Figure 3. Spam coating prevents cell deformation induced by osmotic or chemical manipulation.

(a-d) Kc tissue culture cells transfected with Prom and Spam were stained for Spam surface labeling (red) using anti-Spam antibodies on unpermeabilized, intact cells. Spam-coated (panel a) and uncoated cells were then subjected to osmotic shock. (b-d) Upper panels show low magnification images of cells before and after sequential hypo- and hyper-osmotic shock; lower panels show similar cells at higher magnification but this time after sequential hyper- and then hypo-osmotic shock (reverse order). Hypo-osmotic shock causes dramatic swelling of uncoated cells, while hyper-osmotic treatment of the same preparation leads to extreme shrinking. Notably, the spam-coated cell remains largely unaffected by both treatments. All cells that showed a continuous layer of Spam coating (> 0.5μm thick layer and with no apparent gaps) showed no significant shape changes in response to the osmotic shocks (n=11), while all uncoated cells displayed severe changes in size and shape (n>150). (e) Bar graphs illustrating the increases (left panel) and decreases (right panel) in cell size following hypotonic or hypertonic shock; control, n≥24, spam-coated, n≥8; error bars denote SEM. (f) Spam-coated (red) and uncoated cells were incubated for 90 minutes with latrunculin A. (g) As expected, control cells undergo dramatic changes in cell shape¹³. In contrast, the spam-coated cell (S) remains unaltered. Blue dots and numbers delineate the shape of three sample uncoated cells before (panel f) and after treatment (panel g). Images were captured using epifluorescence and Nomarski interference contrast. (h) Bar graphs illustrating the changes in cell shape (defined as changes in the ratio of cell length over width) following latrunculin treatment in Spam-coated (n=9) and control uncoated cells (n=27); error bars denote SEM.