Cdc42-dependent leading edge coordination is essential for interstitial dendritic cell migration

Abstract

Mature dendritic cells (DCs) moving from the skin to the lymph node are a prototypic example of rapidly migrating amoeboid leukocytes. Interstitial DC migration is directionally guided by chemokines, but independent of specific adhesive interactions with the tissue as well as pericellular proteolysis. Instead, the protrusive flow of the actin cytoskeleton directly drives a basal mode of locomotion that is occasionally supported by actomyosin contractions at the trailing edge to propel the cell's rigid nucleus. We here delete the small GTPase Cdc42 in DCs and find that actin flow and actomyosin contraction are still initiated in response to chemotactic cues. Accordingly, the cells are able to polarize and form protrusions. However, in the absence of Cdc42 the protrusions are temporally and spatially dysregulated, which leads to impaired leading edge coordination. Although this defect still allows the cells to move on 2-dimensional surfaces, their in vivo motility is completely abrogated. We show that this difference is entirely caused by the geometric complexity of the environment, as multiple competing protrusions lead to instantaneous entanglement within 3-dimensional extracellular matrix scaffolds. This demonstrates that the decisive factor for migrating DCs is not specific interaction with the extracellular environment, but adequate coordination of cytoskeletal flow. (Blood. 2009;113: 5703-5710)

Coincidental loss of DOCK8 function in NLRP10-deficient and C3H/HeJ mice results in defective dendritic cell migration

“…In contrast, migration of Nlrp10 −/− DCs in the 3D collagen system was almost completely abrogated (Fig. 1A), similar to DOCK8-or CDC42-deficient DCs (9,10). Further, the Nlrp10 −/− strain exhibited other phenotypes that have been described in DOCK8-deficient mice, such as loss of marginal zone B cells (Fig.…”

“…1 C and D). DOCK8 is a GEF whose primary target is the Rho GTPase CDC42, which in turn regulates endocytosis of antigens in immature DCs (16) and migration of mature DCs to draining LNs (10). Nlrp10 −/− BMDCs efficiently phagocytosed antigen in vitro (4), suggesting that CDC42 activity in immature DCs was intact.…”

“…DOCK8 is a guanine nucleotide exchange factor (GEF) that has two functional domains, DOCK homology region (DHR) 1 and DHR2 (8). In murine DCs, the DHR2 domain has been implicated in regulating the Rho GTPase CDC42 (cell division control protein 42 homolog), which in turn maintains cell polarity of mature DCs during migration (9,10). Furthermore, mice harboring inactivating mutations in Dock8 lack marginal zone B-cell development, long-term antibody production following immunization, and memory CD8 + T-cell responses to viral infections (11,12).…”

Dendritic cell chemotaxis in 3D under defined chemokine gradients reveals differential response to ligands CCL21 and CCL19

“…However, our current understanding of DC chemotaxis is largely qualitative and limited to 2D (16)(17)(18)(19). Furthermore, while it is well established that cell migration requires different mechanisms in 3D vs. 2D (20)(21)(22)(23)(24)(25), nearly all chemotaxis studies to date have been performed in 2D or 2.5D conditions largely because of the lack of model systems in which well defined gradients can be created in 3D while evaluating cell invasion (especially because DC migration is relatively fast compared to the time scale of diffusion for gradient establishment). Here, we describe the chemotactic behavior of DCs in 3D to well defined gradients of CCL21 and CCL19, revealing their chemosensitivity to ligand gradient strength, insensitivity to ligand state (bound vs. soluble) and enhanced response to CCL21 vs. CCL19.…”

“…To observe DC chemotaxis in 3D environments in response to gradients of CCR7 ligands, we used a biological extracellular matrix of 1.5 mg∕mL type I collagen and 10% Matrigel (MG), which had the appropriate stiffness and composition for optimal DC migration (25), while also containing heparan sulfate proteoglycans to allow natural CCL21 binding. This matrix differs from 3D matrices in vivo, which are more complex, composed of higher matrix densities, and heterogeneous in composition and architecture (27); however, physiological processes like tumor cell invasion and dendritic cell migration can be mimicked with close similarity to the in vivo situation in such matrices (21,23,25).We first modified our agarose-based microfluidic gradient culture device (26) to rapidly (within 2 min) establish stable and …”