Identification of a novel Bves function: regulation of vesicular transport

Abstract

Blood vessel/epicardial substance (Bves) is a transmembrane protein that influences cell adhesion and motility through unknown mechanisms. We have discovered that Bves directly interacts with VAMP3, a SNARE protein that facilitates vesicular transport and specifically recycles transferrin and beta-1-integrin. Two independent assays document that cells expressing a mutated form of Bves are severely impaired in the recycling of these molecules, a phenotype consistent with disruption of VAMP3 function. Using Morpholino knockdown in Xenopus laevis, we demonstrate that elimination of Bves function specifically inhibits transferrin receptor recycling, and results in gastrulation defects previously reported with impaired integrin-dependent cell movements. Kymographic analysis of Bves-depleted primary and cultured cells reveals severe impairment of cell spreading and adhesion on fibronectin, indicative of disruption of integrin-mediated adhesion. Taken together, these data demonstrate that Bves interacts with VAMP3 and facilitates receptor recycling both in vitro and during early development. Thus, this study establishes a newly identified role for Bves in vesicular transport and reveals a novel, broadly applied mechanism governing SNARE protein function.

Figure 1

Bves and VAMP3 interact. For co-IP (A), COS-7 cells were transfected with tagged proteins, VAMP3–GFP alone or VAMP3–GFP and full-length Bves–myc. Cell lysates were pulled down with myc and blotted for GFP. In GST pull-down assays (B), the C-terminus of Bves was fused to GST (GST–CT) and was used to pull down transfected VAMP3–GFP from cell lysate. Loading controls (lysate in panel A and LC in panel B) are given.

Figure 2

Bves and VAMP3 colocalize in MDCK cells. Both endogenous Bves (A) and transfected VAMP3–GFP (B) are observed at the membrane and in vesicles. The endogenous distribution of both proteins also demonstrates this same localization pattern (Bves, D; VAMP3, E), and Bves and VAMP3 colocalize in merged images (arrows, C, F). Scale bars are 5 μm.

Figure 3

Endogenous Bves and VAMP3 colocalize in muscle. Bves (A, E) and VAMP3 (B, F) are seen at the cell periphery in adult cardiac (A–C) and skeletal muscle (E–G). Areas of intense colocalization are denoted by white arrows (C, G). Red arrows indicate the area of the fluorescent intensity profile for cardiac (D) and skeletal muscle (H). Scale bars are 20 μm.

Figure 4

Transferrin uptake is attenuated when Bves is disrupted. (A) MDCK and Bves118 cells internalized labelled transferrin for 5, 10, or 20 mins. Transferrin uptake, as measured by the MFI, was significantly decreased in Bves118 cells at all time points. (B) When normalized with COMO values (100%), Bves MO- and VAMP3 (V3) MO-treated caps were impaired in internalization of labelled transferrin/μg of total protein. Transferrin uptake is restored when Bves MO or V3 MO are co-injected with rescue Bves (Bves MOR) or VAMP3 (V3 MOR) RNAs.

Figure 5

Cells stably expressing mutated Bves have decreased integrin recycling. A wounded monolayer of MDCK cells (A–C) internalized FITC-labelled β-1-integrin antibody (A, arrows, intracellular labelling) as cells migrated to close the wound. β-1-Integrin recycling is visualized by the presence of FITC-labelled protein in intracellular compartments. In Bves118 cells, integrin recycling was attenuated (D–F), as seen by decreased intracellular punctate labelling, although integrin expression levels of Bves118 (H, lane 2) cells are consistent with those in MDCK cells (H, lane 1). This decrease in integrin internalization is also seen when directly compared with that in WT-TeNT and mut-TeNT cells (G; Supplementary Figure 6). Cells marked by asterisk (C, F) were counted as integrin-positive (G; Table II). Scale bars are 20 μm.

Figure 6

Cell spreading is attenuated with disruption of Bves or VAMP3 function. Time-lapse analysis indicates that cell spreading, or increase of area prior to polarized cell movement, is decreased in Bves118 cells (C) as compared with that in MDCK cells (A). Similarly, WT-TeNT (G) cells have less cell spreading than mut-TeNT cells (E). Kymographs of cell spreading demonstrate the difference in the degree of cell spreading between control and experimental groups (MDCK (B) vs Bves118 (D); and mut-TeNT (F) vs WT-TeNT (H)). Cell areas (I) and percent increase of cell area (J) for experimental groups and control groups as determined from composite kymographs are given (B, D, F, H). Scale bars are 20 μm.

Figure 7

Bves depletion in X. laevis embryos. Blastopore closure in embryos injected with Bves MO was decreased (B) in comparison to embryos injected with COMO (A). The blastopore is outlined in the bottom embryo in panels A and B for better visualization. Anterior defects are observed in Bves-depleted, stage-35 embryos (C), characterized by disrupted eye morphogenesis and ectodermal outgrowths (arrows). Histological staining demonstrates the BCR remains thickened (E, arrow), whereas the BCR of control embryos has thinned (D, arrow). Also, the involuting HM has become detached from the BCR in Bves-depleted embryos (E, asterisk). Integrin levels in Bves MO- or VAMP3 MO-treated embryos are similar to those in COMO-treated embryos (F). In SEM analysis of HM, COMO-injected embryos display a distinct pattern of overlapped and polarized cells (G, I, white line indicates direction of polarity), whereas Bves MO-injected embryos lack directionality, have increased spaces between cells, and exhibit irregular cell shapes (H, J; quantified in Table IV). Panels I and J show magnified views of the boxed areas in panels G and H, respectively.

Figure 8

Bves- and VAMP3-depleted cells display decreased cell adhesion on FN. HM cells stained with phalloidin-568 from COMO-injected embryos (A) display spread morphologies on FN, while Bves-depleted cells (B) are round. Analysis over time (in minutes) indicates that as an mRFP-labelled, COMO-treated cell moves (C, time 0–30) it maintains a spread phenotype, extending several lamellipodia. Conversely, mGFP-labelled, Bves (E, time 0–30) or VAMP3 (G, Time 0–30)-depleted cells become rounded. Kymographs (D, F, H) depict cell morphology over time, demonstrating the differences in cell shape, lamellipodia number, and cell area, which are quantified in graphs in panels I, J, and K. Scale bars are 100 μm (A, B) and 20 μm (C, E, and G).