Biology subjects

Bouchez, D.

Publications and source records attributed to Bouchez, D..

7 recordsLinked to original sources

bioRxiv2026

The PP2A phosphatase associates with the Arabidopsis TRAPPII tethering complex and dephosphorylates a TRAPPII-derived phosphopeptide in vitro

The transport protein particle II (TRAPPII) complex is a conserved regulator of post-Golgi membrane trafficking. In Arabidopsis, phosphorylation of the TRAPPII-specific subunit TRS120 by SHAGGY-like kinases modulates adaptive growth responses, but the phosphatases that reverse this phosphorylation remain unknown. Here, proteomic analyses identified subunits of Protein Phosphatase 2A (PP2A) in the TRAPPII interactome. PP2A subunits physically associated with TRAPPII, and double-mutant analyses revealed genetic interactions between PP2A and TRAPPII. Loss of TRAPPII function reduced the relative membrane association of PP2A scaffolding subunits. We established an in vitro assay for Arabidopsis PP2A holoenzyme activity using complexes transiently co-expressed and affinity-purified from Nicotiana benthamiana. Structural modelling and interface analysis predicted binding of a phosphorylated peptide encompassing a TRS120 phosphosite cluster at the PP2A catalytic interface, while biochemical assays showed that a PP2A holoenzyme containing the B2 regulatory subunit dephosphorylated this peptide. Together, these findings identify a B2-containing PP2A holoenzyme as a candidate phosphatase for TRS120 and support a model in which antagonistic SHAGGY-like kinase and PP2A activities couple signalling to membrane trafficking during plant development and environmental adaptation.

plant biology↗

bioRxiv2026

A triple fluorescent marker for live imaging of plant cell morphogenesis

Live imaging of plant subcellular structures is key to deciphering the spatiotemporal bases of cellular processes, and their functional impact on growth and morphogenesis at various biological scales. Live imaging of plant cells essentially relies on expression of fluorescent markers labeling cells or subcellular structures of interest. Simultaneous multi-channel imaging of several markers is still not routine practice in plant cell biology, owing to issues linked to genetic or spectral compatibility of markers, differences in expression levels, silencing, toxicity, etc. Here we designed a three-color marker in Arabidopsis thaliana and Capsella rubella, enabling high-resolution live imaging of plant morphogenesis, including labeling of the cell membrane, the nucleus and the microtubule cytoskeleton. Detection of MT arrays involved the development of a MAP4-MBD-based microtubule marker optimized for plant cells. The three-color marker allows visualization of the three-dimensional organization and dynamics of plant microtubules within the intracellular space with unprecedented precision, in various organs including the root and shoot meristems, the leaf, anther, and gynoecium. Our results demonstrate the potential of such single-construct strategy for cell biology studies in plants.

plant biology↗

bioRxiv2025

Actin Counters Geometry to Guide Plant Cell Division

In multicellular organisms, cell division shapes tissue architecture, cell identity, and function. In walled organisms like plants, division plane orientation irreversibly defines tissue topology and is tightly regulated. While divisions often follow the shortest path enclosing cell volume, certain cells deviate, dividing perpendicular to the growth axis. Tissue-scale mechanical stress has been proposed to guide such orientation, but how these cues are integrated remains unclear. Here, we re-examine the role of the actin cytoskeleton in orienting cell division in Arabidopsis root epidermis. Combining cell biology, genetics, pharmacological treatments, 3D segmentation, and modelling, we show that actin is a central molecular actor required to establish cell division orientation against the geometrical rules, highlighting its role in integrating spatial information.

plant biology↗

bioRxiv2025

Semi-automatic quantification of 3D Histone H3 phosphorylation signals during cell division in Arabidopsis root meristems

O_LIPosttranslational modification of histones during the cell cycle is a major process controlling many aspects of cell division. Among the variety of histone modifications, mitotic phosphorylation of histone H3 at serine 10 (H3S10ph) plays a crucial role, particularly in proper chromosome segregation. Here we aimed at precisely quantifying this phosphorylation dynamics during mitosis in plant cells, in order to reveal molecular pathways involved in this process. C_LIO_LIWe describe an analysis pipeline based on 3D image analysis that allows to semi-automatically quantify H3S10 phosphorylation in mitotic Arabidopsis root cells. We also developed a new method for the compensation of signal attenuation in Z, based on measurement of objects of interest themselves. C_LIO_LIWe show that this new attenuation correction method allows significant gains in accuracy and statistical power. Using this pipeline, we were able to reveal small H3S10ph differences between cells treated with hesperadin, an inhibitor of an H3S10ph kinase, or between Arabidopsis mutants affected in PP2A phosphatase activity. C_LIO_LIThis tool opens new avenues to explore such regulation pathway in plants, using the wealth of genetic material available in Arabidopsis. It can also be applied to study other histone post-translational modifications, and more generally to any discrete 3D signals. C_LI

cell biology↗

bioRxiv2023

The cell cycle controls spindle architecture in Arabidopsis by modulating the augmin pathway

To ensure an even segregation of chromosomes during somatic cell division, eukaryotes rely on specific microtubule structures called mitotic spindles. There are, however, striking differences in overall spindle organization among eukaryotic super groups, and in particular little is known about how spindle architecture is determined in plants. As a foundation for our work, we have measured prime characteristics of Arabidopsis mitotic spindles and built a three-dimensional dynamic model of the Arabidopsis mitotic spindle using Cytosim. Next, we identified the cell-cycle regulator CYCLIN-DEPENDENT KINASE B1 (CDKB1) together with its cyclin partner CYCB3;1 as key regulators of spindle shape and organization in Arabidopsis. Loss of CDKB1 function resulted in a high number of astral microtubules that are normally absent from plant spindles, as opposed to animal ones. We identified an augmin complex member, ENDOSPERM DEFECTIVE1 (EDE1), as a substrate of the CDKB1;1-CYCB3;1 complex. A non-phosphorylatable mutant of EDE1 displayed spindles with extended pole-to-pole distance, resembling the phenotypes of cycb3;1 and cdkb1 mutants. Moreover, we found that the mutated EDE1 version associated less efficiently with spindle microtubules. Consistently, reducing the level of augmin in Cytosim simulations largely recapitulated the spindle phenotypes observed in cycb3;1 and cdkb1 mutants. Our results emphasize the importance of cell cycle-dependent phospho-control of the mitotic spindle in plant cells. They also support the validity of our computational model as a framework for the exploration of mechanisms controlling the organization of the spindle in plants and in other species.

developmental biology↗

bioRxiv2022

The phosphoinositide signature guides the final step of plant cytokinesis

Plant cytokinesis, which fundamentally differs from that in animals, involves de novo assembly of a plasma membrane precursor named the cell plate. How the transition from the cell plate to a plasma membrane occurs at the end of the plant cytokinesis remains poorly understood. Here, we describe with unprecedented spatiotemporal precision, the acquisition of plasma membrane identity upon cytokinesis through the lateral patterning of phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 at the newly formed cell plate membrane. We show that during late cytokinesis, opposing polarity domains are formed along the cell plate. Exclusion of PI(4,5)P2 from the leading edge of the cell plate is controlled by SAC9, a putative phosphoinositide phosphatase. SAC9 colocalizes with MAP65-3, a key regulator of the cytokinesis, at the cell plate leading zone and regulates its function. In the sac9-3 mutant, the polar distribution of PI(4,5)P2 at the cell plate is altered, leading to de-novo recruitment of the cytokinesis apparatus and to formation of an additional, ectopic cell plate insertion site. We proposed that PI(4,5)P2 acts as a polar cue to spatially separate the expansion and maturation domains of the forming cell plate during the final steps of cytokinesis. One Sentence SummaryThe phosphoinositide PI(4,5)P2 acts as an hallmark to guide the final step of plant cell division.

cell biology↗

bioRxiv2021

B1-type cyclins control microtubule organization during cell division in Arabidopsis

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support an important role in the regulation of GIP1 by CYCB1s.

cell biology↗