Temperature Drives P granule Formation in Caenorhabditis elegans

Ectotherms are living creatures whose body temperature varies with the environment in which they live. Their physiology and metabolism have to rapidly respond to environmental changes in order to stay viable at across their tolerable thermal range (Lithgow et al. 1994). In nematodes such as Caenorhabditis elegans, temperature is an important factor that defines the fertility of the worm. A feature that delimits an ectotherm’s thermal range is the maximum temperature at which its germ line can produce gametes. How germ cells withstand high environmental stressors such as limiting temperatures is not well understood, especially when considering the thermodynamical principles that dominate the biochemical processes of the cytoplasm (Hyman and Brangwynne 2011). Previous studies in C. elegans have shown that the thermodynamic effects of temperature on the cell cycle rate in nematodes follows an Arrhenius relationship and defines the thermal range where worms can be fertile. At the limits of this relationship a breakdown of the Arrhenius trend is observed (Begasse et al. 2015a). It was hypothesized that some type of discontinuous phase transition occurred in the embryonic cells of C. elegans (Begasse et al 2015). However, it remains unknown if there is the physiological link between a drop off in fertility and the embryonic breakdown of the Arrhenius trend. This work finds the link between a temperature driven phase separation of P granules and fertility. P granules are important for germ line development and the fertility of C. elegans (Kawasaki et al. 1998b). Here it is shown that P granules mix with the cytoplasm upon a temperature quench of 27ºC to T=18ºC and de-mix from the cytoplasm forming droplets upon a temperature downshift of temperature from 18ºC to 27ºC. P granules also show a reversible behavior mixing and de-mixing with changes in temperature in vivo, having a strong dependence of these liquid-like compartments with entropy. These results were further confirmed using a minimally reconstituted, in vitro P granule system and showed that PGL-3, a constitutive component of P granules, can phase separate and form liquid compartments in a similar way as happens in vivo. Additionally, here it is shown that P granule phase separation does not require the chemical activity of other cytoplasmic factors to drive the phase separation of compartments in vivo and in vitro, instead their formation is strongly driven to mix and de-mix with changes in temperature. Furthermore, a binary phase diagram was constructed in order to compare the response of P granules in vivo and in vitro, showing that P granules form and function as a temperature driven liquid phaseseparation. Altogether, this indicates that P granules in vivo and PGL-3 liquid-like compartments in vitro, share the same temperature of mixing and de-mixing which coincides with the fertile temperature range over which Caenorhabditis elegans can reproduce. This suggests that P granule phase separation could define the thermal range of the worm.:Table of Contents 1. Abstract 2. Introduction 2 . 1 . CYTOPLASMIC ORGANIZAT ION 2 . 2 . CYTOPLASMIC PHASE SEPARATIONS 2 . 3 . P GRANULES RESEMBLE L IQUID- L IKE PROPERTI ES 2 . 4 . PHASE CHANGES AND THE CELL CYCLE 3. Aim 4. Methods 4 . 1 . STRAINS 4 . 2 . TEMPERATURE CONTROL 4.2.1. HEATING/COOLING SETUP DEVELOPMENT AND MICROSCOPE STAGE 4.2.2. CONFOCAL SAMPLE HOLDER AND HEATING/COOLING DEVICE 4.2.3. SAMPLE PREPARATION 4.2.4. TEMPERATURE OF THE MICROSCOPE OBJECTIVE 4 . 3 . IN VI VO ASSAYS 4 . 4 . IN VI TRO ASSAY 5. Results 5 . 1 . TEMPERATURE AND P GRANULE PHASE SEPARATION 5 . 2 . P GRANULES ARE TEMPERATURE SENSITIVE COMPARTMENTS 5 . 3 . P GRANULES MIX WITH THE CYTOPLASM AT 27ºC 5 . 4 . P GRANULES DO NOT NEED THE INFLUENCE OF PPTR- 1 TO FORM DROPLETS 5 . 5 . P GRANULES REVERSIBLY MIX AND DE-MIX IN VIVO 5 . 6 . PGL- 3 GRANULES PHASE SEPARATE IN V ITRO AT PHYSIOLOGICAL CONDITIONS 5 . 7 . P GRANULE PHASE SEPARATION IS REVERSIBLE IN VI VO AND IN VI TRO 5 . 8 . AN IN V ITRO PHASE DIAGRAM TO COMPARE THE THERMAL L IMITS OF P GRANULES IN V IVO 6. Discussion 6 . 1 . P GRANULES MIX AND DE-MIX IN A REVERSIBLE MANNER 6 . 2 . CONCENTRATION AND THE SPATIAL CONTROL OF P GRANULES 6 . 3 . THE ROLE OF OTHER CHEMICAL REGULATORS 6 . 4 . ECOLOGICAL RELEVANCE OF P GRANULE PHASE SEPARATION 7. Concluding Remarks 8. Bibliograph