Membrane dynamics correlate with formation of signaling clusters during cell spreading

Abstract

The morphology and duration of contacts between cells and adhesive surfaces play a key role in several biological processes, such as cell migration, cell differentiation, and the immune response. The interaction of receptors on the cell membrane with ligands on the adhesive surface leads to triggering of signaling pathways, which allow cytoskeletal rearrangement, and large-scale deformation of the cell membrane, which allows the cell to spread over the substrate. Despite numerous studies of cell spreading, the nanometer-scale dynamics of the membrane during formation of contacts, spreading, and initiation of signaling are not well understood. Using interference reflection microscopy, we study the kinetics of cell spreading at the micron scale, as well as the topography and fluctuations of the membrane at the nanometer scale during spreading of Jurkat T cells on antibody-coated substrates. We observed two modes of spreading, which were characterized by dramatic differences in membrane dynamics and topography. Formation of signaling clusters was closely related to the movement and morphology of the membrane in contact with the activating surface. Our results suggest that cell membrane morphology may be a critical constraint on signaling at the cell-substrate interface.

Figure 1

Spreading of Jurkat T lymphocytes on antibody-coated substrates. (a) Time-lapse IRM images showing the increasing contact zone as the cell spreads out. Scale bar, 5 μm. (b) Contact area as a function of time for six representative cells. The smooth lines are fits to a tanh function, A(t) ∼ A₀ tanh(αt). (c) Rescaled graphs showing that the spreading of all cells can be described by a common spreading function. (d) Histogram of the spreading rate, α (n = 88). (e) Histogram of the final spread area A₀.

Figure 2

Cells with compromised actin polymerization do not spread efficiently. (a) Spreading rate (α) decreases as the concentration of Lat-A increases (p < 0.01, t-test). (b) The final spread area is unaffected for small concentrations of Lat-A, but is much smaller for higher concentrations (p < 0.05, t-test). (c) Spreading rate is diminished upon inhibition of the activity of NMMII or Rho kinase (ROCK). (d) Final spread area is not affected by inhibition of NMMII or ROCK. The number of cells analyzed was >18 in all conditions.

Figure 3

Spreading in the presence of serum is qualitatively different. (a) Time-lapse IRM images of a T cell spreading on antibody-coated glass substrate in the presence of serum. Scale bar, 5 μm. (b) Contact area of the cell as a function of time. Each graph corresponds to a different cell. The solid gray lines show a fit to a hyperbolic tangent function. (c) Comparison of spreading rate, α, and final spread area, A₀, for serum and serum-free cases shows that these parameters are very similar in the two conditions. (d) Kymographs of four representative sections in serum. (e) Kymographs of two representative sections in serum-free conditions (scale bars, 3 μm, 30 s).

Figure 4

Cell-contact topography is different in the two spreading modes. (a) Denoised IRM images for cells in serum-free media, and (b) in serum containing media. (c) Heat map showing intensity fluctuations relative to shot noise for cells in serum-free media. (d) Heat map showing intensity fluctuations relative to shot noise for cells in serum containing media. (e) Population histograms of relative fluctuation amplitudes, $\stackrel{⌢}{u}$, in serum-free and (f) in serum. Note: The color bars in both cases represent fluctuation amplitudes relative to shot noise. Scale bars, 5 μm; n = 15 for each condition.

Figure 5

Membrane topography and dynamics of contact are correlated with signaling. (a) Left: TIRF image showing ZAP70 clusters; middle: denoised IRM intensity map showing relative membrane height; right: contours in red show ZAP70 clusters superimposed on the height map. Scale bar, 5 μm. (b) ZAP70 clusters (contours in black) superimposed on regions of low fluctuation, shown in four typical zoomed-in sections of the contact zone from four different cells in serum-supplemented medium. Scale bar, 2 μm. (c) Cumulative histogram of the normalized height fluctuations (black, serum-free; red, serum-supplelmented) for pixels belonging to ZAP70 clusters (10 cells for each condition).

Figure 6

Dynamics and spatial distribution of ZAP70 clusters is distinct in the two modes of spreading. (a) Time-lapse TIRF image of T cells expressing YFP-ZAP70 spreading on an antibody-coated glass substrate in serum-free media. The cyan (inner) line represents the boundary of the clustered zone at any given time point. The red (outer) line denotes the final boundary of the fully spread cell. (b) Time-lapse TIRF image of T cells spreading in serum. To facilitate comparison, the three time-lapse images shown are at 40 s, 80 s, and 160 s (corresponding to rescaled times of 1, 2, and 4). (c) TIRF image of a typical YFP-ZAP70 cell in serum-free media. (d) TIRF image of a typical YFP-ZAP70 cell in serum containing media. Scale bars, 5 μm. (e) Intensity-weighted second spatial moment of fluorescent clusters show that ZAP70 clusters are more peripheral in serum-free conditions but uniformly distributed in serum (p < 0.001, Wilcoxon rank test).