Dynamin GTPase stimulation

Shpetner, H. S., and Vallee, R. B. (1992). Dynamin is a GTPase stimulated to high levels of activity by microtubules. Nature 355, 733-735. 

Fig. 1. Dynamin GTPase assay was performed in the presence of truncated amphiphysin. The truncated amphiphysins were designed as shown in (A). Effect of these amphiphysins on dynamin GTPase activity was assayed (B). The data was normalized by the GTPase activity of dynamin. Deletion of the middle domain of amphiphysin (Amph A248-315 and Amph A248-601) resulted in strong stimulation of the dynamin GTPase activity. Binding sites for AP-2 and clathrin are depicted in (A). (Reproduced with permission from Y. Yoshida et al. [2004]. EMBO J. 23, 3483-3491.)...

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... 

Robust Colorimetric Assays for Dynamin s Basal and Stimulated GTPase Activities... 

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). 

The degree of stimulation of dynamin s GTPase activity depends on the composition of the liposomes. The data in Fig. 3A shows that dynamin s GTPase activity is stimulated 100-fold when assayed in the presence of liposomes composed of DOPS, but only 10-fold in the presence of liposomes composed of DOPC PI4,5P2 (90 10 mol%), although dynamin was able to tubulate hposomes of both compositions (Fig. 3B). While we have... 

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. 3. Dynamin s liposome-stimulated GTPase activity varies with liposome composition. (A) Dynamin s GTPase activity is greater when assayed in the presence of liposomes composed exclusively of DOPS, compared to liposomes prepared from DOPC PI4,5P2 (90 10 mol%). Liposomes composed of a higher mol% PI4,5P2 (see Fig. 2) are significantly more effective templates. (B) Negative-stain electron micrographs of dynamin-generated lipid tubules formed on DOPS and DOPC PI4,5P2 liposomes are indistinguishable, despite the observed differences in rates of GTP hydrolysis. Scale bar = 50 nm.

Fig. 4. Dynamin s lipid nanotubule-stimulated GTPase activity is temperature dependent. (A) Time course of GTP hydrolysis by 0.1 fiM dynamin measured in the presence of 400 fiM PI4,5P2-containing lipid nanotubules (LT) at the indicated temperatures. (B) The LT-stimulated rate of GTP hydrolysis of dynamin, measured at 37°, is plotted against the initial concentration of GTP to obtain the Michaelis-Menten constants kcat and Km for dynamin s LT-stimulated GTPase activity. Data shown avg. std. dev. values obtained from triplicate samples performed during single experiment.

Temperature Dependence of Michaelis-Menten Parameters for Dynamin s Basal and Lipid Nanotubule (LT)-Stimulated GTPase Activity. Values Shown Are Average Standard Deviation (n > 3 Independent Experiments)... 

Warnock, D. E., Hinshaw, J. E., and Schmid, S. L. (1996). Dynamin self assembly stimulates its GTPase activity. J. Biol. Chem. 271, 22310-22314. 

Fig. 2. Examples of the dynamin I colorimetric GTPase assay. (A) L-a-phosphatidyl-L-serine (PS) liposome concentration curve. Stimulation of dynamin I GTPase activity was measured as a function of PS concentration using the colorimetric assay. The curve reaches a plateau at about 30 /xg/ml PS in this experiment, but varies between PS preparations. The curve is representative of the level of optimal dynamin activity in the absorbance range 0.4-0.6 OD units. (B) Concentration-dependent effect of compound A on dynamin I GTPase activity stimulated by 40 /xg/ml PS. The effect of the drug alone at increasing concentrations is shown in the open bars. The stimulation by PS + Compound A is shown in the hatched bars. Compound A reduces PS-stimulated dynamin I GTPase activity (hatched bars), until at high concentrations there appears to be stimulation. The solid bars show a subtraction of the other values, which, after eliminating the effect of background phosphate, reveals the full inhibitory curve of compound A.

Here we describe four assays that monitor auxilin-dynamin interactions. Included are a nucleotide-dependent dynamin affinity column, a coprecipitation assay that measures auxilin-dynamin nucleotide-dependent direct binding, a GTPase assay that examines auxilin s ability to inhibit the stimulated rate of GTP hydrolysis, and FRET with FLIM analysis of dynamin-auxilin interaction in cells. 

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. 

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