Direct Imaging of RAB27B-Enriched Secretory Vesicle Biogenesis in Lacrimal Acinar Cells Reveals Origins on a Nascent Vesicle Budding Site

<div><p>This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the “nascent vesicle site,” from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150^Glued, a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules.</p> </div

Schematic model depicting nocodazole's effect on nascent Rab27b-enriched SV formation in LGAC.

<p><b>A.</b> Rab27b-enriched nascent SV bud from the NVS which may be a part of or associated with the TGN. Rab27b-enriched SV undergo microtubule-based transport to the subapical membrane where they join a large subapical pool awaiting stimulation for fusion with apical plasma membrane. <b>B.</b> After depolymerization of microtubules, subapical SV still undergo fusion with the apical membrane and release of content as in non-treated cells. However, newly forming nascent SV appear trapped on the NVS; SV buds are more obvious on the NVS and limited restoration of the subapical pool of SV is seen after CCH-induced depletion of the existing pool.</p

Disruption of the microtubule network hinders nascent YFP-Rab27b-enriched SV production.

<p><b>A</b>. Fixed LGAC expressed YFP-Rab27b for direct fluorescence (<i>green</i>) was co-detected for endogenous p150^Glued (p150; <i>red</i>). Regions on the membrane of apparent SV showing strong co-localization are labeled with <i>solid arrows</i>, whereas regions of SV expressing only one marker are labeled with <i>striped arrows</i>. Actin (<i>white</i>, labeled with fluorescent phalloidin); nuclei (<i>blue</i>, labeled with DAPI). <i>Red asterisk</i> labels the lumen. Bar represents 10 µm, N = 4. <b>B and C.</b> Live LGAC expressing YFP-Rab27b were left untreated (<b>B</b>) or were treated with 33 µM nocodazole (<i>Noc</i>) for 60 m (<b>C</b>). Z-stack images were acquired of cells at resting state, 15 m after CCH stimulation, and 60 m after recovery. The outline of the acinus, which corresponds to the flattened z-stack image of YFP-Rab27b (<i>green</i>) beside it, can be seen in the DIC image. <i>Arrows</i> point to subapical regions with noticeable loss of the SV pool. Insets represent magnifications of the boxed regions; * represents lumen; arrows point to NVS. Bars represent 5 µm, N = 6.</p

Only a subpopulation of basal YFP-Rab27b-enriched membranes partially co-localizes with late Golgi compartments.

<p>Flattened three-dimensional reconstructions of fixed LGAC show YFP-tagged Rab27b expression (green) in parallel with indirect immunofluorescence labeling of different trans-Golgi markers: <b>A.</b> Golgin 97 (<i>Golgin</i>) (N = 4) and <b>B.</b> γ-Adaptin (N = 4). For orientation, overlay images showing co-expression of Rab27b with the trans-Golgi markers are paired with those depicting actin. A schematic at the end of each row offers an interpretation of the expression patterns of the two markers, while an intensity plot of the area marked by the white arrow demonstrates co-localization patterns. Luminal regions (*) were deduced by rotation of three-dimensional stacks and determination of subapical actin orientation around this point, and <i>dashed lines</i> delineate the approximate actin accumulation in the mid-section of stack. <i>Arrowheads</i> point to regions of co-localization between Golgi markers and YFP-Rab27b. Bar represents 5 µm. <b>C.</b> A schematic of two possible origins of the budding SV suggest its association with a late compartment of the Golgi or from a physically separate NVS compartment close to the Golgi stacks. <b>D.</b> Live cell z-stack reconstruction of LGAC doubly transduced with YFP-tagged Rab27b and RFP-tagged GalNAc-T2 show x-z and y-z expression profiles. An intensity plot demonstrates the absence of overlap between the two markers. * represents lumens, <i>A</i> represents apical membrane, <i>BLM</i> represents basolateral membrane, bars represent 5 µm, N = 3.</p

YFP-Rab27b-enriched SV bud from a nascent vesicle site.

<p><b>A.</b> YFP-Rab27b expressed in cultured rabbit LGAC was imaged in live cells which exhibited both basolateral (<i>B</i>) and apical (<i>A</i>) plasma membranes, the latter of which delineated the lumen (<i>L</i>). DIC is shown to confirm location of lumena. Image frames show SV expression at rest and with CCH stimulation. <i>Arrows</i> represent sites of SV fusion with the apical membrane, while the <i>inset</i> shows a higher magnification of a fusion site. Bar represents 10 µm, N = 6. <b>B.</b> Schematic depicts orientation of a cluster of LGAC immediately after CCH washout (0 m). The <i>red box</i> outlines the region of interest for the remainder of the times-series taken during recovery phase, with representative images taken every 5 m. A schematic at 15 m identifies the supposed NVS from which SV appear to bud (N = 5). <i>Arrowheads</i> represent the site of nascent SV emergence; <i>arrows</i> follow the movement of these SV to the apical membrane. Bar represents 10 µm.</p