A distinguishing feature of plant cells is the presence of cortical microtubules adjacent to the plasma membrane (37). It has been noted since the discovery of cortical microtubules that the orientation of cortical microtubules in expanding cells is similar to that of cellulose microfibrils (38). This led to the hypothesis that the deposition of cellulose is oriented by an interaction between cellulose synthase and the microtubules, an idea that was reinforced by many observations of correlations between microtubule and microfibril organization which have been comprehensively and critically reviewed by Baskin (39). In the model of Giddings and Staehelin (40), as recast in an influential textbook (41), the movement of cellulose synthase is constrained by a close association between cortical microtubules and the plasma membrane, much like a bumper car bouncing along between rails of cortical tubulin. It is generally assumed that the energy of polymerization provides the motive force that moves the cellulose synthase complex through the membrane.

However, as noted in a recent critique of the model, there is no direct evidence for involvement of microtubules in microfibril orientation and many inconsistencies mediate against the idea (42). For instance, short treatment of Arabidopsis with the microtubule destabilizing drug oryzalin or the microtubule stabilizing drug taxol caused no apparent change to the orientation of cellulose microfibrils in cells that expanded during the treatment, as visualized by field emission scanning electron microscopy (43, 44). Long treatments caused changes in cellulose orientation but these may have been due to effects on the orientation of cell division. Similarly, when microtubule polymerization was impaired by shifting the temperature-sensitive mor1-1 mutant to non-permissive temperature, cellulose microfibrils exhibited a similar pattern of deposition as in controls (45, 46).

Recently, Paredez and coworkers (47) produced a functional N-terminal YFP fusion to CESA6 that complemented the corresponding mutant in Arabidopsis. When expressed under the native promoter, a substantial amount of the fusion protein accumulates in the Golgi apparatus where it assembles into distinct particles that can be seen to move to the plasma membrane. This is compatible with previous evidence from electron microscopy indicating that cellulose synthase rosettes assemble in the Golgi (48). Within less than a minute of arriving in the plasma membrane, the cellulose synthase particles begin moving in linear paths at a constant rate of about 300 nm min-1, somewhat slower than the rate observed by Hirai and coworkers (49) on tobacco membrane sheets. This is reminiscent of yeast chitin synthase III, in which activity is regulated by a specialized mechanism of vesicle sorting coupled with endocytic recycling (50). In this model, chitin synthase is maintained inside specialized vesicles called chitosomes (TGN/early endosome vesicles) and is trans­ported to the specific sites of function where it becomes activated. Inactivation occurs via endocytosis. Because plant Golgi do not synthesize cellulose, it is apparent that the cellulose synthase complexes observed there are not active but that they become activated upon arrival at the plasma membrane. Rosettes have also been estimated to have only a 20 minutes lifetime in moss (51), which may suggest that they are also dissociated or endocytosed.

When viewed in cells in which the microtubules are labeled with CFP, the YFP-labeled cel­lulose synthase particles can be seen to move along the microtubules. Importantly, inhibition of tubulin polymerization with oryzalin rapidly leads to strong disruptions of the normal patterns of movement of the cellulose synthase particles that aggregate in patterns resem­bling meandering streams. Similarly, treatment of seedlings with Morlin, a novel inhibitor of microtubule treadmilling and membrane attachment, caused stalling of the cellulose syn­thase complexes (52). Thus, from live cell imaging it is readily apparent that microtubules exert a strong effect on the orientation of cellulose synthase movement (which presum­ably reflects cellulose synthesis) (47). However, Paredez and coworkers (47) observed that after relatively long periods of oryzalin treatment, when most or all of the cortical micro­tubules have depolymerized, the cellulose synthase particles resume movement in relatively straight parallel paths. The rigidity of cellulose probably explains why no guidance is nec­essary to ensure that cellulose synthase moves in relatively straight lines. It is not clear what orients the pattern of deposition in these cells but models for the formation of oriented patterns of cellulose based on geometric considerations have been proposed (53) and may be testable in these experimental materials. These observations suggest that both sides of the microtubule-microfibril alignment debate are correct and that the discrepancies and inconsistencies between experiments reflect the limitations of using static imaging methods and different treatment times and conditions. The availability of the new imaging tools outlined here should facilitate a resolution of the matter.

Alignment of GFP-labeled cellulose synthase with microtubules was previously reported by Gardiner and coworkers (54), who used an N-terminal fusion of GFP to the xylem-specific CESA7 (irx3) protein. Because of difficulties viewing the vascular tissues by confocal mi­croscopy, the images of this GFP:CESA7 construct are difficult to discern. However, it appears that the distribution of fluorescence is not uniform and there are bands of fluorescence that are perpendicular to the long axis of the cells. Attempts to colocalize tubulin with CESA7 using immunofluorescence methods (54) indicate a similar pattern. However, the resolu­tion of the images was not high enough to provide a critical analysis. Treatment with the microtubule assembly inhibitor, oryzalin, rapidly reduced the banding pattern. Given the technical limitations of working with xylem-localized markers, the observations of Gardiner and coworkers (54) appear to be entirely consistent with the more recent work of Paredez and coworkers (47).

A surprising twist to the microtubule-cellulose synthase story was the observation that in tobacco protoplasts, inhibition of cellulose synthase activity prevented the development of oriented microtubule arrays (55). These data are consistent with the hypothesis that cellulose microfibrils or cellulose synthase, directly or indirectly, provide spatial cues for cortical microtubule organization. Similarly, microtubule organization in spruce pollen tubes was altered by isoxaben (56), and the orientation of microtubules in Arabidopsis root epidermal cells was disrupted by DCB (46).