Plant Cytokinesis – Insights Gained from Electron Tomography Studies 267
Arabidopsis, pollen from AtSec8 mutants display reduced pollen tube growth
rates, while retaining the ability to complete both post-meiotic and pollen cy-
tokinesis (Cole et al. 2005). These phenotypes are consistent with a role for
Sec3 and Sec8 in polarized exocytosis not only during root hair and pollen
tube expansion, but also during cell plate assembly and growth, since the ex-
pected phenotype for a mutated exocyst complex would be a loss of specificity
in vesicle tethering and a concomitant slowdown in vesicle fusion, but not an
inhibition of the fusion process itself.
In yeast and mammalian cells, regulation of the exocyst tethering com-
plexes is dependent on Rho and Rab GTPases, and on short actin filaments
(Hsu et al. 1999; Guo et al. 2001; Lipschutz and Mostov 2002; Wang et al.
2003). In plants, Rops (Rho-related GTPases of plants) have been localized
to cell plates by means of GFP-tagged AtRop4 (Molendijk et al. 2001). Short
actin filaments, in turn, have been shown to be present at the growing edges
of cell plates (Endle et al. 1998), which is also where the CPAM is seen. Al-
though there is currently no direct evidence localizing Rab-GTPases to cell
plates, their importance for cell plate formation has been inferred from other
studies (Bednarek and Falbel 2002; Falbel et al. 2003). Based on these results,
we propose that the CPAM is the site where plant exocyst complex subunits,
SNARE proteins, and molecules involved in the regulation of vesicle fusion
are stored.
4.5
The CPAM is Enriched in Dynamin-Related Proteins that Assist in the Shaping
of Cell Plate Assembly Intermediates, and the Budding of Clathrin-coated Vesicles
The first microscopic evidence of membrane fusion within the CPAM is
the appearance of vesicles with a bilobed or hourglass-type of morphology
(Fig. 6; Otegui and Staehelin 2000b; Seguí-Simarro et al. 2004). However,
whereas these early, hourglass-shaped vesicle fusion intermediates are rarely
encountered (3 out of>1000 vesicles examined; Seguí-Simarro et al. 2004),
indicating that this is a short-lived intermediate membrane configuration,
other more dumbbell-shaped vesicles with longer connecting necks are more
frequently observed (Fig. 6; Samuels et al. 1995; Rensing et al. 2002; Seguí-
Simarro et al. 2004). A distinguishing feature of these dumbbell-shaped vesi-
cles is that the neck region is often surrounded by a more densely stained
layer, which in the best tomographic samples can be resolved into 40 to 45 nm
wide rings and spirals (Fig. 7A,A′; Otegui et al. 2001; Seguí-Simarro et al.
2004). Similar rings and spirals have been found to pinch the tubules of the
wide tubular network of syncytial-type cell plates (Otegui et al. 2001), and
to be present around the narrow tubular domains of the tubulo-vesicular
network in somatic-type cell plates (Fig. 7B; Seguí-Simarro et al. 2004). Fur-
thermore, the spirals exhibit two configurations, they can be tightly wound
or they can be expanded (Fig. 6). These rings and spirals label with antibod-