A mechanochemical model explains interactions between cortical microtubules in plants.

Microtubules anchored to the two-dimensional cortex of plant cells collide through plus-end polymerization. Collisions can result in rapid depolymerization, directional plus-end entrainment, or crossover. These interactions are believed to give rise to cellwide self-organization of plant cortical microtubules arrays, which is required for proper cell wall growth. Although the cell-wide self-organization has been well studied, less emphasis has been placed on explaining the interactions mechanistically from the molecular scale. Here we present a model for microtubule-cortex anchoring and collision-based interactions between microtubules, based on a competition between cross-linker bonding, microtubule bending, and microtubule polymerization. Our model predicts a higher probability of entrainment at smaller collision angles and at longer unanchored lengths of plus-ends. This model addresses observed differences between collision resolutions in various cell types, including Arabidopsis cells and Tobacco cells.

Spatial organization of plant cortical microtubules: close encounters of the 2D kind.

The shape of plant cells depends on cortical microtubules. Their freedom from central microtubule organizing centres provides a powerful experimental system to study microtubule self-organization. New ideas have emerged from live-cell imaging of microtubules, particularly in the model system Arabidopsis thaliana, revealing the importance of encounters between microtubules in driving self-organization. Encounters are modulated by intrinsic microtubule-assembly dynamics, along with polymer activities that include cortical attachment, bundling and severing. Balancing the activities of microtubule-associated proteins (such as MOR1, CLASP, MAP65s and katanins) that control these processes is crucial for fine-tuning the organization of microtubule arrays. Too much or too little of any given activity tips the balance, with often dramatic effects on array organization, cell morphogenesis and even organ chirality.

CLASP modulates microtubule-cortex interaction during self-organization of acentrosomal microtubules.

CLASP proteins associate with either the plus ends or sidewalls of microtubules depending on the subcellular location and cell type. In plant cells, CLASP's distribution along the full length of microtubules corresponds with the uniform anchorage of microtubules to the cell cortex. Using live cell imaging, we show here that loss of CLASP in Arabidopsis thaliana results in partial detachment of microtubules from the cortex. The detached portions undergo extensive waving, distortion, and changes in orientation, particularly when exposed to the forces of cytoplasmic streaming. These deviations from the normal linear polymerization trajectories increase the likelihood of intermicrotubule encounters that are favorable for subsequent bundle formation. Consistent with this, cortical microtubules in clasp-1 leaf epidermal cells are hyper-parallel. On the basis of these data, we identify a novel mechanism where modulation of CLASP activity governs microtubule-cortex attachment, thereby contributing to self-organization of cortical microtubules.

Mitotic spindle organization by the preprophase band.

In higher plants, the preprophase band (PPB) of microtubules (MTs) forecasts the cell division site prior to mitosis and specifies the organization of MTs into a bipolar prophase spindle surrounding the nucleus. However, the mechanisms governing this PPB-dependent establishment of bipolarity are unclear. Here, we present evidence from live cell imaging studies that suggest a role for the MTs bridging the PPB and the prophase nucleus in mediating this function. Results from drug treatments, along with genetic evidence from null kinesin plants, suggest that these MTs contribute to the bipolarity, orientation, and position of the prophase spindle. Specifically, the absence of these bridge MTs is associated with lack of bipolarity, while non-uniform distributions of bridge MTs correlate with prophase spindle migration, deformation, and enhanced bipolarity toward the region of highest bridge MT density. This behavior does not require actomyosin-based forces, and is enhanced by suppressing MT dynamics with taxol. These observations occur during late prophase, and are coincident with the gradual closing of annular spindle poles. Based on these data, we describe a hypothetical mechanism for bridge MT-dependent organization of prophase spindles.

Functional divergence of the duplicated AtKIN14a and AtKIN14b genes: critical roles in Arabidopsis meiosis and gametophyte development.

Gene duplication is important for gene family evolution, allowing for functional divergence and innovation. In flowering plants, duplicated genes are widely observed, and functional redundancy of closely related duplicates has been reported, but few cases of functional divergence of close duplicates have been described. Here, we show that the Arabidopsis AtKIN14a and AtKIN14b genes encoding highly similar kinesins are two of the most closely related Arabidopsis paralogs, which were formed by a duplication event that occurred after the split of Arabidopsis and poplar. In addition, AtKIN14a and AtKIN14b exhibit varying degrees of coding sequence divergence. Further genetic studies of plants carrying atkin14a and/or atkin14b mutations indicate that, although these two genes have similar functions, there is clear evidence for functional divergence. Although both genes are important for male and female meiosis, AtKIN14a plays a more critical role in male meiosis than AtKIN14b. Moreover, either one of these two genes is necessary and sufficient for gametophyte development, indicating that they are redundant for this function. Therefore, AtKIN14a and AtKIN14b together play important roles in controlling plant reproductive development. Our results suggest that the AtKIN14a and AtKIN14b genes have retained similar functions in gametophyte development and female meiosis, but have evolved partially distinct functions in male meiosis, with AtKIN14a playing a more substantive role.

The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division.

Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.

The kinesin ATK5 functions in early spindle assembly in Arabidopsis.

During cell division, the mitotic spindle partitions chromosomes into daughter nuclei. In higher plants, the molecular mechanisms governing spindle assembly and function remain largely unexplored. Here, live cell imaging of mitosis in Arabidopsis thaliana plants lacking a kinesin-14 (ATK5) reveals defects during early spindle formation. Beginning during prophase and lasting until late prometaphase, spindles of atk5-1 plants become abnormally elongated, are frequently bent, and have splayed poles by prometaphase. The period of spindle elongation during prophase and prometaphase is prolonged in atk5-1 cells. Time-lapse imaging of yellow fluorescent protein:ATK5 reveals colocalization with perinuclear microtubules before nuclear envelope breakdown, after which it congresses inward from the poles to the midzone, where it becomes progressively enriched at regions of overlap between antiparallel microtubules. In vitro microtubule motility assays demonstrate that in the presence of ATK5, two microtubules encountering one another at an angle can interact and coalign, forming a linear bundle. These data indicate that ATK5 participates in the search and capture of antiparallel interpolar microtubules, where it aids in generating force to coalign microtubules, thereby affecting spindle length, width, and integrity.

A minus-end-directed kinesin with plus-end tracking protein activity is involved in spindle morphogenesis.

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.

Arabidopsis thaliana protein, ATK1, is a minus-end directed kinesin that exhibits non-processive movement.

The microtubule cytoskeleton forms the scaffolding of the meiotic spindle. Kinesins, which bind to microtubules and generate force via ATP hydrolysis, are also thought to play a critical role in spindle assembly, maintenance, and function. The A. thaliana protein, ATK1 (formerly known as KATA), is a member of the kinesin family based on sequence similarity and is implicated in spindle assembly and/or maintenance. Thus, we want to determine if ATK1 behaves as a kinesin in vitro, and if so, determine the directionality of the motor activity and processivity character (the relationship between molecular "steps" and microtubule association). The results show that ATK1 supports microtubule movement in an ATP-dependent manner and has a minus-end directed polarity. Furthermore, ATK1 exhibits non-processive movement along the microtubule and likely requires at least four ATK1 motors bound to the microtubule to support movement. Based on these results and previous data, we conclude that ATK1 is a non-processive, minus-end directed kinesin that likely plays a role in generating forces in the spindle during meiosis.