F- and G-actin concentrations in lamellipodia of moving cells

Abstract

Cells protrude by polymerizing monomeric (G) into polymeric (F) actin at the tip of the lamellipodium. Actin filaments are depolymerized towards the rear of the lamellipodium in a treadmilling process, thereby supplementing a G-actin pool for a new round of polymerization. In this scenario the concentrations of F- and G-actin are principal parameters, but have hitherto not been directly determined. By comparing fluorescence intensities of bleached and unbleached regions of lamellipodia in B16-F1 mouse melanoma cells expressing EGFP-actin, before and after extraction with Triton X-100, we show that the ratio of F- to G-actin is 3.2+/-0.9. Using electron microscopy to determine the F-actin content, this ratio translates into F- and G-actin concentrations in lamellipodia of approximately 500 microM and 150 microM, respectively. The excess of G-actin, at several orders of magnitude above the critical concentrations at filament ends indicates that the polymerization rate is not limited by diffusion and is tightly controlled by polymerization/depolymerization modulators.

Figure 1. Dual bleach experiments demonstrate that monomeric GFP-actin recovers in the lamellipodium within 6 s after photobleach and is incorporated into F-actin only at the tip.

A shows the overview before bleach. Bar, 5 µm. B, C, Enlarged images of the region indicated by the white box in A show the bleached region immediately (B) and 20 s after bleach (C). Bar, 3 µm. Following the first bleach of the lamellipodium (green box in A), bleaching was continued in the region outlined by the box in B and C. Graph (D) shows the average intensity, measured in the red rectangular region, over time. The time between the two vertical dashed lines corresponds to the switch period between the end of the initial bleach and the initiation of the second bleach. Note that the intensity in the region marked by the red square stays constant during the second (continuous) bleach of the more distal region.

Figure 2. Selective extraction of G-actin after photobleaching.

A) Overview of a EGFP-actin expressing cell just before photobleach. B, C: Enlarged region indicated in A) about 2 s after bleach (B) and after extraction (C). Bars, 5 µm. Graph shows intensity scans along the lines indicated in B and C. Dark blue line: unbleached region before extraction; yellow: bleached region before extraction; pink: unbleached region after extraction; light blue: bleached region after extraction. Note the preservation of the intensity gradient in the unbleached region before and after extraction.

Figure 3. The fluorescence intensity of EGFP does not change upon cell-lysis in PEG-buffer.

In order to be able to correlate the EGFP-intensities in live and lysed cells, intensity measurements of EGFP-α-tubulin in B16-F1 cells were carried out before (A) and after (B) addition of detergent. In contrast to actin filaments, microtubules can be imaged as single polymers by fluorescence microscopy, thereby making it feasible to measure the intensity of the same fibres before and after extraction. To reduce the influence of the soluble fraction of EGFP-tubulin on the intensity measurements, microtubules in the periphery and close to the substrate were chosen and imaged by TIRF microscopy. To avoid chemical fixatives, PEG was used to stabilize the cytoskeleton. The difference in maximum fluorescence values of microtubules before and after extraction was around 3.9% (mean, standard deviation = 3.0%, 33 measurements in 14 cells). Since this difference is negligible no correction was applied to the experimental data for actin. Ellipses indicate regions for measurements.