Abstract
Purpose: :
Lacrimal gland acinar cells (LGACs) are epithelial cells specialized for exocrine secretory functions. The basal-to-apical transcytosis of dimeric IgA, a key component of the mucosal defense system, is mediated by polymeric immunoglobulin receptor (pIgR), whose extracellular domain is cleaved and released to the apical side as secretory component (SC). The goal of this studyis to characterize the trafficking pathways of pIgR in LGACs.
Methods: :
Primary cultured rabbit LGACs were transduced with Adenovirus (Ad) constructs for the expression of EGFP-pIgR, EGFP-Rab11a, mCherry-myosin Vb tail and/or EGFP-myosin Vc tail. Carbachol, a cholinergic agonist, was used to stimulate the secretion of SC. The release of SC was quantified by western blotting using the LI-COR system. LGACs were incubated with sheep anti-SC serum on ice to bind basolateral pIgR, and then warmed to 37 degrees to activate endocytosis and subsequent trafficking of endocytosed pIgR.
Results: :
EGFP-myosin Vc tail and mCherry-myosin Vb tail showed partial colocalization with pIgR. Rab3D was colocalized with EGFP-myosin Vc tail, whereas Rab11a was colocalized with mCherry-myosin Vb tail. mCherry-myosin Vb tail significantly reduced SC release at resting stage and under carbachol stimulation; EGFP-myosin Vc tail did not affect SC release at resting stage, but significantly reduced SC release under carbachol stimulation. Endocytosed pIgR showed colocalization with EEA1 at 15 min, and showed significant colocalization with Rab11a in the subapical region after 30 min. Endocytosed pIgR was trapped in the compartment labeled by mCherry-myosin Vb tail, but not EGFP-myosin Vc tail.
Conclusions: :
The transcytotic and the secretory pathways both participate in the trafficking of pIgR in LGACs. The transcytotic pathway involves Rab11a and myosin Vb; whereas the secretory pathway involves Rab3D and myosin Vc. pIgR on the basolateral membrane is endocytosed into early endosomes, and then sorted into the transcytotic pathway. A subset of newly biosynthesized pIgR is sorted into the secretory vesicles, which are distinct from the transcytotic pathway.
Keywords: receptors • lacrimal gland • microscopy: confocal/tunneling