A Minus-End–directed Kinesin with Plus-End Tracking Protein Activity Is Involved in Spindle Morphogenesis

Abstract

Diverse kinesin motor proteins are involved in spindle function; however, the mechanisms by which they are targeted to specific sites within spindles are not well understood. Here, we show that a fusion between yellow fluorescent protein (YFP) and a minus-end-directed Kinesin-14 (C-terminal family) from Arabidopsis, ATK5, localizes to mitotic spindle midzones and regions rich in growing plus-ends within phragmoplasts. Notably, in Arabidopsis interphase cells, YFP::ATK5 localizes to microtubules with a preferential enrichment at growing plus-ends; indicating ATK5 is a plus-end tracking protein (؉TIP). This ؉TIP activity is conferred by regions outside of the C-terminal motor domain, which reveals the presence of independent plus-end tracking and minus-end motor activities within ATK5. Furthermore, mitotic spindles of atk5 null mutant plants are abnormally broadened. Based on these data, we propose a model in which ATK5 uses plus-end tracking to reach spindle midzones, where it then organizes microtubules via minus-end-directed motor activity. INTRODUCTIONDuring cell division, the proper segregation of genetic material into daughter cells requires the action of the microtubule spindle apparatus and its associated proteins. The spindle consists of two opposing sets of microtubules oriented with the minus-ends at the poles and the plus-ends at the midzone. The midzone represents the region of overlap between the two halves of the spindle, where microtubule plus-ends terminate at chromosomal kinetochores (kinetochore microtubules) or interdigitate in an antiparallel manner with microtubules from the opposite pole (interpolar microtubules). The spindle midzone is the site of force generation during anaphase spindle elongation (Leslie and Pickett-Heaps, 1983;Khodjakov et al., 2004), and in plants it persists through telophase to form the cytokinetic microtubule apparatus, the phragmoplast (Euteneuer et al., 1982).The assembly and functioning of spindles involve the highly orchestrated activities of diverse microtubule motor proteins. Kinesins convert the energy derived from ATP hydrolysis into translational movement along microtubules (Dagenbach and Endow, 2004;Lawrence et al., 2004). Kinesin-14 family members (previously referred to as C-terminal kinesins), such as Ncd from Drosophila and Kar3p from budding yeast, are unique in that they translocate exclusively toward microtubule minus-ends (Walker et al., 1990). Several Kinesin-14 family members contain microtubule binding sites in their tail regions, which specifies the ability to carry microtubules as cargo along other microtubules; in effect, moving microtubules in relation to one another (Walczak et al., 1997;Narasimhulu and Reddy, 1998;Karabay and Walker, 1999;Matuliene et al., 1999). This finding, in conjunction with subcellular localization and loss-of-function studies, has revealed two distinct roles for Kinesin-14s in spindle functioning. The first role is inferred from studies showing that loss or depletion of various Kinesin-14 family members rescues the s...

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

“…Janson et al (2007) demonstrated that Ase1p localizes to overlapping (Ϫ) ends during interphase and overlapping plus ends in the anaphase spindle, the difference in localization being determined by molecular motors as Klp2p (a minus-end-directed motor) mediates the sliding of newly nucleated MTs along antiparallel MT bundles. In plant cells, ATK5, a plus end tracking protein molecular motor (Ambrose et al, 2005) preferentially localizes at regions of overlap between opposing interpolar MTs, suggesting an increased affinity for plus ends in an antiparallel orientation (Ambrose and Cyr, 2007). In vitro, ATK5 equally coaligns with parallel or antiparallel MTs.…”

Emerging molecular mechanisms that power and regulate the anastral mitotic spindle of flowering plants

“…In the loss-of-function mutants, atk1 and atk5, mitosis is prolonged and the spindles are somewhat broader than in the wild type [Marcus et al, 2003;Ambrose et al, 2005;Ambrose and Cyr, 2007]. In atk5, prophase spindles are longer than those of the wild type [Ambrose and Cyr, 2007], and in atk1, prophase spindles have weak or even absent bipolarity, implying that these kinesins act early in mitosis and that forming the prophase spindle involves a force balance.…”

“…The two proteins share 83% amino acid sequence identity and can crosslink both parallel and antiparallel microtubules in vitro [Ambrose et al, 2005;Ambrose and Cyr, 2007], enabling them in principle to carry out the activities ascribed to this class of kinesin in animal and fungal spindles. ATK5 tracks to the plus end of microtubules in all stages of the cell cycle, independent of the motor domain, but its motor activity is minus-end-directed [Ambrose et al, 2005]. These authors propose that the plus-end tracking targets ATK5 to the spindle midzone, from where it helps to focus the poles by crosslinking parallel microtubules and walking toward the minus ends, as has also been suggested for NCD in Drosophila [Matthies et al, 1996].…”

Mitosis-specific kinesins in Arabidopsis

“…The Arabidopsis genome contains 21 C-terminal kinesins now classified as Kinesin-14, of which seven are transcriptional and up-regulated at mitosis [6,8]. The Ncd-related ATK1 and ATK5 have been shown to move in the minusend direction of MTs and to associate with spindle MTs where they are required for focusing the MTs at the poles, which is similar to Ncd activity ( Figure 2) [33][34][35][36]. Compared with other organisms, the Kinesin-14 group is relatively large.…”

“…Spindle poles are broad in plant cells and poles are focused at the centrosomes in animal cells. Antagonistic forces of Kinesin-14, such as Ncd [81] and Arabidopsis ATK1 [35] and Kinesin-5, such as DmKLP61F [81], HsEg5 or AtTKRP125 establish spindle bipolarity, whereas Kinesin-14 members Ncd, ATK1 and ATK5 focus on the minus-ends of the spindle MTs [32][33][34][35][36]. Furthermore, members of the depolymerizing Kinesin-13 family (DmKLP10A and HsKIF2A) function in mitotic spindle assembly and maintenance [62].…”