The LMP-1::TagRFP(S158T) plasmid was made by replacing the GFP with TagRFP(S158T) coding sequences in a plasmid that expresses an LMP-1::GFP fusion protein . The ~742 bp TagRFP(S158T) cDNA was PCR amplified (primers CACACAACCGGTAGAAAAAATGGTGTCTAAGGGCGAAGAGC and CACACAGAATTCCAAAGCTTGTGGGCTTTTATTAAGTTTGTGCCCCAGTTTG), restriction digested with Age I + Eco RI and ligated into the 2979 bp fragment of pHD43 to make plasmid pHD491. The 3532 bp Sph I + Age I fragment of pHD491 was then ligated to the 3946 bp Sph I + Age I fragment of pPD117.01-LMP-1 to make plasmid pHD499 used to make transgenic lines.
The GFP::CUP-5 and mCherry::CUP-5 plasmids were made by inserting the elt-2 promoter to existing GFP::CUP-5 and mCherry::CUP-5 plasmids (pJF168 and pHD260, respectively). The elt-2 promoter was PCR amplified (primers CACACACCGGGCTGCAGGAATTCACATA and CACACACGCGGATCCATTCTATAAT) from pJM260, and restriction digested with Pst I and Bst UI. Both GFP::CUP-5 and mCherry::CUP-5, pJF168 and pHD260, respectively, were restriction digested with Bmt I, incubated with T4 DNA Polymerase to create a blunt end, and then digested with Pst I. The promoter fragment (5057 bp) was then ligated to the pJF168 fragment (6074 bp) and pHD260 fragment (6227 bp) to make pHD423 and pHD429, used to make transgenic lines.
Nile Red is a lipophilic membrane-permeable dye that diffuses across membranes and fluoresces when it complexes with lipids [24, 26, 36]. For Nile Red staining, adult worms were placed on OP50 bacteria suspended in 60 ug/ml Nile Red, on NGM plates with 60 ng/ml Nile Red (Invitrogen, Carlsbad, CA). Worms were allowed to lay embryos overnight, and embryos were immediately imaged. For imaging of live worm embryos (Nile Red, UV imaging, and fluorescently tagged constructs), embryos were resuspended in M9 buffer and dropped onto 3% agar pads; coverslips were added and sealed with petroleum jelly.
For PGP-2 immunostaining, embryos were dropped onto slides coated with poly-l-lysine (Sigma, St. Louis, MO) and covered with a coverslip. Slides were quickly frozen on a metal plate cooled over dry ice, and coverslips were flicked off the slides. Slides then underwent one 15-minute 100% methanol wash at -20°. Slides were washed twice with PBST (PBS plus 0.1% Tween), and blocked with PBS, 1% nonfat milk, 1% BSA, 0.1% Tween for 2 hours at 4°. Anti-PGP-2 antibody was added at 1:500 dilution in the above blocking buffer and left overnight at 4°, followed by three 10-minute washes in PBST . Secondary anti-rabbit Cy3 antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was added at 1:200 dilution in blocking buffer for two hours at room temperature, followed by three 10-minute washes in PBST. Excessive moisture was wicked away from embryos, and mounting media (Invitrogen) was added prior to imaging.
Confocal images were taken on a Zeiss LSM510 (Carl Zeiss, Oberkochen, Germany) laser scanning confocal, using LSM imaging software. Epifluorescence images were taken on a Leica DMRXA compound microscope (Leica Microsystems, Wetzlar, Germany), using MetaMorph software (Molecular Devices, Sunnyvale, CA).
For colocalization analysis and quantification of organelle size and intensity, MetaMorph (Molecular Devices) was used, and Microsoft Excel was used for statistical analysis. For each embryo measured, two 7 × 7 μm boxes were drawn, and all compartments within each box were measured for area and intensity per unit area. For YP170::GFP and LMP-1::GFP experiments, one z-section for each of five embryos of each genotype was measured. For PGP-2 and UV experiments one z-section of five embryos of each genotype was measured. We used two-tailed t-test p-values to determine significance in all experiments. Figs. 1, 2, 3 and 4 and Additional files were assembled using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA), and Fig. 5 was assembled using Canvas (ACD Systems, Victoria, BC Canada).