Cross-talk between microtubules and microfilaments has been explored in detail for the development of the interdigitated epidermal or pavement cells of Arabidopsis and other plants. During leaf development, epidermal cells with simple shapes undergo co-ordinated and selective, localized expansion to form highly complex and interlocking patterns (Fig. 5A). Outgrowths (lobes) of one cell must be matched with reduced or inhibited growth (necks) of the adjoining cell. This requires both intracellular and as yet unknown intercellular signalling to coordinate growth between adjacent cells (reviewed in Panteris and Galatis 2005; Smith and Oppenheimer 2005).
These selective growth processes involve the cytoskeleton. Microtubules are associated with the development of the necks and the inhibition of growth there (Panteris and Galatis 2005). In Vigna, microtubule bundles are found associated with the non-growing neck regions of the expanding cells (Pan-teris et al. 1993) and as similar events occur in Arabidopsis (Wasteneys et al. 1997; Fu et al. 2002), this suggests that the cell-wall thickening present in the lobe necks might be dependent on the presence of microtubules. This is analogous to microtubule-based wall deposition in other diffusely growing plant cells. Chemical disruption of microtubules in Vigna (Panteris et al. 1993) prevents interdigitation occurring. Microtubule mutants can also show reduced interdigitation (Whittington et al. 2001; reviewed in Kotzer and Wasteneys 2006).
While microtubules are associated with the neck regions where growth is inhibited, diffuse cortical microfilaments are found in the actively growing cell outgrowths of Arabidopsis (Fu et al. 2002) and maize (Frank et al. 2003). Furthermore, microfilament disruption with cytochalasin in Vigna also reduces the waviness of pavement cells (Panteris and Galatis 2005), and various microfilament-related mutants, including those that affect the ARP2/3 complex in Arabidopsis or the brick1 mutant in Zea, also show reduced inter-digitation (Fu et al. 2002; Frank et al. 2003; Li et al. 2003; reviewed in Kotzer and Wasteneys 2006). As both microtubules and microfilaments seem to be required for the developmental processes associated with interdigitation, it is perhaps not surprising that evidence exists in Arabidopsis for cross-talk via the Rop-signalling pathway (Fu et al. 2005; Bannigan and Baskin 2005; Kotzer and Wasteneys 2006).
Rops (Rho of plants) are a plant-specific family of Rho GTPases that act as master switches controlling multiple downstream pathways (Gu et al. 2004). Rops are activated by binding GTP and turn on effector proteins, RICs, that contain CRIB (Cdc42/Rac-interactive binding) motifs (Wu et al. 2001; Fu
Fig. 5 The development of pavement cells in Arabidopsis requires Rop GTPase-dependent signalling to the cytoskeleton, and a feedback loop between microtubules and microfilaments (Fu et al. 2005). A In the leaf primordia, pavement cells have a regular shape but this is modified by co-ordinated cell expansion to form lobes between adjacent cells and an interdigitated morphology. B Microtubule bands (in grey), found connecting the non-growing neck regions alternate with regions of diffuse microfilaments in the tips of growing lobes. This patterning is controlled by Rop-based signalling. Activated Rops promote microfilament through the RIC4 and ARP2/3 pathway and cause the microtubule banding protein RIC1 to dissociate from microtubules leading to their destabilization. Cross-talk between microtubules and microfilament is evident with feedback from the microtubule bundles inhibiting RIC4 activity and microfilament polymerization (Fu et al. 2005) in an unknown way, indicated by a question mark. Feedback from diffuse microfilaments to decrease microtubule stability is evident in root hairs (Saedler et al. 2004, Timmers et al. 2007) and suggested for Zea stomatal development (Panteris et al. 2006). Were these systems also active in pavement cells, a self-reinforcing cycle would result, although it remains unclear whether signalling would come from microfilaments themselves or elements that signal to them (indicated by paired question marks)
et al. 2002). In Arabidopsis epidermal cells, two closely related Rops (Rop2 and Rop4) interact with RIC1 and RIC4 to regulate the cytoskeleton and the formation of pavement cells (Fig. 5B). Rops positively control microfilament formation through activating RIC4 which in turn promotes microfilament polymerization through the WAVE/SCAR and ARP2/3 pathways (Fu et al. 2002). This pathway explains why mutations affecting microfilaments through these complexes reduce pavement cell formation. Rops also negatively control microtubule polymerization because Rop-dependent de-activation of RIC1, a microtubule-associated protein, prevents it from binding to and bundling microtubules (Fu et al. 2002). Thus, the formation of lobed pavement cells is Rop/RIC-dependent and requires signalling to both microfilaments and microtubules (Fig. 5B).
Although microtubules and microfilaments are co-ordinated by the same signalling molecules in this model, this by itself does not imply cross-talk. This has, however, been demonstrated. The status of microtubule polymerization controls RIC4 activity, with microtubule bundling inhibiting Rop/RIC interactions and locally preventing microfilament polymerization. This feedback has been demonstrated by direct, FRET (fluorescence resonance energy transfer)-based measurements of the interactions between Rop2 and RIC4, which increase following both microtubule destabilization or depolymeriza-tion (Fu et al. 2005). How this cross-talk occurs exactly is not yet clear, although in a mechanism analogous to that suggested in Nitella (Collings et al. 1996, Sect. 4.3), it is possible that Rop activity is controlled through the release of some microtubule-associated element (Fu et al. 2005). This factor might directly inactivate RIC4, thus directly blocking microfilament polymerization, or it might prevent Rop activation and indirectly block microfilament polymerization (Fig. 5B, question mark).
In cells showing complex expansion patterns such as pavement cells, microtubules and microfilaments show complementary patterns with microfilament patches being present in sites of active growth. A similar pattern of complementary microtubules and microfilaments, also occurs during the asymmetric divisions of Zea stomatal mother cells, and can be disrupted by latrunculin or mutation of brick1. This patterning "favors the idea that some [MF] nucleating factor(s), but not the [MFs] themselves, is (are) implicated in the depletion of the microtubules" (Panteris et al. 2006).
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