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Blood, Vol. 113, Issue 23, 5703-5710, June 4, 2009
Cdc42-dependent leading edge coordination is essential for interstitial dendritic cell migration
Blood Lämmermann et al.
113: 5703
Supplemental materials for: Lammermann et al
Files in this Data Supplement:
- Video 1. Symmetry-breaking of dendritic cells in response to homogenous CCL19 exposure (MOV, 8.13 MB)
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Dendritic cells (DCs) were injected between a glass cover slip and a layer of agarose containing 2.5 µg/ml CCL19 (experimental setup, Fig. 2A). After initial radial symmetrical expansion, most of the wild-type DCs (left) segregate into leading and trailing edge. Expansion and symmetry breaking occurs equally in Cdc42−∕− DCs (right) and cells develop multiple leading edges. Bright-field microscopy, time-lapse over 33 min 20 s (20 s/frame), objective: LD A-Plan 20×/0.3 Ph1 (Zeiss).
- Video 2. Actin dynamics of dendritic cells (MOV, 4.8 MB)
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Lifeact:GFP transfected dendritic cells (DCs) were injected between a glass cover slip and an agarose layer with uniform CCL19 concentration (experimental setup, Fig. 2A). Polarized wild-type DCs (left) show continuous expansion of F-actin at the leading edge and retrograde actin flow at sites of lateral retraction. Cdc42−∕− DCs (right) show alternating phases of protrusion and contraction at the cell front resulting in multiple competing leading edges. TIRF microscopy, time-lapse over 3 min 2 s (2 s/frame), objective: Plan-FLUAR 100×/1.45 oil objective (Zeiss).
- Video 3. Dendritic cell chemotaxis towards a CCL19 gradient in a planar layer (MOV, 5.45 MB)
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Dendritic cells (DCs) migrate under agarose towards a CCL19 gradient (on top) (experimental setup, Fig. 3A). Wild-type DCs (left) show polarized morphology and perform directed migration with high velocities and directional persistence. Cdc42−∕− DCs (right) also perform chemotaxis with phases of high velocities that are frequently interrupted by phases when multiple leading edges cause cell spinning or complete migratory arrest. Bright-field microscopy, time-lapse over 2 h 5 min (1 min/frame), objective: LD A-Plan 20×/0.3 Ph1 (Zeiss).
- Video 4. Migration of dendritic cells into lymphatic vessels of a whole-mount dermis (ear crawl-in) (MOV, 1.07 MB)
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Dendritic cells (DCs, red) were layered on top of the dermis of ear explants and incubated for 2 h at 37°C, 5% CO2 (experimental setup, Fig. 5A) . A cutout of the confocal z-stacks in Fig. 5B was three-dimensionally reconstructed. Lymphatic vessels were stained with LYVE-1 antibody (green). While wild-type DCs entered the lymphatics (upper panels), Cdc42−∕− DCs were scattered in the dermis (lower panels).
- Video 5. Dendritic cell chemotaxis towards a CCL19 gradient in a 3D collagen gel (MOV, 4.52 MB)
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Dendritic cells (DCs) migrate in a 1.6 mg/ml collagen gel towards a CCL19 gradient (on top) (experimental setup, Fig. 6D). Wild-type DCs (upper panel) perform directed migration with high velocities and directional persistence. In contrast, Cdc42−∕− DCs (lower panel) had a drastically reduced migration perimeter in a 3D environment. Bright-field microscopy, time-lapse over 3 h (2 min/frame), A-Plan 10×/0.25 Ph1 objective.
- Video 6. Multipolar dendritic cells entangle in 3D environments (MOV, 1.18 MB)
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Single dendritic cells (DCs) migrate in a 1.6 mg/ml collagen gel towards a CCL19 gradient (on top) (experimental setup, Fig. 6D). Wild-type DCs (left) funnel all actin flow into few successful protrusions at the cell front. In contrast, Cdc42−∕− DCs (right) entangle, as they lack coordination of the actin flow to the front. DIC microscopy with an inverted Zeiss Axiovert 200M equipped with a EC Plan-NEOFLUAR 40×/0.75 Ph2 objective, time-lapse over 5 min 5 s (5 s/frame).
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