An investigation of imaging approaches for the study of erbb2 distribution and dynamics in the plasma membrane with respect to the distribution of the tetraspanin cd82

Tetraspanin proteins are known to organise in the plasma membrane and form tetraspanin enriched microdomains. The tetraspanin CD82/KAI 1 has been shown previously by ensemble imaging and biochemical methods to associate with ErbB2, and it has been suggested that CD82 may play a role in regulating the distribution and dynamics ofErbB2 in the plasma membrane [1), [2]. CD82 may also play a role in mediating cellular response to Herceptin. In order to investigate the effect of CD82 on ErbB2 distribution and dynamics in the plasma membrane we applied a number of imaging techniques, in particular super resolution and single molecule approaches. We found little effect of CD82 expression on either ErbB2 diffusion or confinement characteristics in MCF7 and SKBR3 cells, and little difference in either the intrinsic clustering of ErbB2 or clustering in response to treatment with a panel of ErbB targeting molecules. We did however demonstrate and quantify the-importance of optimising both the imaging and analysis conditions for dual colour super-resolution imaging, and demonstrated for the first time the combination of super resolution optical fluctuation imaging and single molecule tracking.