Figure 9. Integrin β3 molecules are... | Rockefeller University Press

Figure 9.

$Integrin β3 molecules are temporarily immobilized at the paxillin-enriched islands and perhaps on FA-protein islands in general. (A) Typical results of single-molecule tracking of SeTau647-ACP-integrin β3 (recorded at a 4-ms resolution; large magenta spots; images shown every 0.1 s or 25 frames) simultaneously performed with the live-cell PALM of mEos3.2-paxillin, visualizing the paxillin-enriched islands (green spots; the same PALM image is used for all of the images). Data were obtained by using T24 cells. The magenta spots representing single integrin β3 molecules appear much larger than the green spots representing the paxillin-enriched islands because the spot size in single-molecule imaging is diffraction-limited (≈250 nm for single SeTau647-ACP-integrin β3 molecule: the single-molecule localization precision was ≈21 nm, obtained from the SDs of 15-frame immobile trajectories; n = 30), whereas the FA islands were imaged by PALM. The FAs we consider here are indicated by the white contours in the first frames, which were determined by applying the minimum cross entropy thresholding to the PALM images. (a) Three fluorescently labeled integrin β3 molecules (marked as α, β, and γ) appeared in the FA region. The trajectory of the α molecule is shown. Time 0 is set at the time when the α molecule started exhibiting immobilization in an FA. The immobilization of this molecule lasted for 0.19 s (orange frames). The signal intensities of the magenta spots at times 0.3 and 0.4 s are lower, probably due to the stochastic fluctuation of emitted photon numbers and blinking (off-periods) shorter than 4 ms. (b) An integrin β3 molecule was immobilized from time 0 till the end of the observation, for 1.1 s (270 frames at 250 Hz). Large majorities (>90%) of integrin β3 molecules in the FA were immobile throughout the observation period of 1.1 s, consistent with the previous results reported by Tsunoyama et al. (2018). (B) Distribution of the pixel intensities of the mEos3.2-paxillin signal in the FA indicated by the white contour in the first frame in A-a, normalized by its median pixel intensity. The thermographic color scale for this FA is superimposed (in the linear scale between 0× and 6× of the median intensity). (C) The thermographic PALM image of mEos3.2-paxillin shown in A-a, using the color scale shown in B. The immobilized area identified in the trajectory of the molecule α, as previously defined (Simson et al., 1995), is shown by a green 104-nm diameter circle along with the yellow trajectory (also see the expanded figure on the right). The paxillin pixel signal intensity at the center of the immobilized position was measured (open arrow in B), and after this was done for 60 integrin β3 immobilization events, the histogram in D bottom was generated. (D) Top: Averaged normalized distribution of mEos3.2-paxillin signal pixel intensities (the distributions like that shown in B for a single FA are averaged over 55 FAs). Bottom: Distribution of the normalized mEos3.2-paxillin signal pixel intensities at the centers of the immobilization circles of single integrin β3 molecules (such as the green circle in C; n = 60 events), indicating that integrin molecules are preferentially immobilized on the paxillin-enriched islands (refer to the main text). (E) Typical ultrafast single fluorescent-molecule trajectories of integrin β3 molecules observed at 6 kHz, diffusing outside the FA region (top), entering the FA region (middle), and diffusing and becoming temporarily immobilized inside the FA zone (bottom). The middle figure displays a typical result of simultaneous observations of ultrafast (6 kHz) single-molecule tracking of SeTau647-Halo-integrin β3 (colored trajectory) and ultrafast live-cell PALM of mEos3.2-paxillin (islands; 1 kHz, 10-s integration), showing the entry of integrin β3 from the bulk basal PM into the FA region. Consistent with the FA model of the archipelago architecture of the FA-protein islands in the compartmentalized fluid, integrin molecules continued to exhibit hop diffusion when they entered the FA region outside the paxillin-enriched islands.$

Integrin β3 molecules are temporarily immobilized at the paxillin-enriched islands and perhaps on FA-protein islands in general. (A) Typical results of single-molecule tracking of SeTau647-ACP-integrin β3 (recorded at a 4-ms resolution; large magenta spots; images shown every 0.1 s or 25 frames) simultaneously performed with the live-cell PALM of mEos3.2-paxillin, visualizing the paxillin-enriched islands (green spots; the same PALM image is used for all of the images). Data were obtained by using T24 cells. The magenta spots representing single integrin β3 molecules appear much larger than the green spots representing the paxillin-enriched islands because the spot size in single-molecule imaging is diffraction-limited (≈250 nm for single SeTau647-ACP-integrin β3 molecule: the single-molecule localization precision was ≈21 nm, obtained from the SDs of 15-frame immobile trajectories; n = 30), whereas the FA islands were imaged by PALM. The FAs we consider here are indicated by the white contours in the first frames, which were determined by applying the minimum cross entropy thresholding to the PALM images. (a) Three fluorescently labeled integrin β3 molecules (marked as α, β, and γ) appeared in the FA region. The trajectory of the α molecule is shown. Time 0 is set at the time when the α molecule started exhibiting immobilization in an FA. The immobilization of this molecule lasted for 0.19 s (orange frames). The signal intensities of the magenta spots at times 0.3 and 0.4 s are lower, probably due to the stochastic fluctuation of emitted photon numbers and blinking (off-periods) shorter than 4 ms. (b) An integrin β3 molecule was immobilized from time 0 till the end of the observation, for 1.1 s (270 frames at 250 Hz). Large majorities (>90%) of integrin β3 molecules in the FA were immobile throughout the observation period of 1.1 s, consistent with the previous results reported by Tsunoyama et al. (2018). (B) Distribution of the pixel intensities of the mEos3.2-paxillin signal in the FA indicated by the white contour in the first frame in A-a, normalized by its median pixel intensity. The thermographic color scale for this FA is superimposed (in the linear scale between 0× and 6× of the median intensity). (C) The thermographic PALM image of mEos3.2-paxillin shown in A-a, using the color scale shown in B. The immobilized area identified in the trajectory of the molecule α, as previously defined (Simson et al., 1995), is shown by a green 104-nm diameter circle along with the yellow trajectory (also see the expanded figure on the right). The paxillin pixel signal intensity at the center of the immobilized position was measured (open arrow in B), and after this was done for 60 integrin β3 immobilization events, the histogram in D bottom was generated. (D) Top: Averaged normalized distribution of mEos3.2-paxillin signal pixel intensities (the distributions like that shown in B for a single FA are averaged over 55 FAs). Bottom: Distribution of the normalized mEos3.2-paxillin signal pixel intensities at the centers of the immobilization circles of single integrin β3 molecules (such as the green circle in C; n = 60 events), indicating that integrin molecules are preferentially immobilized on the paxillin-enriched islands (refer to the main text). (E) Typical ultrafast single fluorescent-molecule trajectories of integrin β3 molecules observed at 6 kHz, diffusing outside the FA region (top), entering the FA region (middle), and diffusing and becoming temporarily immobilized inside the FA zone (bottom). The middle figure displays a typical result of simultaneous observations of ultrafast (6 kHz) single-molecule tracking of SeTau647-Halo-integrin β3 (colored trajectory) and ultrafast live-cell PALM of mEos3.2-paxillin (islands; 1 kHz, 10-s integration), showing the entry of integrin β3 from the bulk basal PM into the FA region. Consistent with the FA model of the archipelago architecture of the FA-protein islands in the compartmentalized fluid, integrin molecules continued to exhibit hop diffusion when they entered the FA region outside the paxillin-enriched islands.

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