The Large GTPase Guanylate-Binding Protein-1 (GBP-1) Promotes Mitochondrial Fission in Glioblastoma

Abstract

Glioblastomas (aka Glioblastoma multiformes (GBMs)) are the most deadly of the adult brain tumors. Even with aggressive treatment, the prognosis is extremely poor. The large GTPase Guanylate-Binding Protein-1 (GBP-1) contributes to the poor prognosis of GBM by promoting migration and invasion. GBP-1 is substantially localized to the cytosolic side of the outer membrane of mitochondria in GBM cells. Because mitochondrial dynamics, particularly mitochondrial fission, can drive cell migration and invasion, the potential interactions between GBP-1 and mitochondrial dynamin-related protein 1 (Drp1) were explored. Drp1 is the major driver of mitochondrial fission. While GBP-1 and Drp1 both had punctate distributions within the cytoplasm and localized to regions of the cytoplasmic side of the plasma membrane of GBM cells, the proteins were only molecularly co-localized at the mitochondria. Subcellular fractionation showed that the presence of elevated GBP-1 promoted the movement of Drp1 from the cytosol to the mitochondria. The migration of U251 cells treated with the Drp1 inhibitor, Mdivi-1, was less inhibited in the cells with elevated GBP-1. Elevated GBP-1 in GBM cells resulted in shorter and wider mitochondria, most likely from mitochondrial fission. Mitochondrial fission can drive several important cellular processes, including cell migration, invasion, and metastasis.

Keywords: Dynamin-like Proteins (DLPs); Epidermal Growth Factor Receptor (EGFR); Guanylate-Binding Protein-1 (GBP-1); Translocase of Outer Mitochondrial Membrane 40 (TOMM40); glioblastoma multiforme (GBM); immunofluorescence; mitochondrial dynamin-related protein 1 (Drp1).

Figure 1

GBP-1 localizes to mitochondria in SNB 75 glioblastoma cells by indirect immunofluorescence. (A) GBM cell lines were plated overnight and then serum-starved for 24 h. Cells were either left untreated or treated with 50 ng/mL hEGF or 500 U/mL of hIFN-γ for 24 h. Cell lysates (20 µg) were separated by 8% SDS-PAGE, followed by western blot (WB) for GBP-1 and actin. (B) WB of SNB75 is shown with a lane for MMP-1 expression. (C) SNB75 cells were analyzed by triple-label IF to determine the intracellular location of GBP-1. After incubating with antibodies against GBP-1, TOMM40, and cytochrome c, cells were incubated with appropriate highly cross-adsorbed secondaries, mounted, and images were capture by confocal microscopy at 0.2 µm optical sections. Size bars = 25 µm. (D) SNB75 cells were fractionated into mitochondrial (M) and cytosolic (C) fractions and analyzed for GBP-1 and TOMM40 by WB. Total cell lysates (T) were also provided. (E) SNB75 cells were analyzed by multiphoton confocal microscopy. The image series from one z-stack is shown in Figure 1C. SNB75 images were compiled and analyzed by 3D reconstruction. Visualization of the image from the side shows that the TOMM40 signal (green) for the outer mitochondrial membrane is surrounded by the signal for GBP-1. This indicates that GBP-1 is on the outside of the outer mitochondrial membrane.

Figure 2

GBP-1 localizes to mitochondria in U251 cells. (A) U251 cells were generated to express myc-tagged GBP-1. Control cells (empty vector) and two pools of cells expressing myc-tagged GBP-1 were analyzed by WB for GBP-1 expression. (B) U251+GBP-1 (D4) cells were analyzed by triple-label IF for the localization of GBP-1 using antibodies against myc, TOMM40, and cytochrome c. The panel on the far right shows the overlay of the region of the photomicrograph designated by the box in the cytochrome c panel. Size bar = 20 µm. (C) U251+GBP-1 cells were analyzed by multiphoton confocal microscopy for the expression of TOMM40 and GBP-1. The images were compiled and analyzed by 3D reconstruction. Visualization of the image from the side suggests that the TOMM40 signal (green) for the outer mitochondrial membrane is surrounded by GBP-1 (red). This suggests that GBP-1 is on the outside of the outer mitochondrial membrane.

Figure 3

GBP-1 induces the translocation of Drp1 to the mitochondria. (A) Control (empty vector) and myc GBP-1-expressing U251 cells (D4) were fractionated into mitochondrial (M) and cytosolic fractions (C) and analyzed for Drp1 and TOMM40 by WB. Total cell lysates (T) are also provided. (B) SNB75 cells were fractionated into mitochondrial (M) and cytosolic fractions (C) and analyzed for Drp1 and TOMM40 by WB. The numbers under the blots are the percentage of the protein in each fraction. (C) Control U251 and U251+GBP-1 (D4) cells were plated on coverslips ON and then analyzed for the expressions of GBP-1, Drp1, and TOMM40 by indirect IF. Two representative examples of each cell type are shown.

Figure 4

Drp1 colocalizes with GBP-1 at GBM mitochondria. U251 cells expressing myc GBP-1 (D4) were analyzed for the intracellular location of Drp1 (A), GBP-1 (B), and TOMM40 (C) by triple-label IF. Images were captured at 0.2 µm z-sections. (D) The images of Drp1 and GBP-1 staining are overlayed. The white box delineates the region amplified in (D1). (E) The images of GBP-1 and TOMM40 are overlayed and the white box delineates the region amplified in (E1). (F) The images of Drp1 and TOMM40 are overlayed and the white boxes delineate regions amplified in (F1,F2). (G) The overlay of Drp1, GBP-1 and TOMM40 is shown. The white box delineates the region amplified in (G1).

Figure 5

GBP-1 shortens glioblastoma cell mitochondria. U251 cells with (GBP-1) or without (control) myc-epitope-tagged GBP-1 were stained for TOMM40 and analyzed by confocal microscopy at 0.2 µm optical sections. (A) Representative examples of mitochondrial morphology are presented. Size bar = 10 µm. (B) The lengths of individual mitochondria were measured as described in Methods. (C) The width of individual mitochondria was measured as described. (D) The elongation index of individual mitochondria was calculated by dividing the length by the width. (n = 2; * = p < 0.05; *** = p < 0.001).