264 J.M. Seguí-Simarro et al.
of the CPAM were blunt ended. During the transitional phragmoplast stage,
where the CPAM is absent from the central region of the cell plate, 80 %of
the (+)-ends exhibited a horned morphology, which is typical of disassem-
bling MTs. These results suggest that the CPAM contains factors that stabilize
MT (+)-ends and thereby prolong the life of the CPAM-associated MTs com-
pared to those that terminate outside the CPAM. This stabilization, in turn,
increases the efficiency of vesicle transport along the MTs to the growing cell
plate regions.
To further characterize the spatial relationship between MT (+)-ends and
the cell plate, we used our tomographic database to determine the distance
between the ends of blunt MTs and the closest cell plate membrane (Austin
et al. 2005). This quantitative analysis demonstrated that blunt-ended MTs of-
ten terminate 30 nm from the surface of a cell plate membrane, whereas no
such relationship was detected for the ends of horned and extended MTs. This
finding suggested that the (+)-ends of the metastable MTs could be physi-
cally connected through specific linkers to the cell plate membranes, and that
these linkers could contribute to the stabilization of the blunt-ended MTs. As
showninFig.5AandB,thepostulated 30 nm linkers can even be recognized
in suitably oriented tomographic slice views. Yet to be determined is the mo-
lecular identity of the observed linkers, but two candidate proteins have been
identified. InArabidopsis, the AtEB1 protein is a (+)-end MT-associated pro-
tein (van Damme et al. 2004), which has been localized to the phragmoplast
midline (Mathur et al. 2003). Since evidence from other systems indicates
that EB1 can stabilize MT (+)-ends (Tirnauer et al. 2002), a similar role for
AtEB1 in stabilizing cell plate-associated MTs seems possible. A second can-
didate is AtMAP65-3, an EB1-like protein (van Damme et al. 2004), which,
when mutated, causes a widening of the MT-depleted midline region of the
phragmoplast (Muller et al. 2004).
4.3
The CPAM Scaffold is Probably Composed of Long Coiled-Coil Proteins
AsshowninFig.4,theCPAMcompletelyencompassesthegrowingcellplate
regions and is absent from the maturing cell plate domains (Seguí-Simarro
et al. 2004). In meristem cells, the ribosome-excluding CPAM zone that en-
compasses the tubulo-vesicular cell plate membranes of the solid phragmo-
plast (Fig. 4B) is∼ 160 nm wide (Seguí-Simarro et al. 2004), whereas the
CPAM surrounding the wide tubular network of syncytial-type cell plates
is∼ 100 nm (Otegui et al. 2001). The identity of the scaffolding proteins of
the CPAM is unknown, but in analogy to the composition of the ribosome-
excluding COPII vesicle scaffold and the Golgi matrix (Kang and Staehelin,
submitted), the most likely candidates are long coiled-coil-type proteins simi-
lar to Uso1, p115, and golgins (Gillingham and Munro 2003; Latijnhouwers
et al. 2005). TheArabidopsisgenome contains a total of 252 long coiled-coil