The overarching aims of the proposal are subdivided into four work packages (WPs).
WP1 - In vivo cytoplasm anatomy, dynamics and rheology. To establish a detailed spatiotemporal map of the major structural cytoplasm changes during early embryogenesis, their relationship to cell cycle progression, cell size, shape or fate, I performed the following experiments. I did an in-depth analysis of the structure and organization of bulk cytoplasm from unfertilized to fertilized eggs up to the 16-cell stage, which mark the first fate commitment of vegetal blastomeres in the embryos. I used SBF-SEM imaging, to register the detailed location, density and size of all endomembranes, as well as smaller complexes like ribosomes. I complemented this analysis by using dyes, immunostaining and live imaging to visualize bulk F-actin, microtubules, yolk, mitochondria, and ER. In parallel, to provide a concomitant map of cytoplasm mechanics, we performed active micro-rheological measurements, by actuating injected magnetic beads (~1 µm) in live zygotes and blastomeres, in interphase and metaphase. We modulated parameters such as pull amplitude and duration, and systematically extracted viscoelastic constants, by fitting creep curves with a Jeffrey’s three-element model.
We obtained a spatiotemporal description of cytoplasm organization and changes in material properties across stages showing a mechanical modulation during early embryo development. We observed that cytoplasm mechanical properties change from unfertilized to 16-cell stage, varying from softer to stiffer and more viscous cytoplasm after fertilization up to the 8-cell stage showing a decrease in the elasticity and the viscosity at 16-cell stage. A publication is under preparation as part of the master and PhD works of the PhD student Jiawei Xu, and results have been shared in various conferences.
WP2 - Sea urchin cytoplasm fractions. To investigate the contribution of different cytoplasm elements to cytoplasm mechanics, I proposed to generate cytoplasm fractions by adapting and optimizing previous fragmentation and sedimentation methods. I have obtained complete undiluted crude extracts, cytosolic fraction, yolk purified suspension, pure ER fraction, and ER+yolk fraction from unfertilized egg cytoplasms. These fractions have been accurately characterized with optical microscopy, with the use of dyes or antibodies to label mitochondria, yolk, actin, microtubules or the ER; using TEM (transmission electron microscopy) to obtain finer details of above-mentioned components; and electrophoresis for protein identification and sample comparison. I also used some drugs, such as sodium azide to deplete ATP, or Cytochalasin D to disrupt F-actin filaments, and supplementation with an energy mix (ATP and GTP), to test the behavior of crude extracts under their presence. For fertilized stages, we started to test and adapt the previous protocols to obtain 1-cell and 8-cell stage crude extracts solutions.
I have established robust protocols for obtaining and characterizing individual sea urchin cytoplasm fractions from unfertilized eggs. These protocols and all the know-how generated from this WP is highly valuable for the research developed in the lab and it will be useful for other research groups working in this field. Results have been shared in conferences and a publication including these outcomes has been submitted for peer-review publication.
WP3 - Water-in-Oil (W/O) droplets for bulk solution rheological measurements. In collaboration with researchers from the Laboratoire Jean Perrin-Sorbonne Université (Paris), I developed a quasistatic fabrication method to fabricate cell-like microdroplets encapsulating cytoplasm fractions to examine their rheology.
The main outcome of this WP is the development and implementation of a highly-reproducible setup for obtaining cell-like compartments as a tool to reconstitute cytoplasm mechanics and perform a “mechanical screen” from a minimal set of pre-sorted individual fractions. Results have been shared in several conferences throughout the project lifetime, and a technical brochure of the setup operation has been developed as a guide to fabricate cell-like compartments. The detailed scientific protocol on the methods has been included in the main publication derived from this project.
WP4 - In vitro reconstitution of cytoplasm mechanics from individual intracellular constituents. I performed a “mechanical screen” using cytoplasm fractions enriched in different endomembranes. I encapsulated cytoplasm fractions, from WP2, in cell-like droplets supplemented with magnetic probes, to monitor rheological signatures and compute viscoelastic constants with magnetic tweezers. Using these fractions, I characterized the viscoelastic response of full cytoplasm solution (crude extracts), and of individual constituents (cytosol, yolk granules, ER, and ER+yolk fractions) by applying forces using magnetic tweezers. This has been essential to derive physical understanding for how viscoelasticity of a given fraction may relate to the density, deformation or interaction of suspended components, and its connection with in vivo behavior. I also investigated the effects on crude extract solutions of supplementation with an energy mix, ATP depletion, and F-actin disruption.
I have been able of reconstitute cytoplasm mechanics and recapitulate in vivo cytoplasm signatures contributing to derive quantitative understanding for how the density, deformation or interaction of cytoplasm crowders, define large-scale cytoplasm rheology. I found that the synergy generated by mixing filamentous and granular endomembranes, ER and yolk, promotes solid-like characteristics of the cytoplasm. These original results have been shared in several conferences and are collected in a publication.
