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Membrane phase separation drives organization at B cell receptor clusters
Sarah A. Shelby, Ivan Castello-Serrano, Kathleen C. Wisser, Ilya Levental, Sarah L. Veatch

doi: https://doi.org/10.1101/2021.05.12.443834

 

Supplementary Movie 1-2: Single molecule motions of BCR and PM before (1) and after (2) BCR crosslinking. Time-lapse video showing reconstructed single molecule positions evolving in time for BCR (magenta) and PM (green) for the same PM expressing cell shown in Fig 1. A-C. (1) These localizations were acquired <1min prior to the addition of streptavidin, or 2 min after the addition of streptavidin.  Localizations are drawn on a reconstructed image of all PM localizations for this cell.  The movie is displayed with a frame rate that is 5x slower than real time.  Scale bar is 5 µm.

 

 

Supplementary Movie 3-4: Single molecule motions of BCR and M before (3) and after (4) BCR crosslinking. Time-lapse video showing reconstructed single molecule positions evolving in time for BCR (magenta) and M (green) for the same M expressing cell shown in Fig 1. A-C. These localizations were acquired <1min prior to the addition of streptavidin, or 2 min after the addition of streptavidin.  Localizations are drawn on a reconstructed image of all PM localizations for this cell.  The movie is displayed with a frame rate that is 5x slower than real time.  Scale bar is 5 µm.

 

Supplementary Movie 5: Time lapse of BCR and PM organization before and after BCR cross-linking. Time lapse movie composed of reconstructed images of BCR (magenta) and PM (green) from the same representative cell shown in Fig 1. A-C, shown here at higher time resolution to demonstrate how BCR and PM organization evolve over time. Each sequential image is reconstructed from a window of 750 frames (~20sec) incremented by 250 frames for each image. The entire time lapse spans the length of the live cell experiment and the real time of data acquisition is shown in the bottom-left, where 0 min corresponds to streptavidin addition. Before streptavidin addition, both BCR and PM are relatively uniformly distributed across the plasma membrane. After streptavidin addition, BCR rapidly forms tight clusters. BCR clusters are relatively immobile compared to PM, but do exhibit dynamics that are clearly evident in the time lapse. Scale bar is 5 µm.

 

Supplementary Movie 6: Time lapse of BCR and M organization before and after BCR cross-linking. Time lapse movie composed of reconstructed images of BCR (magenta) and M (green)from the same representative cell shown in Fig 1. A-C. Parameters for reconstruction of individual frames of the time lapse are the same as in Supplementary Movie 3, and the time lapse shows similar organization of BCR and M before and after streptavidin addition. Time before and after streptavidin addition in shown in the bottom left and the scale bar is 5 µm.