F-actin scaffold stabilizes lamellar bodies during surfactant secretion

Abstract

Alveolar type 2 (AT2) cells secrete surfactant that forms a protective layer on the lung's alveolar epithelium. Vesicles called lamellar bodies (LBs) store surfactant. Failure of surfactant secretion, which causes severe lung disease, relates to the manner in which LBs undergo exocytosis during the secretion. However, the dynamics of LBs during the secretion process are not known in intact alveoli. Here, we addressed this question through real-time confocal microscopy of single AT2 cells in live alveoli of mouse lungs. Using a combination of phospholipid and aqueous fluorophores that localize to LBs, we induced surfactant secretion by transiently hyperinflating the lung, and we quantified the secretion in terms of loss of bulk LB fluorescence. In addition, we quantified inter-LB phospholipid flow through determinations of fluorescence recovery after photobleaching. Furthermore, we determined the role of F-actin in surfactant secretion through expression of the fluorescent F-actin probe Lifeact. Our findings indicate that, in AT2 cells in situ, LBs are held in an F-actin scaffold. Although F-actin transiently decreases during surfactant secretion, the LBs remain stationary, forming a chain of vesicles connected by intervesicular channels that convey surfactant to the secretion site on the plasma membrane. This is the first instance of a secretory process in which the secretory vesicles are immobile, but form a conduit for the secretory material.

Disruption of staphylococcal aggregation protects against lethal lung injury

“…Additionally, cytCa 2+ increases occurred not only in alveoli containing MAs, but also in alveoli free of bacteria ( Figure 4H We considered that this spread of cytCa 2+ could have injurious cellular effects as reflected by mitochondrial function (41), surfactant secretion (42), and alveolar barrier integrity (43). Within 15 minutes of MA formation, there was widespread loss of epithelial mitochondrial potential (Figure 5, A-G) and inhibition of alveolar surfactant secretion ( Figure 5, H-M), which we quantified in terms of lamellar body exocytosis (44). After 6 hours, alveolar permeability to intravenously infused high-molecular weight dextran increased markedly ( WT -containing alveoli that were pretreated with BAPTA-AM (Figure 5, E-G, P, and Q), or intranasally instilled with inoculums containing low bacterial counts (Supplemental Figure 5).…”

“…We induced surfactant secretion in isolated, blood-perfused lungs (IPLs) by a single hyperinflation, in which airway pressure was increased from 5 to 15 cm H 2 O for 15 seconds (44).…”

Epithelial Gpr116 regulates pulmonary alveolar homeostasis via Gq/11 signaling

“…GPR116 activation induces cortical F-actin assembly. In vitro and in vivo studies implicate cytoskeletal remodeling processes in the regulation of surfactant secretion and uptake (18,(48)(49)(50)(51)(52)(53)(54)(55)(56). We therefore sought to determine if constitutively active GPR116 CTF caused F-actin organization in HEK cells.…”

“…Actin cytoskeletal remodeling was implicated in the regulation of surfactant secretion and uptake in vitro and in vivo; however, the precise role of actin in surfactant homeostasis remains unclear, as cell and animal studies report both suppression and augmentation of secretion following actin cytoskeletal disruption (18,(48)(49)(50)(51)(52). Tang et al reported that endogenous hGPR116 regulates F-actin stress fiber formation and cell motility in transformed MDA-MB-231 breast cancer cells in a G q / 11 -and Rho-dependent manner (64).…”

“…-dependent pathways (8,(17)(18)(19)(20). While G protein signaling was implicated in surfactant secretion in vitro, mechanisms by which individual GPCRs regulate surfactant homeostasis remain incompletely understood.…”