Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient - PubMed (original) (raw)
Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient
Christian A Wurm et al. Proc Natl Acad Sci U S A. 2011.
Abstract
The translocase of the mitochondrial outer membrane (TOM) complex is the main import pore for nuclear-encoded proteins into mitochondria, yet little is known about its spatial distribution within the outer membrane. Super-resolution stimulated emission depletion microscopy was used to determine quantitatively the nanoscale distribution of Tom20, a subunit of the TOM complex, in more than 1,000 cells. We demonstrate that Tom20 is located in clusters whose nanoscale distribution is finely adjusted to the cellular growth conditions as well as to the specific position of a cell within a microcolony. The density of the clusters correlates to the mitochondrial membrane potential. The distributions of clusters of Tom20 and of Tom22 follow an inner-cellular gradient from the perinuclear to the peripheral mitochondria. We conclude that the nanoscale distribution of the TOM complex is finely adjusted to the cellular conditions, resulting in distribution gradients both within single cells and between adjacent cells.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
STED microscopy enables the analysis of submitochondrial protein distributions. (A) Mitochondria form a branched network within eukaryotic cells. The mitochondria (green) and the microtubule cytoskeleton (red) of PtK2 cells were labeled with antibodies specific for Tom20 and α-tubulin. The nuclei were labeled with DAPI (blue). (Scale bar: 20 μm.) (B) Mitochondria of PtK2 cells labeled with an antiserum against Tom20. STED microscopy (Right) reveals individual Tom20 clusters, which are blurred and not resolvable when using diffraction-limited confocal microscopy (Left). (Scale bar: 2 μm.)
Fig. 2.
Analysis of Tom20 in three different cell lines. (A) Amino acid sequence alignment of Tom20 from the three cell lines. Differences are highlighted in green. (B) Western blots of whole-cell extracts of the three cell lines decorated with the antiserum against Tom20. An antiserum against β-actin was used as a loading control. (C) Overview on the shapes of the mitochondrial networks in PtK2, Vero, and HeLa cells labeled with the Tom20 antiserum. Shown are maximal intensity projections of confocal sections. (D) STED images of mitochondrial tubules of the respective cells labeled with the Tom20 antiserum. Shown are representative images. Note that there was substantial heterogeneity within a single cell line. (E) Staining of the cell lines with the mitochondrial membrane potential-sensitive dye DiOC6. High fluorescence intensity indicates a high membrane potential. The same imaging conditions and color tables were used. (Scale bars: 20 μm in C and E; 2 μm in D.) (F) Oxygen consumption of the three lines, as measured with a Clark-type oxygen electrode.
Fig. 3.
Quantification of the distributions of the Tom20 clusters in HeLa, Vero, and PtK2 cells. For the analysis, cells were grown to ∼50% confluence, chemically fixed, and labeled with an antiserum against Tom20. More than 120 cells of each line, imaged with STED microscopy, were analyzed. (A) Analysis of the diameter of the protein clusters by an autocorrelation algorithm. Note: The diameters determined for the Tom20 clusters are enlarged by the antibodies used for labeling. (B) Analysis of the density of Tom20 clusters in the mitochondria. (C) Analysis of the normalized variance of the local fluorescence signal intensity on the mitochondria, the most sensitive measure for determining differences in the distributions of the labeled protein. In the boxplots, the central lines represent the median, and the edges of the boxes represent the first and the third quartiles, respectively. Notches indicate P = 0.05 for the medians. ***P = 0.001 for the means (paired t test analysis).
Fig. 4.
Growth conditions influence the distribution of the Tom20 clusters. PtK2 cells were seeded to low density (<10% confluence) or high density (>90% confluence) on coverslips. (A) Cells were stained with the membrane potential-sensitive dye DiOC6. (Left) Low confluence. (Right) High confluence. High fluorescence intensity indicates a high membrane potential.(Scale bar: 50 μm.) (B) STED imaging of cells labeled with a Tom20-specific antiserum. Shown are representative images of mitochondria of cells grown to low or high confluence, respectively. (Scale bar: 2 μm.) (C) Boxplots summarizing the quantitative analysis of 200 cells imaged by STED microscopy. Significance levels are as in Fig. 3.
Fig. 5.
Densities of Tom20 clusters and the membrane potentials form radial gradients in microcolonies. PtK2 cells were seeded sparsely on a coverslip and allowed to grow into microcolonies of about 25 cells. (A) Cells were stained with the membrane potential-sensitive dye DiOC6. Shown is a single microcolony. The cross indicates the center, and the dotted line indicates the rim of the microcolony as used for the subsequent analysis. (Scale bar: 50 μm.) (B) Plot of the average DiOC6 fluorescence intensities from the center to the rim of the microcolony shown in A. Blue circles represent 100 bins that pool the ∼4 × 104 individual fluorescence intensity values. The black line indicates the linear fit to the individual fluorescence intensity values. (C) STED image of a microcolony labeled with an antiserum against Tom20. (Scale bar: 50 μm.) (D) Plot of the normalized variance of the fluorescence signal from the center to the rim of the microcolony shown in C. Blue circles represent 100 bins that pool the ∼5 × 106 individual variance values. The black line indicates the linear fit to the individual variance values. The lower variance values at the rim of the colony indicate a higher density of Tom20 clusters.
Fig. 6.
Density of TOM clusters is higher in the perinuclear mitochondria than in mitochondria at the cellular periphery. (A) Graphical representation of all cells of the microcolony shown in Fig. 5_C_. Green indicates cells in which the normalized variance of the fluorescence signal is lower in the perinuclear mitochondria (corresponding to a higher density of the TOM clusters) than in the cellular periphery. (For detailed data, see
Fig. S6
). Gray indicates cells in which the linear fits to the variance values have negative slopes. (Scale bar: 50 μm.) (B) Representative cell used for the analysis shown in C. Its location in the microcolony is indicated by an asterisk in A. The cross indicates the center, and the white line indicates the border of the cell as used for the subsequent analysis. (Scale bar: 20 μm.) (C) The normalized local variance values of the fluorescence signals radiating from the center of the cell to its border. Blue circles indicate 100 bins that pool the ∼3 × 105 individual variance values. The black line indicates the linear fit based on the individual variance values. The positive slope of the curve indicates that the density of the TOM clusters is higher in mitochondria around the nucleus. (D) STED image of a mitochondrion of a Vero cell labeled with an antiserum against Tom22. (Scale bar: 2 μm.) (E) Representative Vero cell labeled with a Tom22 antiserum. (Scale bar: 20 μm.) (F) Analysis of the distribution of Tom22 in the cell shown in E. The analysis was performed as in C.
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