Ceramide Accumulation Uncovers a Cycling Pathway for the cis-Golgi Network Marker, Infectious Bronchitis Virus M Protein

Maceyka, Michael; Machamer, Carolyn E.

To test whether ceramide was likely to mediate the PDMPinduced mislocalization of IBV M, indirect immunofluorescence was performed as above with cells pretreated with either ␤ CA or FB1 for 1 h before the addition of PDMP. To simplify the quantification, the localization of IBV M was classified into one of three staining patterns. Class 1 was the most commonly seen pattern in untreated cells, i.e., tight juxta-nuclear staining that colocalized with Golgi markers. Class 3 was the most commonly seen pattern in PDMP-treated cells, i.e., diffuse staining with prominent nuclear envelope staining that colocalized with ER markers. Class 2 was intermediate between the class 1 and class 3. Whether this pattern represents an overlap of ER and Golgi staining patterns or an increase in IC staining is not clear. For these experiments, coded samples were quantified by counting at least 100 cells for each sample. The experiment was performed five times, with one representative experiment shown (Fig. 7) . Interestingly, when cells were quantified in this way, we observed that both ␤ CA and FB1 caused a slight shift from class 1 to class 2. The significance of this observation is not clear. PDMP dramatically shifted the distribution of IBV M from mainly class 1 in the control cells to mainly class 3. Pretreatment with either ␤ CA or FB1 significantly reduced this shift in the staining pattern of IBV M, suggesting that accumula- Figure 5 . PDMP induced the redistribution of the endogenous IC protein, ERGIC-53, to the ER. Vero cells were incubated with cycloheximide for 1 h and then incubated for 1 h in the presence either 1% isopropanol (control) or 100 M PDMP. Cells were then fixed and prepared for indirect immunofluorescence with antibodies to ERGIC-53. For each experimental group, the Nomarski image is shown on the left and the fluorescence image on the right. Bar, 10 m.