ated as previously described. Cell viability after transduction with Rab27b constructs was tested using the LIVE/DEADH Cell Viability Assay Kit for mammalian cells. For M5C studies, GFP fused to fulllength human myosin 5C and dominant-negative tail of myosin 5C were prepared as described. Amplification of virus constructs was conducted in QBI-HEK cells and, upon display of cytopathological effect, supernatant was collected and purified using CsCl gradient ultracentrifugation as previously described. Viral titers were measured by viral plaque formation in sequential dilutions. For optimal imaging, culture acinar cells were transduced at a MOI of 46 and then incubated for an additional 1824 hours while the protein was expressed, allowing for single transduction efficiencies approximating 7080% and cotransduction efficiencies approximating 5060% per expressed protein. Expression of RFP-tagged late Golgi/TGN marker, N-acetylgalactosaminyltransferase 2, was according to the manufacturer’s instructions as specified for the use of a commercial kit, Bacmam 2.0 Cell Light Golgi. Materials and Methods Reagents For cell culture, MatrigelTM was obtained from Collaborative Biochemicals, 
carbachol and nocodazole Carbamate) from Sigma-Aldrich. Antibodies for immunofluorescence detection of Rab27b were from Novus Biologicals; antibodies to p150Glued and c-Adaptin were from BD Transduction Laboratories; and antibodies to Golgin 97 as well as secondary antibodies and fluorescence affinity probes for RAB27B-Enriched Secretory Vesicle Biogenesis Confocal fluorescence microscopy For fixed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22190001 cell and multi-channel images, cultured acinar cells were ethanol fixed and blocked with 1% BSA prior to incubation with the appropriate primary and secondary antibodies. GLPG-0634 biological activity images were collected by a Zeiss LSM 510 Meta NLO imaging system and translated to tiff files by Adobe Photoshop 7.0. For live cell images and time-lapse series, cells were transduced with YFP-, GFP-, or RFP-tagged constructs and imaged in a closed, 37uC controlled airflow chamber for confocal fluorescence and DIC microscopy. For imaging of cells expressing both YFP- and GFPtagged constructs, the Zeiss LSM 510 META Emission Fingerprinting function allowed for the separation of the two spectra as Lambda Stacks, which were then processed into a digital separation of fluorescence emissions. These images were taken in parallel with non-colocalizing samples. More detailed images were taken by z-stacks, which were reconstructed into a threedimensional image by the Zeiss LSM Projection Tool. Unless otherwise noted, all imaging samples, both fixed and live, are representative data from repeats of at least 4 separate culture preparations of LGAC, harvested from different rabbits on different days and cultured at a cell density of 26106 cells per 35 mm petri dish. For quantitative analysis of the general fluorescence intensities at the described time points, the Zeiss LSM 510 Histogram Tool was used to measure the mean intensity of the subapical region of structurally similar lumens. Fluorescence was measured in acinar clusters from at least 3 random fields per sample, where the samples were from 4 repeats of separate LGAC cultures. This subapical region was defined as an ROI with a radius of approximately half of the radius of the total area of the cells forming the lumen. The subapical fluorescent intensity was then recorded as a percentage of the total intensity within the total area of the cells. Data analysis
