Microfluidics-based side view flow chamber reveals tether-to-sling transition in rolling neutrophils

Abstract

Neutrophils rolling at high shear stress (above 6 dyn/cm2) form tethers in the rear and slings in the front. Here, we developed a novel photo-lithographically fabricated, silicone(PDMS)-based side-view flow chamber to dynamically visualize tether and sling formation. Fluorescently membrane-labeled mouse neutrophils rolled on P-selectin substrate at 10 dyn/cm2. Most rolling cells formed 5 tethers that were 2–30 μm long. Breaking of a single tether caused a reproducible forward microjump of the cell, showing that the tether was load-bearing. About 15% of all tether-breaking events resulted in slings. The tether-to-sling transition was fast (<100 ms) with no visible material extending above the rolling cell, suggesting a very low bending modulus of the tether. The sling downstream of the rolling cell aligned according to the streamlines before landing on the flow chamber. These new observations explain how slings form from tethers and provide insight into their biomechanical properties.

How neutrophils resist shear stress at blood vessel walls: molecular mechanisms, subcellular structures, and cell–cell interactions

“…2). Tethers are long submicrometer-diameter membrane tubes that are generated upon pulling the microvilli from the cell body if the PSGL-1/P-selectin bond experiences a force exceeding ;30 pN [39,72,73]. Tethers form behind the rolling neutrophil and lead to slower and more uniform rolling of neutrophils in response to changes in wall shear stress.…”

“…2A). Tethers do not retract, but detach from the selectin substrate, swing around the cell, and appear as slings in front of the cell [39,73]. Thereby, 15% of all breaking tethers form slings with an average length of 22 mm.…”

“…Thereby, 15% of all breaking tethers form slings with an average length of 22 mm. Slings hang in the streamline following the flow in front of the cell until the origin of the sling at the cell is close to the wall [73]. Then, the neutrophil rolls over the sling and receives a unique adhesive substrate: during tether formation and growth, multiple microvilli are pulled into the tether that appear as discrete PSGL-1 patches along the sling.…”

Modeling membrane nanotube morphology: the role of heterogeneity in composition and material properties

“…Also, there are only a handful of direct experimental observations of membrane heterogeneities along nanotubes in cells [44]. While it is easy to imagine the beaded morphologies in in vitro studies [20,45], it remains to be seen if such morphologies will be observed in cells. And finally, the role of the active transport versus the flow of cytosolic components in governing the stability of these nanotubes is not captured by our model and remains an active focus of our research and modeling efforts.…”

“…Often, multiple beads are observed along a membrane nanotube, suggesting that multiple domains of heterogeneity exist along the nanotube [15,19,73,[44][45][46][47]17]. (Fig.…”