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Exo- and endocytotic membrane trafficking is an essential process for transport of secretory proteins, extracellular glycans, transporters and lipids in plant cells. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco BY-2 cells as a model system, we recently demonstrated that SCAMP2 positive structures containing secretory materials are transported from the Golgi apparatus to the plasma membrane (PM) and/or cell plate. This structure is consisted with clustered vesicles and was thus named the secretory vesicle cluster (SVC). Here, we have utilized the reversible photoswitching fluorescent protein Dronpa1 to trace the movement of SCAMP2 on the PM and cell plate. Activated SCAMP2-Dronpa fluorescence on the PM and cell plate moved into the BY-2 cells within several minutes, but did not spread around PM. This is consistent with recycling of SCAMP2 among endomembrane compartments such as the TGN, PM and cell plate. The relationship between SVC-mediated trafficking and exo- and endocytosis of plant cells is discussed taking into account this new data and knowledge provided by recent reports.
Exo- and endocytosis are essential events for cellular division and expansion. During exocytosis, lipids, proteins and polysaccharides are synthesized and/or modified in the Golgi apparatus and sorted into secretory vesicles at the trans-Golgi network (TGN) for transport to the PM2 or extracellular space. Secretory carrier membrane proteins (SCAMPs) are a group of transmembrane proteins that plays vesicle trafficking between Golgi apparatus and PM in higher eukaryotic cells.3 Recently it was reported that in BY-2 cells, the rice SCAMP1 is localized to the PM and clathrin-coated tubularvesicular structures that were likely the early endosomal compartment.4 The same protein is also targeted to the cell plate in dividing cells.5 We have recently reported that another member of the SCAMP family, SCAMP2 from tobacco, is localized to the TGN, PM, cell plate and previously uncharacterized SVC organelles, which are an intermediate organelle between the TGN and PM.6
Both SCAMP1 and SCAMP2 appear to be recycled between the PM and intracellular compartments. This was suggested by data using stelyl dye FM4-64 as an endocytotic marker, fluorescent-tagged SCAMP proteins and protein trafficking inhibitors such as brefeldin A and 2,3-butanedione monoxime. We reported that SCAMP2 is exported to the PM from dotted structures in the cells, and back from the PM via the acto-myosin pathway but do not transport FM4-64 positive early endosome.6 As SCAMP2 did not localize on multivesicular bodies, endocytic vesicles may be directly transported to TGN or Golgi.6 However, this data was obtained using inhibitors that disrupt the trafficking system, and thus we have now investigated the endocytotic transport in the absence of inhibitors.
Dronpa is a reversible photo-switching fluorescent protein. Using 488 and 405 nm laser light this protein can be converted between fluorescent and non-fluorescent forms within milliseconds.1 In order to test whether SCAMP2 returned to internal compartments from the PM, and to characterize the initial compartment of endocytosis, we expressed Dronpatagged SCAMP2 (SCAMP2-Dronpa) in tobacco BY-2 cells. The fluorescence of SCAMP2-Dronpa was similar to that for SCAMP2-YFP and -mRFP fusions6 (Fig. 1A, upper part). To visualize the endocytic transport of SCAMP2-Dronpa, we first erased the majority of Dronpa fluorescence by illumination with 488 nm laser and then activated the protein at a part of the PM by 405 nm illumination using confocal laser scanning microscope (LSM) (Fig. 1A, upper right part). The fluorescence was then traced by 30 minutes interval up to 90 minutes (Fig. 1A, lower pictures). SCAMP2 signals at the PM did not spread laterally in the PM and decreased over the time. In parallel, signals were detected in the cytosol and some of them appeared as puncta (Fig. 1A, arrowheads). This observation is consistent with our proposal that SCAMP2 is recycled back into the intracellular compartment from the PM, possibly through the TGN without passing through the early endosome.6
During cytokinesis, cell wall materials and membrane proteins accumulate in the cell plate.7–9 It has been shown that clathrin-coated vesicles (CCVs) and their constituents such as adapter proteins and dynamins are associated with cell plate membrane.10 However, it is not clear whether these molecules on the cell plate are re-used in daughter cells or are degraded at the cell plate. We thus investigated the movement of SCAMP2-Dronpa fluorescence on the cell plate during cytokinesis. Fluorescence of SCAMP2-Dronpa within late metaphase cells was first erased, followed by activation of SCAMP2-Dronpa specifically on the cell plate (Fig. 1B). Following a 15 min of incubation, SCAMP2-Dronpa associated fluorescence on the cell plate moved into intracellular structures within daughter cells. This confirmed our previous observation that SCAMP2 was transported to the trans-Golgi/TGN or intracellular structures from the cell plate during the cytokinesis.6
Transmission electron microscope and LSM studies have revealed that CCVs are present in cell plates.10 Recent tomographic observation suggested that early- and late TGNs having CCVs exist not only in the cell plate region but also other places of the plant cell.11 We found that immature SVCs, which might be identical to late TGN, are converted to mature SVCs by budding CCVs.6 Therefore, transport from the Golgi apparatus located inside of the cells to the PM or cell plate is mediated by SVCs, which are generated as immature SVCs from the TGN and converted to mature SVCs by budding CCVs during transport. Eventually, the mature SVC fuses with the PM and/or expanding cell plate (Fig. 2, left), after which CCVs are generated from the expanded cell plate to recycle SCAMPs and other molecules back to the daughter cells.
Our observations of SCAMP2 trafficking and comparison with that for SCAMP1 revealed there are at least two exocytotic mechanisms from TGN in plant cells. Recent analysis of the localization and trafficking of cellulose synthase complexes allowed the detection of an exocytotic compartment called the microtubuleassociated cellulose synthase compartment (MASC). The MASC appeared to be generated from the medial or trans-side of Golgi cistarnae.12 The difference in both size and distribution indicates that the MASC and SVC are distinct compartments. Previous immuno-electron microscopic analysis also suggested that there are at least two distinct export pathways for cell wall glycans from the Golgi.13 Although the relationship between the SVC, the MASC, SCAMP1 containing secretory vesicles, and compartments for transport of distinct cell wall glycans remain unclear, these observations indicated that exocytotic pathways from the Golgi apparatus are quite complex in plant cells.
The endocytotic process for SCAMP1 and SCAMP2 are also thought to be distinct based on differences in the initial endocytosed compartment.6 As several transporters, receptors and other membrane proteins are also internalized from the PM,14,15 it is possible that endocytotic machineries are also complex in plant cells. Isolation and characterization of these exo- and endocytotic compartments and characterization of their regulation in differentiated plant cells is a future challenge to understand the role of the late secretory pathway in plant cells.
We thank Ms. S. Takata in RIKEN Plant Science Center for the construction of plasmids and Dr. Derek B. Goto at Hokkaido University for improving the manuscript. This work was supported in part by grants from the JSPS (No. 17770056 to K.T.) and from Grants-in-Aid for Scientific Research in Priority Areas from MEXT Japan (No. 17078009 to K.M.).
Previously published online: www.landesbioscience.com/journals/psb/article/10075