Lateral mobility of individual integrin nanoclusters orchestrates the onset for leukocyte adhesion

Abstract

Integrins are cell membrane adhesion receptors involved in morphogenesis, immunity, tissue healing, and metastasis. A central, yet unresolved question regarding the function of integrins is how these receptors regulate both their conformation and dynamic nanoscale organization on the membrane to generate adhesion-competent microclusters upon ligand binding. Here we exploit the high spatial (nanometer) accuracy and temporal resolution of single-dye tracking to dissect the relationship between conformational state, lateral mobility, and microclustering of the integrin receptor lymphocyte function-associated antigen 1 (LFA-1) expressed on immune cells. We recently showed that in quiescent monocytes, LFA-1 preorganizes in nanoclusters proximal to nanoscale raft components. We now show that these nanoclusters are primarily mobile on the cell surface with a small (ca. 5%) subset of conformational-active LFA-1 nanoclusters preanchored to the cytoskeleton. Lateral mobility resulted crucial for the formation of microclusters upon ligand binding and for stable adhesion under shear flow. Activation of high-affinity LFA-1 by extracellular Ca(2+) resulted in an eightfold increase on the percentage of immobile nanoclusters and cytoskeleton anchorage. Although having the ability to bind to their ligands, these active nanoclusters failed to support firm adhesion in static and low shear-flow conditions because mobility and clustering capacity were highly compromised. Altogether, our work demonstrates an intricate coupling between conformation and lateral diffusion of LFA-1 and further underscores the crucial role of mobility for the onset of LFA-1 mediated leukocyte adhesion.

Fig. 1.

LFA-1 nanoclusters diffuse randomly on resting monocytes. (A) Schematic description of the SDT experiments. Individual LFA-1 molecules inside nanoclusters were labeled with TS2/4-ATTO520 or L16-ATTO647N using sublabeling conditions. (B) Selected frame from a movie recorded at 100 ms per frame. Bright spots correspond to individual TS2/4-LFA-1 nanoclusters. White dots indicate the perimeter of the cell. (Scale bar: 5 μm.) (C) Selected LFA-1 nanoclusters at different times to illustrate different mobility behavior: fast (Top), stationary (Middle), and slow (Bottom). (D) Representative LFA-1 nanocluster trajectories displaying different lateral mobility, pseudocolor coded according to their apparent mobility: fast (orange), slow (blue), and stationary (gray). (E) Normalized semilog distribution of D values at short-time lags. The vertical arrow indicates the threshold value of diffusion D_th above which the mobile population for CPD analysis has been selected (370 trajectories from 128 cells in multiple experiments). (F) Square displacement plots of the TS2/4 mobile fractions at different time lags as obtained from CPD analysis (157 trajectories).

Fig. 2.

Primed LFA-1 nanoclusters exhibit multiple diffusion profiles on resting monocytes. (A) Representative trajectories of primed LFA-1 nanoclusters labeled with L16-ATTO647N. (B) Normalized semilog distribution of D values at short-time lags of L16⁺-LFA-1 nanoclusters (bars) compared to that of the total (TS2/4) LFA-1 population (dashes) (669 trajectories from 49 cells on multiple experiments). (C) Square displacement plots by fitting the CPD of the L16 mobile fractions at different time lags (380 trajectories). (D) Normalized fractions of stationary (gray), slow (blue), and fast (orange) mobile subpopulations for the total and primed nanoclusters. Error bars represent the standard deviation.

Fig. 3.

The lateral mobility of LFA-1 nanoclusters is affected by extracellular Ca²⁺. (A, C, and E) Diffusion coefficients for the total (TS2/4) LFA-1 population at (A) 0.1 mM Ca²⁺ (bars) compared to 0.4 mM Ca²⁺, Mg²⁺ (dashes); (C) 0.04 mM Ca²⁺ (bars) compared to 0.4 mM Ca²⁺, Mg²⁺ (dashes); (E) binned distribution of D values for different extracellular Ca²⁺ conditions. The vertical arrow points to the lowest binned D value, and the curved arrow highlights the slopes of the D values. (B, D, and F) D values for L16⁺ nanoclusters at (B) 0.1 mM Ca²⁺ (bars) compared to 0.4 mM Ca²⁺, Mg²⁺ (dashes); (D) 0.04 mM Ca²⁺ (bars) compared to 0.4 mM Ca²⁺, Mg²⁺ (dashes); (F) binned distribution of D values for different extracellular Ca²⁺ levels. (G and H) Normalized fractions of stationary, slow, and fast mobile subpopulations for (G) the total (TS2/4), and (H) primed (L16⁺) nanoclusters at different Ca²⁺ levels. Error bars represent the standard deviation (p values are compared to the respective populations at 0.4 mM Ca²⁺, Mg²⁺: ∗  = p < 0.001 compared to the stationary fraction; # = p < 0.00003 compared to the fast fraction; †  = p < 0.03 compared to the stationary fraction; ‡  = p < 0.001 compared to the fast fraction). (I) Binned distribution of D values for TS2/4 (black squares) and L16 (green circles) at (Left) 0.4 mM Ca²⁺, Mg²⁺; (Center) 0.1 mM Ca²⁺; (Right) 0.04 mM Ca²⁺. TS2/4 trajectories: 369 (0.4 mM Ca²⁺, Mg²⁺), 318 (0.1 mM Ca²⁺), and 129 (0.04 mM Ca²⁺). L16 trajectories: 669 (0.4 mM Ca²⁺, Mg²⁺), 656 (0.1 mM Ca²⁺), and 102 (0.04 mM Ca²⁺).

Fig. 4.

The actin cytoskeleton regulates the diffusion of LFA-1 nanoclusters at low Ca²⁺ levels. (A and B) Normalized D distribution for (A) primed (L16⁺) and (B) total (TS2/4) nanoclusters at 0.04 mM Ca²⁺, with cells treated with CytoD (1 μg/mL) (bars), compared to the control (DMSO, 1%) (dashes). (C) Normalized fractions of stationary (gray), slow (blue), and fast (orange) mobile subpopulations for L16 (Left) at 0.04 mM Ca²⁺ in DMSO and after CytoD treatment. For this particular dataset, D_th = 0.002 μm²/s. Due to limited statistics, only the stationary fractions of TS2/4 (Right) in DMSO and after CytoD treatment were estimated. Error bars represent the standard deviation (∗  = p < 0.02 compared to stationary in DMSO; # = p < 0.006 compared to stationary in DMSO). TS2/4 trajectories: 55 (DMSO) and 35 (CytoD). L16 trajectories: 94 (DMSO) and 72 (CytoD).

Fig. 5.

LFA-1 microclustering and cell adhesion under static conditions depends on extracellular Ca²⁺. (A and B) Representative frames of movies recorded in TIRF mode of L16⁺-LFA-1 nanoclusters on monocytes seeded on ICAM-1/BSA micropatterns at (A) 0.4 mM Ca²⁺, Mg²⁺ and (B) 0.04 mM Ca²⁺. Microclusters are surrounded in red, and nanoclusters are highlighted by white arrows. (Scale bars: 5 μm.) (C) Percentage of cells displaying microclusters at different cation conditions. Error bars are the standard deviation from three independent experiments. (D) Intensity distribution of nanoclusters on ICAM-1 regions at 0.04 mM Ca²⁺ (bars) and 0.4 mM Ca²⁺, Mg²⁺ (dashes). (E and F) Interference reflection microscopy of two representative monocytes seeded for 20 min on ICAM-1 substrates at (E) 0.4 mM Ca²⁺, Mg²⁺ and (F) 0.04 mM Ca²⁺. Attachment areas were (153 ± 100) μm² in E and (115 ± 44) μm² in F, thus a reduction of 75% in firm contact area (55 cells inspected in each condition, over 2 separate experiments). (Scale bars: 5 μm.) (G) Histogram of firm cell attachment ratio for monocytes seeded for 20 min on ICAM-1 substrates at 0.04 mM Ca²⁺ (bars) and 0.4 mM Ca²⁺, Mg²⁺ (dashes). The attachment ratio was estimated by dividing the main contact area as measured by interference reflection microscopy by the corresponding bright field image. Mean attachment ratio is 0.70 (σ = 0.19) at 0.4 mM Ca²⁺, Mg²⁺ and 0.55 (σ = 0.16) at 0.04 mM Ca²⁺, thus a reduction to 79% compared to the total cell size at 0.4 mM Ca²⁺. (H) Relative cell adhesion at 0.4 mM Ca²⁺, Mg²⁺ (white) and 0.04 mM Ca²⁺ (black) at different seeding times on ICAM-1 substrates. Results from two independent experiments from 15 different bright field images at each condition.

Fig. 6.

Extracellular Ca²⁺ plays a differential role on LFA-1 adhesiveness under shear-flow conditions. Cell binding to ICAM-Fc coated surfaces quantified as a percentage of maximum binding observed at 1 mM Ca²⁺, Mg²⁺, Mn²⁺ at (A) 0.2 dyn/cm² and (B) 0.5 dyn/cm². Error bars are the standard deviation over average binding calculated over five different areas. One out of two experiments are presented (∗  = p < 0.007 compared to 1 mM Ca²⁺, Mg²⁺, Mn²⁺; # = p < 0.005 compared to 1 mM Ca²⁺, Mg²⁺, Mn²⁺; †  = p < 0.01 compared to 0.04 mM Ca²⁺; ‡  = p < 0.007 compared to 0.4 mM Ca²⁺.