by enzymatic digestion affect Band 3 movement? In the next three experiments, the behavior of one kind of membrane component (linked to a gold sphere) is observed while a different constituent (linked to a 1 mm latex bead) is manipulated by an "optical tweezer":

D. Does moving a Band 3 IMP affect the organization of the underlying cytoskeleton (spectrin)? and conversely,

E. Does perturbing the cytoskeleton affect Band 3 behavior? and, as a control,

F. Does manipulating a Band 3 IMP affect the behavior of a membrane phospholipid?

In Problems C. and D. gold spheres are used to mark one kind of membrane component, while another component linked to the latex bead is manipulated by and optical tweezer. The larger, latex bead can be moved by the laser force field, which does not directly affect the behavior of the gold spheres. Typically, gold is linked to only one membrane component, using an antibody fragment (Fab) that has only one binding site. The latex particles, however, have been coated with many, intact (divalent) IgG antibodies, and hence, they can bind specifically to multiple copies of a membrane antigen. The problems based on these experiments are organized into separate webpages, each containing three frames: the upper frame presents background information about each experiment critical for its interpretation; the lower frame, in turn, is sub-divided into a left-hand compartment containing a labeled image hyperlinked to a video record of the experiment, and into a right-hand compartment presenting questions concerning the image and video record. Begin considering the experiments in sequence by turning the page or jump to the individual problems using the hyperlinks above.

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