An MEK-cofilin signalling module controls migration of human T cells in 3D but not 2D environments

Abstract

T cells infiltrate peripheral tissues to execute immunosurveillance and effector functions. For this purpose, T cells first migrate on the two-dimensional (2D) surface of endothelial cells to undergo transendothelial migration. Then they change their mode of movement to undergo migration within the three-dimensional (3D)-extracellular matrix of the infiltrated tissue. As yet, no molecular mechanisms are known, which control migration exclusively in either 2D or 3D environments. Here, we describe a signalling module that controls T-cell chemotaxis specifically in 3D environments. In chemotaxing T cells, Ras activity is spatially restricted to the lamellipodium. There, Ras initiates activation of MEK, which in turn inhibits LIM-kinase 1 activity, thereby allowing dephosphorylation of the F-actin-remodelling protein cofilin. Interference with this MEK-cofilin module by either inhibition of MEK or by knockdown of cofilin reduces speed and directionality of chemotactic migration in 3D-extracellular matrices, but not on 2D substrates. This MEK-cofilin module may have an important function in the tissue positioning of T cells during an immune response.

Figure 1

Cofilin colocalizes with both dynamic and stable F-actin structures in migrating primary human T cells. (A, B). Confocal microscopy of representative T cells migrating on an ICAM-1-coated coverslip upon the addition of SDF-1α. A single optical x–y section close to the level of the coverslip is shown. (A) F-actin was visualized with fluorescently labelled Phalloidin. (B) F-actin was stained with Phalloidin (green), the uropod was stained with anti-ICAM-3 (blue) and cofilin was visualized with a polyclonal anti-cofilin antiserum (red). The white box marks the enlarged area depicting the lamellipodium. (C) Fluorescence intensity distribution (grey values) across the back–front axis (depicted by the white line in (B)) of the cell shown in (B). (D) Fluorescence intensity distribution across the back–front axis of multiple polarized T cells. The mean grey values±s.d. are shown (n=14).

Figure 2

An Ras-MEK pathway mediates dephosphorylation of cofilin after chemokine receptor triggering in human T cells. (A) Primary human T cells were incubated in the absence or presence of SDF-1α for the indicated time points. Cells were lysed and phosphorylated cofilin (p-Ser3-cofilin) and total cofilin were detected by immunoblotting. Shown is one representative experiment out of three. (B) The levels of phospho-cofilin and total cofilin were detected by immunoblotting as described above, and the cofilin phospho-index was calculated. The mean values±s.d. of three independent experiments are shown. (C) Human T cells were incubated in the absence (upper blot) or presence of Pertussis toxin (PTx, lower blot). Cells were then stimulated with SDF-1α for the indicated time points, lysed and Ras-GTP was precipitated from the lysate with RBD agarose and detected by immunoblotting using an antibody against human Ras. Shown is one representative experiment out of three. (D) Human T cells were transfected either with a cDNA encoding EGFP (EGFP), or with a cDNA encoding an EGFP-fusion protein of dominant-negative Ras (N17-Ras). T cells were incubated in the presence or absence of SDF-1α for 2 min (pERK, pAKT) or 30 min (p-Ser3-cofilin). Then, cells were fixed, permeabilized and stained with either a phospho-Erk (left panel) or a phospho-Akt (middle panel) PE-labelled antibody. P-cofilin was detected with a P-Ser3-cofilin antiserum and PE-conjugated secondary antibodies (right panel). The mean fluorescence intensity for PE of EGFP-positive cells was determined by flow cytometry (see Supplementary Figure S3B). The values obtained for EGFP-transfected cells in the absence of SDF-1α were set 1 and all other values were expressed relative to them. The mean values±s.d. of three independent experiments are shown. ^*P<0.05; ^**P<0.01; NS, not significant. (E) Human T cells were either not treated (no inhibitor) or treated with the indicated inhibitors. Afterwards, cells were incubated in the absence or presence of SDF-1α for the indicated time points and the levels of phospho-cofilin and total cofilin were detected by immunoblotting. In addition, the same blots were probed with two different antisera detecting the phosphorylated forms of Akt and Erk, respectively. Shown is one representative experiment out of three. (F) Human T cells were treated as in (E). The levels of phospho-cofilin and total cofilin were detected by immunoblotting, and the cofilin phospho-index was calculated. The mean values±s.d. of three independent experiments are shown. ^*P<0.05; ^**P<0.01; NS, not significant.

Figure 3

MEK inhibits LIMK1 activity upon chemokine receptor triggering in primary human T cells, thereby enhancing dephosphorylation of cofilin through phosphatase PP2A. (A) T cells were pre-treated or not with the MEK-inhibitor U0126, and then incubated in the absence or presence of SDF-1α for the indicated time points. Cells were lysed and phosphorylated LIMK1 (p-Thr508-LIM kinase) and total LIMK1 were detected by immunoblotting. Shown is one representative experiment out of three. (B) The levels of phospho-LIMK1 and total LIMK1 were detected by immunoblotting as described above, and the LIMK1 phospho-index was calculated. The mean values±s.d. of three independent experiments are shown. (C) Primary human T cells were pre-treated or not with Okadaic acid and incubated in the absence or presence of SDF-1α for the indicated time points. Cells were lysed and phosphorylated cofilin (p-Ser3-cofilin) and total cofilin were detected by immunoblotting. Shown is one representative experiment out of three. (D) The levels of phospho-cofilin and total cofilin were detected by immunoblotting as described above, and the cofilin phospho-index was calculated. The mean values±s.d. of three independent experiments are shown. (E) Resting human T cells were either left untreated or were treated with Okadaic acid. Cells were lysed and phosphorylated PP2A (P-Tyr307-PP2A) was detected by immunoblotting. Shown is one representative experiment out of three.

Figure 4

The GTPase Ras is specifically activated at the leading edge of migrating primary human T cells. (A) Primary human T cells were either transfected with a cDNA encoding EGFP (left panels) or with a cDNA encoding EGFP-Raf-RBD (right panels). Cell migration on ICAM-1-coated coverslips was induced by the addition of SDF-1α. Cells were analysed by confocal microscopy. Shown are single optical x–y sections. The uropod was stained with anti-ICAM-3 (upper panels; red), and cofilin was visualized with a polyclonal anti-cofilin antiserum (upper panels; green). The subcellular distribution of EGFP-Raf-RBD is shown by false colours (lower panels). The white boxes mark the enlarged areas depicting the lamellipodia. EGFP-Raf-RBD is enriched at the edge of the lamellipodium (white arrows). (B) Migration of primary human T cells on ICAM-1-coated coverslips was induced by the addition of SDF-1α. Cells were analysed by confocal microscopy. Total cofilin was detected with a mouse monoclonal antiserum (green), phospho-Ser3-cofilin was detected with a rabbit phospho-Ser3-specific antiserum (red), and F-actin was visualized with Phalloidin (blue). (C) The ratio of phospho-Ser3-cofilin (P-cofilin) versus total cofilin (cofilin) was determined for both the cell front and the cell back as depicted in Supplementary Figure S2D. The ratio determined for the cell back was set 1 and the ratio determined for the cell front was expressed relative to it. The mean values±s.d. are shown (n=19). ^***P<0.001.

Figure 5

Interference with Ras activity impairs chemotactic migration of human T cells in both 2D and 3D environments by reducing speed and directionality. (A, F) Human T cells were either transfected with a cDNA encoding EGFP (Control), or with a cDNA encoding an EGFP-fusion protein of dominant-negative Ras (N17-Ras). Cells were allowed to migrate either across ICAM-1-coated transwell inserts (A) or across a Matrigel layer on the top of a transwell insert (F) in the absence or presence of SDF-1α in the lower compartment. The percentages of migrated EGFP-positive cells were determined. The mean values±s.d. of three independent experiments are shown. ^*P<0.05; ^***P<0.001. (B, G) Time-lapse videomicroscopy of primary human T cells expressing either EGFP (Control) or the N17-Ras-EGFP (N17-Ras)-fusion protein. Cells were allowed to migrate either on an ICAM-1-coated 2D surface of a transwell insert (B) or within a 3D-Matrigel layer on top of a transwell insert (G) in the presence of an SDF-1α gradient. Data are representative of three independent experiments. Tracks of the migrating T cells are shown in red. The pores of the transwell insert (source of the chemokine gradient) are labelled with numbers. The fluorescence intensity of the EGFP-expressing cells is shown by false colours. See also corresponding Supplementary Movies M4–M7. (C, H) Multiple tracks of individual human T cells. The starting points of the recorded cell tracks were artificially set to the same origin. Shown is one representative experiment out of three. (D, I) The velocities and Euclidean distances of individual human T cells were calculated from the recorded tracks. The mean values±s.d. of three independent experiments are shown. ^*P<0.05; ^***P<0.001. (E, J) The directionality of cell migration for each condition was calculated as the Euclidean distance divided by the accumulated distance (see Supplementary Figure S6C). ^*P<0.05.

Figure 6

Inhibition of MEK impairs chemotactic migration of human T cells in 3D but not 2D environments by reducing speed and directionality. (A, F) Human T cells were treated or not with the MEK-inhibitor U0126. Cells were allowed to migrate either across ICAM-1-coated transwell inserts (A) or across a Matrigel layer on top of a transwell insert (F) in the absence or presence of SDF-1α in the lower compartment. The percentages of migrated cells were determined. The mean values±s.d. of three independent experiments are shown. NS, not significant. ^**P<0.01. (B, G) Time-lapse videomicroscopy of primary human T cells migrating on either an ICAM-1-coated 2D surface of a transwell insert (B) or within a 3D-Matrigel layer on top of a transwell insert (G) in the presence of an SDF-1α gradient. Cells were either left untreated (no inhibitor), or were treated with the MEK-inhibitor U0126. Data are representative of three independent experiments. Tracks of the cells are shown in red. The pores of the transwell insert (source of the chemokine gradient) are labelled with numbers. See also corresponding Supplementary Movies M8–M11. (C, H) Multiple tracks of individual human T cells. The starting points of the recorded cell tracks were artificially set to the same origin. Shown is one representative experiment out of three. (D, I) The velocities and Euclidean distances of individual human T cells were calculated from the recorded tracks. The mean values±s.d. of three independent experiments are shown. NS, not significant; ^*P<0.05; ^**P<0.01. (E, J) The directionality of cell migration for each condition was calculated as the Euclidean distance divided by the accumulated distance (see Supplementary Figure S6C). NS, not significant; ^*P<0.05.

Figure 7

Active MEK is required for the actin-flow-driven mode of T-cell movement. (A) Primary human T cells were pre-treated or not with Blebbistatin. Cells were then allowed to migrate in the absence or presence of U0126 on the ICAM-1-coated 2D surface of a transwell insert in the presence of an SDF-1α gradient (see corresponding Supplementary Movie M14). Multiple tracks of individual T cells were recorded by time-lapse videomicroscopy. The starting points of the recorded cell tracks were artificially set to the same origin. Shown is one representative experiment out of three. (B) The velocities of individual human T cells were calculated from the recorded tracks (left panel), the directionalities were calculated as the Euclidean distances divided by the accumulated distances (right panel). NS, not significant; ^***P<0.001.

Figure 8

Knockdown of cofilin in human T cells. (A) Human Jurkat T cells were either transfected without siRNA (Mock), with a ‘scrambled' control siRNA (Control siRNA) or with siRNA against cofilin (cofilin siRNA). Cells were lysed 72 h after transfection and the expression of cofilin and β-tubulin were analysed by immunoblotting. Shown is one representative experiment out of three. (B) Quantification of cofilin protein levels by densitometry. The values obtained for cells transfected with control siRNA were set 100 and the other values were expressed relative to them. The mean values±s.d. of three independent experiments are shown. (C) The F-actin content was determined by flow cytometry using fluorescently labelled Phalloidin. The mean fluorescence values obtained for mock-transfected cells were set 1 and all other values were expressed relative to them. The mean values±s.d. of three independent experiments are shown. ^**P<0.01.

Figure 9

Knockdown of cofilin impairs chemotactic migration of human T cells in 3D but not 2D environments by reducing speed and directionality. (A, F) Human Jurkat T cells were either transfected without siRNA (mock), or with a ‘scrambled' siRNA (control siRNA) or with a cofilin-specific siRNA (cofilin siRNA). Chemotactic migration across transwell inserts (A) or through Matrigel on top of a transwell insert (F) was carried out 72 h after transfection. The numbers of mock-transfected cells, which migrated in the presence of SDF-1α (mock+SDF-1α) were set 100 and all other values were expressed relative to them (absolute numbers for mock+SDF-1α were 25.37±4.63% (A) and 2.97±0.75% (F)). The mean values±s.d. of three (A) or five (F) independent experiments are shown. NS, not significant; ^**P<0.01. (B, G) Human T cells were transfected with siRNA as described above. Then, cells were allowed to migrate on either the ICAM-1-coated 2D surface of a cell culture transwell insert (B) or within a 3D-Matrigel layer on top of a transwell insert (G) in the presence of SDF-1α in the lower compartment, and time-lapse videomicroscopy was performed. The pores of the transwell insert (source of the chemokine gradient) are labelled with numbers. The tracks of the cells are shown in red. See also corresponding Supplementary Movies M15–M18. (C, H) Multiple tracks of individual migrating T cells. The starting points of the recorded cell tracks were artificially set to the same origin. Data are representative of three independent experiments. (D, I) Velocities and Euclidean distances of individual T cells were determined. The mean values±s.d. of three independent experiments are shown. NS, not significant; ^*P<0.05. (E, J) The directionality of cell migration for each condition was calculated as the Euclidean distance divided by the accumulated distance. NS, not significant; ^*P<0.05.

Figure 10

Knockdown of cofilin leads to uncontrolled formation of cell protrusions and thus to a loss of polarity in 3D environments. (A, B) Human Jurkat T cells were transfected with siRNA as described above, and morphologically analysed by time-lapse videomicroscopy. T cells transfected with the control siRNA (control siRNA) as well as T cells transfected with the cofilin-specific siRNA (cofilin siRNA) display the typical ‘hand-mirror-like' morphology of polarized migrating T cells when migrating on a 2D substrate (A). T cells transfected with the control siRNA show a single lamellipodial extension (B, left panel) at the cell front when migrating within a 3D-Matrigel layer, whereas cells transfected with the cofilin-specific siRNA display many thin processes emanating in all directions (B, right panel, black arrows). See also corresponding Supplementary Movie M19. (C) A total of 195 control-siRNA-transfected cells and of 227 cofilin-siRNA-transfected cells were analysed for the presence of multiple cell protrusions and impaired migration within the 3D-Matrigel layer. The mean percentage±s.d. of cells displaying multiple membrane protrusions is shown.