Most leukocytes can roll along the walls of venules at low shear stress (1 dyn/cm 2 ), but neutrophils have the ability to roll at 10-fold higher shear stress in microvessels in vivo 1 , 2 . The mechanisms involved in this shear-resistant rolling are known to involve cell flattening 3 and pulling of long membrane tethers at the rear 4 – 6 . Here, we show that these long tethers do not retract as postulated 6 , 7 , but instead persist and appear as ‘slings’ at the front of rolling cells. We demonstrate slings in a model of acute inflammation in vivo and on P-selectin in vitro , where P-selectin-glycoprotein-ligand-1 (PSGL-1) is presented as discrete sticky patches while LFA-1 is expressed over the entire length on slings. As neutrophils roll forward, slings wrap around the rolling cells and undergo a step-wise peeling from the P-selectin substrate enabled by the failure of PSGL-1 patches under hydrodynamic forces. The ‘step-wise peeling of slings’ is distinct from the ‘pulling of tethers’ reported previously 4 – 6 , 8 . Each sling effectively lays out a cell-autonomous adhesive substrate in front of neutrophils rolling at high shear stress during inflammation.
We introduce quantitative dynamic footprinting microscopy to resolve neutrophil rolling on P-selectin. We show that the footprint of a rolling neutrophil is four times larger than previously thought, the P-selectin-PSGL-1 bonds are relaxed at the leading edge of the rolling cell, compressed under the cell center, and stretched at the trailing edge. Each rolling neutrophil also forms 3-4 long tethers that extend up to 16 μm behind the rolling cell.
Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high-affinity conformation with an ‘open' headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation.
The cyclical protrusion and retraction of the leading edge is a hallmark of many migrating cells involved in processes such as development, inflammation, and tumorigenesis. The molecular identity of signaling mechanisms that control these cycles has remained unknown. Here, we used live cell imaging of biosensors to monitor spontaneous morphodynamic and signaling activities, and employed correlative image analysis to examine the role of cAMP-activated Protein Kinase A (PKA) in protrusion regulation. PKA activity at the leading edge is closely synchronized with rapid protrusion and with the activity of RhoA. Ensuing PKA phosphorylation of RhoA and the resulting increased interaction between RhoA and RhoGDI establishes a negative feedback that controls the cycling of RhoA activity at the leading edge. Thus, cooperation between PKA, RhoA, and a RhoGDI forms a pacemaker that governs the morphodynamic behavior of migrating cells.
The ecology of microbes in the gut has been shown to play important roles in the health of the host. To better understand microbial growth and population dynamics in the proximal colon, the primary region of bacterial growth in the gut, we built and applied a fluidic channel that we call the "minigut." This is a channel with an array of membrane valves along its length, which allows mimicking active contractions of the colonic wall. Repeated contraction is shown to be crucial in maintaining a steady-state bacterial population in the device despite strong flow along the channel that would otherwise cause bacterial washout. Depending on the flow rate and the frequency of contractions, the bacterial density profile exhibits varying spatial dependencies. For a synthetic cross-feeding community, the species abundance ratio is also strongly affected by mixing and flow along the length of the device. Complex mixing dynamics due to contractions is described well by an effective diffusion term. Bacterial dynamics is captured by a simple reaction-diffusion model without adjustable parameters. Our results suggest that flow and mixing play a major role in shaping the microbiota of the colon.
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