The principles by which cortical microtubules self-organize into a global template hold important implications for cell wall patterning. Microtubules move along bundles of microtubules, and neighboring bundles tend to form mobile domains that flow in a common direction. The bundles themselves move slowly and for longer than the individual microtubules, with domains describing slow rotary patterns. Despite this tendency for colinearity, microtubules have been seen to branch off extant microtubules at ;458. To examine this paradoxical behavior, we investigated whether some microtubules may be born on and grow along extant microtubule(s). The plus-end markers Arabidopsis thaliana end binding protein 1a, AtEB1a-GFP, and Arabidopsis SPIRAL1, SPR1-GFP, allowed microtubules of known polarity to be distinguished from underlying microtubules. This showed that the majority of microtubules do branch but in a direction heavily biased toward the plus end of the mother microtubule: few grow backward, consistent with the common polarity of domains. However, we also found that a significant proportion of emergent comets do follow the axes of extant microtubules, both at sites of apparent microtubule nucleation and at cross-over points. These phenomena help explain the persistence of bundles and counterbalance the tendency to branch.

The cell-to-cell movement of Tobacco mosaic virus through plasmodesmata (PD) requires virus-encoded movement protein (MP). The MP targets PD through the endoplasmic reticulum (ER)/actin network, whereas the intercellular movement of the viral RNA genome has been correlated with the association of the MP with mobile, microtubule-proximal particles in cells at the leading front of infection as well as the accumulation of the protein on the microtubule network during later infection stages. To understand how the associations of MP with ER and microtubules are functionally connected, we applied multiple marker three-dimensional confocal and time-lapse video microscopies to Nicotiana benthamiana cells expressing fluorescent MP, fluorescent RNA and fluorescent cellular markers. We report the reconstitution of MP-dependent RNA transport to PD in a transient assay. We show that transiently expressed MP occurs in association with small particles as observed during infection. The same MP accumulates in PD and mediates the transport of its messenger RNA transcript to the pore. In the cellular cortex, the particles occur at microtubuleproximal sites and can undergo ER-associated and latrunculin-sensitive movements between such sites. These and other observations suggest that the microtubule network performs anchorage and release functions for controlling the assembly and intracellular movement of MP-containing RNA transport particles in association with the ER.

Light and dark have antagonistic effects on shoot elongation, but little is known about how these effects are translated into changes of shape. Here we provide genetic evidence that the light/gibberellin-signaling pathway affects the properties of microtubules required to reorient growth. To follow microtubule dynamics for hours without triggering photomorphogenic inhibition of growth, we used Arabidopsis thaliana light mutants in the gibberellic acid/DELLA pathway. Particle velocimetry was used to map the mass movement of microtubule plus ends, providing new insight into the way that microtubules switch between orthogonal axes upon the onset of growth. Longitudinal microtubules are known to signal growth cessation, but we observed that cells also self-organize a strikingly bipolarized longitudinal array before bursts of growth. This gives way to a radial microtubule star that, far from being a random array, seems to be a key transitional step to the transverse array, forecasting the faster elongation that follows. Computational modeling provides mechanistic insight into these transitions. In the faster-growing mutants, the microtubules were found to have faster polymerization rates and to undergo faster reorientations. This suggests a mechanism in which the light-signaling pathway modifies the dynamics of microtubules and their ability to switch between orthogonal axes.