Matching Patterns of Gene Expression to Mechanical Stiffness at Cell Resolution through Quantitative Tandem Epifluorescence and Nanoindentation

Cell differentiation has been associated with changes in mechanical stiffness in single-cell systems, yet it is unknown whether this association remains true in a multicellular context, particularly in developing tissues. In order to address such questions, we have developed a methodology, termed quantitative tandem epifluorescence and nanoindentation, wherein we sequentially determine cellular genetic identity with confocal microscopy and mechanical properties with atomic force microscopy. We have applied this approach to examine cellular stiffness at the shoot apices of Arabidopsis (Arabidopsis thaliana) plants carrying a fluorescent reporter for the CLAVATA3 (CLV3) gene, which encodes a secreted glycopeptide involved in the regulation of the centrally located stem cell zone in inflorescence and floral meristems. We found that these CLV3-expressing cells are characterized by an enhanced stiffness. Additionally, by tracking cells in young flowers before and after the onset of GREEN FLUORESCENT PROTEIN expression, we observed that an increase in stiffness coincides with this onset. This work illustrates how quantitative tandem epifluorescence and nanoindentation can reveal the spatial and temporal dynamics of both gene expression and cell mechanics at the shoot apex and, by extension, in the epidermis of any thick tissue.Morphogenesis is a complex process that results from the coordinated actions of many genes and gene products across developing tissues and organs. Because shape is a function of the structural elements of cells, the molecular and genetic control of growth and morphogenesis must rely on the regulation of the mechanics of these elements. In this context, cell differentiation has been linked with mechanical stiffness in animal single-cell systems (Collinsworth et al., 2002; Balland et al., 2006; Engler et al., 2006; Darling et al., 2008), although the direct measurement of cell mechanics in growing animal tissues remains elusive (Blanchard and Adams, 2011; Davidson, 2011).In plants, growth involves a delicate mechanical balance: it is powered by turgor pressure and contained by cell wall stiffness (Cosgrove, 1986). Several groups have recently achieved mechanical measurements made at a subcellular resolution in plants (Milani et al., 2011; Peaucelle et al., 2011; Fernandes et al., 2012; Radotić et al., 2012; Routier-Kierzkowska et al., 2012) using scaled-down indentation methods (Geitmann, 2006; Hayot et al., 2012; Milani et al., 2013; Routier-Kierzkowska and Smith, 2013), wherein one quantifies the force needed to push down on a sample to a prescribed depth. These studies have revealed spatiotemporal patterns of stiffness, notably in tissues (Milani et al., 2011; Peaucelle et al., 2011; Fernandes et al., 2012; Routier-Kierzkowska et al., 2012).However, these measurements have not been associated directly with cell identity. This association would become feasible if mechanical measurements were combined with optical imaging of fluorescent reporters. Such a combination, termed nanoindentation coupled to inverted optical microscopy, has already been developed for single animal cells and for thin plant tissues, (Rotsch and Radmacher, 2000; Routier-Kierzkowska and Smith, 2014), but it cannot be extended to thick tissues because they are opaque, making it impossible to simultaneously observe the tissue surface optically with an inverted microscope and probe it mechanically. To circumvent this difficulty, we have developed a methodology involving the use of three microscopes to image the same sample: (1) an atomic force microscope (AFM), which is a nanoindentation system for obtaining stiffness maps of the surface of a sample; (2) an AFM-coupled upright epifluorescence macroscope to precisely identify the points to be probed; and (3) a confocal microscope to determine cell fate at cellular resolution, which may in turn be correlated with the stiffness maps. We call this methodology quantitative tandem epifluorescence and nanoindentation (qTEN), and we use it to probe the shoot apical meristem (SAM) of Arabidopsis (Arabidopsis thaliana), which is a good model system in which to investigate morphogenesis.The SAM is located at the growing tip of the shoot and consists of distinct functional zones (Ha et al., 2010). One of these zones is the slow-dividing central zone (CZ), which can be defined by the expression of the CLAVATA3 (CLV3) signaling glycopeptide. Through cell division, cells exit the CZ into the surrounding peripheral zone (PZ). In the PZ, cells proliferate rapidly, and some become incorporated into organ primordia, thus yielding all aerial organs of the plant. Recent work on the SAM has revealed patterns of mechanical properties (Milani et al., 2011; Peaucelle et al., 2011; Kierzkowski et al., 2012; Braybrook and Peaucelle, 2013), but it is still unclear how these patterns are related to the activity of the SAM or to its functional zonation. Here, we analyze the dynamics of such a mechanical pattern in vivo and show that it is spatially and temporally related to stem cell fate.     

Molecular phylogeny and ecogeography of Onobrychis viciifolia Scop. (Fabaceae) based on nrDNA ITS sequences and genomic ISSR fingerprinting

In this study 40 specimens from 8 representative Iranian populations of Onobrychis viciifolia SCOP. were collected from their natural habitats. The specimens were biometrically assessed using 43 quantitative and 15 qualitative morphological characters. At each sample site we recorded ecogeographical variables. In order to evaluate the difference between the variation due to phenotypic responses and genetic adaptation, we generated nucleotide sequence data from the internal transcribed spacer of the nuclear rDNA genes (ITS) and carried out genomic fingerprints using inter‐simple sequence repeat PCR (ISSR). Cluster analysis of morphological characters divided the eight populations into three major groups. Leaf length, leaflet length and width, calyx length, plant height, and stem length were introduced as diagnostic characters for three different morphological groups. Furthermore, canonical correspondence analysis (CCA) of ecogeographical data showed correlations between morphological variations and ecogeographical factors. Clay%, saturation percentage (SP), total neutralizing value (TNV%), texture, minimum temperature, and geographic separation were the main environmental variables associated with morphological groups of O. viciifolia. ISSR analysis revealed three main groups which are in correspondence with morphological groups, indicating that the three morphological groups have been shaped by genetic and phenotypic responses to environmental conditions. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Use of mitochondrial and nuclear genes to infer the origin of two endemic pigeons from the Canary Islands

DNA nucleotide sequences from two mitochondrial genes (cytochrome b and NADH dehydrogenase subunit 2) and the nuclear intron 7 of β-fibrinogen were obtained to infer the phylogenetic origin of the two endemic Canarian pigeons: Bolle’s Pigeon (Columba bollii) and Laurel Pigeon (C. junoniae). Phylogenetic analyses of mitochondrial and nuclear genes based on maximum parsimony, maximum likelihood and Bayesian inference all converged into a congruent topology: C. bollii clusters together with the Wood Pigeon (C. palumbus) which is common in Europe and Asia, while C. junoniae was found near the base of the clade that includes other species of the genus Columba from the Old World. Laurel Pigeon probably represents an old lineage that might have colonized the Canary Islands a long time ago (20 My) while Bolle’s Pigeon might have arrived on the archipelago much later during the Upper Miocene (5 My). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Leaf herbivory and nutrients increase nectar alkaloids

Correlations between traits may constrain ecological and evolutionary responses to multispecies interactions. Many plants produce defensive compounds in nectar and leaves that could influence interactions with pollinators and herbivores, but the relationship between nectar and leaf defences is entirely unexplored. Correlations between leaf and nectar traits may be mediated by resources and prior damage. We determined the effect of nutrients and leaf herbivory by Manduca sexta on Nicotiana tabacum nectar and leaf alkaloids, floral traits and moth oviposition. We found a positive phenotypic correlation between nectar and leaf alkaloids. Herbivory induced alkaloids in nectar but not in leaves, while nutrients increased alkaloids in both tissues. Moths laid the most eggs on damaged, fertilized plants, suggesting a preference for high alkaloids. Induced nectar alkaloids via leaf herbivory indicate that species interactions involving leaf and floral tissues are linked and should not be treated as independent phenomena in plant ecology or evolution.     

In vitro organogenesis and alkaloid accumulation in Datura innoxia

The kinetics of tropane alkaloids accumulation in different organs such as roots, leaves, stems, flowers and seeds of Datura innoxia was investigated by GC-MS. Twenty-six tropane alkaloids were detected. The ester derivatives of tropine (3alpha-tigloyloxytropine and 3-tigloyloxy-6-hydroxytropine) are the major compounds. Undifferentiated callus were established from the stem explants of Datura innoxia using Murashige and Skoog (MS) medium supplied with 6-benzylaminopurine (BA, 1 mg l(-10) and indole-3-acetic acid (IAA, 0.5 mg l(-1)) in combination for 6 weeks. Callus differentiation was initiated by subculture onto solid MS medium, free from hormones, for more than 10 months. Initially, shoots were formed after four weeks from subculture. Further subculturing in basal MS medium without growth regulators initiated the rooting of a shooty callus after 6 weeks. Investigation of the alkaloid content of the unorganized and organized callus revealed that callus (either green or brown) yielded only trace amounts of alkaloids. On the other hand, re-differentiated shoots contained mainly scopolamine while re-differentiated roots biosynthesized hyoscyamine as the main alkaloid.     

Sequential protein recruitment in C. elegans centriole formation

Formation of the microtubule-based centriole is a poorly understood process that is crucial for duplication of the centrosome, the principal microtubule-organizing center of animal cells . Five proteins have been identified as being essential for centriole formation in Caenorhabditis elegans: the kinase ZYG-1, as well as the coiled-coil proteins SAS-4, SAS-5, SAS-6, and SPD-2 . The relationship between these proteins is incompletely understood, limiting understanding of how they contribute to centriole formation. In this study, we established the order in which these five proteins are recruited to centrioles, and we conducted molecular epistasis experiments expanding on earlier work. We find that SPD-2 is loaded first and is needed for the centriolar localization of the four other proteins. ZYG-1 recruitment is required thereafter for the remaining three proteins to localize to centrioles. SAS-5 and SAS-6 are recruited next and are needed for the presence of SAS-4, which is incorporated last. Our results indicate in addition that the presence of SAS-5 and SAS-6 allows diminution of centriolar ZYG-1. Moreover, astral microtubules appear dispensable for the centriolar recruitment of all five proteins. Several of these proteins have homologs in other metazoans, and we expect the assembly pathway that stems from our work to be conserved.     

Muc4-ErbB2 complex formation and signaling in polarized CACO-2 epithelial cells indicate that Muc4 acts as an unorthodox ligand for ErbB2

Muc4 serves as an intramembrane ligand for the receptor tyrosine kinase ErbB2. The time to complex formation and the stoichiometry of the complex were determined to be <15 min and 1:1 by analyses of Muc4 and ErbB2 coexpressed in insect cells and A375 tumor cells. In polarized CACO-2 cells, Muc4 expression causes relocalization of ErbB2, but not its heterodimerization partner ErbB3, to the apical cell surface, effectively segregating the two receptors. The apically located ErbB2 is phosphorylated on tyrosines 1139 and 1248. The phosphorylated ErbB2 in CACO-2 cells recruits the cytoplasmic adaptor protein Grb2, consistent with previous studies showing phosphotyrosine 1139 to be a Grb2 binding site. To address the issue of downstream signaling from apical ErbB2, we analyzed the three MAPK pathways of mammalian cells, Erk, p38, and JNK. Consistent with the more differentiated phenotype of the CACO-2 cells, p38 phosphorylation was robustly increased by Muc4 expression, with a consequent activation of Akt. In contrast, Erk and JNK phosphorylation was not changed. The ability of Muc4 to segregate ErbB2 and other ErbB receptors and to alter downstream signaling cascades in polarized epithelial cells suggests that it has a role in regulating ErbB2 in differentiated epithelia.     

Phylogenetic relationships based on two mitochondrial genes and hybridization patterns in Anatidae

We produced DNA sequence data from two mitochondrial genes (cytochrome b and the NADH dehydrogenase subunit 2) to reconstruct the phylogenetic relationships among 121 species of the Anseriformes (waterfowls including ducks, geese, swans, the magpie goose and screamers). Phylogenetic analyses converged into a congruent topology and defined several well-supported clades. We calibrated a molecular clock and reconstructed ancestral biogeographical areas using Bayesian inference supporting an austral continental (Gondwanaland) origin of the waterfowls. Ducks, swans and geese might have diversified during the Miocene (23–5 Myr ago) reaching northern distributions in Holarctic and Afrotropical regions. The evolution of hybridization patterns in Anseriformes has been investigated using a cladistic analysis (morphology), which may underestimate or overestimate the phylogenetic divergence among species, or restricted only to ducks. Using a phylogenetic framework, genetic-based distances and a Bayesian time calibration, our data support the hypothesis based on immunological distances of slow rate of appearance of reproductive incompatibilities in waterfowls compared with other vertebrates and the view that these birds may be like frogs in having lost their interspecific hybridization potential more slowly than mammals.