Modelling biochemical gradients in vitro to control cell compartmentalization in a microengineered 3D model of the intestinal epithelium

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they formed and sensed by the epithelium is still not fully understood. Here we present a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and biomolecular gradients of the ISC niche (v). Biochemical gradients are formed through the hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of precise spatiotemporally controlled gradients, we employ light-sheet fluorescence microscopy and in-silico modelling. The ISC niche biochemical gradients applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.

Enzymatic degradation is an effective means to reduce aflatoxin contamination in maize

Background Aflatoxins are carcinogenic compounds produced by certain species of Aspergillus fungi. The consumption of crops contaminated with this toxin cause serious detrimental health effects, including death, in both livestock and humans. As a consequence, both the detection and quantification of this toxin in food/feed items is tightly regulated with crops exceeding the allowed limits eliminated from food chains. Globally, this toxin causes massive agricultural and economic losses each year. Results In this paper we investigate the feasibility of using an aflatoxin-degrading enzyme strategy to reduce/eliminate aflatoxin loads in developing maize kernels. We used an endoplasmic reticulum (ER) targeted sub-cellular compartmentalization stabilizing strategy to accumulate an aflatoxin-degrading enzyme isolated from the edible Honey mushroom Armillariella tabescens and expressed it in embryo tissue in developing maize kernels. Three transgenic maize lines that were determined to be expressing the aflatoxin-degrading enzyme both at the RNA and protein level, were challenged with the aflatoxin-producing strain Aspergillus flavus AF13 and shown to accumulate non-detectable levels of aflatoxin at 14-days post-infection and significantly reduced levels of aflatoxin at 30-days post-infection compared to nontransgenic control Aspergillus-challenged samples. Conclusions The expression of an aflatoxin-degrading enzyme in developing maize kernels was shown to be an effective means to control aflatoxin in maize in pre-harvest conditions. This aflatoxin-degradation strategy could play a significant role in the enhancement of both US and global food security and sustainability.

ER-localized phosphatidylethanolamine synthase plays a conserved role in lipid droplet formation

The asymmetric distribution of phospholipids in membranes is a fundamental principle of cellular compartmentalization and organization. Phosphatidylethanolamine (PE), a nonbilayer phospholipid that contributes to organelle shape and function, is synthesized at several subcellular localizations via semi-redundant pathways. Previously, we demonstrated in budding yeast that the PE synthase Psd1, which primarily operates on the mitochondrial inner membrane, is additionally targeted to the ER. While ER-localized Psd1 is required to support cellular growth in the absence of redundant pathways, its physiological function is unclear. We now demonstrate that ER-localized Psd1 sub-localizes on the ER to lipid droplet (LD) attachment sites and show it is specifically required for normal LD formation. We also find that the role of phosphatidylserine decarboxylase (PSD) enzymes in LD formation is conserved in other organisms. Thus, we have identified PSD enzymes as novel regulators of LDs and demonstrate that both mitochondria and LDs in yeast are organized and shaped by the spatial positioning of a single PE synthesis enzyme.

Cue-specific remodeling of the neuronal transcriptome through intron retention programs

Sub-cellular compartmentalization through the nuclear envelope has for a long time been primarily considered a physical barrier that separates nuclear and cytosolic contents. More recently, nuclear compartmentalization has emerged to harbor key regulatory functions in gene expression. A sizeable proportion of protein-coding mRNAs is more prevalent in the nucleus than in the cytosol reflecting the existence of mechanisms to control mRNA release into the cytosol. However, the biological relevance of the nuclear retention of mRNAs remains unclear. Here, we provide a comprehensive map of the subcellular localization of mRNAs in mature neurons and reveal that transcripts stably retaining introns are broadly targeted for nuclear retention. We systematically probed these transcripts upon neuronal stimulation and found that sub-populations of nuclear-retained transcripts are bi-directionally regulated in response to cues: some appear targeted for degradation while others undergo splicing completion to generate fully mature mRNAs which are exported to the cytosol to increase functional gene expression. Remarkably, different forms of stimulation mobilize distinct groups of intron-retaining transcripts and this selectivity arises from the activation of specific signaling pathways. Overall, our findings uncover cue-specific control of intron retention as a major regulator of acute remodeling of the neuronal transcriptome.

ER-localized phosphatidylethanolamine synthase plays a conserved role in lipid droplet formation

The asymmetric distribution of phospholipids in membranes is a fundamental principle of cellular compartmentalization and organization. Phosphatidylethanolamine (PE), a nonbilayer phospholipid that contributes to organelle shape and function, is synthesized at several subcellular localizations via semi-redundant pathways. Previously, we demonstrated in the yeast Saccharomyces cerevisiae that the PE synthase Psd1, which primarily operates on the mitochondrial inner membrane, is additionally targeted to the endoplasmic reticulum (ER). While ER-localized Psd1 is required to support cellular growth in the absence of redundant pathways, its physiological function at the ER is unclear. We now demonstrate that ER-localized Psd1 sub-localizes on the ER to lipid droplet (LD) attachment sites and further show it is specifically required for normal LD formation. We also find that the role of PSD enzymes in LD formation is conserved in other organisms. Thus, we have identified PSD enzymes as novel regulators of LDs and demonstrate that both mitochondria and LDs in yeast are organized and shaped by the spatial positioning of a single PE synthesis enzyme.

mRNA distribution in skeletal muscle is associated with mRNA size

Skeletal muscle myofibers are large and elongated cells with multiple and evenly distributed nuclei. Nuclear distribution suggests that each nucleus influences a specific compartment within the myofiber and implies a functional role for nuclear positioning. Compartmentalization of specific mRNAs and proteins has been reported at the neuromuscular and myotendinous junctions, but mRNA distribution in non-specialized regions of the myofibers remains largely unexplored. We report that the bulk of mRNAs is enriched around the nucleus of origin and that this perinuclear accumulation depends on recently transcribed mRNAs. Surprisingly, mRNAs encoding large proteins – giant mRNAs – are spread throughout the cell and do not exhibit perinuclear accumulation. Furthermore, by expressing exogenous transcripts with different sizes we found that size contributes to mRNA spreading independently of mRNA sequence. Both these mRNA distribution patterns depend on microtubules and are independent of nuclear dispersion, mRNA expression level and stability, and the characteristics of the encoded protein. Thus, we propose that mRNA distribution in non-specialized regions of skeletal muscle is size selective to ensure cellular compartmentalization and simultaneous long-range distribution of giant mRNAs.

Nuclei are mobile processors enabling specialization in a gigantic single-celled syncytium

In multicellular organisms, the specification, coordination, and compartmentalization of cell types enable the formation of complex body plans. However, some eukaryotic protists such as slime molds generate diverse and complex structures while remaining in a multinucleated syncytial state. It is unknown if different regions of these giant syncytial cells have distinct transcriptional responses to environmental encounters, and if nuclei within the cell diversify into heterogeneous states. Here we performed spatial transcriptome analysis of the slime mold Physarum polycephalum in the plasmodium state under different environmental conditions, and used single-nucleus RNA-sequencing to dissect gene expression heterogeneity among nuclei. Our data identifies transcriptome regionality in the organism that associates with proliferation, syncytial substructures, and localized environmental conditions. Further, we find that nuclei are heterogenous in their transcriptional profile, and may process local signals within the plasmodium to coordinate cell growth, metabolism, and reproduction. To understand how nuclei variation within the syncytium compares to heterogeneity in single-nucleated cells, we analyzed states in single Physarum amoebal cells. We observed amoebal cell states at different stages of mitosis and meiosis, and identified cytokinetic features that are specific to nuclei divisions within the syncytium. Notably, we do not find evidence for predefined transcriptomic states in the amoebae that are observed in the syncytium. Our data shows that a single-celled slime mold can control its gene expression in a region-specific manner while lacking cellular compartmentalization, and suggests that nuclei are mobile processors facilitating local specialized functions. More broadly, slime molds offer the extraordinary opportunity to explore how organisms can evolve regulatory mechanisms to divide labor, specialize, balance competition with cooperation, and perform other foundational principles that govern the logic of life.

Triggered reversible disassembly of an engineered protein nanocage

Protein nanocages play crucial roles in sub-cellular compartmentalization and spatial control in all domains of life and have been used as biomolecular tools for applications in biocatalysis, drug delivery, and bionanotechnology. The ability to control their assembly state under physiological conditions would further expand their practical utility. To gain such control, we introduced a peptide capable of triggering conformational change at a key structural position in the largest known encapsulin nanocompartment. We report the structure of the resulting engineered nanocage and demonstrate its ability to on-demand disassemble and reassemble under physiological conditions. We demonstrate its capacity for in vivo encapsulation of proteins of choice while also demonstrating in vitro cargo loading capabilities. Our results represent a functionally robust addition to the nanocage toolbox and a novel approach for controlling protein nanocage disassembly and reassembly under mild conditions.

Learning the molecular grammar of protein condensates from sequence determinants and embeddings

Intracellular phase separation of proteins into biomolecular condensates is increasingly recognized as a process with a key role in cellular compartmentalization and regulation. Different hypotheses about the parameters that determine the tendency of proteins to form condensates have been proposed, with some of them probed experimentally through the use of constructs generated by sequence alterations. To broaden the scope of these observations, we established an in silico strategy for understanding on a global level the associations between protein sequence and phase behavior and further constructed machine-learning models for predicting protein liquid–liquid phase separation (LLPS). Our analysis highlighted that LLPS-prone proteins are more disordered, less hydrophobic, and of lower Shannon entropy than sequences in the Protein Data Bank or the Swiss-Prot database and that they show a fine balance in their relative content of polar and hydrophobic residues. To further learn in a hypothesis-free manner the sequence features underpinning LLPS, we trained a neural network-based language model and found that a classifier constructed on such embeddings learned the underlying principles of phase behavior at a comparable accuracy to a classifier that used knowledge-based features. By combining knowledge-based features with unsupervised embeddings, we generated an integrated model that distinguished LLPS-prone sequences both from structured proteins and from unstructured proteins with a lower LLPS propensity and further identified such sequences from the human proteome at a high accuracy. These results provide a platform rooted in molecular principles for understanding protein phase behavior. The predictor, termed DeePhase, is accessible from https://deephase.ch.cam.ac.uk/.