Membrane fission is associated with the breakage of a tiny nanometer-scale membrane neck connecting two separating/dividing membrane compartments at the late stages of division. Severing this neck in a timely and leakage-free manner is critical for normal functioning of endomembrane systems, hence membrane fission is performed by specialized and tightly-regulated protein machinery assembling on the neck. While our current mechanistic understanding of fission, in life and disease, is heavily based upon in vitro reconstitution approaches, such approaches rarely (if at all) reproduce confined and crowded environment of the neck. Instead, in vitro reconstitution has been mostly performed using large (sub-micron to micron scale) membrane templates of various physico-chemical properties, resulting in controversial outcomes and precluding rigorous mechanistic analysis of fission. This project is focused on creation of the next- generation in vitro approaches that reconstruct and quantify membrane fission at physiological length/time scales. We will combine nanotechnology with modern biophysical approaches and protein engineering to solve the long-standing puzzle of membrane fission mediated by the proteins of dynamin superfamily, which are intimately involved in intracellular fusion/fission and directly linked to various human pathologies. We will approach this problem from several different angles: - We will perform single-molecule analysis of dynamin oligomerization on membrane surfaces with precisely (2 nm) calibrated curvature (10-1 to 10-2 nm range) to identify and characterize elementary mechano-chemical units assembled by dynamin. We will determine (i) the pathways of dynamin oligomerization/self-assembly on a curved membrane surface, (ii) the size/geometrical arrangement of minimal oligomers capable of cooperative GTP hydrolysis and (iii) the effects of membrane curvature on self-assembly and GTPase activity of small dynamin oligomers. - We will assess membrane activity of individual dynamin oligomers (dimers and higher order multimers) at nano-confined membrane templates to determine how the force fields produced by dynamin are coupled to lipid rearrangements throughout fission. We will (i) measure the local forces produced by different dynamin oligomers and quantify associated membrane deformations and instabilities, and (ii) determine pathway(s) of lipid rearrangements and their dependence on the size/geometry of dynamin complexes and geometrical/mechanical parameters of membrane templates. - We will analyze effects of auxiliary proteins and critical mutations of dynamins, compare the self- assembly and fission pathways for different members of dynamin superfamily to distinguish general and protein-specific parameters (perhaps, even specific pathways) of membrane fission and unravel molecular mechanisms behind functional evolution and regulation of dynamin fission machinery.
) This project is expected to (i) provide critical mechanistic insights to membrane fission, the core membrane process critical for function and maintenance of cellular organelles; and (ii) develop general-use experimental tools for in vitro analysis of proteins mediating membrane fission. Alterations (e.g. point mutations) in such proteins, primarily the dynamin superfamily, have been linked to been linked to neurological disorders in humans, including epilepsy, centronuclear myopathy and others. One of the major hurdles in developing therapeutics against these pathologies is the extreme complexity of the associated protein interactomes. Dynamins, through their specialized domains, interact with a large variety of proteins involved in membrane remodeling and signaling networks. The effects of the mutations associated with the different pathologies are equally complex. Many mutations also do not display a distinct dominant negative phenotype, and seem to be affecting kinetics of membrane fission through yet unidentified mechanisms. The knowledge and experimental tools generated by this project are expected to advance our mechanistic understanding of the malfunctions of dynamins directly associated with membrane fission, distinguish them from those associated with other functions of dynamins, and develop in vitro diagnostic tools to analyze the kinetic effects of dynamin mutations in the context of variable lipid/protein environment at biologically relevant length- and time- scales. This knowledge will be critical for developing new therapeutic approaches for treating diseases related to mutations in dynamin genes.
