Sec5 and Exo84 mediate distinct aspects of RalA-dependent cell polarization

Abstract

Metastasis is a complex process during which several gross cellular changes occur. Cells must dissociate from the tumor mass and gain the ability to degrade extracellular matrix and migrate in order to ultimately attach and form a satellite tumor. Regulation of the actin cytoskeleton is an indispensible aspect of cell migration, and many different factors have been implicated in this process. We identified interactions between RalA and its effectors in the Exocyst complex as directly necessary for migration and invasion of prostate cancer tumor cells. Blocking RalA-Exocyst binding caused significant morphological changes and defects in single and coordinated cell migration.

Figure 1. RalA-Exocyst interactions are required for Matrigel invasion.

The invasion index of indicated cell types was determined and normalized to invasion of parental PC-3 cells. Invasion was determined by average # Matrigel invaded cells/average # migrated cells. Asterices, p<0.0001.

Figure 2. Coordinated cell movement is dependent on RalA-Exocyst interactions.

Confluent monolayers of indicated cell types were scratched and images were collected either (A) 20 and 40 hours, or (B) 24 and 48 hours later.

Figure 3. RalA-Sec5 and -Exo84 interactions differentially affect velocity and persistence of single cell migration.

(A) Colloidal gold-coated coverslips were stained with crystal violet after allowing random migration of indicated cell types at low density. (B) Time-lapse photography was used to determine velocities of individual migrating cells. For each cell type, 5 cells from 4 fields of view were quantified during a 3-hour period. (C) From time-lapse photography images, distances between cell positions at the start and end of the in the 3-hour period were quantified and used to determine directional persistence. Directional persistence is defined as total distance migrated/net displacement. Asterices, p<0.0001.

Figure 4. Cell morphology and Exocyst localization is differentially affected by uncoupling RalA from Sec5 or Exo84.

(A) Indicated cell lines were labeled for phalloidin (f-actin). (B) The long and short axes of 50 cells of each type were measured and plotted. (C) Indicated cell lines were labeled with phalloidin (f-actin, green) and Sec15 (red). Representative images are shown. Bars, 10 µm. Asterices, p<0.0001.

Figure 5. Mutant Sec5 capable of binding mutant RalA rescues morphological and functional defects.

(A) RalA^38R cells were labeled with phalloidin (green) and Sec15 (red) and RalA^38R cells co-expressing GFP and Sec5^27E were labeled with Sec15 (red). (B) Matrigel invasion of RalA mutant cells alone and co-expressing Sec5^27E. Bars, 20 µm. Asterices, p<0.0001.

Figure 6. RalA-Exocyst interactions affect Rac1 activation through SH3BP1 localization.

(A) Active Rac1 was isolated with GST-Pac binding domain bound to glutathione beads. Samples were visualized by western blot in triplicate and normalized to total Rac1 and expressed as percent of total Rac1. (B) PC-3 parental cells were transfected with mCherry-SH3BP1 only or together with myc-RalA^72L, RalA^38R, or RalA^47E and labeled with antibodies specific for myc. Asterisk, p<0.05. Bar, 20 µm.