Chapter 13 Visualization of Dynamins
Introduction
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.
Section snippets
Self-Assembly of Dynamins
Purified dynamin in high salt exists as a tetramer/monomer (Binns et al., 1999) and dilution into low salt conditions (<50 mM NaCl) forms ring and spiral structures (Hinshaw and Schmid, 1995). In addition, incubation with GDP/BeF, under physiological salt conditions, results in dynamin rings and spirals (Carr and Hinshaw, 1997). To make spirals, dynamin (∼0.2 mg/ml) in HCB100 (Hepes Column Buffer (20 mM Hepes, pH 7.2, 1 mM MgCl2, 2 mM EGTA, 1 mM DTT) with 100 mM NaCl) is incubated with 1 mM
Discussion
In vitro studies of any protein require that the protein behave in a manner similar to in vivo preparations. For example, dynamin spirals and dynamin–lipid tubes observed in vitro are similar to dynamin structures observed at the necks of invaginating pits in nerve synapses (Evergren et al., 2004, Koenig and Ikeda, 1989, Takei et al., 1995). Also the large Dnm1 structures seen in vitro coincide with the mitochondrial constriction sites seen in wild-type yeast (Bleazard et al., 1999, Ingerman et
Summary
The tools presented in this chapter have been used to characterize the structural and biochemical properties of dynamins. The versatility in microscopic techniques allows for visualization of dynamins with varied shapes, including ring, spiral, and helical oligomers. Negative stain allows for structures to be examined quickly; however, larger structures may flatten, as was observed with Dnm1. Cryo-EM helps eliminate flattening, as shown with Dnm1, allows the specimen to be viewed in a more
Acknowledgments
The authors thank Ye Fang and Dr. Jan Hoh (JHU) for assistance in acquiring AFM results and Dr. Blair Bowers (NHLBI/NIH) for work with rotary shadowing. We also thank Dr. Dan Sackett (NICHD/NIH) for help with light scattering and Dr. Edward Egelman (UVa) for his collaboration on image reconstruction of ΔPRD dynamin tubes using the IHRSR method.
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The mechanoenzymatic core of dynamin-related protein 1 comprises the minimal machinery required for membrane constriction
2015, Journal of Biological ChemistryCitation Excerpt :Tubes that were not uniformly decorated with protein were omitted. To quantify Drp1 oligomerization, a sedimentation assay was conducted similar to what has been described previously (34, 35). Large oligomers formed by Drp1 samples, in the presence of ligands, were found in the pellet after a medium speed centrifugation.
A helical processing pipeline for EM structure determination of membrane proteins
2011, MethodsCitation Excerpt :Phoelix mainly operates in Fourier space and works best for filaments that provide diffraction patterns with several strong layer lines. In contrast, the Iterative Helical Real Space Reconstruction method (IHRSR) operates in real space and has been shown to work well for helical filaments that diffract weakly and contain a high level of disorder [59–63]. Appion encourages the use of independent methods as every dataset is different and therefore responds differently to various protocols.
Dimeric endophilin A2 stimulates assembly and GTPase activity of dynamin 2
2011, Biophysical JournalCitation Excerpt :We performed negative-stain transmission electron microscopy (TEM) on dynamin 2 in the presence and absence of endophilin when diluted into 80 mM NaCl buffer. In the presence of endophilin, dynamin forms significantly more fully formed and thicker ring-like structures than in its absence (Fig. 8) (15,35). The fact that endophilin engenders more fully formed dynamin rings could account for the higher dynamin GTPase activities observed in the presence of endophilin.
Vesicle scission: Dynamin
2011, Seminars in Cell and Developmental BiologyCitation Excerpt :The highly conserved GTPase domain of dynamin is relatively large (∼300 residues) when compared to regulatory GTPases such as Ras (∼180 residues), but conserves all core structural elements of a G protein (G1–G4) that are necessary for guanine nucleotide binding and hydrolysis [31]. Various extensions and insertions in the sequence confer additional biochemical properties and functionalities unique to dynamin [32,33]. By virtue of its relatively low binding affinity for guanine nucleotides and the spontaneous acceleration of GTP hydrolysis rate upon higher-order self-assembly, dynamin serves as its own GEF (guanine nucleotide exchange factor) and GAP (GTPase activating protein) during the GTP hydrolysis cycle.
Multiple modes of endophilin-mediated conversion of lipid vesicles into coated tubes: Implications for synaptic endocytosis
2010, Journal of Biological ChemistryHelical crystallization of soluble and membrane binding proteins
2010, Methods in EnzymologyCitation Excerpt :As a result, proteins that promote membrane deformation can easily bind and often tubulate liposomes, producing ordered helical arrays that are suitable for cryo-EM structural studies. Such arrays have been observed with dynamins (Chen et al., 2004; Mears and Hinshaw, 2008; Sweitzer and Hinshaw, 1998; Zhang and Hinshaw, 2001), Snx9 (Yarar et al., 2008), amphiphysin (Peter et al., 2004), endophilin (Gallop et al., 2006), epsin (Ford et al., 2002), epsin homology domain proteins (Daumke et al., 2007), and Escherichia coli RNA polymerase (Opalka et al., 2000; Polyakov et al., 1998). In the next section, we describe procedures for generating helical crystals of dynamin and botulinum toxin from liposome templates and briefly discuss general approaches for extending this methodology to other membrane-associating proteins.