Dynamics of Thymocyte-Stromal Cell Interactions Visualized by Two-Photon Microscopy Philippe Bousso, Nirav R. Bhakta, Richard S. Lewis, and Ellen Robey

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Online Movies

In each example, images were taken in a single focal plane every 10 seconds. The actual duration of each movie is indicated below. All of the movies were acquired using 2-photon microscopy at 37°C.

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• Movie S1    size = 1,851KB

  Active crawling of DP thymocytes occurs in intact fetal thymic lobes reseeded with GFP-expressing T cell precursors. Day 15 fetal thymocytes were retrovirally transduced to express GFP and used to repopulate thymocyte-depleted day 15 fetal thymic lobes. After 6 days of fetal thymic organ culture, lobes were subjected to TPLSM imaging at 3°C. Green (GFP-expressing thymocytes, red, membrane labeling). At this time point, 85% of the thymocytes have a CD4⁺ CD8 ⁺ phenotype. The edges of the tissue are visible in this movie. Duration=17 min

• Movie S2

  The three-dimensional tissue architecture can promote thymocyte crawling. Reaggregate thymic organ cultures were formed using thymocytes from F5-TCR microglobulin^-/- mice and wild-type thymic stromal cell. This movie shows the behavior of thymocytes located inside and at the border of the tissue. The dashed line indicates the limit of the tissue. Note that virtually all thymocytes at the surface of the lobe are rounded and non-motile. By contrast, a large fraction of thymocytes located inside the tissue display an elongated shape and are highly motile. Duration=8 min.

• Movie S3    size = 1,245KB

  Example of clusters of thymocytes associating with selecting (wild-type) stromal cells. Note that some thymocytes remain attached to the stromal cell on the left while actively crawling over its surface. In this example, both dynamic and stable associations can be seen on the same stromal cell. Duration=21 min.

• Movie S4    size = 1,475KB

  This movie shows three thymoytes making very stable interactions with selecting stromal cells. Duration=33 min.

• Movie S5

  An example of an encounter of a thymocyte and a stromal cell, resulting in a stable interaction. A thymocyte crawls in from the left, makes an initial contact with a stromal cell in the center, continues crawling, contacts another stromal cell, and returns to the stromal cell in the center. After the second contact the thymocyte stops and remains associated with the stromal cell for the remainder of the movie. Duration=18 min.

• Movie S6    size = 1,504KB

  Another example of a stable association in which a thymocyte makes initial contact with a selecting stromal cell and stops crawling. A thymocyte crawls in from the left, stops upon contact with the stromal cell and becomes partially enclosed by the stromal cell. Duration=23 min.

• Movie S7

  Example of a stable interaction that ends when the thymocyte suddenly detaches and begins crawling off to the right. Duration=20 min.

• Movie S8

  Another example of cell detachment (top right) following a stable interaction with a selecting stromal cell. Duration=14 min.

• Movie S9

  Example of a dynamic association in which the thymocyte remains attached to the stromal cell while actively crawling over its surface. Duration=16 min.

• Movie S10

  Example of a dynamic association associated with rapid morphological changes of the stromal cell, resulting in the partial enclosure of the thymocyte. Duration=17 min.

• Movie S11

  This movie shows several thymocytes making very dynamic contacts with a selecting stromal cell. Duration=33 min.