Figure 4
Visualization, diffusion and dimerization of Alexa546-labeled SNAP-D2L receptors and on membrane protrusions using HF-treated slides.
(a) Representative images of a single CHO cell stably expressing the SNAP-D2L receptor and seeded on HF-treated glass slides, labeled with Alexa546-BG and visualized by TIRF-M. The insert corresponds to higher magnification image of the area in the small white box. (b) Plot of mean square displacement (MSD ± s.d.) versus the time interval (δt) of receptor particles that were tracked in a. The plot is linear (r2 = 0.99 – linear fit (blue)), over a 3-s timescale, which is consistent with receptor movement following a random walk. (c) The distribution of the diffusion coefficients of the analyzed receptor particles (n) are shown (n = 4409, 5 cells - HF-treated slide; n = 4409, 8 cells - non HF-treated slide) and revealed no evidence for anomalous diffusive behavior as a result of HF-treatment. (d) Representative intensity distribution of fluorescent spots identified over the first 10-frame time window of TIRF-illumination. Data were fitted with a mixed Gaussian model (sum of two Gaussian functions). (e) Representative TIRF-M images of CHO cells stably transfected with SNAP-D2L receptor, incubated in iso-osmotic (300 mOsm) (left) and hypo-osmotic PBS (108 mOsm) (right) for 2 h and labeled with Alexa546-BG. (f,g) Imaging of a region of membrane protrusions of CHO cells stably transfected with the SNAP-D2L receptor, incubated in hypo-osmotic (108 mOsm) PBS for 2 h and labeled with Alexa546-BG in epi-illumination (f) and TIRF-illumination (g). (h) Representative images of one membrane protrusion of a CHO cell stably expressing the labeled SNAP-D2L receptor and visualized by TIRF-M. (f) Plot of mean square displacement (MSD ± s.d.) versus the time interval (δt) of receptor particles that were tracked in (h). The plot is linear (r2 = 0.99 – linear fit (blue)), over a 2,5-s time scale and the calculation of the average diffusion coefficient Dlat of 0.077 ± 0.007 μm2 s−1 (mean ± s.d., 16 regions of membrane protrusions of 8 cells) revealed no evidence for anomalous diffusive behavior in the membrane protrusions. Scale bars, 10 μm.
