Nurni Ravi, Aparna et al. (2021) International Worm Meeting "PP1/SDS-22 phosphatase is required for germ plasm segregation in the one-cell C. elegans embryo"

Araten, Alison, Miller, Stephanie, Kim, Amelia, Nurni Ravi, Aparna, Han, Bingjie, Griffin, Erik

[International Worm Meeting, 2021]

We are interested in understanding the mechanisms that control the polarization of cytoplasmic factors during asymmetric cell division. During the asymmetric division of the C. elegans zygote, the RNA-binding protein MEX-5 recruits PLK-1 kinase to the anterior cytoplasm (Nishi, 2008). In turn PLK-1 phosphorylates the RNA-binding protein POS-1, inhibiting POS-1 retention in the anterior cytoplasm (Han, 2018). As a result, POS-1 is retained and accumulates in the posterior cytoplasm, likely reflecting the activity of a phosphatase that counteracts PLK-1 phosphorylation. Through an RNAi screen to identify the phosphatase that enables POS-1 retention in the posterior, we identified the catalytic subunits (GSP-1 and GSP-2) and a regulatory subunit (SDS-22) of PP1 phosphatase. In sds-22(RNAi) embryos, MEX-5, PLK-1 and upstream PAR polarities are established normally. In contrast, POS-1 retention in the posterior cytoplasm is reduced in sds-22(RNAi) embryos, causing a weakening and delay in POS-1 segregation. Strikingly, we find that SDS-22 is similarly required for PIE-1 segregation and for P granule formation/stability, suggesting to a central role in germplasm organization. Whereas PLK-1 is enriched in the anterior, endogenously-tagged SDS-22::GFP is symmetrically distributed throughout the cytoplasm. We propose that PLK-1 kinase and PP1/SDS-22 phosphatase act in opposition to control germ plasm retention and that spatial variation in their relative activities drives the segregation of factors such as POS-1 to the posterior cytoplasm.

Barbieri, Sofia et al. (2021) International Worm Meeting "The dynamic partnered dance between PLK-1 and MEX-5: interpreting gradient formation with computational modelling."

Gotta, Monica, Barbieri, Sofia, Griffin, Erik E., Nurni Ravi, Aparna

[International Worm Meeting, 2021]

In C. elegans embryos, PLK-1 is pivotal for cell division and it orchestrates polarity establishment together with its binding partner, MEX-5. To achieve this, their localization/activity must be precisely regulated. MEX-5 enrichment at the anterior cytoplasm results from a change in its diffusivity following uneven phosphorylation along the embryo axis. We know PLK-1 relocalization to the anterior depends on MEX-5. However, the biological and physical mechanisms behind the dynamics of this protein are still poorly described. To address this, PLK-1 and MEX-5 gradient formation was measured in two CRISPR strains and significant discrepancies were revealed between the two proteins in terms of: 1) gradient steepness, as PLK-1 forms a less steep gradient compared to MEX-5; 2) dynamics, with PLK-1 gradient establishment delayed and slower; 3) diffusivity, as PLK-1 diffusion coefficient does not correspond to MEX-5's one from anterior to posterior. To shed light on PLK-1 dynamics, and how it is intertwined to MEX-5, we developed a novel Monte Carlo simulation framework able to recreate the protein motions in the C. elegans one-cell embryo. Thanks to our computational approach, we were able to postulate on the biological mechanisms behind MEX-5 and PLK-1 dynamics during the whole cell division, from early embryos to the steady-state before cytokinesis. The simulations succeed in reproducing PLK-1 gradient formation, in agreement with experimental measurements, if: 1) PLK-1 binds to phosphorylated MEX-5; 2) the binding is triggered after a defined time delay; 3) PLK-1 dynamically interacts with MEX-5, leading to a continuous replenishment of a pool of unbound PLK-1. The Monte Carlo framework we propose can eventually be applied to other polarity-related factors or mutants in which polarization is perturbed, to understand if it can be traced back to a failure in PLK-1 localization. Finally, conditions where gradient formation is altered, like after stress, can be simulated.

Ravi, Bhavya et al. (2019) International Worm Meeting "C. elegans egg-laying behavior is controlled by an internal, stretch-dependent homeostat that is gated by external sensory input."

Kang, Lijun, Ravi, Bhavya, Collins, Kevin, Zhao, Jian

[International Worm Meeting, 2019]

The C. elegans egg-laying circuit is a simple, well-characterized neural circuit which drives a two-state behavior comprising ~20-minute inactive states that are punctuated by ~2-minute active states where eggs are laid. The circuit is comprised of two serotonergic Hermaphrodite Specific Neurons (HSNs) which release serotonin and NLP-3 neuropeptides to promote the egg-laying active state, and cholinergic ventral cord motoneurons which synapse onto a set of vulval muscles that contract to release eggs. We have been investigating how external and internal sensory signals converge on the circuit to determine when and where animals lay eggs. Using calcium imaging in behaving animals, we have recently found the accumulation of eggs in the adult uterus renders the postsynaptic vulval muscles sensitive to presynaptic HSN input (Ravi et al. 2018). Sterilization or acute electrical silencing of the vulval muscles inhibits presynaptic HSN activity. Reversal of muscle silencing triggers a homeostatic increase in HSN activity and egg release that maintains ~12-15 eggs in the uterus. These results show that egg-laying behavior in C. elegans is driven by an internal, stretch-dependent homeostat that scales serotonin motor neuron activity in response to postsynaptic muscle feedback. Parallel to mechanical activation, HSN activity is also regulated by external sensory input. Inhibitory neurotransmitters and peptides released from sensory neurons signal through receptors on HSN that activate the conserved, Pertussis Toxin-sensitive G protein, G?o/ GOA-1. In recent unpublished results, we find that mutations that increase inhibitory G?o signaling in HSN reduce the frequency of Ca2+ transients in both the HSNs and in the postsynaptic vulval muscles, extending the duration of the egg-laying inactive state. Conversely, mutations that eliminate inhibitory G?o signaling in HSN increase the frequency of Ca2+ transients in HSNs and vulval muscles, reducing the interval between egg-laying active states. Interestingly, animal sterilzation suppressed this increase of Ca2+ activity in the vulval muscles but not HSNs, showing the stretch homeostat acts outside of HSN. Patch clamp recordings show the HSNs are hyperpolarized in egl-10 mutants with increased G?o signaling and depolarized in transgenic animals where G?o is inhibited in HSN with Pertussis Toxin. Thus, inhibitory G?o signaling in HSN maintains a bi-stable state of cell electrical excitability that keeps the egg-laying circuit off in unfavorable environmental conditions and/or when the uterus is without eggs to lay. Ravi B, Garcia J, and Collins KM (2018). J. Neuroscience. 38 (28), 6283-6298.