Investigating the Secretory Pathway of the Baculovirus-Insect Cell System Using a Secretory Green Fluorescent Protein

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• 作者：Philip R. LeDuc ; Erik M. Whiteley ; Gang Bao ; and Michael J. Betenbaugh
• 刊名：Biotechnology Progress
• 出版年：2000
• 出版时间：October 2000
• 年：2000
• 卷：16
• 期：5
• 页码：716 - 723
• 全文大小：260K
• 年卷期：v.16,no.5(October 2000)
• ISSN：1520-6033

文摘

The secretory pathway is important in actively transporting proteins into theextracellular environment of eucaryotic cells. In this study a green fluorescent protein(GFP) mutant engineered to contain a secretion signal was used as a model proteinin order to visualize the secretion process inside insect cells. Fluorescent microscopyindicated that significant amounts of secreted green fluorescent protein (sGFP)accumulated in High-Five, Trichoplusia ni, cells following infection with a baculovirusvector containing the gene under the polyhedrin promoter. Laser scanning confocalmicroscopy was used to reconstruct whole cell images of the infected High-Five cellsat multiple days postinfection. While the protein was widely distributed at 2 dayspostinfection, certain intracellular regions appeared to contain higher or lowerconcentrations of the sGFP. A layer by layer examination indicated pockets in whichsGFP was absent, and these appear to be vesicles that have recently released thesGFP or are not yet accumulating sGFP. By 3 days postinfection, the sGFP in somecells was concentrated in a number of widely dispersed globules, which may representthe vesicle remnants of a deteriorating secretory pathway. In contrast, nonsecretedGFP was more uniformly distributed in the cells than sGFP and did not accumulatein vesicles. In addition to GFP, the lectins wheat germ agglutinin (WGA) andconcanavalin A (ConA), which have affinities for sugar residues, were used to examinethe secretory pathway. The WGA, which is a Golgi marker, was distributed aroundthe nucleus prior to infection but then was found to be polarized in one region of thecell following the baculovirus infection. The expansion of other cellular compartmentsfollowing the baculovirus infection may have caused a change in intracellulardistribution of the Golgi. While some of the sGFP was found to colocalize with theWGA label, much of the sGFP was outside this Golgi region. In contrast, ConA labeling,which was not as specific as WGA, was found throughout the cell both before andafter infection similar to the sGFP distribution. These studies demonstrate thatconfocal visualization of fluorescent proteins can be used as an in vivo tool forexamining secretory processing in insect cells.