Two Microtubule-associated Proteins of Arabidopsis MAP65s Promote Antiparallel Microtubule Bundling

Abstract

The Arabidopsis MAP65s are a protein family with similarity to the microtubule-associated proteins PRC1/Ase1p that accumulate in the spindle midzone during late anaphase in mammals and yeast, respectively. Here we investigate the molecular and functional properties of AtMAP65-5 and improve our understanding of AtMAP65-1 properties. We demonstrate that, in vitro, both proteins promote the formation of a planar network of antiparallel microtubules. In vivo, we show that AtMAP65-5 selectively binds the preprophase band and the prophase spindle microtubule during prophase, whereas AtMAP65-1-GFP selectively binds the preprophase band but does not accumulate at the prophase spindle microtubules that coexists within the same cell. At later stages of mitosis, AtMAP65-1 and AtMAP65-5 differentially label the late spindle and phragmoplast. We present evidence for a mode of action for both proteins that involves the binding of monomeric units to microtubules that "zipper up" antiparallel arranged microtubules through the homodimerization of the N-terminal halves when adjacent microtubules encounter. INTRODUCTIONIn higher plant cells, interphase microtubules occur predominantly at the cell cortex as ordered bundles in close association with the plasma membrane. These so-called cortical microtubules (CMTs) play crucial roles in cell morphogenesis (Wasteneys and Fujita, 2006). In rapidly elongating cells, CMTs are linear bundles that are usually transversely oriented relative to the longest cell axis. When cell expansion slows down, the transverse organization is lost and CMTs become oblique (Lloyd, 1994;Dixit and Cyr, 2004). CMTs are highly dynamic and show higher (de)-polymerization rates than interphase mammalian microtubules (Shaw et al., 2003;Dixit et al., 2006). In contrast to animal and yeast microtubules (MTs), CMTs do not emanate from a well-defined nucleating center. Instead, the MT nucleation activity in interphase plant cells mostly occurs at dispersed sites along pre-existing MTs at the cell's cortex (Murata et al., 2005) in a ␥-tubulin-dependent manner (Pastuglia et al., 2006). This MT-dependent nucleation results in branching patterns of CMTs (Murata et al., 2005) that are subsequently resolved into coaligned CMTs. After nucleation, the majority of the MTs are released from their nucleation site, move in the cell cortex by a hybrid treadmilling mechanism leading to polymer interactions (Shaw et al., 2003), and are incorporated into bundles (Dixit and Cyr, 2004). Significantly, the CMT bundles are not anchored and consequently both MT ends are dynamic. Thus, the ordered patterns of CMT arrays are not correlated with the patterns of the CMT nucleation sites (Dixit et al., 2006) and depend on a yet-to-be-discovered mechanism.To accommodate for the transverse network observed in expanding cells, CMTs form bundles. How these bundles are organized is not clear, and for instance, the polarity of CMTs within bundles is not yet solved. Using an in vitro model that provides good access to the cortex combined with the hoo...

The rise and fall of the phragmoplast microtubule array

“…In vitro experiments on two Arabidopsis MAP65 isoforms show that the proteins first bind to MTs in the monomeric form, and the dimerization process adds a zippering effect to crosslink anti-parallel MTs [26]. Moreover, it has been…”

Phosphorylation of MAP65-1 by Arabidopsis Aurora Kinases Is Required for Efficient Cell Cycle Progression

“…The arrow marks the autophosphorylation of Aurora1. Gaillard et al, 2008;Fache et al, 2010;Sasabe et al, 2011). We therefore used this system to analyze the colocalization between MAP65-1 and Aurora 1, as well as the effect of Aurora-phospho-mutations on the localization of MAP65-1 throughout mitosis.…”

“…All of them have a similar structural organization as PRC1/Ase1: an N-terminal region that comprises the dimerization domain required for the bundling activity, and a C-terminal domain required for MT binding Smertenko et al, 2008). The MT-binding domain is composed of two distinct parts: the N-terminal part adopts a spectrin fold, which is responsible for the binding to MTs Subramanian et al, 2010), and an unfolded C terminus, which is highly divergent between the different MAP65 isoforms and carries several phosphorylation sites (Smertenko et al, 2006(Smertenko et al, , 2008Gaillard et al, 2008). Although the role of the unfolded C terminus is still not well understood, several studies indicate that it could be involved in MT polymerization and bundling properties, and contribute to the differences in MAP65s intracellular targeting (Van Damme et al, 2004a;Mao et al, 2005b).…”

Preprophase band formation and cortical division zone establishment: RanGAP behaves differently from microtubules during their band formation

“…Next, we compared the location of MTs and MAP65, which is an MT-associated protein family whose isotypes have been found in the PPBs. [58][59][60][61][62] We used anti-BY2MAP65 53 which cross-reacted with a single band of the crude extract of onion root tips (Fig. 1C).…”