Plant Kinesins Research Articles

Why are ATP-driven microtubule minus-end directed motors critical to plants? An overview of plant multifunctional kinesins.

In plants, microtubule and actin cytoskeletons are involved in key processes including cell division, cell expansion, growth and development, biotic and abiotic stress, tropisms, hormonal signalling as well as cytoplasmic streaming in growing pollen tubes. Kinesin enzymes have a highly conserved motor domain for binding microtubule cytoskeleton assisting these motors to organise their own tracks, the microtubules by using chemical energy of ATP hydrolysis. In addition to this conserved binding site, kinesins possess non-conserved variable domains mediating structural and functional interaction of microtubules with other cell structures to perform various cellular jobs such as chromosome segregation, spindle formation and elongation, transport of organelles as well as microtubules-actins cross linking and microtubules sliding. Therefore, how the non-motor variable regions specify the kinesin function is of fundamental importance for all eukaryotic cells. Kinesins are classified into ~17 known families and some ungrouped orphans, of which ~13 families have been recognised in plants. Kinesin-14 family consisted of plant specific microtubules minus end-directed motors, are much diverse and unique to plants in the sense that they substitute the functions of animal dynein. In this review, we explore the functions of plant kinesins, especially from non-motor domains viewpoint, focussing mainly on recent work on the origin and functional diversity of motors that drive microtubule minus-end trafficking events.

Identifying Pseudomonas syringae Type III Secreted Effector Function via a Yeast Genomic Screen.

Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as the promiscuity of individual T3SEs that can have multiple host targets. To overcome these challenges, we generated and characterized a collection of yeast strains stably expressing 75 T3SE constructs from the plant pathogen Pseudomonas syringae. This collection is devised to facilitate heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. Among 75 T3SEs tested, we identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media. We utilized Pathogenic Genetic Array (PGA) screens to identify potential host targets of P. syringae T3SEs. We focused on the acetyltransferase, HopZ1a, which interacts with plant tubulin and alters microtubule networks. To uncover putative HopZ1a host targets, we identified yeast genes with genetic interaction profiles most similar (i.e., congruent) to the PGA profile of HopZ1a and performed a functional enrichment analysis of these HopZ1a-congruent genes. We compared the congruence analyses above to previously described HopZ physical interaction datasets and identified kinesins as potential HopZ1a targets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1, illustrating the utility of our T3SE-expressing yeast library to characterize T3SE functions.

Importin-β Directly Regulates the Motor Activity and Turnover of a Kinesin-4

Spatiotemporal regulation of kinesins is essential formicrotubule-dependent intracellular transport. In plants, cell wall deposition depends on the FRA1 kinesin, whose abundance and motility are tightly controlled to match cellular growth rate. Here, we show that an importin-β, IMB4, regulates FRA1 activity in a developmental manner. IMB4 physically interacts with a PY motif in the FRA1 motor domain and inhibits its motility by preventing microtubule binding, while also protecting FRA1 against proteasome-mediated degradation, thus providing a mechanism to couple the motility and stability of FRA1. This regulatory mechanism is likely to be broadly applicable, based on the conservation of the PY motif in the motor domains of plant and animal kinesins and the direct interaction of multiple plant kinesins with IMB4. Together, our data establish IMB4 as a multi-functional regulator of FRA1 and reveal a mechanism for how plants control the magnitude of cargo transport needed for cell wall assembly.

The Rice Kinesin Oskch1 is a Dynamic Cross-Linker of Actin Filaments and Microtubules

The interaction between microtubules and actin filaments impacts many processes during plant growth and development including cell division and expansion. Despite numerous reports of co-localization between the two filament types in the past, only recently plant kinesins with a calponin-homology domain (KCH) were identified as putative dynamic cross-linkers. OsKCH1, a KCH from rice, has been described to interact with cortical microtubules and actin filaments both in vivo and in vitro. However, it remained unsolved whether this interaction is static or dynamic. Here, we show that OsKCH1 drives the active sliding of actin filaments along microtubules in vitro. We found that the sliding velocities corresponded to the velocities of microtubules propelled along OsKCH1 immobilized on glass substrates. This suggests that the motor interacts statically with actin filaments and dynamically with microtubules. We propose that kinesins with a calponin-homology domain play an essential role in the dynamic interaction between microtubules and actin filaments in plant cells.

Rice kinesin O12 is identical to kinesin OsKCH1

Since 2004, we have been conducting molecularlevel in vitro studies for the biochemical characterization of kinesins encoded on the rice genome. Some kinesins have previously been characterized, and their biochemical and crystallographic studies have been published (1–4). The kinesin we named as ‘O12’ is one of the several rice kinesins. To express kinesin O12 using an Escherichia coli expression system, we used a plasmid (accession number: AK065586; clone name: J013034O12) that was supplied by the National Institute of Agrobiological Science. We determined the unique enzymatic properties of kinesin O12 in 2005 and reported its biochemical characterization at meetings in 2006 (5, 6) and 2007 (7). At that stage, O12 was recognized as a novel kinesin. We believe that we characterized the biochemical properties of the rice kinesin O12 before any other research groups. Subsequently, we published an article detailing additional biochemical studies of kinesin O12 in The Journal of Biochemistry in 2011 (8). The aim of this study was to apply the unique characteristics of rice plant kinesins to the field of bionanotechnology. For instance, we observed an interesting property that the ATPase activity of the motor domain of kinesin O12, which does not contain a calponin homology domain, is regulated by actin and may be applicable in the regulation of a molecular shuttle based on the kinesin motor protein. As mentioned so far, our series of studies on the rice kinesins have been performed independently, on the basis of our original ideas since 2004.

Characterization of Tubulin Derived from Ginkgo Biloba and its Interaction with Rice Plant Kinesin

Previously, we have expressed novel Rice plant specific kinesins and studied their biochemical characterization. The plant kinesins showed very unique properties. Especially ATPase activities of the kinesins were relatively much lower than that of conventional kinesin. Moreover, the kinesins did not show motile activity on the microtubules prepared from porcine brain. Although the structure of tubulin is well conserved, it is demonstrated that the plant tubulin has different characteristics from tubulin derived from animal. Therefore, plant kinesin may be more compatible with plant tubulin than animal tubulin.In this study, we prepared the plant tubulin from the pollen of Ginkgo biloba. Native tubulin was prepared from ground pollen by acetone treatment methods and purified with DEAE, Mono Q column and Gel filtration chromatography. We have also cloned cDNA of the α/β tubulin from the Ginkgo biloba leaves by RT-PCR. The recombinant α/β tubulins were expressed by E. coli. bacterial expression system. The recombinant tubulins were purified by Co2+ chelating column.Polymerization of the purified plant tubulin to microtubule was monitored by measuring the increase of absorption at 350 nm. Negative staining electron microscopic analysis also revealed the microtubule configuration. ATPase activity of the Rice plant kinesin, K16 was activated by plant tubulin more significantly than that of animal tubulin. These results suggested that plant kinesin is more compatible with plant kinesins than kinesin derived from animal.

The novel functions of kinesin motor proteins in plants

Kinesin superfamily proteins are important microtubule-based motor proteins with a kinesin motor domain that is conserved among all eukaryotic organisms. They are responsible for unidirectionally transporting various cargos, including membranous organelles, protein complexes, and mRNAs. They also play critical roles in mitosis, morphogenesis, and signal transduction. Most kinesins in plants are evolutionarily divergent from their counterparts in animals and fungi. The mitotic kinesins in the plant kinesin-5 and kinesin-14 subfamilies appear to be similar to those in fungi and animals. However, others with nonmotor sequences are unique to plants. The kinesins affect microtubule organization, organelle distribution, vesicle transport, and cellulose microfibril order. Ultimately, plant kinesins contribute directly or indirectly to cell division and cell growth in various tissues. Here, we review a novel function of kinesins with transcription activation activity in regulating gibberellin biosynthesis and cell growth. These findings will open exciting new areas of kinesin research.

Functions of the Arabidopsis kinesin superfamily of microtubule-based motor proteins

Plants possess a large number of microtubule-based kinesin motor proteins. While the kinesin-2, 3, 9, and 11 families are absent from land plants, the kinesin-7 and 14 families are greatly expanded. In addition, some kinesins are specifically present only in land plants. The distinctive inventory of plant kinesins suggests that kinesins have evolved to perform specialized functions in plants. Plants assemble unique microtubule arrays during their cell cycle, including the interphase cortical microtubule array, preprophase band, anastral spindle and phragmoplast. In this review, we explore the functions of plant kinesins from a microtubule array viewpoint, focusing mainly on Arabidopsis kinesins. We emphasize the conserved and novel functions of plant kinesins in the organization and function of the different microtubule arrays.

Preparation and Polymerization of Tubulin of Ginkgo Biloba and its Interaction with Rice Plant Kinesin

Previously, we have expressed novel rice plant specific kinesins and studied their biochemical characterization. The plant kinesins showed very unique properties. Especially ATPase activities of the kinesins were relatively much lower than that of conventional kinesin. Moreover, the kinesins did not show motile activity on the microtubules prepared from porcine brain. Although the structure of tubulin is well conserved, it is demonstrated that the plant tubulin has different characteristics from tubulin derived from animal.

A kinesin with calponin-homology domain is involved in premitotic nuclear migration

Interaction and cross-talk between microtubules and actin microfilaments are important for numerous processes during plant growth and development, including the control of cell elongation and tissue expansion, but little is known about the molecular components of this interaction. Plant kinesins with the calponin-homology domain (KCH) were recently identified and associated with a putative role in microtubule-microfilament cross-linking. The putative biological role of the rice KCH member OsKCH1 is addressed here using a combined approach with Tos17 kch1 knock-out mutants on the one hand, and a KCH1 overexpression line generated in tobacco BY-2 cells. It is shown that OsKCH1 is expressed in a development and tissue-specific manner in rice and antagonistic cell elongation and division phenotypes as a result of knock-down and overexpression are reported. Further, the dynamic repartitioning of OsKCH1 during the cell cycle is described and it is demonstrated that KCH overexpression delays nuclear positioning and mitosis in BY-2 cells. These findings are discussed with respect to a putative role of KCHs as linkers between actin filaments and microtubules during nuclear positioning.

Dynamic Bridges—A Calponin-Domain Kinesin From Rice Links Actin Filaments and Microtubules in Both Cycling and Non-Cycling Cells

Interaction and cross-talk between microtubules and actin microfilaments are important for the cell axis and polarity during plant cell growth and development, but little is known about the molecular components of this interaction. Plant kinesins with a calponin-homology domain (KCHs) were recently identified and associated with a putative role in microtubule-microfilament cross-linking. KCHs belong to a distinct branch of the minus end-directed kinesin subfamily and so far have only been identified in land plants including the mosses. Here we report the identification of a new KCH from rice (Oryza sativa), OsKCH1, and show that OsKCH1 is associated with cortical microtubules and actin microfilaments in vivo. Furthermore, OsKCH1 is shown to bind to micro-tubules and actin microfilaments in vitro in a domain-dependent way. Additionally, this unique type of kinesin is shown to oligomerize both in vivo and in vitro. These findings are discussed with respect to a general role for KCHs as linkers between actin filaments and microtubules in both cell elongation and division.

Cotton GhKCH2, a Plant-specific Kinesin, is Low-affinitive and Nucleotide-independent as Binding to Microtubule

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, M396-734 corresponding to amino acids Q396-N734 was fused with 6xHis-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of M396-734 is not activated by Ca2+, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 microM, much higher than that of other kinesins reported. The cosedimentation assay indicated that M396-734 could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

Preparation and Characterization of a Novel Rice Plant–Specific Kinesin

Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. The kinesin family is classified into subfamilies. Kinesin species derived from vertebrates have been well characterized. In contrast, plant kinesins have yet to be adequately characterized. In this study, we expressed the motor domain of a novel rice plant-specific kinesin, K16, in Escherichia coli, and then determined its enzymatic characteristics and compared them with those of kinesin 1. Our findings demonstrated that the rice kinesin motor domain has different enzymatic properties from those of well known kinesin 1.

Cytoskeletal Motors in Arabidopsis. Sixty-One Kinesins and Seventeen Myosins

Cytoskeletal motor proteins are ATPases that use the energy released from ATP hydrolysis to move along the cytoskeletal elements of microtubules and actin microfilaments. Found among all eukaryotic organisms, kinesins are microtubule-based motor proteins with a conserved kinesin motor domain, and myosins are actin microfilament-based motor proteins with a conserved myosin motor domain. Cytoskeletal motor proteins directly contribute to the organization of various cytoskeletal arrays during cell division and cell growth in plant tissues. They are also responsible for the motility of molecules and organelles, and the segregation of genetic materials during mitosis and meiosis. In the genome of the model plant Arabidopsis (Arabidopsis thaliana), there are at least 61 genes encoding kinesins and 17 genes encoding myosins. Most Arabidopsis kinesins and all myosins are evolutionarily divergent from their counterparts in animals and fungi. Little is known about the functions of most plant kinesins and myosins. Arabidopsis kinesins form a number of subfamilies. The mitotic kinesins in the BIMC/Kinesin-5 and the NCD/Kinesin-14 subfamilies appear to be similar to those in fungi and animals. Others, however, are very divergent, as their nonmotor sequences are unique to plants. Some of Arabidopsis kinesins are associated with microtubules, mitochondria, Golgi stacks, and vesicles. They affect microtubule organization, organelle distribution, and vesicle transport, respectively. Ultimately, Arabidopsis kinesins contribute directly or indirectly to cell division and cell growth in various tissues. Arabidopsis myosins are classified into two subfamilies: class VIII and class XI. The class XI myosins are associated with various organelles/vesicles. Functions of Arabidopsis myosins are still elusive. Future efforts will be devoted to deciphering not only the functions of these motors by molecular genetics but also the molecular mechanisms underlying how these roles are played.

An Internal Motor Kinesin Is Associated with the Golgi Apparatus and Plays a Role in Trichome Morphogenesis inArabidopsis

Members of the kinesin superfamily are microtubule-based motor proteins that transport molecules/organelles along microtubules. We have identified similar internal motor kinesins, Kinesin-13A, from the cotton Gossypium hirsutum and Arabidopsis thaliana. Their motor domains share high degree of similarity with those of internal motor kinesins of animals and protists in the MCAK/Kinesin13 subfamily. However, no significant sequence similarities were detected in sequences outside the motor domain. In Arabidopsis plants carrying the T-DNA knockout kinesin-13a-1 and kinesin-13a-2 mutations at the Kinesin-13A locus, >70% leaf trichomes had four branches, whereas wild-type trichomes had three. Immunofluorescent results showed that AtKinesin-13A and GhKinesin-13A localized to entire Golgi stacks. In both wild-type and kinesin-13a mutant cells, the Golgi stacks were frequently associated with microtubules and with actin microfilaments. Aggregation/clustering of Golgi stacks was often observed in the kinesin-13a mutant trichomes and other epidermal cells. This suggested that the distribution of the Golgi apparatus in cell cortex might require microtubules and Kinesin-13A, and the organization of Golgi stacks could play a regulatory role in trichome morphogenesis. Our results also indicate that plant kinesins in the MCAK/Kinesin-13 subfamily have evolved to take on different tasks than their animal counterparts.

TETRASPORE encodes a kinesin required for male meiotic cytokinesis in Arabidopsis.

A key step in pollen formation is the segregation of the products of male meiosis into a tetrad of microspores, each of which develops into a pollen grain. Separation of microspores does not occur in tetraspore (tes) mutants of Arabidopsis thaliana, owing to the failure of male meiotic cytokinesis. tes mutants thus generate large 'tetraspores' containing all the products of a single meiosis. Here, we report the positional cloning of the TES locus and details of the role played by the TES product in male cytokinesis. The predicted TES protein includes an N-terminal domain homologous to kinesin motors and a C-terminus with little similarity to other proteins except for a small number of plant kinesins. These include the Arabidopsis HINKEL protein and NACK1 and two from tobacco (Nishihama et al., 2002), which are involved in microtubule organization during mitotic cytokinesis. Immunocytochemistry shows that the characteristic radial arrays of microtubules associated with male meiotic cytokinesis fail to form in tes mutants. The TES protein therefore is likely to function as a microtubule-associated motor, playing a part either in the formation of the radial arrays that establish spore domains following meiosis, or in maintaining their stability.