Dynamin assembly

Carr, J. F., and Hinshaw, J. E. (1997). Dynamin assembles into spirals under physiological salt conditions upon the addition of GDP and gamma-phosphate analogues. J Biol. Chem. 272, 28030-28035. 

Soulet, F., Yarar, D., Leonard, M., and Schmid, S. L. (2005). SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis. MoL Biol. Cell 16, 2058-2067. 

The analysis of dynamin helices formed on lipid tubules provides a picture of what happens to a dynamin assembly upon GTP hydrolysis, as dynamin adopts different conformations in the presence of different nucleotides. While the GTP bound state of dynamin (observed in the presence of GTP7S) forms a tight helix with a spacing of 11 nm, in the GDP bound state the spacing has nearly doubled to 20 nm (Stowell et al, 1999). 

Hinshaw, J. E., and Schmid, S. L. (1995) Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding [see comments]. Nature. 374, 190-192. 

In the kiss-and-run mode exocytosis and endocytosis are directly coupled to each other, while in the case of classical complete vesicle fusion, exocytosis and slow clathrin-mediated endocytosis are timely and spatially separated. However, it appears that also in the latter case exocytosis and endocytosis occur coordinated, as both are stimulated by an increase of the cytoplasmic calcium concentration. It has been shown that after calcium entry the enzyme phospho-inositol-5 kinase Iy, which is enriched in the synapse, catalyzes the synthesis of phosphatidylinos-itol (4,5)-bisphosphate and that this mechanism is important for synaptic vesicle trafficking (Di Paolo et al. 2004). As many proteins involved in clathrin-mediated endocytosis are recruited to the plasma membrane by binding to phosphatidylinosi-tol (4,5)-bisphosphate (e.g., amphiphysin, dynamin, epsin, AP-180, and AP-2) it is attractive to speculate that elevated levels of calcium mediate the recruitment of en-docytic proteins to the plasma membrane by this mechanism. The increased level of phosphatidylinositol (4,5)-bisphosphate could be in part degraded by synaptojanin that thereby initiates the disassembly of the clathrin coat. Hence, calcium-induced transient increases in the level of phosphatidylinositol (4,5)-bisphosphate appear to play a central role for coupling exocytosis to clathrin-mediated endocytosis. In addition, it has been demonstrated that calcium also leads to the dephosphorylation of endocytic proteins as amphiphysin, dynamin, and synaptojanin, which in vitro is important for efficient coat assembly (Cousin and Robinson 2001). 

Kim, Y.W., Park, D.S., Park, S.C., Kim, S.H., Cheong, G.W., and Hwang, I., 2001b, Arabidopsis dynamin-like 2 that binds specifically to phosphatidylinositol 4-phosphate assembles into a high-molecular weight complex in vivo and in vitro. Plant Physiol. 127 1243-1255. 

Dynamin is a microtubule-associated GTP-binding protein. Dynamins are activated by proteinkinase C. They have a PH, a pleckstrin-homology domain and bind inositol phospholipids which aaivate the GTPase of dynamin. Dynamins are involved in microtubule assembly and vesicular traffic. 

A specialized cell-matrix contact point, which is stracturaUy distinct from focal adhesion complexes. See Linder, S. and Aepfelbacher, M., Podo-somes adhesion hot-spots of invasive cells. Trends Cell Biol. 13, 376-385, 2003 McNiver, M.A., Baldassarre, M., and Buccione, R., The role of dynamin in the assembly and function of podosomes and invadopodia. Front. Biosci. 9, 1944-1953, 2004 Linder, S., and Kopp, R, Podosomes at a glance, J. Cell Sci. 118, 2079-2082, 2005. 

Dynamin 1, a member of the dynamin family of large GTPases. Mammalian dynamin 1 forms ring-like assemblies aroimd the necks of budding synaptic vesicles. StmeturaUy, it can be subdivided into five domains. The high-resolution X-ray structure of the GTPase domain from Rattus norvegicus was described in 2005 [K. Takei et al.. Nature 1995, 374,186 ... 

Palacios, F., Schweitzer, J. K., Boshans, R. L., and D Souza-Schorey, C. (2002). ARF6-GTP recruits Nm23-Hl to facihtate dynamin-mediated endocytosis during adherins junctions assembly. Nat. Cell Biol. 4, 929-935. 

Shpetner and Vallee, 1992 Warnock et al, 1996). We believe that these reported ranges of dynamin s GTPase activity reflect, in part, differences in assay conditions and assembly templates. 

Here we describe a simple, colorimetric assay to measure GTP hydrolysis by dynamin and discuss some of the variables that can affect measurements of dynamin s basal and assembly-stimulated GTPase activity. 

In the absence of GTP, or in the presence of nonhydrolyzable analogues of GTP, dynamin will self-assemble onto and tubulate hposomes. This activity has been observed with liposomes of varied composition, including those formed solely using dioleoylphosphatidylserine (Sweitzer and Hinshaw, 1998), or Folch fraction I, a brain lipid extract (Takei et al, 2001). Correspondingly, dynamin s GTPase activity is stimulated 10-100-fold when measured in the presence of liposomes. We refer to this as dynamin s liposome-stimulated GTPase activity. 

Liposome-stimulated GTPase assays are performed as described for basal except that the final concentration of dynamin used in the assay is lower (typically 0.1-0.5 iiM). Liposomes are added from a 20x stock solution to the dynamin in assay buffer just prior to mixing this 2x dyna-min-liposome stock with an equal volume of the 2x GTP stock in assay buffer to initiate the incubation. The remainder of the assay is as described above except that time points are taken more frequently and typically over a 0-15 min time course. The data in Fig. 2A shows that dynamin s GTPase activity could be stimulated > 100-fold upon assembly onto a liposome template composed of DOPC PI4,5P2 cholesterol (80 15 5 mol%), with maximum stimulation occurring at >150 /iM lipid (Fig. 2A). As previously described (Tuma and Collins, 1994), the concentration dependence for dynamin was sigmoidal, indicating positive cooperativity at low concentrations of dynamin (Fig. 2B). 

Fig. 2. Dynamin s GTPase activity is stimulated by assembly onto PI4,5P2-containing liposomes. (A) Dependence of the rate of GTP hydrolysis by dynamin on the concentration of liposomes, assayed at fixed concentrations of dynamin (0.5 jxM) and GTP (1 mM). (B) Dependence of the rate of GTP hydrolysis by dynamin on the concentration of dynamin in the assayed at fixed concentrations of liposomes (25 M) and GTP (1 mM). Note the sigmoidal shape of the curve indicating cooperativity.

Fig. 1. Schematic depiction of the domain structure of SNX9 and the association between SNX9 and dynamin. Dimeric SNX9 is shown with its SH3-, PX-, and B AR-domains. Dynamin dimers (Dyn) are bound to the SH3-domains. The initial membrane binding occurs via the PX- and BAR-domains of SNX9. Through its unstructured region (curved black line), SNX9 has the abihty to attach to the clathrin/AP-2 lattice. The lower part of the picture illustrates that SNX9 and dynamin are thought to co-assemble at the neck of a forming clathrin-coated vesicle.

Dynamin hydrolyzes GTP into GDP and inorganic phosphate (Pi). Its GTPase activity can be stimulated by a number of protein or lipid effectors, or by its own self assembly. This method uses sonicated L-a-phosphatidyl-L-serine (PS, 100%) liposomes. PS liposomes facihtate dynamin self assembly (Lin et al, 1997 Tuma et al, 1993) and an optimal concentration of PS liposomes will stimulate maximal dynamin GTPase activity in vitro (Barylko et al, 2001) at very low cost. The protocol offers advantages in speed and... 

Dynamin self-assembly stimulates its basal GTPase activity 10-100-fold in vitro (Barylko et al, 2001 Stowell et al., 1999 Tuma and Collins, 1994 Warnock et al., 1996). This is due to activation of dynamin s intramolecular GTPase activating protein (GAP, amino acids 618-752 of dynamin), which becomes activated upon dynamin self-assembly (Muhlberg et al., 1997 Sever etal., 1999). Most assays that measure assembly-stimulated GTP hydrolysis by dynamin use templates such as lipids to promote dynamin self-assembly (Barylko et al., 2001 Song et al., 2004 Stowell et al., 1999). In contrast, we have developed a soluble assay that reconstitutes assembly-stimulated GTP hydrolysis by simply adding recombinant isolated GAP domain to unassembled dynamin (Sever et al., 1999). Thus, recombinant GAP domain, on its own, stimulates GTP hydrolysis by dynamin approximately 100-fold (Sever etal., 1999). Pre-incubation of dynamin with auxilin prior to addition of the recombinant GAP domain abolishes GAP s ability to stimulate GTP hydrolysis. The GAP-stimulated assay that we routinely use is described. 

---