Periodic Reporting for period 1 - CYTOMECH (Reconstituting Cytoplasm Mechanics)
Reporting period: 2024-06-01 to 2026-05-31
Summary of the context and overall objectives of the project
The cytoplasm is a complex and crowded medium, that exhibits a rich rheology thought to regulate essential cellular processes ranging from molecular diffusion, to vesicular transport and cell division. Cytoplasm elements include macromolecules and organelles of various sizes, densities and shapes, as well as cytoskeletal networks. These may behave as active, dense heterogenous suspensions close to jamming or as percolated polymer gels, to endow the cytoplasm with unique rheological properties. To date, how cytoplasm composition, organization and activity set its rheology, and how in turn these properties impact fundamental cellular functions remain poorly understood.
The project, Reconstituting Cytoplasm Mechanics (CYTOMECH), advances our understanding for how the mechanical properties of the cytoplasm are regulated during early embryonic development. CYTOMECH hypothesizes that endomembrane vesicles, tubules, and sheets form a densely packed suspension that largely determines cytoplasm rheology at these larger scales. Using sea urchin embryos as a model system, the project combined advanced live imaging with in vivo magnetic tweezers to characterize how the organization of endomembranes and the mechanical properties of the cytoplasm change throughout embryonic development. By means of an in vitro approach, we reconstituted cytoplasm mechanics by encapsulating cytoplasm extracts enriched in different endomembrane components within cell-like compartments, enabling a systematic mechanical analysis of their contribution. The fundamental breakthroughs of the action were addressed through four research objectives (RO):
RO1 - To map cytoplasm anatomy, dynamics and rheology.
RO2 - To extract and characterize cytoplasm fractions.
RO3 - To experimentally develop Water-in-Oil (W/O) droplets for bulk solution rheological measurements.
RO4 - To reconstitute cytoplasm mechanics from individual intracellular constituents and measure rheological properties.
The project, Reconstituting Cytoplasm Mechanics (CYTOMECH), advances our understanding for how the mechanical properties of the cytoplasm are regulated during early embryonic development. CYTOMECH hypothesizes that endomembrane vesicles, tubules, and sheets form a densely packed suspension that largely determines cytoplasm rheology at these larger scales. Using sea urchin embryos as a model system, the project combined advanced live imaging with in vivo magnetic tweezers to characterize how the organization of endomembranes and the mechanical properties of the cytoplasm change throughout embryonic development. By means of an in vitro approach, we reconstituted cytoplasm mechanics by encapsulating cytoplasm extracts enriched in different endomembrane components within cell-like compartments, enabling a systematic mechanical analysis of their contribution. The fundamental breakthroughs of the action were addressed through four research objectives (RO):
RO1 - To map cytoplasm anatomy, dynamics and rheology.
RO2 - To extract and characterize cytoplasm fractions.
RO3 - To experimentally develop Water-in-Oil (W/O) droplets for bulk solution rheological measurements.
RO4 - To reconstitute cytoplasm mechanics from individual intracellular constituents and measure rheological properties.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
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.
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.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
CYTOMECH has contributed to establish a biophysical description of cytoplasm fluid mechanics in space and time, combining concepts, methodologies and tools from soft-matter physics, engineering and biology.
This project has advanced our understanding for how cells may tune their cytoplasm fluid mechanical properties for proper embryo development. CYTOMECH merges biology, soft-matter physics and engineering and has generated high-quality knowledge with strong impact in fundamental science, technology, and society, even beyond the scope and duration of the project, with an intricate connection between them. Our results establish a breakthrough biophysical description of the cytoplasm showing for the first time that the rheology of bulk cytoplasm is dominated by a composite system made of yolk granular suspension and ER filamentous membranes, that endows solid-like behavior and glassy dynamics to the cytoplasm at short time scales, but flows at longer time scales to facilitate cellular reorganization. The cutting-edge technological development of setups, for droplet fabrication or for precise force measurements/applications with magnetic tweezers, may constitute solid roots to attract the interest of companies with innovation and technology transfer impact. CYTOMECH has already improved current methods for cytoplasm fragmentation by optimizing protocols, tuning factors like centrifugation force or time, increasing the efficiency and reducing costs coming from older standardized steps.
This project has advanced our understanding for how cells may tune their cytoplasm fluid mechanical properties for proper embryo development. CYTOMECH merges biology, soft-matter physics and engineering and has generated high-quality knowledge with strong impact in fundamental science, technology, and society, even beyond the scope and duration of the project, with an intricate connection between them. Our results establish a breakthrough biophysical description of the cytoplasm showing for the first time that the rheology of bulk cytoplasm is dominated by a composite system made of yolk granular suspension and ER filamentous membranes, that endows solid-like behavior and glassy dynamics to the cytoplasm at short time scales, but flows at longer time scales to facilitate cellular reorganization. The cutting-edge technological development of setups, for droplet fabrication or for precise force measurements/applications with magnetic tweezers, may constitute solid roots to attract the interest of companies with innovation and technology transfer impact. CYTOMECH has already improved current methods for cytoplasm fragmentation by optimizing protocols, tuning factors like centrifugation force or time, increasing the efficiency and reducing costs coming from older standardized steps.