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Spatiotemporal Quantification of Subcellular ATP Levels in a Single HeLa Cell During Changes in Morphology

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Spatiotemporal Quantification of Subcellular ATP Levels in a Single HeLa Cell During Changes in Morphology

Rika Suzuki et al. Sci Rep.

Abstract

The quantitative relationship between change in cell shape and ATP consumption is an unsolved problem in cell biology. In this study, a simultaneous imaging and image processing analysis allowed us to observe and quantify these relationships under physiological conditions, for the first time. We focused on two marginal regions of cells: the microtubule-rich 'lamella' and the actin-rich 'peripheral structure'. Simultaneous imaging and correlation analysis revealed that microtubule dynamics cause lamellar shape change accompanying an increase in ATP level. Also, image processing and spatiotemporal quantification enabled to visualize a chronological change of the relationships between the protrusion length and ATP levels, and it suggested they are influencing each other. Furthermore, inhibition of microtubule dynamics diminished motility in the peripheral structure and the range of fluctuation of ATP level in the lamella. This work clearly demonstrates that cellular motility and morphology are regulated by ATP-related cooperative function between microtubule and actin dynamics.

Figures

Figure 1
Figure 1. Inhibition of cytoskeletal dynamics induces an increase in local ATP.
(a,d) Typical images of the cellular morphology at the beginning of the observation. The gray-shaded regions represent 8 automatically divided compartments, while the gray line depicts the cellular morphology at the end of the observation. Scale bar represents 30 μm. (b,e) Typical image of the spatiotemporal behavior of intracellular relative ATP levels in the compartment indicated by the arrowhead in the left figure. The horizontal axis indicates time, the vertical axis indicates position, and pseudo color indicates relative ATP level. Each color lookup table is linear and covers the full range of the data. The gray bar indicates the duration of inhibition. (c,f) Comparisons of relative ATP level before and after inhibition at the central and edge parts. When cells were treated with Latrunculin A, relative ATP level increased only at the edge (n = 6). On the other hand, Taxol-treated cells showed an increase in relative ATP level at both the central and the edge parts (n = 6). Error bar represents standard deviation (s.d.).
Figure 2
Figure 2. Structure at the cell edge: the lamella and the peripheral structure are distinguished.
(a) Typical EB3-Venus (left), FM4-64 (middle), and merged (right) images at the edge of a HeLa cell. EB3 probe diffused throughout the cytosol. (b) Typical Venus-actin (left), FM4-64 (middle), and merged (right) images at the edge of a HeLa cell. The peripheral structure of the cell edge is constructed of actin filaments. (c) Typical images of the (mseCFP + Venus) fluoresce of ATeam (left), FM4-64 (middle), and DIC (right) at the edge of a HeLa cell. The peripheral structure at the cell edge was detectable from the (mseCFP+Venus) signal. Scale bar represents 5 μm.
Figure 3
Figure 3. Microtubule dynamics cause a change in cell shape, which is accompanied by an increase in the ATP level at the cell edge.
(a) Typical time courses of EB3 density (green), the number of newly generated lamella areas (orange), and the relative ATP level (pink) within an ROI. (b) Cross correlation function between newly generated lamella areas vs. EB3 density. Positive correlations were observed at −7 min, −2.5 min, and 3 min (12 ROIs from 6 cells). (c) An average of waveforms in the duration between before and after the timing of a peak in newly generated lamella areas (solid line, 9 events from 6 cells). EB3 density increased about 1 min before the peak, and this increase in the lamella area accompanied an increase in the relative ATP level. No apparent peak was observed in the average of waveforms from randomly shuffled sequences (broken lines). Error bar represents standard error of mean (s.e.m.).
Figure 4
Figure 4. Both actin and microtubule dynamics affect cellular ATP levels at the cell edge.
(a) Typical tracks of protrusion length and ATP levels at the edges of HeLa cells under physiological conditions (9 examples from 5 cells). The horizontal axis indicates protrusion length, the vertical axis indicates relative ATP levels, and the pseudo color indicates time. (b–d) Typical tracks of HeLa cells treated with (b) Latrunculin A (left, 2 examples from 2 cells), (c) Taxol (middle, 2 examples from 2 cells), or (d) 2-DG (right, 2 examples from 2 cells). During the last 5 min of observation (pink), the tracks of Latrunculin A-treated cells lost their movement, while a reduced range of variation in relative ATP levels was observed in the tracks of Taxol-treated cells. Cells treated with 2-DG showed decreased motility as well as ATP levels. (eh) Ranges of measured values in the last 5 min as percentages of the total timeframe. Markers indicate the mean values, while bars indicate the ranges. (i,j) Statistical analysis of (left) width and (right) average values of the ranges of (i) protrusion length and (j) relative ATP levels (4 ROIs from 2 cells for Latrunculin A, 7 ROIs from 3 cells for Taxol, 6 ROIs from 2 cells for 2-DG, and 15 ROIs from 6 cells for control).

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