Computational identification of prion-like RNA-binding proteins that form liquid phase-separated condensates

MOTIVATION: Eukaryotic cells contain different membrane-delimited compartments, which are crucial for the biochemical reactions necessary to sustain cell life. Recent studies showed that cells can also trigger the formation of membraneless organelles composed by phase-separated proteins to respond to various stimuli. These condensates provide new ways to control the reactions and phase-separation proteins (PSPs) are thus revolutionizing how cellular organization is conceived. The small number of experimentally validated proteins, and the difficulty in discovering them, remain bottlenecks in PSPs research. RESULTS: Here we present PSPer, the first in-silico screening tool for prion-like RNA-binding PSPs. We show that it can prioritize PSPs among proteins containing similar RNA-binding domains, intrinsically disordered regions and prions. PSPer is thus suitable to screen proteomes, identifying the most likely PSPs for further experimental investigation. Moreover, its predictions are fully interpretable in the sense that it assigns specific functional regions to the predicted proteins, providing valuable information for experimental investigation of targeted mutations on these regions. Finally, we show that it can estimate the ability of artificially designed proteins to form condensates (r=-0.87), thus providing an in-silico screening tool for protein design experiments. AVAILABILITY AND IMPLEMENTATION: PSPer is available at bio2byte.com/psp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.status: publishe

Biophysical properties of heterochromatin in totipotent mouse embryos

Totipotency is an incredibly plastic and transient state during the development of an embryo, which in mouse is restricted to the zygote and 2-cell stage. Indeed, totipotent embryos, are unique in terms of their chromatin architecture, metabolism and transcriptional status. Studying this short window of development is thus very important not only to better understand how normal development at these early stages occurs, but also to obtain conceptual and methodological tools to better manipulate the potency of cells in culture. During my PhD I first studied the metabolism of totipotent cells by measuring the mitochondrial membrane potential of 2-cell embryos and comparing it to pluripotent inner cell mass and differentiated trophectoderm of blastocysts. My results show, that although these two embryo stages differ in their oxygen consumption and mitochondria matrix shape, there are no major changes in mitochondrial membrane potential. The main objective of my PhD was the characterization of the biophysical properties of heterochromatin during the process of chromocenter formation that occurs at the 2-cell stage. I first identified a set of core heterochromatic proteins and revealed their higher potential to phase separate based on an in silico analysis. Using a wide variety of microscopy techniques, I showed that pericentromeric heterochromatin transitions from a liquid state to a more solid or gel like state during the process of chromocenter formation. Overall, my work contributes to a better understanding of the features that characterise totipotency as well as developing state of the art tools to study the biophysical properties of constitutive heterochromatin at these early stages of development

Insights Into the Non-Osmoregulatory Function of a Pollen-Specific Mechanosensitive Ion Channel

Pollen, the male gametophyte of flowering plants, delivers the sperm cells to the ovule to carry out sexual reproduction. During this process, the pollen grain undergoes dramatic physical changes. Survival requires careful control of cell mechanics, particularly the balance between protoplast expansion and cell wall resistance. One control mechanism is the use of a mechanosensitive (MS) ion channel, MscS-Like (MSL)8. This pollen-specific protein was previously shown to be essential for pollen survival during hydration and was proposed to function as a tension-gated osmoregulator. However, direct proof of osmoregulation during initial hydration has not yet been found. In fact, studies of the role in plants have suggested that there are functions of MSL proteins beyond osmotic regulation, such as MSL10 which functions to promote programmed cell death signaling, particularly after a hypo-osmotic shock.In this work, mathematical modeling alongside in vitro experiments show that MSL8 is likely not acting via osmoregulation during early hydration. Instead, we conclude that channel function stiffens the cell wall, thus preventing overexpansion. This follows the previously published finding that there are changes in the cell wall (callose) of pollen tubes lacking MSL8. However, an initial survey performed here did not identify any cell wall-related mutants with an msl8-5-like hydration phenotype. Moreover, attempts to understand MSL8 protein interactions hit a roadblock due to strong induction of cell death when MSL8 is highly expressed ectopically. MSL10 – which is known to induce programmed cell death – did not rescue the reduced viability of msl8-5 pollen when expressed under the MSL8 promoter. This supports the idea that it is not a universal channel or signaling function inherent to MSLs that is maintaining pollen mechanics. In fact, it is likely the MSL8 channel function, not a separate signaling function, that is lethal to the plant. Ectopic expression of a previously published variant of MSL8 with a “blocked” channel does not result in strong induction of death and MSL8 with a constitutively open channel causes near-complete obliteration of male fertility. In the last chapter, the structure and function of the N-terminus of MSL8 was examined. We found that this domain of the protein, similar to other MSL proteins, is predicted to be intrinsically disordered. Purification of the MSL8 N-terminus found that it readily phase separates even in high salt and at low concentrations. This may allow for regulation of the protein in the pollen grain as it transitions from desiccated (high osmolyte concentration) to hydrated (low osmolyte concentration) conditions. Overall, these findings indicate that MSL8, contrary to our original hypothesis, may be functioning and regulated in pollen using mechanisms beyond tension-gated osmoregulation