Electron Tomographic Analysis of Somatic Cell Plate Formation in Meristematic Cells of Arabidopsis Preserved by High-Pressure Freezing[W]

Abstract

We have investigated the process of somatic-type cytokinesis in Arabidopsis (Arabidopsis thaliana) meristem cells with a three-dimensional resolution of~7 nm by electron tomography of high-pressure frozen/freeze-substituted samples. Our data demonstrate that this process can be divided into four phases: phragmoplast initials, solid phragmoplast, transitional phragmoplast, and ring-shaped phragmoplast. Phragmoplast initials arise from clusters of polar microtubules (MTs) during late anaphase. At their equatorial planes, cell plate assembly sites are formed, consisting of a filamentous ribosomeexcluding cell plate assembly matrix (CPAM) and Golgi-derived vesicles. The CPAM, which is found only around growing cell plate regions, is suggested to be responsible for regulating cell plate growth. Virtually all phragmoplast MTs terminate inside the CPAM. This association directs vesicles to the CPAM and thereby to the growing cell plate. Cell plate formation within the CPAM appears to be initiated by the tethering of vesicles by exocyst-like complexes. After vesicle fusion, hourglass-shaped vesicle intermediates are stretched to dumbbells by a mechanism that appears to involve the expansion of dynamin-like springs. This stretching process reduces vesicle volume by~50%. At the same time, the lateral expansion of the phragmoplast initials and their CPAMs gives rise to the solid phragmoplast. Later arriving vesicles begin to fuse to the bulbous ends of the dumbbells, giving rise to the tubulo-vesicular membrane network (TVN). During the transitional phragmoplast stage, the CPAM and MTs disassemble and then reform in a peripheral ring phragmoplast configuration. This creates the centrifugally expanding peripheral cell plate growth zone, which leads to cell plate fusion with the cell wall. Simultaneously, the central TVN begins to mature into a tubular network, and ultimately into a planar fenestrated sheet (PFS), through the removal of membrane via clathrin-coated vesicles and by callose synthesis. Small secondary CPAMs with attached MTs arise de novo over remaining large fenestrae to focus local growth to these regions. When all of the fenestrae are closed, the new cell wall is complete. Few endoplasmic reticulum (ER) membranes are seen associated with the phragmoplast initials and with the TVN cell plate that is formed within the solid phragmoplast. ER progressively accumulates thereafter, reaching a maximum during the late PFS stage, when most cell plate growth is completed.

Exploring the ESCRTing machinery in eukaryotes

“…Preliminary results from our laboratory and Martin Hülskamp's group suggest that putative components of the ESCRT machinery are necessary for the proper execution of cytokinesis in Arabidopsis (V. Winter et al, unpublished; C. Spitzer et al, unpublished). This putative ESCRT function is corroborated by recent findings that MVBs are enriched at the newly formed cell plate during cytokinesis [15,16]. The identification of mutants that affect components of the ESCRT-machinery, of suitable markers for the characterization of MVB maturation, and of their endogenous cargos should shed light on the cellular processes that MVB are involved with.…”

“…MVBs might also be involved in recycling membranes. Although the presence of MVBs and their roles as prevacuolar compartments for lytic and storage vacuoles, in vacuolar receptor recycling, and in internalization and secretion of arabinogalactan-rich glycoproteins have been described [2,4,5,[13][14][15][16]18], only a few reports have identified components of the ESCRT-machinery in plants [53,54,67]. Here we show by cross-species comparisons that most protein entry components and all the ESCRT-components are present in Arabidopsis and rice.…”

“…Thus, MVBs act as an intermediate station for cargo on the way to degradation, intracellular recycling and secretion ( Figure 1). Although MVBs have been described in plants, little is known about their biogenesis, cargos and function [2,4,[13][14][15][16]. These studies show that plant MVBs are prevacuolar compartments and are labeled by vacuolar sorting receptor (VSR) antibodies such as BP80 [5,17].…”

Membrane fusion process and assembly of cell wall during cytokinesis in the brown alga, Silvetia babingtonii (Fucales, Phaeophyceae)

“…He described a close association of a tubular ER network with the nascent cell plate, surrounding the latter structure. Recent electron tomographic analysis of cell plate formation in meristematic cells of Arabidopsis prepared by high-pressure freezing showed that few ER membranes associated with the cell plate during the initial stages of the cell plate formation, but that this increased towards cell plate completion (Seguí-Simarro et al 2004). However, there is no direct evidence that ER contributes to the membranous structures involved in cell plate formation.…”

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae)

“…Electron tomography, which is a powerful electron microscopic technique for getting a nano-scale three-dimensional analysis, has been used in the ultrastructural studies of cytokinesis of land plants (Otegui and Staehelin 2004, Segui-Simarro et al 2004, Haas and Otegui 2007. This technique will be also useful for analysis of tiny PD structure, in order to investigate their ultrastructure and the process of their formation.…”