Automated Quantification of Subcellular Particles in Myogenic Progenitors.

Fluorescence microscopy is a powerful tool enabling the visualization of protein localization within cells. In this article, we outline an automated and non-biased way to detect and quantify subcellular particles using immunocytochemistry, fluorescence microscopy, and the program CellProfiler. We discuss the examination of two types of subcellular particles: messenger ribonucleoprotein (mRNP) granules, namely processing bodies and stress granules, and autophagosomes. Fluorescent microscopy Z-stacks are acquired and deconvolved, and maximum intensity images are generated. The number of subcellular particles per cell is then quantified using the described CellProfiler pipeline. We also explain how to isolate primary myoblast progenitor cells from mice, which were used to obtain the presented results. Last, we discuss the critical parameters to be considered for each of these techniques. Both mRNP granules and autophagosomes play important roles in sequestering intracellular cargo, such as messenger RNAs and RNA-binding proteins for mRNP granules and cytoplasmic waste for autophagosomes. The methods outlined in this article are widely applicable for studies relating to subcellular particle formation, localization, and flux during homeostasis, following stimuli, and during disease. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence microscopy of messenger ribonucleoprotein granules in primary myoblasts Alternate Protocol: Immunofluorescence microscopy of autophagosomes in primary myoblasts Support Protocol: Isolation of primary myoblasts from mice Basic Protocol 2: Automated quantification of subcellular particles.

TDP43 ribonucleoprotein granules: physiologic function to pathologic aggregates.

Ribonucleoprotein (RNP) assemblies are ubiquitous in eukaryotic cells and have functions throughout RNA transcription, splicing, and stability. Of the RNA-binding proteins that form RNPs, TAR DNA-binding protein of 43 kD (TDP43) is of particular interest due to its essential nature and its association with disease. TDP43 plays critical roles in RNA metabolism, many of which require its recruitment to RNP granules such as stress granules, myo-granules, and neuronal transport granules. Moreover, the presence of cytoplasmic TDP43-positive inclusions is a pathological hallmark of several neurodegenerative diseases. Despite the pervasiveness of TDP43 aggregates, TDP43 mutations are exceedingly rare, suggesting that aggregation may be linked to dysregulation of TDP43 function. Oligomerization is a part of normal TDP43 function; thus, it is of interest to understand what triggers the irreversible aggregation that is seen in disease. Herein, we examine TDP43 functions, particularly in RNP granules, and the mechanisms which may explain pathological TDP43 aggregation.

Mechanisms and Regulation of RNA Condensation in RNP Granule Formation.

Ribonucleoprotein (RNP) granules are RNA-protein assemblies that are involved in multiple aspects of RNA metabolism and are linked to memory, development, and disease. Some RNP granules form, in part, through the formation of intermolecular RNA-RNA interactions. In vitro, such trans RNA condensation occurs readily, suggesting that cells require mechanisms to modulate RNA-based condensation. We assess the mechanisms of RNA condensation and how cells modulate this phenomenon. We propose that cells control RNA condensation through ATP-dependent processes, static RNA buffering, and dynamic post-translational mechanisms. Moreover, perturbations in these mechanisms can be involved in disease. This reveals multiple cellular mechanisms of kinetic and thermodynamic control that maintain the proper distribution of RNA molecules between dispersed and condensed forms.

Local Translation in Growth Cones and Presynapses, Two Axonal Compartments for Local Neuronal Functions.

During neural development, growth cones, very motile compartments of tips of axons, lead axonal extension to the correct targets. Subsequently, presynapses, another axonal compartment with vigorous trafficking of synaptic vesicles, emerge to form functional synapses with postsynapses. In response to extracellular stimuli, the immediate supply of proteins by local translation within these two axonal compartments far from cell bodies confers high motility of growth cones and active vesicle trafficking in presynapses. Although local translation in growth cones and presynapses occurs at a very low level compared with cell bodies and even dendrites, recent progress in omics and visualization techniques with subcellular fractionation of these compartments has revealed the actual situation of local translation within these two axonal compartments. Here, the increasing evidence for local protein synthesis in growth cones and presynapses for axonal and synaptic functions has been reviewed. Furthermore, the mechanisms regulating local translation in these two compartments and pathophysiological conditions caused by dysregulated local translation are highlighted.

FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions.

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.

Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity.

Neuronal inclusions of aggregated RNA-binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation-prone, yeast prion-like, low sequence-complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in-cell phosphorylation sites across FUS LC We show that both phosphorylation and phosphomimetic variants reduce its aggregation-prone/prion-like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self-interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS-associated cytotoxicity. Hence, post-translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.

An atlas of chromatoid body components.

The genome of male germ cells is actively transcribed during spermatogenesis to produce phase-specific protein-coding mRNAs and a considerable amount of different noncoding RNAs. Ribonucleoprotein (RNP) granule-mediated RNA regulation provides a powerful means to secure the quality and correct expression of the requisite transcripts. Haploid spermatids are characterized by a unique, unusually large cytoplasmic granule, the chromatoid body (CB), which emerges during the switch between the meiotic and post-meiotic phases of spermatogenesis. To better understand the role of the CB in male germ cell differentiation, we isolated CBs from mouse testes and revealed its full RNA and protein composition. We showed that the CB is mainly composed of RNA-binding proteins and other proteins involved RNA regulation. The CB was loaded with RNA, including pachytene piRNAs, a diverse set of mRNAs, and a number of uncharacterized long noncoding transcripts. The CB was demonstrated to accumulate nascent RNA during all the steps of round spermatid differentiation. Our results revealed the CB as a large germ cell-specific RNP platform that is involved in the control of the highly complex transcriptome of haploid male germ cells.