During immune surveillance and inflammation, leukocytes exit the vasculature through transient openings in the endothelium without causing plasma leakage. However, the exact mechanisms behind this intriguing phenomenon are still unknown. Here we report that maintenance of endothelial barrier integrity during leukocyte diapedesis requires local endothelial RhoA cycling. Endothelial RhoA depletion in vitro or Rho inhibition in vivo provokes neutrophil-induced vascular leakage that manifests during the physical movement of neutrophils through the endothelial layer. Local RhoA activation initiates the formation of contractile F-actin structures that surround emigrating neutrophils. These structures that surround neutrophil-induced endothelial pores prevent plasma leakage through actomyosin-based pore confinement. Mechanistically, we found that the initiation of RhoA activity involves ICAM-1 and the Rho GEFs Ect2 and LARG. In addition, regulation of actomyosin-based endothelial pore confinement involves ROCK2b, but not ROCK1. Thus, endothelial cells assemble RhoA-controlled contractile F-actin structures around endothelial pores that prevent vascular leakage during leukocyte extravasation.

The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology. Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling.

Summary Although many proteins, receptors, and viruses are transported rearward along filopodia by retrograde actin flow[1-3], it is less clear how molecules move forward in filopodia. Myosin-X (Myo10) is an actin-based motor hypothesized to use its motor activity to move forward along actin filaments to the tips of filopodia[4]. Here we use a sensitive total internal reflection fluorescence (TIRF) microscopy system to directly visualize the movements of GFP-Myo10. This reveals a novel form of motility at or near the single-molecule level in living cells wherein extremely faint particles of Myo10 move in a rapid and directed fashion towards the filopodial tip. These fast forward movements occur at ∼600 nm/s over distances of up to ∼10 μm and require Myo10 motor activity and actin filaments. As expected for imaging at the single-molecule level, the faint particles of GFP-Myo10 are diffraction-limited, have an intensity range similar to single GFP molecules, and exhibit stepwise bleaching. Faint particles of GFP-Myo5a can also move towards the filopodial tip, but at a slower characteristic velocity of ∼250 nm/s. Similar movements were not detected with GFP-Myo1a, indicating that not all myosins are capable of intrafilopodial motility. These data indicate the existence of a novel system of long-range transport based on the rapid movement of myosin molecules along filopodial actin filaments.

Plakoglobin (PG) is a member of the Armadillo family of adhesion͞ signaling proteins and has been shown to play a critical role in the organization of desmosomes and tissue integrity. Because dissolution of intercellular junctions is frequently an initial step in the onset of epithelial cell migration, we examined whether loss of PG promotes cell motility by compromising adhesive strength. Keratinocyte cultures established from PG؊͞؊ mice exhibited weakened adhesion and increased motility in transwell migration assays; both were restored by reintroducing PG through adenoviral infection. Interestingly, single PG؊͞؊ cells also exhibited increased motility, which was suppressed by reintroducing PG, but not the closely related ␤-catenin. Whereas both N-and C-terminally truncated PG deletion mutants restored adhesion, only N-terminally deleted PG, but not C-terminally deleted PG, suppressed single-cell migration. Furthermore, both the chemical inhibitor PP2 and dominant-negative Src tyrosine kinase inhibited single-cell motility in PG؊͞؊ cells, whereas constitutively active Src overcame the inhibitory effect of PG. These data demonstrate that PG strengthens adhesion and suppresses motility in mouse keratinocytes, through both intercellular adhesion-dependent and -independent mechanisms, the latter of which may involve suppression of Src signaling through a mechanism requiring the PG C terminus.

Directed cell migration in native environments is influenced by multiple migratory cues. These cues may include simultaneously occurring attractive soluble growth factor gradients and repulsive effects arising from cell-cell contact, termed contact inhibition of locomotion (CIL). How single cells reconcile potentially conflicting cues remains poorly understood. Here we show that a dynamic crosstalk between epidermal growth factor (EGF) mediated chemotaxis and CIL guide metastatic breast cancer cell motility, whereby cells become progressively insensitive to CIL in a chemotactic input-dependent manner. This balance is determined via integration of protrusion-enhancing signaling from EGF gradients and protrusion-suppressing signaling induced by CIL, mediated in part through EphB. Our results further suggest that EphB and EGF signaling inputs control protrusion formation by converging onto regulation of phosphatidylinositol 3-kinase (PI3K). We propose that this intricate interplay may enhance the spread of loose cell ensembles in pathophysiological conditions such as cancer, and possibly other physiological settings.