The complexity of mitochondrial outer membrane permeability and VDAC regulation by associated proteins

Abstract

Previous studies have shown that class II β-tubulin plays a key role in the regulation of oxidative phosphorylation (OXPHOS) in some highly differentiated cells, but its role in malignant cells has remained unclear. To clarify these aspects, we compared the bioenergetic properties of HL-1 murine sarcoma cells, murine neuroblastoma cells (uN2a) and retinoic acid - differentiated N2a cells (dN2a). We examined the expression and possible co-localization of mitochondrial voltage dependent anion channel (VDAC) with hexokinase-2 (HK-2) and βII-tubulin, the role of depolymerized βII-tubuline and the effect of both proteins in the regulation of mitochondrial outer membrane (MOM) permeability. Our data demonstrate that neuroblastoma and sarcoma cells are prone to aerobic glycolysis, which is partially mediated by the presence of VDAC bound HK-2. Microtubule destabilizing (colchicine) and stabilizing (taxol) agents do not affect the MOM permeability for ADP in N2a and HL-1 cells. The obtained results show that βII-tubulin does not regulate the MOM permeability for adenine nucleotides in these cells. HL-1 and NB cells display comparable rates of ADP-activated respiration. It was also found that differentiation enhances the involvement of OXPHOS in N2a cells due to the rise in their mitochondrial reserve capacity. Our data support the view that the alteration of mitochondrial affinity for ADNs is one of the characteristic features of cancer cells. It can be concluded that the binding sites for tubulin and hexokinase within the large intermembrane protein supercomplex Mitochondrial Interactosome, could be different between muscle and cancer cells.

Keywords: Adenylate kinase; Glycolysis; Mitochondria; OXPHOS; Tubulin; Warburg effect.

Fig. 1

Western blot analysis for the presence of total β and βII-tubulin in HL-1 cells (a, b) as well as the levels of free and polymerized total β- and βII-tubulin in these tumor cell line (c); here, lower panel shows the representative immunoblot test for free and polymerized total β and βII tubulin in HL-1 cell. Upper panel shows a densitometric quantification of the total β and βII tubulin in the soluble and insoluble fractions of HL-1 cells. Error bars are the mean ± SE from 3 separate experiments; *p < 0.05 when compared to total β tubulin in HL-1 cells; **p < 0.005 when compared to βII-tubulin in HL-1 cells

Fig. 2

Confocal immunofluorescence imaging of the mitochondrial VDAC1 protein (green), βII-tubulin (red), nucleus (blue) and their colocalization in HL-1 tumor cells; bars are 10 μm

Fig. 3

Western blot (WB) analysis of the expression levels of βI-, βII- and βIII-tubulin in undifferentiated and RA-differentiated N2a cells (a) as well as the representative WB images (b). Error bars are the mean ± SE from 5 independent experiments

Fig. 4

Confocal immunofluorescence imaging of the mitochondrial VDAC1 protein (red), βII-tubulin (green) and their colocalization in undifferentiated (uN2a) and RA-differentiated (dN2a) cells (a) distribution of βI- (red) (b) and βIII-tubulin isoforms (green) (c) in uN2a and dN2a cells. The cell nuclei were stained with DAPI (blue); bars are 10 μm

Fig. 5

The effects of taxol and colchicine on mitochondrial bioenergetics in HL-1 cardiac tumor cells. a Basal respiration – V₀, responses to treatment with 2.5 μM oligomycin, FCCP and antimycin A. b Effects of taxol and colchicine on proton leak, ATP linked respiration, maximal respiratory capacity and mitochondrial respiratory reserve capacity in HL-1 cells compared to control (DMSO treated) cells. Data are shown as mean ± SEM (n = 4). Significance stars depict changes in mitochondrial respiration compared to DMSO treated control cells: * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 6

Confocal microscopy imaging of VDAC1 and hexokinase-2 (HK-2) along with their colocalization in HL-1 cells (a); and colocalization of VDAC1 with HK-2 in undifferentiated N2a (uN2a) and RA-treated N2a cells (dN2a) (b) the cell nucleus (blue, DAPI), HK-2 (red), VDAC1 (green); bars are 10 μM

Fig. 7

Analysis of the coupling of hexokinase (HK) catalyzed processes with OXPHOS in permeabilized HL-1 cells as well as in undifferentiated (uN2a) and RA-treated N2a cells (dN2a). The efficiency of the coupling between HK and OXPHOS was expressed by the glucose index (I_GLU). Here: Vo – basal respiration; glu – glucose; and Cyt c – cytochrome c. All data points are the mean from 5 independent experiments; error bars are SEM. *- significant difference, p < 0.05

Fig. 8

Apparent Km values and rates of maximal (Vm) ADP-activated respiration for HL-1 cells (a) as well as for undifferentiated (uN2a) and RA-treated N2a cells (dN2a) (b); bars are SEM, n = 7