Dynamin Overview

The dynamic process of membrane trafficking within eukaryotic cells is a complex process requiring specialized protein and lipid complexes. The focus of our laboratory is to study the structure and function of trafficking components by high-resolution electron microscopy techniques. Presently, we are examining a 100 kDa GTPase called dynamin which is involved in the fission event of receptor-mediated endocytosis, caveolae internalization and trafficking in and out of the Golgi. We have shown in vitro that dynamin self assembles in spirals under conditions that favor a GTP transition state (figure 1) (Carr & Hinshaw, 1997) and can self-assemble onto liposomes and form tubes that resemble the necks of constricted coated pits. Upon addition of GTP the dynamin tubes constrict and fragment demonstrating dynamin is capable of generating a force on the underlying lipid bilayer (figure 2) (Sweitzer & Hinshaw, 1998).

To further understand the mechanism of dynamin we have calculated a three-dimensional map of dynamin tubular crystals in the constricted state using cryo-electron microscopy (figure 3) and helical reconstruction methods (Zhang & Hinshaw, 2001). The 20 Å map reveals a T-shaped dimer consisting of three prominent densities: leg (gold), stalk (blue) and head (green) (figure 4). Based on biochemical and crystallographic data, we predict the pleckstrin homology domain of dynamin resides in the leg density, the GTPase domain in the head density and the GTPase Effector domain and middle domain in the stalk density. The structure suggests that the dense stalk and head regions rearrange when GTP is added, a rearrangement that generates a force on the underlying lipid bilayer and thereby leads to membrane constriction. In 2004 we solved the structure of dynamin in the non-constricted state using single particle reconstruction methods (figure 5) (Chen et al., 2004). Comparison of the 3D maps in the constricted and non-constricted states indeed shows a large conformational change in the GED domain and suggests GED interacts in trans with the GTPase domain to cause membrane constriction. These results indicate that dynamin is a force-generating 'contrictase' that may ultimately result in membrane fission.

Dynamin-Related Proteins and Mitochondria Morphology

Additional dynamin family members have been implicated in numerous fundamental cellular processes, including other membrane fission events, anti-viral activity, cell plate formation and chloroplast biogenesis. Among these proteins, self-assembly and oligomerization into ordered structures (i.e. rings and spirals) is a common characteristic and, for the majority, essential for their function. While they are continually being implicated in diverse functions of the cell, we would like to know if a common mechanism of action exists among all the dynamin family members. To address this problem, we examined Drp1 (Dnm1), a dynamin related protein involved in mitochondria fission. This work was a collaboration with Dr. Jodi Nunnari at UC Davis. We have shown that Dnm1 assembles into large spirals, 100 nm in diameter compared to the 50 nm dynamin spirals (figure 6) (Ingerman et al., 2005,). The large Dnm1 spirals remarkably resemble mitochondria constriction sites observed in yeast. These results suggest that the structural properties of dynamin family members are uniquely tailored to fit their function. In addition, the binding of Dnm1 to its partner Mdv1 depends on the assembly state of Dnm1: Mdv1 readily binds Dnm1 in the spiral state only (Naylor et al., 2005).

The Laboratory of Cell Biochemistry and Biology of the National Institute of Diabetes and Digestive and Kidney Diseases is part of the National Institutes of Health, Bethesda, MD, USA. General inquiries may be addressed directly to Jenny Hinshaw, Bldg. 8, Rm. 419, 8 Center Dr MSC 0850, NIH Bethesda, MD 20892, USA. TEL:301-594-0842. Email Jenny Hinshaw at jennyh@helix.nih.gov or the webmaster at dkwebmaster@extra.niddk.nih.gov.