RalB mobilizes the exocyst to drive cell migration

Abstract

The Ras family GTPases RalA and RalB have been defined as central components of the regulatory machinery supporting tumor initiation and progression. Although it is known that Ral proteins mediate oncogenic Ras signaling and physically and functionally interact with vesicle trafficking machinery, their mechanistic contribution to oncogenic transformation is unknown. Here, we have directly evaluated the relative contribution of Ral proteins and Ral effector pathways to cell motility and directional migration. Through loss-of-function analysis, we find that RalA is not limiting for cell migration in normal mammalian epithelial cells. In contrast, RalB and the Sec6/8 complex or exocyst, an immediate downstream Ral effector complex, are required for vectorial cell motility. RalB expression is required for promoting both exocyst assembly and localization to the leading edge of moving cells. We propose that RalB regulation of exocyst function is required for the coordinated delivery of secretory vesicles to the sites of dynamic plasma membrane expansion that specify directional movement.

FIG. 1.

siRNA specificity and efficiency in NRK cells. NRK cells were transfected with the indicated siRNAs. After 48 h, whole-cell extracts were analyzed by immunoblotting with the indicated antibodies. ERK1/2 is shown as a loading control.

FIG. 2.

RalB is required for cell migration. (A) RalB depletion inhibits wound healing. Confluent monolayers of NRK cells transfected with the indicated siRNA were wounded 36 h posttransfection (T0), and healing was followed over time. T12 and T24 refer to 12 and 24 h postwounding. The panels shown are representative of three independent experiments. (B) Quantitative cell migration assays. NRK cells were transfected as above with the indicated siRNAs. At 48 h posttransfection, cells were delivered to Boyden chambers containing 10% (left) or 0.5% (right) serum. After 20 h, the cultures were fixed and cell migration was assessed by crystal violet staining as described in Materials and Methods. Error bars represent standard deviations from the mean from three independent experiments. Statistically significant differences are indicated by an asterisk (P < 0.05).

FIG. 3.

The exocyst is required for cell migration. (A) Depletion of exocyst subunits inhibits wound healing. Confluent monolayers of NRK cells transfected with the indicated siRNA were wounded (T0), and healing was followed over time as described in the legend to Fig. 2A. Panels shown are representative of three independent experiments. (B) Quantitative cell migration assays. Following transfection with the control siRNAs (left columns) or siRNA depleting Sec5 (middle columns), or RLIP76 (right columns), NRK cell migration in Boyden chambers was assayed as described in the legend to Fig. 2B.

FIG. 4.

RalB is activated upon release of cell-cell contacts and forms a complex with Sec5. (A) Exponentially growing NRK cells were transfected with myc-tagged RalB or myc-tagged-RalA. Forty-eight hours later, confluent monolayers were scratched extensively to generate a large number of cell edges no longer involved in cell-cell contacts. Cell extracts were prepared either immediately (for simplicity, they are labeled I, for immobile) or 1 h later (M, migrating) and assayed for relative amounts of activated RalA and RalB with immobilized gluthathione S-Sepharose-Ral-binding domain (34). (B) Myc-RalB was immunoprecipitated from the same cell extracts derived in the experiment shown in panel A and evaluated for coprecipitation of endogenous Sec5.

FIG. 5.

Migration triggers RalB-dependent stabilization of the exocyst complex. (A) Migration promotes the exocyst assembly. Monolayers of NRK cells were scratched to liberate cell edges as shown in Fig. 4. Endogenous Sec8 was immunoprecipitated from cell extracts prepared immediately (I, for immobile) or 3 h after wounding (M, migrating) and assessed for coprecipitation of endogenous Sec5 and Sec10. A representative experiment of three independent experiments is shown. (B) The amount of Sec10 and Sec5 recovered in the immunoprecipitates was quantified with a Luminescent Image analyzer and normalized for the amount of Sec8. Each experiment was repeated three times. The amount of Sec5 and Sec10 associated with Sec8 in immobile cells is defined as 100%. (C) Migration-induced exocyst assembly is RalB dependent. NRK cells were transfected with control siRNAs (targeting luciferase; siLuc), or siRNA against RalA or RalB (siRalA or siRalB), and exocyst assembly was evaluated as in described in the legend to panel A. (D) Quantitative analysis of exocyst assembly was performed as described for panel B. The amount of Sec5 and Sec10 associated with Sec8 in siLuc-transfected cells is defined as 100%. (E) Exocyst complex components were immunodetected in lysates of cells depleted of RalA or RalB by siRNA. siLuc was used as a siRNA control. Actin was used as a loading control.

FIG. 6.

RalB recruits Sec6 and Exo70 to the leading edge of motile cells. Monolayers of NRK cells were wounded 36 h posttransfection with the indicated siRNAs. Cells were fixed 3 h after wounding, and the indicated proteins were detected with specific antibodies. F-actin was visualized with FITC-conjugated phalloidin. Exo70 (A) and Sec6 (B) can be observed recruited at the leading edge, as opposed to two other proteins resident on membranes, Rab6 and the transferring receptor (C). Arrowheads indicate enrichment of Exo70 or Sec6 at the leading edge of the plasma membrane.

FIG. 7.

Golgi disruption blocks migration but not Exocyst recruitment. (A) NRK cells were wounded in the presence of increasing concentrations of brefeldin A, and the consequences on cell migration were monitored as described in the legend to Fig. 2. Experiments were performed on collagen where migration was faster than on plastic to avoid extended exposure to BFA. (B) Cells were treated as in described for panel A. Three hours postwounding, cells were fixed and stained with the indicated antibodies.