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 (TM) 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

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

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

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

TPX2-LIKE PROTEIN 3 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 thaliana, these are termed α and β 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 PROTEINS 2 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 non-redundantly 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

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 offsprin

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

Comparison of echocardiography with tissue Doppler imaging and magnetic resonance imaging with delayed enhancement in the assessment of children with hypertrophic cardiomyopathy

Introduction : In children with hypertrophic cardiomyopathy (HCM) there often occurs a non-ischemic pattern of myocardial fibrosis, which could be the cause of impaired left ventricular (LV) diastolic function assessed by tissue Doppler imaging (TDI). The aim of the study was to determine the prevalence of myocardial fibrosis in children with HCM, and to evaluate its relationship with echocardiographic parameters including LV diastolic dysfunction. Material and methods : Sixty-three children with HCM, mean age 12.2 ±4.5 years, underwent magnetic resonance imaging (MRI) and echocardiographic study from January 2010 to April 2014. The results of MRI, echocardiography, and TDI velocities were analyzed and compared between children with and without myocardial fibrosis. Moreover, correlations between the results of echocardiography and MRI were assessed. Results : Our results showed a significant correlation between magnetic resonance and echocardiographic measurements of septal wall thickness, posterior wall thickness, LV mass and left atrial dimension. Children with myocardial fibrosis (60%) had a significantly thicker interventricular septum (21.3 vs. 1.8 mm; p < 0.0001) and larger left atrial dimension (36.7 vs. 27.8 mm; p = 0.0004) and volume index (42.0 vs. 26.6 ml/m²; p = 0.0011). Tissue Doppler imaging demonstrated significantly decreased lateral E’ (9.02 vs. 13.53 cm/s; p < 0.0001) and septal E (7.05 vs. 9.36 cm/s; p = 0.0082) velocities and a significantly increased transmitral lateral (10.34 vs. 6.68, p = 0.0091) and septal (13.1 vs. 9.8; p = 0.046) E/E ratio in children with myocardial fibrosis. Conclusions : Myocardial fibrosis in children with hypertrophic cardiomyopathy was associated with markers for disease severity such as larger septum thickness, enlargement of the left atrium as well as impairment of left ventricular diastolic function. Tissue Doppler imaging is a helpful tool to detect the presence of left ventricular diastolic dysfunction in children with hypertrophic cardiomyopathy and myocardial fibrosis