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A conserved C-terminal domain of EFA6-family ARF6-guanine nucleotide exchange factors induces lengthening of microvilli-like membrane protrusions

Valérie Derrien^1,*, Carole Couillault^2,*, Michel Franco³, Stéphanie Martineau², Philippe Montcourrier⁴, Rémi Houlgatte² and Philippe Chavrier^1,

¹ Laboratoire de la Dynamique de la Membrane et du Cytosquelette, UMR 144, Centre National de la Recherche Scientifique, Institut Curie, Section Recherche. 26 rue d'Ulm, 75241 Paris Cedex 5, France
² Centre d'Immunologie INSERM/CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France
³ Institut de Pharmacologie Moléculaire et Cellulaire, UPR411, CNRS, 660 route des Lucioles, Sophia-Antipolis, 06650 Valbonne, France
⁴ CNRS UMR 5539, Université Montpellier II, 34095 Montpellier Cedex 5, France
^* These authors contributed equally

View larger version (60K): [in a new window]   Fig. 1. A family of evolutionary conserved EFA6-related proteins. (A) Predicted amino acid sequence of EFA6B. Regions corresponding to the Sec7 domain, the PH domain and the putative coiled-coil motif at the C-terminus are highlighted in yellow, green and purple, respectively. The predicted coiled-coil motif was identified using COILS (Lupas et al., 1991). (B) The Sec7 domains of human ARNO (Chardin et al., 1996), EFA6A (Perletti et al., 1997), EFA6C [AL136559 (Wiemann et al., 2001)], EFA6B (this study), EFA6D [KIAA0942 (Nagase et al., 1999)], ARF-GEP100 (Someya et al., 2001), D. melanogaster CG6941, C. elegans Y55D9A.1 and S. cerevisiae Sec7 (Achstetter et al., 1988) were aligned using Clustal W (Thompson et al., 1994). The 10 helices (denoted A-J) of the ARNO Sec7 domain crystal structure are shown (Cherfils et al., 1998; Goldberg, 1998). Invariant and conserved residues are highlighted in green and yellow, respectively. Two highly conserved regions that together form the active site are underlined and denoted Motif 1 and Motif 2. The invariant glutamate residue in Motif 1 is highlighted in blue. Conserved residues in EFA6 family proteins are highlighted in purple, whereas conserved residues in other ARF GEFs that diverge in EFA6 family members are shown in red. (C) Sec7 domains were aligned using Clustal W and a phylogenetic-type tree was calculated from the alignment using NJplot (Perriere and Gouy, 1996; Thompson et al., 1994).

View larger version (41K): [in a new window]   Fig. 9. Overall organization of EFA6 family GEFs. Alignment of the Sec7 domain, PH domain and C-terminal region sequences. (A) A schematic structure of the EFA6 family and ARNO GEFs. Sec7 domains, PH domains and putative coiled-coil motifs are depicted by red, blue and yellow boxes, respectively. (B) Amino-acid sequences of the PH domains of human EFA6 family members, D. melanogaster CG6941 gene product, C. elegans Y55D9A. 1 and human beta IV 1 spectrin (Berghs et al., 2000) were aligned using Clustal W. Conserved residues in the seven PH domains are highlighted in green. Residues of the spectrin PH domain involved in interactions with inositol (1,4,5)P3 phosphates (Hyvonen et al., 1995), and which are conserved in EFA6 family PH domains, are shown in bold. Invariant or similar residues amongst EFA6 family PH domains are highlighted in blue. (C) Amino-acid sequences of the conserved C-terminal regions of EFA6-family GEFs were aligned using Clustal W. Invariant residues are highlighted in green. Putative coil-coiled motifs identified using COILS are underlined. The terminus of C-terminal deletion variants of EFA6A (designed CT1 to 4) is indicated by an exclamation mark.

View larger version (60K): [in a new window]   Fig. 2. mRNA and protein expression analysis of EFA6-like genes. Poly(A)+ RNA from the indicated human tissues was hybridised with probes derived from EFA6A (A), EFA6B (B) and EFA6C (C) cDNAs. The blots were also probed with ß-actin cDNA to ensure that loading was equal between the different tissues (data not shown). H, heart; B, brain; P, placenta; L, lung; Li, liver; S, skeletal muscle; K, kidney; Pa, pancreas; Sp, spleen; T, thymus; Pr, prostate; Te, testis; O, ovary; Si, small intestine; C, colon; PB, peripheral blood. D shows immunoprecipitations from lysates of Jurkat human T lymphocytes (lanes 1 and 5), BHK cells (lanes 2 and 6) or BHK cells overexpressing N-terminally VSV-G-tagged EFA6B (lanes 3, 4, 7) using anti-EFA6B (lanes 1-3), anti VSV-G tag (lane 4) or anti-Sec10 irrelevant antibodies (lanes 5-7). EFA6B and VSVG-EFA6B in the immunoprecipitates were detected with anti-EFA6B antibodies (lanes 1-3 and 5-7). Alternatively, VSVG-EFA6B was detected with an anti-VSVG antibody (lane 4). EFA6B (predicted molecular weight 116.34 kDa) migrates as a 180 kDa species in SDS-PAGE (arrows). Asterisks denote heavy (H) and light (L) immunoglobulin chains.

View larger version (18K): [in a new window]   Fig. 3. Measurement of [35S]GTPS binding to myristoylated ARF6 (mARF6) (A) or mARF1 (B) in the absence ([UNK]) or presence of purified recombinant EFA6A () or EFA6B/245-1056 (). Spontaneous GTPS binding on mARF1 in low free Mg2+ concentration (1 µM) ().

View larger version (130K): [in a new window]   Fig. 4. EFA6A and EFA6B colocalise with ARF6 in microvilli-like structures at the plasma membrane. (A-D) Localization of C-terminally HA-tagged ARF6 and EGFP-EFA6A (A,B), or -EFA6B (C,D) in BHK cells. Micrographs show that surface microvilli-like structures are enriched in ARF6 and EFA6A or EFA6B (arrows). The focus was set at the level of the dorsal plasma membrane. Bar, 10 µm.

View larger version (115K): [in a new window]   Fig. 5. Dynamics of EFA6A ruffles and microvilli-like protrusions. (A) BHK cell expressing EGFP-EFA6A at the start of a time-lapse series. The box indicates the region of focus displayed as frames in B. Epifluorescence frames were obtained every 30 seconds. The arrows point to a ruffle that persisted during the entire series. Double-head arrows and arrowheads point to ruffles that formed or collapsed during the series, respectively.

View larger version (86K): [in a new window]   Fig. 6. Effect of EFA6A and EFA6B variants on cortical actin cytoskeleton morphology. BHK cells were transfected with EGFP-tagged EFA6A (A-C); EFA6A variants corresponding to the isolated PH domain (EFA6A-PH, D-F); the C-terminal region including the PH domain (EFA6A-PHCTer, G-I); the C-terminal region with the PH domain deleted (EFA6A-CTer, J-L), EFA6B (M-O); or the C-terminal region and adjacent PH domain of EFA6B (P-R). After fixation, cells were stained with Texas-Red-conjugated phalloidin to visualize actin filaments (B,E,H,K,N,Q). Localization of EGFP constructs is shown in panels A,D,G,J,M,P. Panels C,F,I,L,O,R show the superimposed images (EGFP constructs are show in green, and F-actin is shown in red). Confocal sections were taken in the dorsal plane of the cells. Bar, 10 µm.

View larger version (169K): [in a new window]   Fig. 7. Scanning EM analysis of microvilli-like protrusions in BHK cells expressing EFA6 or EFA6-PHCTer. BHK cells were transfected to express a CD25-derived transmembrane protein (see Materials and Methods) alone (A,B) or together with EFA6A (C,D) or EFA6A-PHCTer (E,F). Transfected cells were identified using anti-CD25-coated latex beads. EFA6A-expressing cells exhibited microvilli with a comparable length but the microvilli were present at a higher density than in control cells. EFA6A-PHCTer-expressing cells show a `hedgehog-like' morphology with numerous, long finger-like structures covering the cell surface. The arrows point at lengthened microvilli structures laying down on the surface of EFA6A-PHCTer-expressing cells. Double-head arrows indicate smaller erected microvilli-like protrusions. Bars, 2 µm.

View larger version (108K): [in a new window]   Fig. 8. Localization of EGFP-EFA6A-PHCT1 (A), PHCT2 (B), PHCT3 (C) and PHCT4 (D) in BHK cells. (These constructs, which include the PH domain, start at position 349 and their C-terminus is indicated, see also Fig. 9C). Expression of PHCT1-3 constructs induced the formation of finger-like protrusions on the dorsal cell surface. By contrast, deletion of the conserved C-terminal coiled-coil motif in PHCT4 prevented the induction of these structures, and this construct accumulated in short microvilli-like protrusions. Confocal sections were taken in the dorsal plane of the cells. Bar, 10 µm.