Dynamins play a crucial role in numerous membrane remodeling events throughout eukaryotic cells and have a relatively low nucleotide affinity and high rate of GTP hydrolysis. The propensity of dynamins to self-assemble and stimulate their own GTPase activity distinguishes them from other GTPases. The founding member, dynamin, regulates vesicle scission at the plasma membrane, endosome, and trans-Golgi network during endocytosis and caveolae internalization (Hinshaw, 2000). The dynamin-related protein (Drp1/Dnm1/ADL2B) is involved in mitochondrial fission, while mitofusins (Mfn1 and Mfn2) and OPA1/Mgm1 control fusion of the outer and inner mitochondrial membranes, respectively (Hoppins et al., 2007). Other dynamin family members control peroxisome (Vps1/Drp1) division as well as chloroplast division and cell wall formation in plants (ARC5/ADLs/Phragmoplastin) (Hong et al., 2003; Otegui et al., 2001; Praefcke and McMahon, 2004).
To achieve these varied tasks, all dynamins contain three conserved domains essential for function: a highly conserved GTPase domain, a middle domain, and a GTPase effector domain (GED) (Fig. 1). Each domain is required for self-assembly of dynamins into functional, oligomeric structures (Ingerman et al., 2005; Ramachandran et al., 2006; Smirnova et al., 1999; Song et al., 2004). In addition to these conserved motifs, dynamins contain other functional domains specific to the cellular mechanism associated with each protein (Fig. 1).
Dynamin, the family member studied most extensively, has an additional pleckstrin-homology (PH) domain and a proline-rich domain (PRD) (Fig. 1). The PH domain serves to target dynamin to negatively charged lipids (Klein et al., 1998; Tuma et al., 1993; Zheng et al., 1996), which may concentrate dynamin at the necks of invaginating pits during endocytosis (Achiriloaie et al., 1999; Artalejo et al., 1997; Lee et al., 1999). The PRD interacts with SH3-domain containing proteins, including endophilin, amphiphysin, intersectin, and cortactin. These dynamin partners all serve to help regulate vesicle endocytosis (Dawson et al., 2006; Schmid et al., 1998). Other dynamin family members contain transmembrane (TM) domains (mitofusin, Opa1/Mgm1), a mitochondrial targeting sequence (MTS; OPA1/Mgm1) and additional inserts whose functions remain unknown (see B-insert in Dnm1/Drp1) (Fig. 1). All of these domains are tailored to the cellular function associated with the individual proteins while maintaining the conserved GTPase, middle, and GED topology. For mitofusins, the TM domains anchor the protein in opposing membranes and likely act as tethers during mitochondrial fusion (Koshiba et al., 2004) in a mechanism believed to be similar to SNARE fusion events (Choi et al., 2006). The MTS found in Mgm1/OPA1 is essential for targeting the protein to the intermembrane space in mitochondria, where it is responsible for fusion events at the inner mitochondrial membrane and regulating cristae structure (Frezza et al., 2006; Meeusen et al., 2006; Meeusen and Nunnari, 2005). Some of the smallest dynamin-related proteins are the Mx proteins, which are involved in viral resistance (Haller and Kochs, 2002). The GTPase, middle, GED topology is maintained with little added sequence and no additional domains. Of all the dynamin family members studied to date, MxA contains the minimal set of domains essential for the function of dynamins.
Large oligomers of dynamins formed upon self-assembly, are amenable to visualization using various microscopic techniques. Specifically, transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning transmission electron microscopy (STEM) have been used to examine dynamins. To quantify the assembly state of the entire sample, biochemical techniques are also an essential tool. For dynamins, sedimentation assays provide a measure of the oligomeric state, while light scattering experiments provide a measure of conformational changes in dynamin structures due to GTP hydrolysis. When combined with high-resolution imaging techniques, these methods provide a complementary representation of dynamin self-assembly and structural properties, giving a more complete interpretation.
In this chapter, we will focus on three dynamin family members: human dynamin 1, yeast Dnm1, and human MxA. Despite differences in sequence, all three proteins contain similar structural features that can be attributed to the conserved GTPase–middle–GED topology. Each protein oligomerizes in low-salt conditions or with nucleotide analogs and forms helical arrays in the presence of lipid. In the absence of lipid, both dynamin and Dnm1 assemble into spirals while MxA forms curved filaments and rings (Fig. 2). For dynamin and Dnm1, the oligomeric state is tailored to its function: dynamin forms structures with sizes comparable to the size of necks at budding vesicles (~50 nm) (Hinshaw and Schmid, 1995), while Dnm1 forms significantly larger structures required for mitochondrial fission with sizes comparable to diameters observed at mitochondrial constriction sites (~100 nm) (Ingerman et al., 2005). Furthermore, both Dnm1/Drp1 and MxA proteins have an apparent affinity for lipid despite lacking a PH domain. Therefore, the polymers of dynamins may preferentially interact with lipid bilayers due to their inherent curvature. Comparing the similarities and differences in dynamin family members using a combination of biochemical and imaging techniques provides the opportunity to understand the relationship between conserved and unique protein domains associated with varied cellular functions.