Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration

Abstract

Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function-associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.

Figure 1.

Association of MyH9 with LFA-1. (A) Human primary T lymphocytes were allowed to adhere to cover glasses coated with ICAM-1 (IC-1) or poly-l lysine (PLL) ± CXCL-12. Cells were suspended in either L-15/2 mg/ml glucose or 20 mM Hepes, 150 mM NaCl, 5 mM MgCl₂, 1 mM EGTA, and 2 mg/ml glucose, as indicated. Migrating T lymphocytes were tracked over a 20-min period, and time-lapse DIC images were acquired every 5 s to generate movies (Videos S1–4). The bottom left corner of each image shows a randomly selected region at threefold magnification. Bar, 100 μm. (B and C) LFA-1 immunoprecipitates obtained from bound cells (using TS2/4 antibody) were analyzed for possible LFA-1 binding partners. MyH9 was identified by silver staining (n = 3) (B) and mass spectrometry (C). The spectrum of one of the MyH9 peptides obtained by nanospray-ion trap tandem mass spectrometry is shown in C. (D) PCR amplification of MyH9, MyH10, and MyH14 cDNAs from human PBMCs and T lymphocytes. Reverse-transcribed cDNA from human skeletal muscle served as a positive control. (E) LFA-1 immunoprecipitates obtained for each of the four substrate conditions were subjected to Western blotting with the indicated antibodies.

Figure 2.

MyH9 mediates LFA-1 de-adhesion at the uropod. (A) Human T lymphocytes were transfected with MyH9-GFP or β-actin–GFP, and cell migration on ICAM-1 and CXCL-12 was analyzed by time-lapse fluorescence microscopy at 37°C (see also Video S5 for MyH9-GFP and Video S6 for β-actin–GFP). Arrows mark the direction of migration. Two-dimensional images and three-dimensional histograms of fluorescence intensity and cell surface distribution are shown in a pseudo-color scale (from low [black] to high [red]). Bar, 20 μm. (B and C) Human T lymphocytes were pretreated with DMSO or 50 μM blebbistatin and allowed to migrate on ICAM-1– and CXCL-12–coated cover glass for 20 min at 37°C (B). The polarization index of cells was calculated as the ratio of x to y, where x is the longest distance across cells (from head to tail) and y is the greatest width perpendicular to x (C). Bar, 25 μm (B). (D) T lymphocytes were pretreated with DMSO or 50 μM blebbistatin for 2 h and stimulated with CXCL-12 for 20 min. LFA-1 immunoprecipitates (TS2/4 antibody) were then obtained and subjected to silver staining. (E and F) Human T lymphocytes were pretreated with DMSO or 50 μM blebbistatin for 1 h at 37°C, and cell migration on ICAM-1/CXCL-12–coated cover glasses was tracked over a 30-min period. Each line represents one cell. Experiments were repeated on T lymphocyte preparations from three independent donors.

Figure 3.

Selective suppression of MyH9 protein with siRNA. Human primary T lymphocytes were transfected with Cy3-conjugated control siRNA only or Cy3-conjugated control siRNA plus MyH9 siRNA at a 1:10 ratio and allowed to migrate on ICAM-1/CXCL-12 cover glasses. (A) An overlay of DIC and Cy3 fluorescence images is shown. Bar, 20 μm. (B) Quantification of tail detachment, as determined by the polarization index. The polarization index was measured on Cy3⁺ cells. *, P < 0.0001 for MyH9 siRNA (n = 19) versus control siRNA only. (C) T lymphocyte cell lysates were analyzed for MyH9 and β-actin levels by Western blot. Experiments were repeated on T lymphocyte preparations from three independent donors.

Figure 4.

MyH9–LFA-1 association is not required for CXCL-12–induced LFA-1 activation. (A) T lymphocytes were stimulated with (filled histograms) or without (open histograms) 100 ng/ml CXCL-12 for the indicated time period. Cells were labeled with KIM127 antibody and subjected to flow cytometry. Experiments were performed in triplicate with cells from three different donors. Representative histograms showing the time course of KIM127 epitope expression are shown. (B) Human T lymphocytes adherent to ICAM-1–coated slides were stimulated with 100 nM CXCL-12. Nonadherent cells were removed, and the number of bound cells was counted. Data are expressed as mean ± SEM of three experiments. The experiment is representative of results from three independent donors. (C) Lysates obtained from CXCL-12–stimulated T lymphocytes were subjected to α_L (TS2/4 antibody) and β₂ (CBR LFA-1/2 antibody) immunoprecipitation, and then to silver staining. Mouse IgG antibody served as a negative control. (D) T lymphocytes were pretreated with 50 μM blebbistatin or DMSO and analyzed as described above (A). A representative result of three separate experiments is shown. (E) Data represent the mean instantaneous velocities of 10 representative T lymphocytes without (Control) or with 50 μM blebbistatin (Blebbistatin), respectively, in a parallel-plate flow chamber coated with CXCL-12 and ICAM-1 at a wall shear stress of 0.5 dyn/cm². T lymphocyte trajectory was monitored for 2 s before the first contact between cells and substrate (arrow) and arrest (time 0). The time interval between data points is 33 ms. All data are expressed as mean ± SEM.

Figure 5.

MyH9–LFA-1 association is not required for the localization of active LFA-1 at the leading edge. (A) Human primary T lymphocytes were pretreated for 1 h with DMSO or 50 μM blebbistatin, incubated on cover glasses coated with ICAM-1 and CXCL-12 for 30 min, and processed for dual immunofluorescence labeling with AL-57 (green), an antibody specific for the active human α_L I domain, and TS2/4 (red). Nuclei of blebbistatin-treated cells were counterstained with DAPI (blue). Bar, 20 μm. (B) T lymphocytes treated with DMSO, 50 μM, or 200 μM blebbistatin for 1 h were allowed to migrate on ICAM-1/CXCL-12–coated surfaces. DIC time-lapse images were obtained at an acquisition rate of one frame per second. A narrow rectangular cursor was drawn on the time-lapse image stack (left, white line) so that the long axis of the rectangle was aligned with the direction of cell movement, which was determined by viewing the series as a movie. The rectangle was 4 pixels in width and long enough for complete motion analysis. This region of interest was then taken from each image in the time-lapse series, and the images were pasted side-by-side in a montage to form the kymograph picture. Each cycle of contractions was marked with white arrows (right). Bar, 10 μm. (C) T lymphocytes treated with DMSO (Con), 50 μM, 100 μM, or 200 μM blebbistatin for 1 h were allowed to migrate on ICAM-1/CXCL-12–coated surfaces. The polarization index of cells was calculated as the ratio of x to y, where x is the longest distance across cells (from head to tail) and y is the greatest width perpendicular to x. 30 cells for each group were analyzed.

Figure 6.

Uropodal adhesion depends on LFA-1 avidity regulation, and uropodal detachment is mediated by MyH9. (A–C) Human T lymphocytes were pretreated with DMSO or 50 μM blebbistatin for 1 h and allowed to migrate on ICAM-1/CXCL-12–coated surfaces for 20 min. Then cells were treated with 20 μM BIRT377, 1 μM XVA143, or an equivalent concentration of DMSO for the indicated times. The cell polarization index was then determined. Data are expressed as mean ± SEM of three experiments, each performed in triplicate. Representative DIC images from time-lapse movies are shown in C. (D) T lymphocytes were pretreated with DMSO or blebbistatin and allowed to settle on the ICAM-1/CXCL-12–coated cover glasses for 20 min in L-15 plus 2 mg/ml glucose. Cells were then treated with 20 μM BIRT377, 1 μM XVA143, or DMSO. After removal of nonadherent cells from the cover glasses, bound cells were counted. Data are expressed as mean ± SEM of three experiments.

Figure 7.

High affinity LFA-1 is not required for uropodal adhesion. (A and B) Human primary T lymphocytes were pretreated with blebbistatin and incubated on cover glasses coated with ICAM-1 and CXCL-12 for 20 min. Cells were then processed for dual immunofluorescent labeling with TS2/4 antibody and KIM127 (A) or m24 (B) antibody and subjected to TIRF microscopy. Ratio images were generated by subtracting the background and dividing KIM127 (A) or m24 (B) intensity by TS2/4 intensity. Line profiles of LFA-1 intensity are presented along the front (Head)-to-back (Tail) line (A). (B) From three independent experiments, a total of 48 (KIM127/TS2/4) and 35 (m24/TS2/4) cells was randomly selected, carefully analyzed, and scored for the presence of KIM127 or m24 staining enriched at the anterior region, the posterior region, or both, based on the ratio images. Each bar represents the percentage of total cells scored. The black portion of each bar is the fraction of cells scored for the anterior region dominant staining of KIM127 or m24. The white portion of each bar is the fraction of the cells that showed even distribution of staining. The gray portion of each bar is the fraction of cells scored for the posterior region dominant staining of KIM127 or m24. Bar, 20 μm.