The recirculation of leukocytes is essential for proper immune responses. However, the molecular mechanisms that regulate the entry of leukocytes into the lymphatics remain unclear. Here we show that plexin-A1, a principal receptor component for class III and class VI semaphorins, was crucially involved in the entry of dendritic cells (DCs) into the lymphatics. Additionally, we show that the semaphorin Sema3A, but not Sema6C or Sema6D, was required for DC transmigration and that Sema3A produced by the lymphatics promoted actomyosin contraction at the trailing edge of migrating DCs. Our findings not only demonstrate that semaphorin signals are involved in DC trafficking but also identify a previously unknown mechanism that induces actomyosin contraction as these cells pass through narrow gaps.
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- Supplementary Text and Figures (2M)
Supplementary Figures 1–7 and Supplementary Methods
- Supplementary Video 1 (2M)
Normal to sense direction in response to chemokines in plexin-A1−/− DCs. DCs from wild-type or plexin-A1−/− mice were allowed to adhere to fibronectin-coated cover-slips and placed in a CCL21 gradient in a Zigmond chamber. DC trafficking was recorded every 30 sec by a confocal time-lapse video microscope.
- Supplementary Video 2 (4M)
Plexin-A1−/− DCs exhibit impaired transmigration across a lymphatic EC-monolayer. BMDCs derived from wild-type or plexin-A1−/− mice were added to lymphatic EC monolayers, and interactions between DCs and the lymphatic ECs were recorded every 30 sec by a time-lapse video microscope. The yellow dotted lines show the cellular junctions of the ECs. White arrows indicate DCs that were contacting the lymphatic ECs.
- Supplementary Video 3 (980K)
Impaired transmigration in plexin-A1−/− DCs. CFSE-labeled DCs derived from wild-type or plexin-A1−/− mice were added to lymphatic EC monolayers, incubated for 45 min, fixed, and then stained with Alexa 546-conjugated phalloidin. Confocal microscope images were obtained with an optical section separation (Z-interval) of 0.22 μm. Twelve Z-stack images were reconstituted into a 3Dimage using Imaris 3D software. Wild-type DCs penetrated from the apical to basal sides, but plexin-A1−/− DCs could not reach the basal side.
- Supplementary Video 4 (980K)
Sema3A acts on the rear side of migrating DCs. Two-dimensional DC chemotaxis assays using EZ-TAXIScan were performed, in which recombinant Sema3A or human IgG was applied to the opposite side of CCL21. DC migration was recorded every 30 sec by a time-lapse video microscope.
- Supplementary Video 5 (192K)
Plexin-A1 localizes to the back of migrating DCs. Plexin-A1-EGFP fusion protein-expressed BMDCs treated with LPS were suspended in type I collagen (3 mg/ml) containing 2% FCS and then placed on one side of the Zigmond chamber to cover the stage with gel. After gel was polymerized, CCL21was added to the other side. DC locomotion was examined at 1-min intervals by a confocal time-lapse video microscope.
- Supplementary Video 6 (1M)
Sema3A enhances the velocity of DC migration in 3D-collagen matrices. BMDCs treated with LPS for 12 h were suspended in type I collagen (3 mg/ml) containing 2% FCS with either a human IgG or Sema3A-Fc and then placed on one side of the Zigmond chamber to cover the stage with gel. The cells were incubated at 37C for 30 min to polymerize the matrix, and then RPMI containing 0.5% BSA with CCL21 (5 μg/ml) was added to the other chamber. After a 20-min incubation, DC locomotion was examined at 1-min intervals by a confocal time-lapse video microscope.