Uptake of a Fluorescent Marker in Plant Cells Is Sensitive to Brefeldin A and Wortmannin

Abstract

We assessed FM1-43 [ N -(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl)pyridinium dibromide] as a fluorescent endocytosis marker in intact, walled plant cells. At 4 ؇ C, FM1-43 stained the plasma membrane, and after 30 to 120 min of incubation at 26 ؇ C, FM1-43 labeled cytoplasmic vesicles and then the vacuole. Fluorimetric quantitation demonstrated dye uptake temperature sensitivity ( ‫ف‬ 65% reduction at 16 ؇ C, Ͼ 90% at 4 ؇ C). FM1-43 uptake in suspension cells was stimulated more than twofold by brefeldin A and inhibited ‫ف‬ 0.4-fold by wortmannin. FM1-43 delivery to the vacuole was largely inhibited by brefeldin A, although overall uptake was stimulated, and brefeldin A treatment caused the accumulation of large prevacuolar endosomal vesicles heavily labeled with FM1-43. Three-dimensional time lapse imaging revealed that FM1-43-labeled vacuoles and vesicles are highly dynamic. Thus, FM1-43 serves as a fluorescent marker for imaging and quantifying membrane endocytosis in intact plant cells. INTRODUCTIONEndocytosis in animal cells begins with the budding of invaginated coated pits from the plasma membrane into coated vesicles, which then uncoat and fuse with the endocytic organelles (Gruenberg et al., 1989;Gruenberg and Maxfield, 1995). By this action, both membrane proteins and fluid phase molecules are sequestered from the cell exterior into intracellular organelles, from where they can recycle to the plasma membrane or be transported to other destinations, such as the lysosomes. Thus, endocytosis serves to balance membrane secretion and to remove, transport, or degrade receptor ligand complexes from the cell surface (Goldstein et al., 1985). Although clathrin coatdependent endocytosis is involved in receptor internalization, clathrin-independent endocytosis pathways also contribute to internalization. These include uptake via nonclathrin-coated caveolae (Anderson, 1998), macropinocytosis (Swanson, 1989;Hewlett et al., 1994), and phagocytosis (Aderem and Underhill, 1999).Our understanding of animal cell endocytosis was advanced by the use of fluorescent and electron-opaque markers that can be internalized Maxfield, 1988, 1989;Griffiths et al., 1989). The use of fluid phase markers has permitted the development of assays that reconstitute endocytic vesicle fusion (Braell, 1987(Braell, , 1992Gruenberg et al., 1989;Emans et al., 1995;Emans and Verkman, 1996) and led to the identification of many proteins that catalyze and control fusion, such as the small GTPase rab5 (Gorvel et al., 1991) and its effector proteins (Christoforidis et al., 1999a(Christoforidis et al., , 1999bMcBride et al., 1999).In plants, there is evidence that endocytosis is likely to occur (Low and Chandra, 1994; for review, see Battey et al., 1999;Marcote et al., 2000). Endocytosis is postulated to counterbalance membrane secretion (Samuels and Bialputra, 1990) and permit cell volume to respond to changes in osmolality (Thiel et al., 1998;Kubitscheck et al., 2000). However, the study of plant cell endocytosis has been hampered by t...

A Membrane Microdomain-Associated Protein, Arabidopsis Flot1, Is Involved in a Clathrin-Independent Endocytic Pathway and Is Required for Seedling Development

“…In addition, GFP-Flot1 was also found to colocalize partially with FM4-64. Given that this FM4-64 dye is internalized, via endocytosis from the plasma membrane, and is considered to be a bona fide marker for the endocytic pathway in plant cells (Emans et al, 2002;Chintagari et al, 2006), our findings offer strong support for the hypothesis that Flot1 participates in an endocytic pathway.…”

Unstable F-Actin Specifies the Area and Microtubule Direction of Cell Expansion in Arabidopsis Root Hairs

“…To test this hypothesis, we loaded Arabidopsis root hairs with 2 M of the amphiphilic styryl dye FM4-64, which is internalized by endocytosis (Fischer-Parton et al, 2000). (A similar amphiphilic styryl dye, FM 1-43, was discussed by Emans et al [2002].) The loading of root hairs with this dye, pretreated and grown in 0.5 M CD, was not inhibited as expected.…”

Rice SCAMP1 Defines Clathrin-Coated,trans-Golgi–Located Tubular-Vesicular Structures as an Early Endosome in Tobacco BY-2 Cells

“…FM4-64, a fluorescent styryl dye that is internalized via endocytosis from the PM to the lytic vacuole, serves as a marker for the endocytic pathway in plant cells (Emans et al, 2002;Tse et al, 2004;Lo and Jiang, 2006). We previously demonstrated that FM4-64 colocalizes with the PVC marker YFP-BP-80 but not the Golgi marker GONST1-YFP in transgenic BY-2 cells (Tse et al, 2004), indicating that the secretory and endocytic pathways merge in PVCs.…”

“…In addition, the fluorescent dyes FM4-64 and FM1-43 have been successfully used to study the endocytic pathway in plant cells. After their insertion in the PM, these styryl dyes pass through endosomal compartments on their way to the tonoplast (Vida andEmr, 1995, Kim et al, 2001;Ueda et al, 2001;Emans et al, 2002;Tse et al, 2004;Dettmer et al, 2006). Therefore, colocalization of proteins with internalized FM4-64 may be used to judge whether a particular compartment labeled by a specific protein is a putative endosomal compartment in plant cells.…”