Rab11-FIP3 and FIP4 interact with Arf6 and the exocyst to control membrane traffic in cytokinesis

Abstract

The dual Rab11/Arf binding proteins, family of Rab11-interacting proteins FIP3 and FIP4 function in the delivery of recycling endosomes to the cleavage furrow and are, together with Rab11, essential for completion of abscission, the terminal step of cytokinesis. Here, we report that both FIP3 and FIP4 bind Arf6 in a nucleotide-dependent manner but exhibit differential affinities for Rab11 and Arf6. Both FIP3 and FIP4 can form ternary complexes with Rab11 and Arf6. Arf6 is localised to the furrow and midbody and we show that Arf6-GTP functions to localise FIP3 and FIP4 to midbodies during cytokinesis. Exo70p, a component of the Exocyst complex, also localises to the furrow of dividing cells and interacts with Arf6. We show that depletion of Exo70p leads to cytokinesis failure and an impairment of FIP3 and Rab11 localisation to the furrow and midbody. Moreover, Exo70p co-immunoprecipitates FIP3 and FIP4. Hence, we propose that FIP3 and FIP4 serve to couple Rab11-positive vesicle traffic from recycling endosomes to the cleavage furrow/midbody where they are tethered prior to fusion events via interactions with Arf6 and the Exocyst.

Figure 1

FIP3 binds Arf6 and Rab11. The binding characteristics of FIP3 were examined in a series of in vitro experiments. The C-terminus of FIP3 (residues 300–759) or Rip11 (residues 490–652) were expressed in bacteria as hexa–his fusions and incubated with 50 μl of beads loaded with 5 μg GST alone, or GST-Rabs/Arfs as indicated for 1 h. After washing, the beads were boiled in SDS–PAGE sample buffer and levels of associated FIP3 or Rip11 determined by immunoblotting. Data from a representative experiment is shown in (A). In the experiment shown in (B). increasing concentrations of FIP3-containing beads were incubated with 5 μg of Arf5, Arf6 or Rab11, and the binding determined by quantitative immunoblotting. The data shown is from three experiments of this type (mean±s.d.). (C) HeLa cells were transfected with HA-tagged Arf1, Arf5 or Arf6 as described. After 48 h, cells were lysed and an anti-HA immunoprecipitation performed. The immunoprecipitates were probed for the presence of endogenous FIP3. The result of a typical experiment is shown. In the experiment shown in (D), 50 μl of beads loaded with 5 μg of GST or GST-Arf6 were incubated with recombinant FIP3 in the presence of increasing amounts of Rab11. The ability of Rab11 to displace FIP3 from the Arf6-loaded beads was determined by immunoblotting. The experiment shown is typical of three experiments of this type. In order to determine whether FIP3 can form a ternary complex with Arf6 and Rab11, we performed the experiment shown in (E). GST-Arf6 was incubated with recombinant FIP3 (residues 300–759) or Rip11 (residues 490–652). The upper panel shows a Coomassie stain, revealing the interaction of FIP3 with Arf6, but not Rip11. These beads were incubated with recombinant Rab11, washed and then boiled in SDS–PAGE buffer and immunoblotted for Rab11 (middle panel) or Rip11 (lower panel).

Figure 2

FIP4 has distinct GTPase preferences. Increasing concentrations of FIP4-containing beads (residues 227–557) were incubated with 5 μg of Arf5, Arf6 or Rab11, and the binding determined by quantitative immunoblotting. The data shown in (A) is from three experiments of this type, with the mean±s.d. shown. In order to determine whether FIP4 can form a ternary complex with Arf6 and Rab11, we performed the experiment shown in (B). GST-Arf6 was incubated with bacterially expressed FIP4 (residues 227–557) or Rip11 (residues 490–652). The upper panel shows a Coomassie stain demonstrating equivalent loads of Arf6 in each of the three conditions; the presence of FIP4 was demonstrated by immunoblotting (middle panel). These beads were incubated with bacterially expressed Rab11, washed and then boiled in SDS–PAGE buffer and immunoblotted for Rab 11 (lower panel). As shown, Rab11 was only recovered in the GST-Arf6 beads in the presence of FIP4. (C) GST, GST-Arf6 or GST-Arf6-T44N expressed in bacteria were loaded on glutathione–sepharose and incubated with either bacterially expressed FIP3 (upper panel) or FIP4 expressed in CHO cells (lower panel). The beads were washed and boiled in SDS–PAGE buffer and immunoblotted for FIP3 or FIP4 as indicated. Start refers to the starting material (100 ng of FIP3, or 50% of the start volume of cell lysate for FIP4). Data are representative of three experiments of this kind. (D) HeLa cells were transfected with HA-tagged Arf1, Arf5 or Arf6 as described. After 48 h, cells were lysed and an anti-HA immunoprecipitation performed. The immunoprecipitates were probed for the presence of endogenous FIP4. The result of a typical experiment is shown.

Figure 3

The Rab11 and Arf6 binding sites are spatially distinct. Beads loaded with GST, GST-FIP3 or GST-FIP3-I737E were incubated with either Rab11 or Arf6 as described. After incubation, the beads were washed and boiled in SDS–PAGE buffer and subjected to immunoblot analysis with anti-Arf6 (middle panel) or anti-Rab11 (lower panel). Similar data using GST-FIP4 constructs are shown in the adjacent panel.

Figure 4

The distribution of Arf6 during the cell cycle. HeLa cells were plated onto glass coverslips and 12 h later fixed and stained with anti-Arf6 antibodies. Shown are representative images at different stages of the cell cycle. Arf6 staining is pseudo-coloured green, DNA is pseudo-coloured blue.

Figure 5

Arf6 controls midbody localisation of FIP3 and FIP4. (A) HeLa cell were cotransfected with GFP-FIP3 (green) and HA-Arf6, HA-Arf6-Q67L or HA-Arf6-T27N (red). Cells were fixed and imaged. Yellow represents the degree of the overlap. Note that expression of Arf6-Q67L results in a marked accumulation of FIP3 at the midbody, and that 79% of cells expressing Arf6-T27N exhibited no FIP3 in the midbody. (B) HeLa cells stably expressing GFP-FIP3 were transfected with either wild-type Arf6 or Arf6-Q67L. Shown are representative images of the FIP3 localisation in the furrow/midbody regions of such cells. Note that for clarity, the HA staining is not shown in panels B–D. (C) As panel A, except cells were stained for endogenous Rab11 distribution. (D) As panel A, except GFP-FIP4 was used. Note that FIP4 accumulation in the midbodies was abrogated by overexpression of Arf6-T27N. In all, >80% of cells expressing Arf6-T27N exhibited little or no FIP4 staining in the midbody.

Figure 6

Real-time analysis of Rab11, Arf6 and FIP3 movement to the furrow and midbody. HeLa cells stably expressing either FIP3-YFP/CFP-Rab11 (A) or FIP3-YFP/Arf6-CFP (B) were plated on collagen-coated coverslips and imaged using time-lapse microscopy (see Supplementary movies 1 and 2). Images were collected every 1.4 min for 2.5 h. Note that Rab11 always colocalizes with FIP3, while Arf6 appears to be recruited directly to the midbody (asterisk). FIP3 at the midbody is a small fraction of the total cellular FIP3, and is not clearly evident in these nonconfocal images.

Figure 7

FIP4 exhibits a similar distribution to Arf6 during the cell cycle. Cells plated as outlined in Figure 5 were stained for FIP4 (red) or DNA (blue). The images shown are DeltaVision reconstructions and are typical of many images of this type. Scale bar 5 μm.

Figure 8

Exo70p interacts with FIPs and is required for cytokinesis. (A) Representative fields of HeLa cells stained with antibodies against Exo70 (green) and α-tubulin (red). DNA is stained using DAPI (blue). (B) HeLa cells were transfected with either siRNA designed to knock down Exo70p or a scrambled siRNA, and lysates prepared after 48 h. Lysates were immunoblotted with antibodies specific for Exo70p, FIP3 or Rab11, and a representative experiment is shown. (C) Representative fields of HeLa cells transfected with either mock or Exo70 siRNA. At 48 h after transfection, cells were immunostained with anti-Exo70 antibodies (green) and DAPI (blue). (D) Quantification of the effect of Exo70 RNAi on levels of binucleate cells. n refers to the numbers of cell counted and compares cells transfected with Exo70p siRNA, scrambled siRNA or mock-transfected cells. The difference between Exo70p siRNA-treated cells and both control groups is significant (P<0.05). (E–F) Lysates of CHO cells expressing either GFP-FIP3 (E) or GFP-FIP4 (F) were used in an experiment to determine whether antibodies specific for Exo70 or random IgG can co-immunoprecipitate FIP3 or FIP4. Shown are representative experiments in which an aliquot of cell lysate, and the immunoprecipitated material were immunoblotted for FIP3 or FIP4. Data shown are representative of three experiments of this type.

Figure 9

Exo70p depletion results in mislocalisation of FIP3 and Rab11 in the furrow. (Top panels) Representative fields of HeLa cells transfected with either scrambled or Exo70 siRNA. At 48 h after transfection, cells were immunostained with anti-FIP3 (green) or anti-Exo70p (red) antibodies. The bright field image at the right shows the presence of the furrow and midbody. (Bottom panels) Representative field of HeLa cells transfected with Exo70p siRNA and immunostained for FIP3 (green) or Rab11 (red) (cf. bright field image at right). The asterisk indicates the position of the midbody. For comparison, a cell transfected with a scrambled siRNA and stained for Rab11 is shown at the left.

Figure 10

A model for FIP function in cytokinesis. (1) Rab11 is activated by GTP loading, which serves to recruit FIP3 to vesicles derived from recycling endosomes in the region of the centrosome. (2) These vesicles move along microtubules into the furrow (or later midbody) via interaction with a motor protein. (3) In the furrow, the FIP3/Rab11/vesicle encounters active Arf6, perhaps localised within microdomains on the plasma membrane; the interaction between Rab11/FIP3/Arf6 may serve to tether the vesicle to the plasma membrane via interaction with the Exocyst complex. (4) In the midbody at late stages of cytokinesis (represented here by a dashed line), we suggest that clusters of FIP3 vesicles may become tethered at the midbody ring just prior to abscission. See text for details.