Fattaccioli, J. | bioRxiv Science

bioRxiv2019

Design of a comprehensive microfluidic and microscopic toolbox for the ultra-wide spatio-temporal study of plant protoplasts development and physiology

BackgroundOne of the main features of plant cells is their strong plasticity, and their propensity to regenerate an organism from a single cell. Plant protoplasts are basic plant cells units in which the pecto-cellulosic cell wall has been removed, but the plasma membrane is intact. One of the main features of plant cells is their strong plasticity, which in some species, can be very close from what is defined as cell totipotency. Methods and differentiation protocols used in plant physiology and plant biology usually involve macroscopic vessels and containers that make difficult, for example, to follow the fate of the same protoplast all along its full development cycle, but also to perform continuous studies of the influence of various gradients in this context. These limits have hampered the precise study of regeneration processes. ResultsHerein, we present the design of a comprehensive, physiologically relevant, easy-to-use and low-cost microfluidic and microscopic setup for the monitoring of Physcomitrella patens (P. patens) growth and development on a long-term basis. The experimental solution we developed is made of two parts (i) a microfluidic chip composed of a single layer of about a hundred flow-through microfluidic traps for the immobilization of protoplasts, and (ii) a low-cost, light-controlled, custom-made microscope allowing the continuous recording of the moss development in physiological conditions. We validated the experimental setup with three proofs of concepts: (i) the kinetic monitoring of first division steps and cell wall regeneration, (ii) the influence of the photoperiod on growth of the protonemata, and (iii) finally the induction of leafy buds using a phytohormone, cytokinin. ConclusionsWe developed the design of a comprehensive, physiologically relevant, easy-to-use and low-cost experimental setup for the study of P. patens development in a microfluidic environment. This setup allows imaging of P. patens development at high resolution and over long time periods.

plant biology↗

bioRxiv2018

A multiparametric, quantitative and high-throughput assay to quantify the influence of target size on phagocytic uptake

Phagocytosis by macrophages represents a fundamental process essential for both immunity and tissue homeostasis. It consists in the uptake of pathogenic or cellular targets larger than 0.5m. For the biggest particles, the phagocytic process involves a massive reorganization of membrane and actin cytoskeleton as well as an important intracellular deformation, all in a matter of minutes. The study of the role of the size of objects in their phagocytosis has lead to contradictory results in the last decades.\n\nWe designed a method using confocal microscopy, automated image analysis and databases for fast quantitative analysis of phagocytosis assays. It yields comprehensive data on the cells and targets geometric and fluorescence intensity parameters, automatically discriminates internalized from external targets, and stores the relationship between a cell and the targets it has engulfed. We used two types of targets, solid polystyrene beads and liquid lipid droplets, to investigate the influence of size on the phagocytic uptake of macrophages. The method made it possible, not only to perform phagocytic assays with functionalized droplets and beads of different sizes, but to use polydisperse particles to further our understanding of the role of size in phagocytosis.\n\nThe use of monodisperse and polydisperse objects shows that while smaller monodisperse objects are internalized in greater numbers, objects of different sizes presented simultaneously are internalized without preferred size. Throughout results, the total surface engulfed by the cell appeared to be the main factor limiting the uptake of particles, regardless of their nature or size. A meta-analysis of the literature reveals that this dependence in surface is consistently conserved throughout cell types, targets nature or activated receptors.

biophysics↗