Mechanical Forces Impeding Exocytotic Surfactant Release Revealed by Optical Tweezers

Abstract

The release of surfactant from alveolar type II cells is essential to lower the surface tension in the lung and to facilitate inspiration. However, the factors controlling dispersal and diffusion of this hydrophobic material are still poorly understood. Here we report that release of surfactant from the fused vesicle, termed lamellar body (LB), resisted mechanical forces applied by optical tweezers: At constant trapping force, the probability to expand LB contents, i.e., to "pull" surfactant into the extracellular fluid, increased with time after LB fusion with the plasma membrane, consistent with slow fusion pore expansion in these cells. Elevations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) had a similar effect. Inasmuch as surfactant did not disintegrate in the extracellular space, this method permitted for the first time the determination of elastic and recoil properties of the macromolecular complex, yielding a spring constant of approximately 12.5 pN/ micro m. This is the first functional evidence that release of hydrophobic material is mechanically impeded and occurs in an "all-or-none" fashion. This mode of release is most probably the result of cohesive forces of surfactant, combined with adhesive forces and/or retaining forces exerted by a constrictive fusion pore acting as a regulated mechanical barrier, withstanding forces up to 160 pN. In independent experiments equiaxial strain was exerted on cells without optical tweezers. Strain facilitated surfactant release from preexisting fused vesicles, consistent with the view of mechanical impediments during the release process, which can be overcome by cell strain.

Fusion-activated Ca 2+ entry via vesicular P2X 4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes

“…As outlined above, the fusion pore acts as a mechanical barrier for release, resulting in a significant delay between pore opening and content release (16,27). Content release can be directly observed by FM1-43-labeled surfactant that forms tubular or spherical protrusions extending into the extracellular space (Movie S3) on extrusion from fused LBs.…”

“…Moreover, the vesicle content of LBs has properties of solid-state matter that does not disperse in aqueous solutions (i.e., lung surfactant). Thus, slow fusion pore expansion often delays full release, and surfactant "clogs" the fusion pore (16,27), preventing full collapse or ready retrieval of fused vesicles ("kiss-and-run").…”

“…LBs fuse with the plasma membrane in a sequential fashion for up to 20 min after stimulation (23 (25). After LB fusion with the plasma membrane, surfactant, a water-insoluble bulky complex, largely remains entrapped within the fused vesicles (26) in which the fusion pores behave as regulated valves or mechanical barriers for release (16,27). As a result, in vitro full content release can be delayed for minutes up to hours (28).…”

Actin coating and compression of fused secretory vesicles are essential for surfactant secretion: a role for Rho, formins and myosin II

“…Given the apparent stability of LB fusion pores, full widening into the PM is probably constrained. Hence, it is conceivable that fusion pores act as mechanical barriers for vesicle content release (Haller et al, 2001b;Singer et al, 2003) and additional mechanisms are required to promote efficient vesicle cargo discharge.…”

“…Rather, surfactant is so insoluble, that it might remain entrapped within the fused vesicle for many minutes after the onset of exocytosis and the fusion pore apparently acts as a mechanical barrier for the release of the large, macromolecular vesicle cargo entity, which is released as one single complex as observed by live-cell microscopy (Dietl and Haller, 2005;Dietl et al, 2001;Haller et al, 2001a;Singer et al, 2003). Upon fusion of the LB with the PM, fusion pore expansion in these cells is slow (Haller et al, 2001a;Singer et al, 2003). Hence, it is conceivable that secretion of surfactant is not merely a passive process, but requires additional mechanisms to be effective.…”