Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes

Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors--and how this cross talk influences physiological processes--is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein-mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein-mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors

Cell wall mediated regulation of plant cell morphogenesis : pectin esterification and cellulose crystallinity

La morphogenèse cellulaire est une composante fondamentale du développement d’un organisme. Toute cellule végétale est entourée de parois régulant sa morphogenèse. Cette matrice extra-cellulaire est principalement composée de polysaccharides. Afin de montrer le lien entre la forme et la fonction d’une cellule il est primordial de comprendre la façon dont ces polysaccharides sont modifiés durant le développement et l’expansion cellulaire. Chez les plantes, la mécanique de l'expansion cellulaire est principalement régulée par la cellulose, le biopolymère le plus abondant sur Terre. Comme les microfibrilles de cellulose présentent une forte résistance à la traction le long de leur axe d’orientation principal, elles réguleraient le processus d'expansion en conférant des propriétés mécaniques aux composants pariétaux qui contrôlent l’ampleur et la directivité de la croissance expansive au niveau subcellulaire. Les homogalacturonanes, type de pectine le plus abondant des parois cellulaires primaires, sont des biopolymères également susceptibles d’agir sur l’expansion cellulaire. La distribution spatiale et le degré d’estérification des pectines homogalacturonanes affectent les propriétés mécaniques de la paroi et par conséquent le pattern d’expansion. J'ai utilisé une approche génétique combinée à des stratégies novatrices de biochimie, de biomécanique et d’imagerie, afin de comprendre comment la dynamique spatio-temporelle de la cellulose et des homogalacturonanes régule l'expansion et la morphogenèse cellulaires. Pour ce faire, je me suis basé sur l’étude de deux types de cellules épidermiques de formes différentes: celles du cotylédon et de l'hypocotyle d'Arabidopsis thaliana. J’ai prouvé que la formation des ondulations des cellules fondamentales du cotylédon nécessite des modifications spatiales et temporelles des microfibrilles de cellulose et des pectines déméthylestérifiées. Ces modifications régulent la rigidité mécanique de la paroi péricline à deux moments distincts : lors de l’initiation de la formation du lobe et lors de son expansion ultérieure. L’initiation de la formation du lobe requiert une augmentation de la rigidité de la paroi péricline au niveau des potentielles saillies de l’ondulation, et ce par une accumulation locale de pectines déméthylestérifiées. L’expansion ultérieure est quant à elle contrôlée par le degré de cristallinité de la cellulose et par l’alignement perpendiculaire des microfibrilles tangentiellement aux saillies de l’ondulation de la paroi péricline. Durant l'élongation et l'expansion anisotrope des cellules épidermiques de l’hypocotyle, la cellulose et les pectines homogalacturonanes jouent des rôles distincts lors de chaque phase d'élongation. Durant la première phase de développement, une réduction du taux de pectines déméthylestérifiées diminue la rigidité de la paroi et accélère l’élongation des cellules. Lors de la seconde phase du développement, une réduction de la cristallinité de la cellulose diminue la vitesse d’élongation de l’hypocotyle. À partir de l’étude des deux systèmes cellulaires, nous pouvons conclure que, contrairement à l’hypothèse acceptée de longue date, la cellulose ne serait pas un élément essentiel au déclenchement d’évènements morphogénétiques mais qu’elle jouerait plutôt un rôle au sein de mécanismes de rétroaction accentuant le processus de morphogenèse. De plus, la morphogenèse induite par des contraintes joue un rôle clé lors des étapes initiales et serait dépendante du degré d’estérification des pectines. Mes expériences permettent de corréler les données de mécanique cellulaire expérimentale à la biologie cellulaire fonctionnelle et à la génétique.Cellular morphogenesis is a fundamental underpinning of development. All cells in the plant kingdom are surrounded by walls that govern shape formation. This extracellular matrix is composed mainly of polysaccharides. How these polysaccharides are modified during cellular development to regulate cell expansion, and thus cell shape, must be understood to link form with function. In plants, the mechanical aspect of cell expansion is known to be mainly influenced by cellulose, the most abundant biopolymer on Earth. Because cellulose microfibrils exhibit a strong tensile strength along their long axis, they may be used to control the expansion process by conferring mechanical properties to the cell wall material that determine the directionality and the magnitude of expansive growth at subcellular level. Another wall polymer that may influence cell expansion is homogalacturonan pectin, the most abundant type of pectin in the primary wall. The spatial distribution and esterification status of homogalacturonan pectin may affect the mechanical aspects of the wall and, therefore, the expansion pattern. I used a genetic approach combined with novel biochemical, biomechanical and imaging strategies to study the impact of the spatio-temporal dynamics of cellulose and homogalacturonan pectin during cell expansion and shape formation. I investigated cell shape formation in two differently shaped types of epidermal cells: those of the cotyledon and of the hypocotyl of Arabidopsis thaliana. I show that undulation formation in pavement cells of the cotyledon requires spatial and temporal changes of cellulose microfibrils and demethyl-esterified pectin. These changes regulate the mechanical stiffness of the periclinal wall at two different stages: lobe initiation and subsequent expansion. Lobe initiation involves an increase in the stiffness of the periclinal wall at the prospective neck region of the undulation through a local accumulation of demethyl-esterified pectin. The subsequent expansion is controlled by the degree of cellulose crystallinity and the perpendicular alignment of the microfibrils at the tangent of the neck side of the undulation at the periclinal wall. During the elongation process and the anisotropic expansion of the epidermal hypocotyl cells, cellulose and homogalacturonan pectin make distinct contributions in each developmental phase of the elongation. During the first developmental phase, reduction in the proportion of demethyl-esterified pectin decreases the wall stiffness and accelerates the elongation. A reduction in the cellulose crystallinity decreases the elongation of the hypocotyl at the second developmental phase. It may be concluded from the two cell systems that cellulose, contrary to a long-established hypothesis, may not be essential for the initiation of morphogenetic events and their function may be reassigned to the feedback-mediated augmentation of cell shaping processes. Moreover, stress-induced shape formation plays a key role during the initiating steps and it is likely to be dominated by the degree of pectin esterification. My data link experimental cell mechanics to functional cell biology and genetics

Exploiting artistic cues to obtain line labels for free-hand sketches

Artistic cues help designers to communicate design intent in sketches. In this paper, we show how these artistic cues may be used to obtain a line labelling interpretation of freehand sketches, using a cue-based genetic algorithm to obtain a labelling solution that matches design intent. In the paper, we show how this can be achieved from off-line or paper based sketches, thereby allowing designers greater flexibility in the choice of sketching medium.peer-reviewe

Pectin chemistry and cellulose crystallinity govern pavement cell morphogenesis in a multi-step mechanism

Author Posting. ©American Society of Plant Biologists, 2019. This article is posted here by permission of [publisher] for personal use, not for redistribution. The definitive version was published in Altartouri, B., Bidhendi, A. J., Tani, T., Suzuki, J., Conrad, C., Chebli, Y., Liu, N., Karunakaran, C., Scarcelli, G., & Geitmann, A. Pectin chemistry and cellulose crystallinity govern pavement cell morphogenesis in a multi-step mechanism. Plant Physiology, 181(1), (2019): 127-141, doi:10.1104/pp.19.00303.Simple plant cell morphologies, such as cylindrical shoot cells, are determined by the extensibility pattern of the primary cell wall, which is thought to be largely dominated by cellulose microfibrils, but the mechanism leading to more complex shapes, such as the interdigitated patterns in the epidermis of many eudicotyledon leaves, is much less well understood. Details about the manner in which cell wall polymers at the periclinal wall regulate the morphogenetic process in epidermal pavement cells and mechanistic information about the initial steps leading to the characteristic undulations in the cell borders are elusive. Here, we used genetics and recently developed cell mechanical and imaging methods to study the impact of the spatio-temporal dynamics of cellulose and homogalacturonan pectin distribution during lobe formation in the epidermal pavement cells of Arabidopsis (Arabidopsis thaliana) cotyledons. We show that nonuniform distribution of cellulose microfibrils and demethylated pectin coincides with spatial differences in cell wall stiffness but may intervene at different developmental stages. We also show that lobe period can be reduced when demethyl-esterification of pectins increases under conditions of reduced cellulose crystallinity. Our data suggest that lobe initiation involves a modulation of cell wall stiffness through local enrichment in demethylated pectin, whereas subsequent increase in lobe amplitude is mediated by the stress-induced deposition of aligned cellulose microfibrils. Our results reveal a key role of noncellulosic polymers in the biomechanical regulation of cell morphogenesis.Natural Sciences and Engineering Research Council of Canada Canada Research Chair Program Marine Biological Laboratory NIH R01GM100160 Canada Foundation for Innovation University of Saskatchewan Government of Saskatchewan Western Economic Diversification Canada National Research Council (Canada) Canadian Institutes of Health Researc

Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels

Multiplexed, real-time fluorescence detection at the single-molecule level is highly desirable to reveal the stoichiometry, dynamics, and interactions of individual molecular species within complex systems. However, traditionally fluorescence sensing is limited to 3-4 concurrently detected labels, due to low signal-to-noise, high spectral overlap between labels, and the need to avoid dissimilar dye chemistries. We have engineered a palette of several dozen fluorescent labels, called FRETfluors, for spectroscopic multiplexing at the single-molecule level. Each FRETfluor is a compact nanostructure formed from the same three chemical building blocks (DNA, Cy3, and Cy5). The composition and dye-dye geometries create a characteristic F\"orster Resonance Energy Transfer (FRET) efficiency for each construct. In addition, we varied the local DNA sequence and attachment chemistry to alter the Cy3 and Cy5 emission properties and thereby shift the emission signatures of an entire series of FRET constructs to new sectors of the multi-parameter detection space. Unique spectroscopic emission of each FRETfluor is therefore conferred by a combination of FRET and this site-specific tuning of individual fluorophore photophysics. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs statistically selected to minimize classification errors, measured using an Anti-Brownian ELectrokinetic (ABEL) trap which provides precise multi-parameter spectroscopic measurements. The ABEL trap also enables discrimination between FRETfluors attached to a target (here: mRNA) and unbound FRETfluors, eliminating the need for washes or removal of excess label by purification. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs selected to minimize classification errors.Comment: 43 pages, 6 figures, 13 Supplementary figures, 3 Supplementary tables, 5 Supplementary note

A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases