|M.Sc Student||Margolis Moran|
|Subject||Self-Assembly-Activity Relationship Study of the GTPase|
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Dganit Danino|
|Full Thesis text|
The exact mechanism of dynamin action in endocytosis is still unclear. Two distinct models have been proposed to explain its function in this process: one suggests that dynamin functions as a mechanochemical enzyme that uses GTP hydrolysis to generate a force on the membrane that directly drives membrane fission; the other suggests that dynamin, like small GTPases, functions as a signaling switch for other factors that mediate the fission event.
The goal of this research was to elucidate the function of dynamin in endocytosis by studying the relationship between its self-assembly and GTPase activity, the two key components of its action, using advanced cryo-TEM technique and biochemical assays.
As was already known, wild-type dynamin oligomerizes and self-assembles onto liposomes in helical structures which lead to the formation of protein-coated tubes; we showed that this oligomerization is not affected by the GTPase domain mutants that were examined, but is abolished by a mutation in the middle domain. This supports previous findings that the middle domain is involved in the self-assembly of dynamin, but for the first time shows the region in the middle domain responsible for the self-assembly.
The addition of GTP to preformed wild-type dynamin-coated tubes resulted in constriction of the tubes and in the disassembly of dynamin from the lipid membrane. This strengthens the model that refers to dynamin as a mechanochemical enzyme that generates force on the membrane by hydrolyzing GTP, yet no fission occurred in vitro.
GTPase activity of wild-type dynamin in the absence or in the presence of liposomes was highly stimulated when lipid membranes are present. As for the GTPase domain mutants, in both cases no GTP hydrolysis was observed, most likely because of the mutants' inability to bind GTP. As for the middle domain mutant, GTP binding and hydrolysis were not damaged, but no stimulation occurred in the presence of liposomes because of the inability of this mutant to oligoemrize onto lipid membranes.
In addition, preliminary work was done on dynamin binding partner, the SH3-domain protein amphiphysin. When dynamin and amphiphysin were incubated together in the presence of liposomes they created lipid tubes of the same dimensions like the lipid tubes formed by dynamin or amphiphysin alone. Additionally, in the absence of liposomes, the two proteins create ring-like complexes under conditions none of the proteins assembles separately. These complexes resemble in their dimensions those of dynamin when bound to lipid.