Dynamin assay

BARS concentration by some 5-fold to 15-fold, based on the calculations that the intracellnlar concentration is around 20 //g/ml and on the assumption that 5-10% of the cell volume is injected. Prior to injection, the protein is mixed with 0.4 mg/ml fluorescein isothiocyanate (FITC)- or tetramethyl-rhodamine B isothiocyanate (TRITC)-labeled dextran (Molecnlar Probes) as a marker for the microinjected cells (Bonazzi et al., 2005). To give an example, in stndies of basolateral and apical transport (using the vesicular stomatitis virus glycoprotein and p75, respectively), the proteins were microinjected 1 h after the beginning of the 20° incubation in the transport assay. After injection, the cells were allowed to recover for 1 h before proceeding with the experimental protocol (Bonazzi et al, 2005). The BARS (p50-2) and dynamin (DYN2) antibodies were injected at 4.5 mg/ ml, 3 h before farther experimental procedures. 

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

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. 

We and others have previously described dynamin GTPase assays that... 

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.

To initiate GTPase assays, 175 /rl of the dynamin/GED mixtures in PCR tubes are transferred to 37° and 25 //I of 4 mM GTP, 5mM MgCl2 in H2O is added and rapidly mixed. A 0 min time point is taken while the mixtures are still on ice by transferring 17.5 //I of dynamin/GED and 2.5 of GTP stock to a microtiter well containing 5 //I EDTA. 20-/ul aliquots are removed for each subsequent time point and the colorimetric determination of Pi released is performed as described above. Additional controls include dynamin alone and GED alone, subjected to the same dialysis (Fig. 5A). 

Damke, H., Muhlberg, A. B., Sever, S., Sholly, S., Wamock, D. E., and Schmid, S. L. (2001). Expression, purification, and functional assays for self-association of dynamin-1. Meth. Enzymol. 329, 447-457. 

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 GTPase activity was measured essentially by the method of Barylko (2001). GTPase assay was performed in cytosolic buffer (25 mM Hepes-KOH, pH 7.2, 25 mM KCl, 2.5 mM magnesium acetate, 100 mM potassium glutamate) in 2.0 ml microcentrifuge tubes. One hundred /ul of reaction mixture contained protein and lipid at the following concentrations. 

Rapid Purification of Native Dynamin I and Colorimetric GTPase Assay... 

SH3 domains. The McMahon lab suggested the combination of the amphi-physin II—SH3 domain and 1.2 M NaCl to elute dynamin (Vallis et al, 1999). We use an extract from 200 g of sheep brain to purify dynamin in 3 days with a recovery of 8-15 mg of protein at high purity (greater than 98%). The purification of 15 mg of dynamin I provides enough sample for about 50,000 GTPase assays (at 0.3 fig dynamin per sample). 

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