UC San Diego

TAT peptide transduction domain (PTD)-mediated cellular uptake permits the delivery of therapeutic macromolecules into cells in vitro and in vivo. Further understanding of the molecular players responsible for transduction will allow for enhanced efficiency and specificity of this tool, in order to take advantage of the enormous potential it holds. To this end, I have sought to elucidate the roles of glycans expressed on the cell surface and small GTPases in the process of macropinocytotic uptake of transducible peptides and proteins. Glycans, heparan sulfate proteoglycans in particular, have been postulated to mediate transduction by a variety of potential mechanisms. I have found, by multiple measures, that transduction occurs efficiently in the absence of glycosaminoglycans and sialic acids. My data preclude the conclusion that TAT -mediated transduction is dependent on heparan sulfate. Instead they support a model where glycans (through charge based interactions) increase the amount of TAT that binds to the cell surface and is therefore transduced, but are mechanistically dispensible for PTD-mediated uptake. Uptake of TAT PTD is known to occur via induction of macropinocytotic uptake, followed by escape from macropinocytic vesicles into the cytoplasm. However, the molecular basis for this process remains inadequately defined. I have found that TAT induces Rac1-dependent membrane ruffling and macropinocytosis. In addition, actin within TAT-induced membrane ruffles colocalized with Rab34, a small GTPase previously implicated in macropinocytosis. Furthermore, overexpression of Rab34 and Rab5 mutants altered the efficiency of transduction and macropinocytosis of TAT. Together, my data support a model whereby TAT induces membrane ruffling and macropinocytosis via Rac1 and Rab34, independent of its binding to glycans. These macropinosomes are then trafficked through Rab34 and Rab5-positive endosomal compartments. This knowledge further strengthens the role of macropinocytosis in TAT transduction and gives some of the first indications about trafficking and endocytic markers associated with TAT macropinosomes. Further study of the induction and trafficking of TAT macropinosomes may allow us to increase transduction efficiency by either enhancing the induction of macropinocytotic uptake or altering trafficking of macropinosomes to enhance endosomal escape.