Interaction between PAR proteins and active mechanics in the one cell stage C. elegans embryo


Supervising PI

Stephan Grill (website)

ESR1 Victoria Yan

Project Description

Background

In the cytoplasm of the cell, proteins observed in liquid-like phases are thought to contribute to compartmentalization of the cytoplasm. This is thought to enhance the construction of essential cellular architecture for functional deformations to drive morphogenesis especially in development. F-actin filaments make up the key architecture that is the cell’s cortex. It is unclear whether demixing of cytoskeletal components could also play a role in partitioning actin nucleators to the membrane during the formation of an actomyosin cortex.

Objectives

The ESR will determine which markers of polarity control which mechanical activities of the cellular cytoskeleton.

To comprehensively capture the kinetic behaviour of Par proteins during polarization, CRISPR/Cas9 technology (collaboration with the Boxem group) will be used to tag endogenous Par proteins (together with the Goehring group) as well as members of the actomyosin cell cortex (Myosin, Actin via Lifeact) with newly optimized fluorescent tag, to make the system amenable for a midplane analysis. We will identify which active mechanical properties of the actomyosin cortex are under control of the PAR pathway, by determining the turnover of cortical components in relation to the PAR state of the cortex via FRAP. We will use theory to describe PAR pattern formation via a coupling a thin film active viscous fluid to a reaction-diffusion model of PAR protein behaviour. We will challenge our model through a series of milder-to-stronger RNAi experiments of components of actomyosin cortex (myosin) and the PAR system (PAR-2, PAR-4, PAR-6). For each perturbation all components are imaged in terms of concentration and velocity fields, and our theoretical description should, only by adjusting amounts and without other changes in model parameters, predict the full spatiotemporal evolution of the polarization process.

Summary of Results

In the cytoplasm of the cell, proteins observed in liquid-like phases are thought to contribute to compartmentalization of the cytoplasm. This is thought to enhance the construction of essential cellular architecture for functional deformations to drive morphogenesis especially in development. F-actin filaments make up the key architecture that is the cell’s cortex. It is unclear whether demixing of cytoskeletal components could also play a role in partitioning actin nucleators to the membrane during the formation of an actomyosin cortex.

To study this, I use the C. elegans embryo to examine potential condensation of actin nucleators, combing high spatio-temporal resolution live-imaging in TIRF and SIM-TIRF microscopy, as well as quantitative image analysis. I observed cortical F-actin network condensation with nucleators. The F-actin phases assemble and disassemble in a steady-state size distribution, to achieve coordinated actin polymerization during development.

References

  1. Goehring NW, Grill SW. Cell polarity: mechanochemical patterning. Trends Cell Biol. 2013 Feb;23(2):72–80.
  2. Naganathan SR, Fürthauer S, Nishikawa M, Jülicher F, Grill SW. Active torque generation by the actomyosin cell cortex drives left-right symmetry breaking. eLife. 2014;3:e04165.
  3. Fürthauer S, Strempel M, Grill SW, Jülicher F. Active chiral processes in thin films. Phys Rev Lett. 2013 Jan 25;110(4):048103.
  4. Goehring NW, Trong PK, Bois JS, Chowdhury D, Nicola EM, Hyman AA, et al. Polarization of PAR proteins by advective triggering of a pattern-forming system. Science. 2011 Nov 25;334(6059):1137–41.
  5. Goehring NW, Hoege C, Grill SW, Hyman AA. PAR proteins diffuse freely across the anterior-posterior boundary in polarized C. elegans embryos. J Cell Biol. 2011 May 2;193(3):583–94.
  6. Bois JS, Jülicher F, Grill SW. Pattern formation in active fluids. Phys Rev Lett. 2011 Jan 14;106(2):028103.
  7. Goehring NW, Chowdhury D, Hyman AA, Grill SW. FRAP analysis of membrane-associated proteins: lateral diffusion and membrane-cytoplasmic exchange. Biophys J. 2010 Oct 20;99(8):2443–52.
  8. Mayer M, Depken M, Bois JS, Jülicher F, Grill SW. Anisotropies in cortical tension reveal the physical basis of polarizing cortical flows. Nature. 2010 Sep 30;467(7315):617–21.