Confocal Microscopy-based Linescan Methodologies for Intra-Golgi Localization of Proteins

Abstract

Localization of resident Golgi proteins to earlier ( cis) or later ( trans) Golgi compartments has traditionally required quantitative immunocytochemistry and electron microscopy, which are inaccessible to many researchers. For this reason, light microscopy has often been used, initially for localization of Golgi glycotransferases and, more recently, for other Golgi proteins (e.g., Arf1, GBF1, Rab6). Quantitation of light microscopic intra-Golgi localization can be problematic. We describe here a novel quantitative light microscopic methodology using linescans crossing the Golgi ribbon. Our method determines a localization for the unknown protein in a one-dimensional coordinate system in which 0.0 corresponds to localization of a cis marker and 1.0 to localization of a trans marker. We also describe a variant of this methodology in which Golgi morphology is simplified by nocodazole-induced dispersal into ministacks, allowing a fully automated analysis. In our assay, β1,4-galactosyltransferase-YFP and Golgin97 localize similarly to trans markers, whereas p115, GBF1, and p58-YFP are similarly near other cis markers. The medial Golgi protein α1,3–1,6-mannosidase II gives an intermediate localization in this assay. These methodologies may prove useful in instances where electron microscopy is technically difficult as well as when rapid analysis of large numbers of samples is required.

Rab18 and Rab43 have key roles in ER-Golgi trafficking

“…3C). Similar results were obtained in a fully automated version of this assay (Dejgaard et al, 2007) which required nocodazole treatment of cells to simplify Golgi geometry (see supplementary material Fig. S2B).…”

“…We have recently developed a quantitative light microscopic assay that permits us to localize the major pool of transfected proteins to early or late Golgi regions (Dejgaard et al, 2007) (Fig. 3A,B).…”

“…The cis-Golgi marker p115 was visualized using a Cy5-tagged secondary antibody (blue) and the trans-Golgi marker TGN38 was visualized using a Cy3-tagged secondary antibody (red). GFP constructs were scored for localization within the Golgi as described (Dejgaard et al, 2007). ( for Rab33b) agreed closely with previous immuno-electron microscopic localizations.…”

Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway

“…1). NZ treatment induces the formation of Golgi ministacks that, despite their small size, preserve the structural and molecular polarity (cis-to-trans) of the Golgi ribbon (Dejgaard et al, 2007;Ho et al, 1989), which in turn facilitates colocalization analysis. In NZ-treated cells, LPP3 overlapped with all these markers to a variable extent (Sec31A .KDELr .GM130 .Golgin 97) (Fig.…”

Arf activation at the Golgi is modulated by feed-forward stimulation of the exchange factor GBF1

“…Primate cells express five Arf proteins (Arf1, 3, 4, 5 and 6) that are classified into three classes (class I: Arf1 and Arf3; class II, Arf4 and Arf5; and class III, Arf6). Class I and II Arfs are found differentially distributed through the Golgi (Dejgaard et al, 2007;Chun et al, 2008;Manolea et al, 2010). Arf proteins exert their regulatory effect through cycles of GTP binding and hydrolysis, induced by Arf guaninenucleotide-exchange factors (GEFs) and Arf GTPase-activating proteins (GAPs).…”