TPX2-LIKE PROTEIN3 Is the Primary Activator of α-Aurora Kinases and Is Essential for Embryogenesis.

Aurora kinases are key regulators of mitosis. Multicellular eukaryotes generally possess two functionally diverged types of Aurora kinases. In plants, including Arabidopsis (Arabidopsis thaliana), these are termed α- and ß-Auroras. As the functional specification of Aurora kinases is determined by their specific interaction partners, we initiated interactomics analyses using both Arabidopsis α-Aurora kinases (AUR1 and AUR2). Proteomics results revealed that TPX2-LIKE PROTEINS2 and 3 (TPXL2/3) prominently associated with α-Auroras, as did the conserved TPX2 to a lower degree. Like TPX2, TPXL2 and TPXL3 strongly activated the AUR1 kinase but exhibited cell-cycle-dependent localization differences on microtubule arrays. The separate functions of TPX2 and TPXL2/3 were also suggested by their different influences on AUR1 localization upon ectopic expressions. Furthermore, genetic analyses showed that TPXL3, but not TPX2 and TPXL2, acts nonredundantly to enable proper embryo development. In contrast to vertebrates, plants have an expanded TPX2 family and these family members have both redundant and unique functions. Moreover, as neither TPXL2 nor TPXL3 contains the C-terminal Kinesin-5 binding domain present in the canonical TPX2, the targeting and activity of this kinesin must be organized differently in plants.

Cytoplasmic MTOCs control spindle orientation for asymmetric cell division in plants.

Proper orientation of the cell division axis is critical for asymmetric cell divisions that underpin cell differentiation. In animals, centrosomes are the dominant microtubule organizing centers (MTOC) and play a pivotal role in axis determination by orienting the mitotic spindle. In land plants that lack centrosomes, a critical role of a microtubular ring structure, the preprophase band (PPB), has been observed in this process; the PPB is required for orienting (before prophase) and guiding (in telophase) the mitotic apparatus. However, plants must possess additional mechanisms to control the division axis, as certain cell types or mutants do not form PPBs. Here, using live imaging of the gametophore of the moss Physcomitrella patens, we identified acentrosomal MTOCs, which we termed "gametosomes," appearing de novo and transiently in the prophase cytoplasm independent of PPB formation. We show that gametosomes are dispensable for spindle formation but required for metaphase spindle orientation. In some cells, gametosomes appeared reminiscent of the bipolar MT "polar cap" structure that forms transiently around the prophase nucleus in angiosperms. Specific disruption of the polar caps in tobacco cells misoriented the metaphase spindles and frequently altered the final division plane, indicating that they are functionally analogous to the gametosomes. These results suggest a broad use of transient MTOC structures as the spindle orientation machinery in plants, compensating for the evolutionary loss of centrosomes, to secure the initial orientation of the spindle in a spatial window that allows subsequent fine-tuning of the division plane axis by the guidance machinery.

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

Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1-a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants-is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases.

GAP Activity, but Not Subcellular Targeting, Is Required for Arabidopsis RanGAP Cellular and Developmental Functions.

The Ran GTPase activating protein (RanGAP) is important to Ran signaling involved in nucleocytoplasmic transport, spindle organization, and postmitotic nuclear assembly. Unlike vertebrate and yeast RanGAP, plant RanGAP has an N-terminal WPP domain, required for nuclear envelope association and several mitotic locations of Arabidopsis thaliana RanGAP1. A double null mutant of the two Arabidopsis RanGAP homologs is gametophyte lethal. Here, we created a series of mutants with various reductions in RanGAP levels by combining a RanGAP1 null allele with different RanGAP2 alleles. As RanGAP level decreases, the severity of developmental phenotypes increases, but nuclear import is unaffected. To dissect whether the GAP activity and/or the subcellular localization of RanGAP are responsible for the observed phenotypes, this series of rangap mutants were transformed with RanGAP1 variants carrying point mutations abolishing the GAP activity and/or the WPP-dependent subcellular localization. The data show that plant development is differentially affected by RanGAP mutant allele combinations of increasing severity and requires the GAP activity of RanGAP, while the subcellular positioning of RanGAP is dispensable. In addition, our results indicate that nucleocytoplasmic trafficking can tolerate both partial depletion of RanGAP and delocalization of RanGAP from the nuclear envelope.

Combined linkage and association mapping reveals CYCD5;1 as a quantitative trait gene for endoreduplication in Arabidopsis.

Endoreduplication is the process where a cell replicates its genome without mitosis and cytokinesis, often followed by cell differentiation. This alternative cell cycle results in various levels of endoploidy, reaching 4× or higher one haploid set of chromosomes. Endoreduplication is found in animals and is widespread in plants, where it plays a major role in cellular differentiation and plant development. Here, we show that variation in endoreduplication between Arabidopsis thaliana accessions Columbia-0 and Kashmir is controlled by two major quantitative trait loci, ENDO-1 and ENDO-2. A local candidate gene association analysis in a set of 87 accessions, combined with expression analysis, identified CYCD5;1 as the most likely candidate gene underlying ENDO-2, operating as a rate-determining factor of endoreduplication. In accordance, both the overexpression and silencing of CYCD5;1 were effective in changing DNA ploidy levels, confirming CYCD5;1 to be a previously undescribed quantitative trait gene underlying endoreduplication in Arabidopsis.

Ectopic expression of Kip-related proteins restrains root-knot nematode-feeding site expansion.

The development of nematode feeding sites induced by root-knot nematodes involves the synchronized activation of cell cycle processes such as acytokinetic mitoses and DNA amplification. A number of key cell cycle genes are reported to be critical for nematode feeding site development. However, it remains unknown whether plant cyclin-dependent kinase (CDK) inhibitors such as the Arabidopsis interactor/inhibitor of CDK (ICK)/Kip-related protein (KRP) family are involved in nematode feeding site development. This study demonstrates the involvement of Arabidopsis ICK2/KRP2 and ICK1/KRP1 in the control of mitosis to endoreduplication in galls induced by the root-knot nematode Meloidogyne incognita. Using ICK/KRP promoter-GUS fusions and mRNA in situ hybridizations, we showed that ICK2/KRP2, ICK3/KRP5 and ICK4/KRP6 are expressed in galls after nematode infection. Loss-of-function mutants have minor effects on gall development and nematode reproduction. Conversely, overexpression of both ICK1/KRP1 and ICK2/KRP2 impaired mitosis in giant cells and blocked neighboring cell proliferation, resulting in a drastic reduction of gall size. Studying the dynamics of protein expression demonstrated that protein levels of ICK2/KRP2 are tightly regulated during giant cell development and reliant on the presence of the nematode. This work demonstrates that impeding cell cycle progression by means of ICK1/KRP1 and ICK2/KRP2 overexpression severely restricts gall development, leading to a marked limitation of root-knot nematode development and reduced numbers of offspring.

Functional modules in the Arabidopsis core cell cycle binary protein-protein interaction network.

As in other eukaryotes, cell division in plants is highly conserved and regulated by cyclin-dependent kinases (CDKs) that are themselves predominantly regulated at the posttranscriptional level by their association with proteins such as cyclins. Although over the last years the knowledge of the plant cell cycle has considerably increased, little is known on the assembly and regulation of the different CDK complexes. To map protein-protein interactions between core cell cycle proteins of Arabidopsis thaliana, a binary protein-protein interactome network was generated using two complementary high-throughput interaction assays, yeast two-hybrid and bimolecular fluorescence complementation. Pairwise interactions among 58 core cell cycle proteins were tested, resulting in 357 interactions, of which 293 have not been reported before. Integration of the binary interaction results with cell cycle phase-dependent expression information and localization data allowed the construction of a dynamic interaction network. The obtained interaction map constitutes a framework for further in-depth analysis of the cell cycle machinery.

Systematic localization of the Arabidopsis core cell cycle proteins reveals novel cell division complexes.

Cell division depends on the correct localization of the cyclin-dependent kinases that are regulated by phosphorylation, cyclin proteolysis, and protein-protein interactions. Although immunological assays can define cell cycle protein abundance and localization, they are not suitable for detecting the dynamic rearrangements of molecular components during cell division. Here, we applied an in vivo approach to trace the subcellular localization of 60 Arabidopsis (Arabidopsis thaliana) core cell cycle proteins fused to green fluorescent proteins during cell division in tobacco (Nicotiana tabacum) and Arabidopsis. Several cell cycle proteins showed a dynamic association with mitotic structures, such as condensed chromosomes and the preprophase band in both species, suggesting a strong conservation of targeting mechanisms. Furthermore, colocalized proteins were shown to bind in vivo, strengthening their localization-function connection. Thus, we identified unknown spatiotemporal territories where functional cell cycle protein interactions are most likely to occur.

Endomembrane trafficking overarching cell plate formation.

By contrast to other eukaryotic kingdoms, plant cytokinesis is an inside-out process. A coordinated action of cytoskeletal transitions and endomembrane trafficking events builds a novel membrane compartment, the cell plate. Deposition of cell wall polymers transforms the lumen of this membrane compartment into a new cross wall, physically separating the daughter cells. The characterization of tethering complexes acting at discrete phases during cell plate formation and upstream of vesicle fusion events, the presence of modulators directing secretion and recycling during cytokinesis, as well as the identification and temporal recruitment of the endocytic machinery, provides a starting point to dissect the transitions in endomembrane trafficking which shape this process. This review aims to integrate recent findings on endomembrane trafficking events which spatio-temporally act to construct the cell plate.

Emission spectra profiling of fluorescent proteins in living plant cells.

BACKGROUND: Fluorescence imaging at high spectral resolution allows the simultaneous recording of multiple fluorophores without switching optical filters, which is especially useful for time-lapse analysis of living cells. The collected emission spectra can be used to distinguish fluorophores by a computation analysis called linear unmixing. The availability of accurate reference spectra for different fluorophores is crucial for this type of analysis. The reference spectra used by plant cell biologists are in most cases derived from the analysis of fluorescent proteins in solution or produced in animal cells, although these spectra are influenced by both the cellular environment and the components of the optical system. For instance, plant cells contain various autofluorescent compounds, such as cell wall polymers and chlorophyll, that affect the spectral detection of some fluorophores. Therefore, it is important to acquire both reference and experimental spectra under the same biological conditions and through the same imaging systems. RESULTS: Entry clones (pENTR) of fluorescent proteins (FPs) were constructed in order to create C- or N-terminal protein fusions with the MultiSite Gateway recombination technology. The emission spectra for eight FPs, fused C-terminally to the A- or B-type cyclin dependent kinases (CDKA;1 and CDKB1;1) and transiently expressed in epidermal cells of tobacco (Nicotiana benthamiana), were determined by using the Olympus FluoView™ FV1000 Confocal Laser Scanning Microscope. These experimental spectra were then used in unmixing experiments in order to separate the emission of fluorophores with overlapping spectral properties in living plant cells. CONCLUSIONS: Spectral imaging and linear unmixing have a great potential for efficient multicolor detection in living plant cells. The emission spectra for eight of the most commonly used FPs were obtained in epidermal cells of tobacco leaves and used in unmixing experiments. The generated set of FP Gateway entry vectors represents a valuable resource for plant cell biologists.

A kaleidoscopic view of the Arabidopsis core cell cycle interactome.

Although protein-protein interaction (PPI) networks have been shown to offer a systems-wide view of cellular processes, only a few plant PPI maps are available. Recently, the core cell cycle of Arabidopsis thaliana has been analyzed by three independent PPI technologies, including yeast two-hybrid systems, bimolecular fluorescence complementation and tandem affinity purification. Here, we merge the three interactomes with literature-curated and computationally predicted interactions, paving the way for a comprehensive picture of the plant core cell cycle machinery. Platform-specific interactions unveil the strengths and weaknesses of each detection method and give insights into the nature of the interactions among cell cycle proteins. Moreover, comparison of the obtained data reveals that a complete interactome can only be obtained when multiple techniques are applied in parallel.

A high-throughput bimolecular fluorescence complementation protein-protein interaction screen identifies functional Arabidopsis CDKA/B-CYCD4/5 complexes.

The eukaryotic cell cycle is a process controlled by protein assemblies, of which the key subunits are serine-threonine cyclin-dependent kinases (CDKs). Timely association and dissociation of these assemblies ensure that the cell division program is executed correctly. The challenge to unravel the rules of the plant cell cycle results from the multiplicity of the process-regulating genes that emerged through genome duplications during the evolution of flowering plants. Despite the increasing knowledge on the plant cell cycle control, little is known about the composition of the different CDK-Cyclin complexes and their spatio-temporal occurrence. The binary interactions of the previously annotated 58 Arabidopsis thaliana core cell cycle proteins were tested in two high-throughput protein-protein interaction (PPI) assays: the bimolecular fluorescence complementation (BiFC) and the yeast two-hybrid. The resulting PPI network was integrated with available cycle phase-dependent gene expression data and subcellular localization information, revealing distinct cell cycle clusters acting at different cell division stages. Additionally, the BiFC assay revealed that three D-type cyclins, CYCD4;1, CYCD4;2 and CYCD5;1, form active kinase complexes with CDKA;1 and CDKB1;1 in vivo because they induce cell divisions in differentiated tobacco (Nicotiana benthamiana) epidermal cells. We demonstrate that these complexes promote cell proliferation in Arabidopsis and we discuss their putative mode of action in plant development.

CDKB1;1 forms a functional complex with CYCA2;3 to suppress endocycle onset.

The mitosis-to-endocycle transition requires the controlled inactivation of M phase-associated cyclin-dependent kinase (CDK) activity. Previously, the B-type CDKB1;1 was identified as an important negative regulator of endocycle onset. Here, we demonstrate that CDKB1;1 copurifies and associates with the A2-type cyclin CYCA2;3. Coexpression of CYCA2;3 with CDKB1;1 triggered ectopic cell divisions and inhibited endoreduplication. Moreover, the enhanced endoreduplication phenotype observed after overexpression of a dominant-negative allele of CDKB1;1 could be partially complemented by CYCA2;3 co-overexpression, illustrating that both subunits unite in vivo to form a functional complex. CYCA2;3 protein stability was found to be controlled by CCS52A1, an activator of the anaphase-promoting complex. We conclude that CCS52A1 participates in endocycle onset by down-regulating CDKB1;1 activity through the destruction of CYCA2;3.

The PRA1 gene family in Arabidopsis.

Prenylated Rab acceptor 1 (PRA1) domain proteins are small transmembrane proteins that regulate vesicle trafficking as receptors of Rab GTPases and the vacuolar soluble N-ethylmaleimide-sensitive factor attachment receptor protein VAMP2. However, little is known about PRA1 family members in plants. Sequence analysis revealed that higher plants, compared with animals and primitive plants, possess an expanded family of PRA1 domain-containing proteins. The Arabidopsis (Arabidopsis thaliana) PRA1 (AtPRA1) proteins were found to homodimerize and heterodimerize in a manner corresponding to their phylogenetic distribution. Different AtPRA1 family members displayed distinct expression patterns, with a preference for vascular cells and expanding or developing tissues. AtPRA1 genes were significantly coexpressed with Rab GTPases and genes encoding vesicle transport proteins, suggesting an involvement in the vesicle trafficking process similar to that of their animal counterparts. Correspondingly, AtPRA1 proteins were localized in the endoplasmic reticulum, Golgi apparatus, and endosomes/prevacuolar compartments, hinting at a function in both secretory and endocytic intracellular trafficking pathways. Taken together, our data reveal a high functional diversity of AtPRA1 proteins, probably dealing with the various demands of the complex trafficking system.