Synthetic lipid (DOPG) vesicles accumulate in the cell plate region but do not fuse

Abstract

The cell plate is the new cell wall, with bordering plasma membrane, that is formed between two daughter cells in plants, and it is formed by fusion of vesicles (approximately 60 nm). To start to determine physical properties of cell plate forming vesicles for their transport through the phragmoplast, and fusion with each other, we microinjected fluorescent synthetic lipid vesicles that were made of 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) into Tradescantia virginiana stamen hair cells. During interphase, the 60-nm wide DOPG vesicles moved inside the cytoplasm comparably to organelles. During cytokinesis, they were transported through the phragmoplast and accumulated in the cell plate region together with the endogenous vesicles, even inside the central cell plate region. Because at this stage microtubules are virtually absent from that region, while actin filaments are present, actin filaments may have a role in the transport of vesicles toward the cell plate. Unlike the endogenous vesicles, the synthetic DOPG vesicles did not fuse with the developing cell plate. Instead, they redistributed into the cytoplasm of the daughter cells upon completion of cytokinesis. Because the redistribution of the vesicles occurs when actin filaments disappear from the phragmoplast, actin filaments may be involved in keeping the vesicles inside the developing cell plate region.

Figure 1.

Synthetic lipid vesicles labeled with Bodipy FC12-HPC in microinjection buffer, imaged with cryo-TEM. Bar = 200 nm.

Figure 2.

Synthetic lipid vesicles move in the cytoplasm of interphase cells. A, Time series of an interphase stamen hair cell of T. virginiana injected with fluorescent lipid (DOPG) vesicles. Time is in minutes after injection; bar = 10 μm. B, Time series of moving vesicles in the cytoplasm. Time is in seconds; bar = 5 μm. Some vesicles move together for a while (asterisk, arrow, and arrowhead) and then separate from each other. The fluorescent spot pointed to with an arrow is probably a cluster of three vesicles. C, Graph showing the average velocity (± se) of injected lipid vesicles (n = 60 in five cells) and organelles visible with DIC (n = 61 in five cells) in the same cells. The vesicles and organelles were followed for at least 17 s, with 1.97-s intervals. The average velocity was calculated from the displacement of vesicles or organelles over the time that they were followed.

Figure 3.

Synthetic vesicles accumulate in the cell plate region. A to C, Time series of a T. virginiana stamen hair cell (as seen with DIC; A) that was coinjected at anaphase with fluorescently labeled synthetic lipid vesicles (B) and 10-kD Alexa-568 dextran (C). Lipid vesicles accumulate in the whole cell plate region while it is being produced (between the white arrowheads). Fluorescently labeled dextran is distributed throughout the accessible cytoplasmic volume, excluding the cell plate (between the white arrows). Time is in minutes after injection; scale bar = 10 μm.

Figure 4.

FM4-64 does not label the synthetic lipid vesicles in vitro and in vivo, but the accumulation of injected lipid vesicles in the cell plate region coincides with FM4-64 labeling of the cell plate. A and A1, Bodipy FC12-HPC-labeled DOPG vesicles (A) incubated for 1.5 h with 2 μm FM4-64 in microinjection buffer are not labeled with FM4-64 (A1). Bar = 10 μm. Injected fluorescently labeled DOPG vesicles (B) are not labeled with FM4-64 (B1) in a cell with a phragmoplast. The vesicles marked with white circles do not colocalize with FM4-64 labeling. The cell plate and plasma membrane, however, are clearly labeled with FM4-64. Bar = 5 μm. C, The cell plate is labeled by injected lipid vesicles and with FM4-64. The synthetic lipid vesicles accumulate in the region of the cell plate at the same time as the cell plate is labeled with FM4-64; both labels correspond to the cell plate visible in DIC microscopy. Time is in minutes; bar = 10 μm; n = 3 cells. FM4-64 concentration is 2 μm (in A1, B1, and C), added to the surrounding medium 15 min before injection during anaphase. D, Fluorescence intensity profiles of vesicles, FM4-64, and Alexa-568 dextran (10 kD) through the phragmoplast. The line profiles were taken from cells in late telophase, before the cell plate was attached to the parental cell wall. Three regions of 1 × 4 μm perpendicular to the phragmoplast were selected from the images. The average fluorescence intensity of each horizontal row of pixels was plotted versus its vertical position. The accumulation of vesicles in the cell plate region coincides with the FM4-64 labeling. However, the synthetic vesicles in the cell plate show a broader peak of fluorescence than the FM4-64 labeling. The dextran clearly does not enter the cell plate region.

Figure 5.

Injected vesicles do not hinder or delay cell plate formation. The duration of cell plate formation in noninjected cells, in cells injected with microinjection buffer, and in cells injected with fluorescent vesicles was measured from the moment of its visible appearance until its attachment to the parental cell wall. The moment of cell plate attachment was chosen as the moment when the cell plate touched the parental cell wall and appeared straight. Data shown with se.

Figure 6.

Synthetic vesicles accumulate at the same time in the whole cell plate region when injected at late telophase and are not redistributed from the central region of the torus-shaped phragmoplast. A, Vesicle accumulation after injection during late telophase when the cell plate was already partially formed (white arrowheads). Time is in minutes after injection; bar = 10 μm. B, Injected synthetic vesicles remain localized to the central cell plate region although the phragmoplast is already torus shaped (indicated by white arrowheads). This is visible with DIC microscopy and Alexa-568 dextran labeling (white arrowheads). Bar = 10 μm, 46 min after injection.

Figure 7.

Bodipy FC12-HPC alone does not label the cell plate. A cell below a dividing one was injected with fluorescent synthetic vesicles. No labeling of the cell plate is visible. The probe only accumulates in the cell plate when it is incorporated into vesicles. Insert, Detail of the phragmoplast of the cell that neighbors the injected cell imaged at 32 min after injection, showing no incorporation of Bodipy FC12-HPC into the cell plate. Time is in minutes; bar = 20 μm.

Figure 8.

Redistribution of DOPG lipid vesicles after the cell plate is attached to the parental cell wall. The fluorescence of microinjected synthetic vesicles (A) is redistributed to the cytoplasm of the daughter cells while the labeling with FM4-64 (B) stays in the cell plate region. C, A merged image of A and B. The cell plate is visible with DIC microscopy (D). The FM4-64 concentration is 2 μm and was added to the surrounding medium 5 min before vesicle injection during telophase. Time is in minutes; time 0 is arbitrarily chosen; bar = 10 μm.

Figure 9.

Comparison of vesicle and FM4-64 labeling in a phragmoplast and cell plate region. A, The width of the maximum fluorescence intensity peak of injected vesicles and FM4-64 in the phragmoplast over time. The width of the peak is the width at the one-half maximal height of a Gauss curve fitting. B, The peak fluorescence intensity of the Gauss curve fitting of injected vesicles and FM4-64 in the phragmoplast and in the cytoplasm over time.

Figure 10.

A, Median plane of a tobacco BY-2 suspension cell at late telophase, transformed with GFP∷FABD to visualize the actin cytoskeleton. Actin is present in the whole phragmoplast including the central part. Bar = 10 μm. B, Median plane of the tobacco BY-2 suspension cell at late telophase, transformed with GFP∷MBD to visualize the microtubule cytoskeleton. Microtubules are absent from the central part of the phragmoplast. Bar = 10 μm (courtesy Dr. T. Ketelaar).