Article Figures & Data
Figures
- Figure 1.
Phragmoplast Initials.
(A) Shoot apical meristem cell. EM micrograph showing a vesicle cloud surrounded by the CPAM. Spindle MTs extend from the segregating chromosomes (chr) to the CPAM.
(B) to (G) Root meristematic cell.
(B) Model view of a root meristematic cell phragmoplast initial with its associated vesicles and CPAM.
(C) Detail of the region boxed in (B). Cell plate–forming vesicles (green small-dark (sdv) and large-light (llv); see also Figure 2) are seen both outside and inside the CPAM, whereas the dumbbell vesicles are confined to the CPAM. ER is excluded from the CPAM region.
(D) to (G) Enlargements of small-dark (sdv) (D), large-light (llv) (E), and dumbbell ([F] and [G]) vesicles.
Bars in (A) to (C) = 200 nm; bar in (G) = 50 nm for (D) to (G).
- Figure 2.
Two Different Types of Vesicles in the Vicinity of the Cell Plate.
The black arrows in (A) point to small-dark vesicles, and the white arrows point to large-light vesicles. Examples of small, dense vesicles ([B] and [C]) and large-light vesicles ([D] and [E]). Arrows in (C) and (E) point to L-shaped complexes (see also Figures 4A to 4H). cp, cell plate. Bar in (A) = 200 nm; bar in (E) = 50 nm for (B) to (E).
- Figure 3.
Vesicle Diameter Distribution during Cytokinetic Stages.
Histograms of the size distribution of cell plate–forming vesicles during different stages of cell plate formation. The individual bars indicate the number of vesicles per 5-nm diameter interval. Closed bars, small-dark vesicles; shaded bars, large-light vesicles.
- Figure 4.
Tomographic Analysis of Putative Vesicle Tethering Complexes and Their Distribution in Relation to a CPAM and a Cell Plate.
(A) to (D) Vesicle-associated L-shaped complex (beige arrows) as seen in a series of tomographic slices taken every five slices.
(E) to (H) Slicer-tilted image showing a face-on view of the complex, which is depicted in modeled form in (F) to (H) with no tilt (F), 45° tilt (G), and 90° tilt (H).
(I) to (L) Tomographic slice images of two vesicles connected by a Y-shaped tethering complex (purple arrows). The upper vesicle also carries an L-shaped complex (yellow arrow).
(M) to (P) Slicer-tilted image showing a face-on view of the Y-shaped tether, which is depicted in modeled form in (N) to (P) with no tilt (N), 45° tilt (O), and 90° tilt (P).
(Q) to (S) Face-on views of Y-shaped tethering complexes as seen in tomographic slices.
(T) to (V) Images of glutaraldehyde-fixed purified Sec6/8 tethering complexes from mammalian brain (courtesy of J. Heuser, with permission).
(W) Distribution of L- and Y-shaped complexes around a CPAM containing a TVN stage cell plate (cp). Note that whereas the vesicles with L-shaped complexes (circles) are seen both outside and inside the CPAM, Y-shaped tethers (squares) are found only within the CPAM.
Bar in (F) = 50 nm for (A) to (P); bar in (Q) = 25 nm for (Q) to (V); bar in (W) = 500 nm.
- Figure 5.
Changes in Width of the CPAM Associated with the Different Types of Cell Plates.
The mean distance (±sd) corresponds to the average distance between the cell plate and the closest 200 ribosomes. The dashed line marks the minimum threshold distance measured in stages in which no CPAM could be detected.
- Figure 6.
Formation of Dumbbell Vesicles from Freshly Fused Hourglass Vesicles and Initial Cell Plate Assembly Steps.
(A) to (K′) Tomographic slice (A) and model (F) of an hourglass-shaped vesicle fusion profile. Arrows point to compact dynamin-like springs; arrowheads point to individual loops of the expanded dynamin-like spring. Insets in (B) and (K), tomographic face-on views of compact and dynamin-like springs, respectively.
(B) to (E) Tomographic slices of dumbbell vesicles with dynamin-like springs.
(G) to (J) Modeled dumbbell vesicles with dynamin-like springs at different stages during elongation.
(K) Tomographic image of a TVN-stage cell plate tubule with an expanded dynamin-like spring.
(K′) Model of K structures.
(L) to (O) Cell plate intermediates showing how the fusion of vesicles to the dumbbell ends gives rise to TVN-type cell plate networks.
(P) An early TVN network consisting of isolated dumbbell-derived intermediates. cp, cell plate.
Bars in (A) to (O) = 50 nm; bar in (P) = 200 nm.
- Figure 7.
Solid Phragmoplast with CPAM and TVN Stage Cell Plate.
(A) Shoot apical meristem cell. Side view of a nearly complete TVN cell plate (cp) with its associated CPAM and phragmoplast MTs. Arrows point to clathrin-coated vesicles. Arrowhead points to a small dumbbell not yet integrated within the cell plate. m, mitochondria.
(B) Face-on view of the same TVN. Asterisk marks the initials of a future planar sheet region.
(C) Projection of 10 consecutive tomographic slices (2 nm thick each) through a TVN-type cell plate and its associated CPAM. Note that the MT (+) ends (arrows) terminate within the CPAM, where only very few ribosomes can occasionally be seen. The asterisk indicates a small thin sheet domain. m, mitochondria.
(D) Detail of a fusion profile (arrow) in a TVN-type cell plate showing the increased staining of the cell plate contents.
(E) Modeling of the ribosome-excluding CPAM. The ribosomes (r) define the outer boundary of the cocoon-like CPAM, which encompasses the TVN-type cell plate (cp). Isolated dumbbells (arrows) also are surrounded by a CPAM.
Bars in (A), (B), and (E) = 500 nm; bar in (C) = 200 nm; bar in (D) = 50 nm.
- Figure 8.
TN Stage Cell Plates.
(A) and (D) Shoot apical meristematic cell.
(B) and (C) Root meristematic cell.
(A) Tomographic slice of a TN. Arrows point to sites of early callose accumulation. Note the absence of a CPAM around a cell plate (cp) as evidenced by trans cell plate channels (delineated with brackets) filled with ribosomes.
(B) Note the lower density of MTs compared with the TVN stage (Figure 7A) and their greater separation from the cell plate. ccv, clathrin-coated vesicles; cp, cell plate.
(C) and (D) Face-on views of modeled early and late TN-type cell plates. The callose-induced tubule widening leads to increasingly large planar fenestrated domains (asterisks). ccb, clathrin-coated buds; ccv, clathrin-coated vesicles.
Bars = 200 nm.
- Figure 9.
Tomographic Images of the PGZ and the PFS Cell Plates.
Tomographic images ([A] and [B]) and models ([C] to [E]) of half of a PGZ/PFS cell plate.
(A) Shoot apical meristematic cell. The leftmost part of the cell plate (PGZ) is still actively growing and exhibits a mixture of features typical of TVN and TN stage cell plates. The PFS lacks a CPAM except over large fenestra (arrowhead). ccb, clathrin-coated buds; cw, cell wall; m, mitochondria.
(B) Detail of a PFS cell plate region (cp) where a dense dynamin-like ring (arrow) is found constricting a clathrin-coated bud (ccb).
(C) Side model view of the PGZ and PFS regions. Note the convergence of MTs and vesicles over the PGZ region and the high density of clathrin-coated buds (ccb) and vesicles (ccv) over the PFS. cw, cell wall; g, Golgi stack; m, mitochondria; v, vesicle.
(D) and (E) CPAMs are seen to encompass the PGZ region and to cover individual fenestrae of the PFS. Note the focusing of MTs to the CPAM regions where the MT (+) ends are embedded. Brackets mark the same fenestrae in (C) to (E). ccb, clatrin-coated bud; cw, cell wall.
Bars in (A) and in (C) to (E) = 500 nm; bar in (B) = 50 nm.
- Figure 10.
Changes in the Organization of the ER during Cell Plate Formation.
(A) and (B) TVN stage.
(A) Side view of the cell plate (cp). Note the limited number of sites where ER–TVN cell plate interaction occurs. Similarly, few ER cisternae are seen between the solid phragmoplast MTs.
(B) 45° tilt view of the TVN shown in (A). At the cell plate surface the ER cisternae branch, giving rise to very narrow ER tubules that cross the cell plate (arrows).
(C) and (D) Association of ER membranes with PGZ and PFS cell plate regions. Dashed line marks the transition between them.
(C) Side view. Many cytoplasmic ER tubules are seen intermingled with MTs over the PGZ region (left side), but little direct interaction of ER tubules with cell plate (cp) membranes is observed. By contrast, in the PFS zone (right), numerous ER tubules appear closely associated with the cell plate. Small white spheres correspond to ER-bound ribosomes.
(D) 45° tilt view of (C). For better viewing of the numerous cell plate–crossing tubular ER regions, the corresponding membrane domains have been highlighted with a red collar (arrows), and the top layer of ER membranes is displayed in a semitransparent mode.
(E) Detail of a contact site (arrow) between an ER tubule and a PFS-type cell plate (cp). Note the membrane-bridging elements.
Bars in (A) to (D) = 500 nm; bar in (E) = 200 nm.
- Figure 11.
Changes in Cell Plate Surface Area and Volume during Different Stages of Somatic-Type Cell Plate Formation.
The numbers in the bars indicate the mean cell plate surface area (A) and volume (B) per 1 μm3 ± sd, and the numbers next to the black arrows reflect the net growth unit cell plate surface area (A) or volume (B) in terms of small vesicle equivalents. The bracketed numbers reflect the predicted numbers of small/large (in black/gray, respectively) vesicle equivalents required for these changes given the vesicle ratios documented in Figure 3. The gray dotted arrow indicates net loss changes in cell plate surface area in terms of clathrin-coated vesicle equivalents. As a reference, the estimated final cell wall surface area and volume values also are displayed.
- Figure 12.
Model of Plant Somatic-Type Cytokinesis.
(A) Phragmoplast assembly phase during late anaphase. Phragmoplast initials arise from opposite sets of polar spindle MTs. Vesicles (v) travel along MTs toward the assembly sites, defined by the presence of CPAMs. Inside the CPAMs, the first cell plate initials are produced through the formation of dumbbell-shaped (db) vesicle intermediates (see also Figure 13). Chr, chromosomes; ne, nuclear envelope; pm, plasma membrane.
(B) Solid phragmoplast phase. After fusion of vesicles to the dumbbell ends and joining of the enlarged phragmoplast initials, a TVN cell plate arises within the CPAM that now extends across the entire interzone between the two sets of opposing MTs that form the solid phragmoplast. N, nucleus; ne, nuclear envelope; pm, plasma membrane; v, vesicles.
(C) Transitional phragmoplast phase. As the central CPAM and associated MTs disassemble, a new CPAM with MTs arises at the edge of the cell plate, giving rise to the ring-shaped phragmoplast and the PGZ. In parallel, the TVN cell plate is converted into a TN through callose synthesis in the cell plate lumen. N, nucleus; ne, nuclear envelope; pm, plasma membrane; v, vesicles.
(D) Ring-shaped phragmoplast phase. As the central cell plate domain is converted to a PFS, secondary CPAMs and associated MTs reform over the remaining large fenestrae, focusing cell plate growth to these regions. The ring-shaped phragmoplast and CPAM define the PGZ, which expands centrifugally until the cell plate reaches and fuses with the plasma membrane (pm). N, nucleus; ne, nuclear envelope; pm, plasma membrane; v, vesicles.
- Figure 13.
Hypothetical Model Explaining How Golgi-Derived, Cell Plate–Forming Vesicles Can Give Rise to Large Vesicles and to Dumbbells.
Small, Golgi-derived vesicles outside the CPAM interact through an unknown tethering complex before fusion and give rise to large-light vesicles. Vesicles that enter the CPAM before fusion first become tethered through an exocyst-like complex and then give rise to an hourglass intermediate. This hourglass-type vesicle is converted into a dumbbell vesicle with the help of dynamin springs. First, GTP-dynamin monomers are assembled into tight springs around the hourglass vesicle necks. GTP hydrolysis causes expansion of the dynamin spring, which elongates the neck to form a dumbbell. New GTP-dynamin springs are assembled next to the bulbous ends of the dumbbell, and the expansion/stretching process is repeated. During each neck elongation step, the volume of the vesicle is reduced by expulsion of water. The resulting concentration of the vesicle contents (pectic polysaccharides and xyloglucans) could lead to polysaccharide gelling and thereby stabilize the elongated vesicle architecture.
Additional Files
Supplemental Data
Files in this Data Supplement:
- Video Sequence 1 - Tethering complexes. This movie shows a series of tomographic slices through two tethered vesicles with an L-shaped complex (upper vesicle) and an exocyst-like Y-shaped complex (arrows).
- Video Sequence 2 - Tubulo-vesicular network (TVN) cell plate. This movie corresponds to a model of a TVN cell plate rotated 360o, where the 3-D arrangement of the cell plate, phragmoplast MTs, and CPAM is clearly shown.
- Video Sequence 3 - Tubular network (TN). This movie shows a TN cell plate where vesicular domains are almost absent. Note the lower density of MTs and their greater distance from the cell plate.
- Video Sequence 4 - Peripheral growth zone (PGZ) / Planar fenestrated sheet (PFS). In this movie, a PGZ domain (left) is seen fused with the plasma membrane. Most of the MTs and vesicles are concentrated around the PGZ. The PFS (center-right) is almost devoid of MTs and vesicles, which are focused only to the large fenestrae. Mitochondria, Golgi stacks, and vacuoles are present near the cell plate. Note the increase in the number of clathrin-coated vesicles with respect to the previous stages.
