DIFFUSION AND LOCALIZATION OF G PROTEIN βγ SUBUNITS IN DICTYOSTELIUM CELLS: ROLE OF PHOSDUCIN LIKE PROTEIN 1 Ruchira1, M.Blaauw2, M.A.Hink1 , P.J.M.van Haastert2, A.J.W.G.Visser1 Introduction Results and Discussion Heterotrimeric GTP-binding proteins (G proteins), composed of the subunits Diffusion measurements were made in the membrane and cytosol of wild type Gα, Gβ and Gγ mediate chemotaxis and multicellular development in cells and in the cytosol of phlp1-null cells (Table 1). Dictyostelium (1). On activation G-protein dissociates into α and βγ subunits. Table 1: Average diffusion coefficients and standard deviations for Gβ-GFP and Gγ-GFP in Amongst other proteins, phosducin is thought to modulate Gβγ activity by wild type cells and phlp1-null cells. During measurement cells were incubated in buffer. binding to free Gβγ and blocking its association with Gα subunits, effectors or membranes. Recently, three phosducin-like protein (phlp) genes have been Subunit Wild type cells Phlp1-null cells discovered in Dictyostelium (2). Of the three, disruption of the phlp1 gene Membrane Cytosol Cytosol strongly impairs G-protein signalling. Phlp1 deletion causes Gβ-GFP and Gγ- (µm2/s) (µm2/s) (µm2/s) GFP, which are membrane associated in wild type cells, to become cytosolic. Gβ-GFP 0.31 ± 0.2 13 ± 5 11 ± 5 It has been proposed that phlp1 facilitates proper folding of Gβ or assembly of Gγ-GFP 0.40 ± 0.3 14 ± 6 20 ± 6 Gβ into Gβγ complex (2). Gβ-GFP Gγ-GFP Data from the measurements in membrane were fit to a model describing diffusion of two species. 46 ± 18% of both, Gβ-GFP and Gγ-GFP showed slow A B diffusion, indicating association of these subunits with the membrane. Data from the measurements in cytosol were fit to a model describing free diffusion of a single species. An offset term was added to take into account slow intensity fluctuations that were probably caused by cellular and intracellular movement (4). Gβ-GFP and Gγ-GFP show similar diffusion characteristics in wild type cells. Wild type However, in phlp1-null cells, the diffusion coefficient distribution of Gγ-GFP shows a shift to higher diffusion coefficient values (Fig 2). C D 0.60 0.45 Fraction 0.30 0.15 Phlp1 - null 0.00 Fig 1: Fluorescence confocal images showing the localization of Gβ-GFP and Gγ-GFP in 0 5 10 15 20 25 30 35 wild type cells (A and B) and in phlp1-null cells (C and D). Images were obtained with the 2 Zeiss ConfoCor 2TM, LSM 510 combination setup. Bar = 10 µm. D (µm /s) Fig 2: Diffusion coefficient distributions of Gβ-GFP (green) and Gγ-GFP(blue) in phlp1-null cells. Aim & Approach Different diffusion characteristics for Gβ-GFP and Gγ-GFP indicate that in phlp1- The aim of the present experiments was to determine if Gβ-GFP and Gγ-GFP in null cells Gγ and Gβ are not associated. The slow diffusion of Gβ in phlp1-null phlp1-null cells were free in the cytoplasm or were interacting with intracellular cells can be due to interaction with an intracellular moiety or aggregation. moieties or aggregating. Using the technique of FCS (fluorescence correlation spectroscopy) the diffusion times of Gβ-GFP and Gγ-GFP have been determined Future Directions in wild type and phlp1-null cells. Gγ-GFP diffusion characteristics will be determined in Gβ-null cells and PRINCIPLE FCS (3): compared to that in phlp1-null cells. Using PCH (5) (Photon Counting Histogram) Autocorrelation the possibility of aggregation of Gβ-GFP in phlp1-null cells will be examined. 1.06 Fluorescence 1.05 function References Intensity 1.04 G (t) 1.03 1.02 1. Parent, C.A. and Devreotes, N.A. (1999) Science 284: 765 – 770 1.01 2. Blaauw, M., Knol, J.C., Kortholt, A., Roelofs, J., Ruchira, Postma, M., Visser, A.J.W.G. Tim e (s) 1 and van Haastert, P.J.M. (2003) EMBO J. 22: 5047 - 5057 0.0 0.1 1.0 10.0 100.0 1000.0 Intensity fluctuations Time (log scale) ms 3. Hess S.T., Huang, S., Heikal, A. A. and Webb, W.W. (2002) Biochemistry 41: 697 - 705 In FCS a small open volume element (<1fL) is created by a focused laser 4. Brock, R., Vamosi, G., Vereb, G. and Jovin, T.M. (1999) Proc. Natl. Acad. Sci. USA 96: beam. Fluctuations in the fluorescence intensity due to diffusion of fluorescent 10123-10128. molecules in and out of this volume are monitored and autocorrelated. The 5. Chen, Y., Muller, J., So, P.T. and Gratton, E. (1999) Biophys. J. 77: 553-567 normalized autocorrelation function provides information about the number of The investigations were supported by the Research Council for Earth and Life Sciences (ALW) with financial aid from the fluorescing particles and their dynamics. Netherlands Organization for Scientific Research (NWO). 1 MicroSpectroscopy Centre, Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands. Telephone: +31 317 484701 e-mail: email@example.com 2 Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.