Microtubules Contribute to Tubule Elongation and Anchoring of Endoplasmic Reticulum,Resulting in High Network Complexity in Arabidopsis

The endoplasmic reticulum (ER) is a network of tubules and sheet-like structures in eukaryotic cells. Some ER tubules dynamically change their morphology, and others form stable structures. In plants, it has been thought that the ER tubule extension is driven by the actin-myosin machinery. Here, we show that microtubules also contribute to the ER tubule extension with an almost 20-fold slower rate than the actin filament-based ER extension. Treatment with the actin-depolymerizing drug Latrunculin B made it possible to visualize the slow extension of the ER tubules in transgenic Arabidopsis (Arabidopsis thaliana) plants expressing ER-targeted green fluorescent protein. The ER tubules elongated along microtubules in both directions of microtubules, which have a distinct polarity. This feature is similar to the kinesin- or dynein-driven ER tubule extension in animal cells. In contrast to the animal case, ER tubules elongating with the growing microtubule ends were not observed in Arabidopsis. We also found the spots where microtubules are stably colocalized with the ER subdomains during long observations of 1,040 s, suggesting that cortical microtubules contribute to provide ER anchoring points. The anchoring points acted as the branching points of the ER tubules, resulting in the formation of multiway junctions. The density of the ER tubule junction positively correlated with the microtubule density in both elongating cells and mature cells of leaf epidermis, showing the requirement of microtubules for formation of the complex ER network. Taken together, our findings show that plants use microtubules for ER anchoring and ER tubule extension, which establish fine network structures of the ER within the cell.The endoplasmic reticulum (ER) is a complex network composed of tubules and sheet structures. The ER network’s morphology changes dynamically by elongation and shrinkage of tubules, sheet expansion, and sliding junctions. For example, an ER tubule elongates straight forward from a cisterna and subsequently, fuses to another cisterna, producing a linkage between two cisternae. If an elongating tubule fails to fuse to another cisterna, the tubule contracts into the original cisterna. However, the ER has stable anchoring points that associate with other cellular structures, such as the plasma membrane or cytoskeleton. When an elongating ER tubule reaches an association point, it forms a stable ER anchor (i.e. establishment of the ER anchoring points forms stable ER tubules). Hence, increasing the number of ER anchoring points produces fine ER meshwork.ER dynamics in eukaryotes depend on the cytoskeleton. In plants, major contributors for ER organization are actin filaments (Quader et al., 1989; Knebel et al., 1990; Lichtscheidl and Hepler, 1996; Sparkes et al., 2009a) and the actin-associated motor proteins (myosins; Prokhnevsky et al., 2008; Peremyslov et al., 2010; Ueda et al., 2010). However, it had generally been thought that microtubules are not involved in ER organization in plants, because microtubule-depolymerizing drugs do not induce obvious changes in the ER network (Quader et al., 1989; Knebel et al., 1990; Lichtscheidl and Hepler, 1996; Sparkes et al., 2009a). Nevertheless, involvement of microtubules in plant ER organization has been suspected from several electron microscopy observations that showed microtubules located close to the ER membrane in Vicia faba guard cells, Nicotiana alata pollen tubes, and Funaria hygrometrica caulonemata (Lancelle et al., 1987; Hepler et al., 1990; McCauley and Hepler, 1992).Foissner et al. (2009) have suggested that microtubules are involved in motility and orientation of cortical ER in Characean algae (Nitella translucens, Nitella flexilis, Nitella hyalina, and Nitella pseudoflabellata) internodal cells. Characean cortical ER is spatially separated from inner cytoplasmic streaming by the middle layer of fixed chloroplasts. The cortical ER forms a tight meshwork of predominantly transverse ER tubules that frequently coalign with microtubules, and microtubule depolymerization reduces the transverse ER tubules and increases mesh size (Foissner et al., 2009). Consistently, Hamada et al. (2012) have shown in Arabidopsis (Arabidopsis thaliana) that microtubule depolymerization increases mesh size in young elongating cells. In addition, stable ER tubule junctions are often colocalized with cortical microtubules (Hamada et al., 2012), suggesting that microtubules stabilize ER tubule junctions to form fine ER meshes. Oryzalin-induced ER nodulation (Langhans et al., 2009) was not observed in our experimental conditions.Here, we showed that ER tubules elongate along microtubules in plant cells. In addition, we revealed that the ER is stably anchored to defined points on cortical microtubules. The stable anchoring points are the basis of various ER shapes, such as three-way, two-way, or dead-end ER tubules. These microtubule-ER interactions, together with the actin-myosin system, contribute to ER network organization.