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First published online 27 November 2007
doi: 10.1242/jcs.017640

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ADF/cofilin family proteins control formation of oriented actin-filament bundles in the cell body to trigger fibroblast polarization

¹ MRC-Laboratory Molecular Cell Biology, UCL, London, WC1E 6BT, UK
² Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA
³ Department of Biology, UCL, London, WC1E 6BT, UK

View larger version (48K): [in this window] [in a new window]   Fig. 1. Cell polarization in chick embryo fibroblasts. (A) Still images from a phase-contrast movie of fibroblast polarization. Time is hours:minutes:seconds. Lat A was added at –1:07:03 for just over 1 hour: within minutes in the drug, the cell rounded up (–1:00:44 to 0:00:00) and remains rounded and retracted in Lat A (0:00:00). Lat A was washed out at 0:00:00. The cell re-spreads (0:00:00 to 0:15:00), then a small stable localized retraction of the cell edge occurs (by 0:35:00, black arrow). The retracted area elaborates to form the cell rear (0:58:30, black arrows) then net protrusion and cell migration occur in the direction of the white arrow (1:06:30; net protrusion occurring for 6 minutes by this time – see E). Further migration then occurs (1:27:02). Red and green dots (in 0:15:00 to 1:27:02) mark the position of the fully re-spread cell (at 0:15:00) at the presumptive location of where the rear initiates and front forms, respectively, determined from the movie. (B) Trace of the cell in A showing direction of polarity (arrow) before and after cell repolarization. (C) Quantification of the cell population showing direction of polarity for individual cells before and after repolarization (n=15 live-cell movies). (D) Quantification of cell polarization with time after washout (% total of 15 live cells). (E) Line graph showing cell retraction to form the cell rear and protrusion at the cell front of the live cell in A measured from the red (see above) and green (see above) dot, respectively, during cell polarization (the process of re-spreading is not shown on the graph). Note that net protrusion lags net retraction. (F) Histogram of time (to nearest minute) that individual cells in the population initiate cell polarization, as identified by initial, stable, localized cell-edge retraction (n=15 live-cell movies). (G) Delay in individual cells between initial cell-edge retraction and net protrusion at the cell front (n=15 live-cell movies). Bar, 10 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Two main types of actin organization are detectable in cells during cell polarization. Cells were treated with 1 µm Lat A for 1 hour (A), then washed in medium (B-E) (see Materials and Methods) to initiate cell polarization, then fixed at various time points and stained with Alexa-Fluor-594-conjugated phalloidin to visualize actin organization. (A) Rounded-up cell, after 1 hour of Lat A treatment, prior to initiation of cell polarization with actin aggregates (note that no visible actin-filament bundles are present). (B,B') Re-spread cells early in cell polarization: actin is organized as a non-oriented actin-filament meshwork (B) or non-oriented actin-filament bundles (B'). Red arrows (B') indicate different directions of unaligned filament bundles. (C,C') Re-spread cells later in cell polarization: actin is organized as oriented actin-filament bundles. Bundles are more than 70% (C) to 100% (C') aligned with each other (red arrow, C') ±0-30°, as in fully polarized and moving cells (Cramer et al., 1997) (and see E). (D) The cell edge is retracted (arrow) and actin remains organized as oriented actin-filament bundles. (E, fully polarized cell) Actin-filament bundles within the cell body are oriented in the direction of migration. (F) Quantitative analysis of actin organization in fixed and stained polarizing cells at fixed time points during the cell-polarization assay (0-60 minutes after Lat A washout). (G) Stacked histogram illustrating actin organization in fixed cells as a function of cell shape during cell polarization. Graphs (F,G) are expressed as mean ± s.e.m. (n=600 cells per time point, triplicate experiments). Bar, 10 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Formation of oriented actin-filament bundles precedes initial, localized cell-edge retraction, the first visible sign of cell polarization. Cells expressing GFP-actin were imaged live by spinning-disc confocal microscopy during cell polarization. (A) Still images from a time-lapse movie of a single polarizing cell (time in hours:minutes:seconds, indicates time after washout of Lat A). This movie was representative of a total of 22 live GFP-actin-expressing cells analyzed that polarized. A meshwork of actin increases in visibility (0:02:20 to 0:06:01) and, by 0:29:34, oriented actin-filament bundles have formed (also see enlarged views beneath). The initial, permanent break in cell symmetry then occurs (by 00:36:39, arrow). Cell-edge retraction then elaborates around the bundles (ongoing at 00:49:59, arrows) forming a polarized cell with a defined front and rear, and the cell migrates (to 1:06:13, the end of the movie-sequence taken). Red and green dots (0:29:34 to 1:06:13) mark the position of the fully re-spread cell (at 15 minutes, image not shown) at the presumptive locations of where the rear initiates and front forms, respectively. Small fluctuations at the cell edge, which do not permanently break cell symmetry, cause slight differences in the position of the re-spread cell at 0:15:00 (image not shown) and 0:29:34. (B) Stacked histogram illustrating actin organization in live cells expressing GFP-actin as a function of cell shape during cell polarization (n=22 live-cell movies). (C) Actin organization in cells that polarized (22/24 cells) scored 60 seconds before the initial break in cell symmetry occurred (light-grey bar), identified in each movie, and in cells in which polarization naturally failed (2/24 cells) during the course of observation (up to 2 hours) (dark-grey bar). Note that, in all (22/22) cases, oriented actin-filament bundles formed prior to initiation of cell polarization. (D) Histogram of the time interval between oriented actin-filament-bundle formation and initiation of cell-edge retraction in 22 individual live cells. (E) Histograms of time taken to form oriented actin-filament bundles (to 70% total actin bundles) (upper panel) and to initiate cell-edge retraction (lower panel) in 22 individual live cells. (F) Detailed analysis of elaboration of cell-edge retraction (n=22 live cells expressing GFP-actin). Cells either: (i) initiate and elaborate cell-edge retraction (arrows) end-on to bundles; or (ii) initiate retraction in one location, off-set to bundles, then (i) elaborate further from this site; or (iii) initiate retraction in two locations, off-set to bundles, then (i) elaborate further between the two sites of initiation. Note that, irrespective of where retraction initiates, common to all is that retraction then elaborates around one end of oriented actin-filament bundles to form the cell rear (iv). (G) Distance that the cell-edge retracted (red) and protruded (green) measured at the presumptive location of where the rear initiates (A, red dot) and front forms (A, green dot), respectively, during cell polarization from the re-spread stage (0:15:00) for the cell in A. As in Fig. 1, net retraction precedes net protrusion. Bar, 10 µm.

View larger version (76K): [in this window] [in a new window]   Fig. 4. Actin-filament disassembly is required for formation of oriented actin-filament bundles and cell polarization. Cells infected and expressing GFP-actin (A-C) were imaged live by spinning-disc confocal time-lapse microscopy during normal cell polarization (after Lat A washout; A); or Lat A was washed off, cells left in culture medium for 15 minutes, then 0.5 µm jasplakinolide added and cells left in the continued presence of jasplakinolide (B,C); or Lat A was washed off uninfected cells, then 0.5 µm jasplakinolide added 15 minutes later and cells left in the continued presence of jasplakinolide then fixed in a time-course assay and stained for actin (D,E). (A-C) Time is hours:minutes:seconds and indicates total time after washout of Lat A. (A) As expected, in the control, actin initially forms a meshwork (by 0:12:00) then forms oriented actin-filament bundles (by 0:17:00). Stable retraction of the cell edge occurs (by 0:27:00) and the cell is fully polarized and migrating by 0:58:40. (B) In the jasplakinolide-treated cell, at 0:19:00 (19 minutes after Lat A washout), jasplakinolide has been present for the last 4 (0:15:00-0:19:00) minutes and, at 0:55:30, for the last 40 minutes, 30 seconds (0:15:00-0:40:30). Both formation of oriented actin-filament bundles and cell polarization fails. Note that delocalized protrusions still occur in the presence of jasplakinolide, as expected and similar to non-polarized cells (Cramer, 1999b) (see text for further details). (C, 1:07:30, last 0:52:30 in jasplakinolide) Prolonged treatment with 0.5 µm jasplakinolide causes formation of actin aggregates and disrupted bundles, as documented previously (see text). Note that this occurs well after the specific block in formation of oriented actin bundles, shown in B and D. (D, jasplakinolide added at 15 minutes after Lat A washout) Quantification of formation of oriented actin-filament bundles in cells fixed and stained for actin. (E, jasplakinolide added at 15 minutes after Lat A washout) Quantification of polarized cells in cells fixed at 30 and 60 minutes after washout of Lat A. Note the failure of jasplakinolide-treated cells to form oriented actin-filament bundles (D) and to polarize (E) when compared to the control cells. Graphs are expressed as mean ± s.e.m. for three independent experiments (n=600 cells counted for each condition). Bar, 10 µm.

View larger version (81K): [in this window] [in a new window]   Fig. 5. ADF colocalizes with F-actin in the cell body during cell polarization. During cell polarization, cells were extracted with detergent (see Materials and Methods) to remove soluble ADF then fixed and co-stained with Alexa-Fluor-594-conjugated phalloidin (A,B, green) and anti-chick ADF antibody (C,D, red), and the two respective staining patterns were merged (E,F). (A,C,E,E') A re-spread cell with a non-oriented meshwork of F-actin. (B,D,F-F'') A cell with localized cell-edge retraction and oriented actin-filament bundles. Boxed areas in the merged images (E,F) are enlarged in E'-F'', respectively, and show colocalization (orange/yellow) of ADF and the actin meshwork (E') and in a proportion (F'), but not all (F''), of the oriented actin-filament bundles. Bar, 6 µm in A for A-F; 1.3 µm in E',F',F''.

View larger version (39K): [in this window] [in a new window]   Fig. 6. AC is required for formation of oriented actin-filament bundles and cell polarization. Cell polarization was initiated in chick heart fibroblasts by washing out Lat A. Cells were then incubated live for 15, 25, 30 or 60 minutes after the washout, fixed and stained with Alexa-Fluor-380-conjugated or FITC-conjugated phalloidin. (A) Control cells expressing GFP only. (B) Cells expressing inactive pseudo-phosphorylated AC only (XAC1 E3 mutant). (C) Cells expressing LIMK1 EE508 only to block AC function. (D) Rescue: cells co-expressing LIMK1 EE508 and active, non-phosphorylatable AC (XAC1 A3 mutant). Cells expressing GFP only were identified by GFP expression (inset in A); LIMK-EE508-expressing cells by GFP expression off a separate promoter (insets in C and right insets in D); and XAC1-E3- and XAC1-A3-expressing cells by anti-XAC-antibody staining (insets in B and left insets in D), which has minimal cross-reactivity with endogenous AC (Shaw et al., 2004). Note the lack of formation of oriented actin-filament bundles in cells in which AC function is blocked (B,C). (E) Quantification of the formation of oriented actin-filament bundles in the cell population. (F) Quantification of cell polarization in the cell population. Graphs are expressed as mean ± s.e.m. of triplicate experiments (n=600 cells per condition). Bar, 10 µm.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Model for forming the cell rear and front during fibroblast polarization. Prior to cell polarization in these cells, delocalized cell protrusions occur around the entire cell periphery (A,B, blue band). F-actin is enriched in protrusions, as expected. Prior to cell polarization (A,B), within the cell body, actin initially assembles to form non-oriented meshwork/bundles (A, black lines) and subsequently forms oriented actin-filament bundles (B, black lines). Oriented actin-bundle formation triggers localized cell-edge retraction, the first visual break in cell symmetry (C, thin arrow). Cell-edge retraction elaborates around one end of oriented actin-filament bundles (D, jagged edge) to form the cell rear, leaving a stable protrusion (D, blue band) at the opposing end of the bundles. Once cell-edge retraction is elaborated (D,E), net protrusion at the front and cell migration occurs (E). AC (A-E, red dots) localizes to polymerized actin within the cell body early in this process (A) and is appropriately localized, spatially and temporally, to promote formation of oriented actin-filament bundles (B, black lines) prior to cell polarization.

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