Isolation of GFP-labeled endoplasmic reticulum (GFP-ER) from tobacco cultured cell, BY-2.
To visualize the endoplasmic reticulum (ER) in tobacco cultured cell BY-2, the cells which aretransformed with a chimeric gene constructed from the signal peptide of pumpkin 2S albumin,
GFP and the ER-retention signal (HDEL) have been prepared and shown a strong and stable
fluorescence on the ER network (Mitsuhashi et al., 2000). As reported in various plant cells, thedistribution, morphology and behavior of GFP-ER in BY-2 cells also alter spatially and temporally.

In interphase cells, GFP-ER is distributed throughout cytoplasmic strands emanating from thenuclear region and penetrating the vacuole (Fig. 1A). The GFP-ER in cytoplasmic strands moves
and streams actively with an average velocity of about 7 μm/sec ranging from 4 to 9 μm/sec. In
the cortical region, GFP-ER is arranged into mesh-like structures consisting of tubes and sacks(Fig. 1B), which are less mobile, but show shrinking, elongation and contraction-like motions.
These movements and streaming are suppressed by actin-depolymerization drugs, but not by
microtubule-depolymerization ones, indicating that the actin cytoskeleton is involved in thesemotions of ER. In mitotic cells, some portions of GFP-ER gather in the spindle (Fig. 1C), at theequatorial plane in the phragmoplast (Fig. 1D) and around daughter nuclei (Fig. 1D). Because
the ER-retention signal peptide, HDEL, is incorporated into nuclear envelopes, perhaps due to thefusion of ER into them (Napier et al., 1992), bright GFP signals are also observed around nuclei(Fig. 1E and F).

In this section, the isolation procedure of GFP-ER from BY-2 cells not synchronized isintroduced. The following procedures are carried out at 0 to 4 oC. A solution A containing 8 mM
EGTA, 1 mM MgCl2, 1% casein, 0.3 M sucrose, 0.5 mM DTT, 1 mM PMSF, 50 µg/ml leupeptin
and 40 mM PIPES-KOH (pH 7.0) is chilled on ice and centrifuged at 27,000xg for 5 min toremove undissolved casein. Three volumes [?] of the solution A are added to one volume ofprotoplasts prepared from BY-2 cells. The mixture is homogenized with a hand-operated Downs
homogenizer. The homogenization is performed carefully, with observation and checking of thedegree of breakage of the protoplasts using light microscopy. The homogenate is first centrifuged
at 500xg for 3 min. The supernatant is further centrifuged at 12,000xg for 10 min. The resultant
supernatant containing microsomal and cytosol fractions is applied on 0.6 M, 1.0 M and 1.5 M
discontinuous sucrose density gradient made up in a solution B containing 5 mM EGTA, 1 mM
MgCl2, 0.5 mM DTT, 1 mM PMSF, 50 µg/ml leupeptin and 30 mM PIPES-KOH (pH 7.0), andcentrifuged at 86,000xg for 1.5 hr. Interfaces between 0.6 M and 1.0 M, and 1.0 M and 1.5 M
sucrose are recovered and analyzed using fluorescent microscopy or immunoblotting with anantibody against GFP. Almost [all?] of GFP-ER are recovered in the interface between 1.0 and 1.5
M sucrose (GFP-ER fraction; Fig. 1G). To further remove cortical proteins, 3 M KCl or KI areadded to ER fraction at final concentrations of 0.5 M or 0.4 M, respectively. The mixture is stoodon ice for 10 to 20 min and then centrifuged at 140,000xg for 20 min. The pellet is suspended inthe solution B supplemented with 0.3 M sucrose with a glass-Teflon homogenizer.

References
Mitsuhasi N, Shimada T, Mano S, Nishimura M, Hara-Nishimura I (2000) Characterization oforganelles in the vacuolar-sorting pathway by visualization with GFP in tobacco BY-2 cells. Plant
Cell Physiol. 41: 993-1001.
Napier RM, Fowke LC, Hawes C, Lewis M, Pelham HRB (1992) Immunological evidence thatplants use both HDEL and KDEL for targeting proteins to the endoplasmic reticulum. J. Cell Sci.
102: 261-271.

Fig. 1ABCEFGD

The distribution of GFP-ER in loving BY-2 cells and isolated GFP-ER.
A, GFP-ER around nucleus and in cytoplasmic strands emanating from nuclear region in aninterphase cell. B, GFP-ER in the cortical region in an interphase cell. C, GFP-ER in the spindle ina metaphase cell. D, GFP-ER in the phragmoplast in a telophase cell. The GFP-ER accumulates inthe equatorial plane of phragmoplast and around daughter nuclei. E, GFP signals in nuclear
envelops in isolated nuclei. F, Nomarski light microscopic image of E. G, isolated GFP-ER by thesucrose density gradient ultra-centrifugation. Bras represents 10 μm.