Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression

Figure 1. Kv1.3 participates in the proliferation of preadipocytes. 3T3-L1 preadipocytes express Kv1.3, and genetic ablation of the channel alters cell proliferation. (A) Representative immunofluorescence confocal image of Kv1.3 in 3T3-L1 preadipocytes. The scale bar represents 20 µm. (B) Kv1.3 silencing in 3T3-L1 preadipocytes. Cells were infected with Kv1.3 shRNA (Kv1.3KD) or scramble shRNA (SCR) lentivirus. β-actin was used as a loading control. Noninfected 3T3-L1 cells were called wild-type (WT) cells. (C) Quantification of the efficiency of Kv1.3 silencing. The data are the mean ± SE (n ≥ 3). * p < 0.05, ** p < 0.01 (one-way ANOVA and post hoc Tukey test for multiple comparisons). (D) Cells were trypsinized, and their size was analyzed with an automatic cell counter. Kv1.3 shRNA (KD). The data are the mean ± SE (n = 16). *** p < 0.001 (Student’s t-test). (E) Representative bright field images showing 3T3-L1 preadipocytes. An equal number of cells were seeded and cultured in the absence of growth factors for 24 h. Cells were imaged at 0 and 48 h after the addition of 10% NCS. Note the lower confluence achieved after 48 h by Kv1.3KD preadipocytes than wild-type preadipocytes. (F) The…

Figure 2. Kv1.3 facilitates the G1/S transition of the cell cycle in preadipocytes. Serum-starved resting cells were incubated for the indicated time after serum readdition. (A) Cell cycle analysis of 3T3-L1 preadipocytes was performed with propidium iodide. Representative histograms at 0, 6, 12, 18 or 24 h after serum readdition. The cells exhibit two blue peaks corresponding to the G0/G1 (left) and G2 (right) phases. The cell population in purple corresponds to cells in the S phase. Left panels, wild-type preadipocytes; right panels, Kv1.3KD preadipocytes. (B) The % of cells in the G0/G1 phase, % of cells in the S phase and % of cells in the G2 phase for wild-type (black) and Kv1.3KD (white) preadipocytes. The data are the mean ± SE (n = 4–10 independent experiments). Two-way ANOVA indicated p < 0.001 in the G0/G1 phase and p < 0.01 in the S phase between wild-type and Kv1.3KD preadipocytes. (C) protein expression of Kv1.3, cyclin E, cyclin D1 and cyclin A. (D) Relative protein expression from the data in C. The values were relative to 0 h for each condition and normalized to the level of β-actin, which was used as a loading control (wild-type, black columns; Kv1.3KD, white columns). The…

Figure 3. Kv1.3 accumulates at perinuclear mitochondria during the G1/S transition. (A) Subcellular fractionation of 3T3-L1 wild-type preadipocytes to obtain the membranous (Mb) and mitochondrial (Mit) fractions. The samples were probed for Kv1.3, Na+/K+ ATPase (a membrane marker) and TIMM50 (a mitochondrial marker). (B) Electron micrograph showing mitochondria of 3T3-L1 wild-type preadipocytes. Kv1.3 was labeled with 18 nm immunogold particles (black arrowhead) and was located at the inner mitochondrial membrane. The scale bar represents 200 nm. (C–H) Cells were either in the G0/G1 or the G1/S phase following serum deprivation or serum readdition for 12 h, respectively. Representative confocal images showing Kv1.3 and mitochondria in wild-type preadipocytes fixed in the G0/G1 (C–E) and G1/S (F–H) phase. Ea-Eb and Ha-Hb are magnified images of E and H, respectively. Ea and Ha show distal regions, and Eb and Hb show perinuclear regions. Yellow indicates colocalization of Kv1.3 (green) and mitochondria (red). The scale bar represents 20 µm. (I) Pearson’s coefficient of colocalization between Kv1.3 and mitochondria. The data are the mean ± SE (n > 30). *** p < 0.001. (J,K) Bull’s eye analysis…

Figure 4. Kv1.3 regulates the mitochondrial fusion/fission equilibrium during the G1/S transition. Confocal images showing mitochondria in cells fixed in the G0/G1 (A–F) and G1/S (G–L) phase for WT (A–C,G–I) and Kv1.3KD preadipocytes (D–F,J–L). The scale bar represents 20 µm. Images were processed (tubeness and skeleton) to perform morphometric analysis of mitochondria. (M) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. (N) Number of mitochondrial particles per µm2. (O) The form factor describes the particle shape complexity and was computed as the average (perimeter)2/(4π·area). A circle corresponds to a minimum value of 1. The data are the mean ± SE (n > 30). *, p < 0.05; **, p < 0.01 (one-way ANOVA). (P) Protein expression of Kv1.3, dynamin-related protein 1 (Drp1) and mitofusin 2 (Mtf2) during the G0/G1 and G1/S phase in WT and Kv1.3KD 3T3-L1 preadipocytes. β-actin was used as a loading control. (Q,R) Electron micrographs showing mitochondria of WT (Q) and Kv1.3KD (R) 3T3-L1 preadipocytes during the G1/S phase. Note that, unlike those in Kv1.3KD, mitochondria in wild-type cells were highly fused.

Figure 5. Kv1.3 regulates the mitochondrial membrane potential during the cell cycle. Ablation of Kv1.3 impairs the mitochondrial membrane potential. (A) TMRM intensity in wild-type (black bar) and Kv1.3KD (white bar) 3T3-L1 preadipocytes was analyzed with flow cytometry. The data are the mean ± SE (n = 3), * p < 0.05 (Student’s t-test). (B) TMRM intensity, as determined by confocal imaging. The data are the mean ± SE (n > 30 cells), ***, p < 0.001 (Student’s t-test). (C–F) Representative confocal images showing MitoTracker and TMRM intensity in wild-type (C,D) and Kv1.3KD (E,F) cells. MitoTracker green (C–E) was used to create a mask to detect mitochondrial particles, and the mean intensity was analyzed in the TMRM channel (D–F). The color scale on the right represents TMRM intensity values. The scale bar represents 20 µm. Bottom panels represent 3× magnification of the insets in D and F. The white insets are magnified images of the regions at the bottom.

Figure 6. Representative cartoon summarizing the participation of the mitochondrial Kv1.3 (mitoKv1.3) in the proliferation of preadipocytes. Kv1.3 would facilitate the G1/S transition of the cell cycle in preadipocytes accumulating at perinuclear mitochondria. The elucidation of a putative mitochondrial-nuclear communication during this phase of the cell cycle in which Kv1.3 would participate deserves much effort. During the G1/S transition, Kv1.3 would contribute to the mitochondrial fusion/fission equilibrium controlling the mitochondrial membrane potential. Ablation of Kv1.3 (Kv1.3KD) would impair mitochondrial dynamics during cell cycle progression. Kv1.3KD, 3T3-L1 preadipocytes, with a genetic ablation of Kv1.3. Green dots, Kv1.3 channels; magenta, mitochondrial network.