Theory for weakly polydisperse cytoskeleton filaments

The ability of actins and tubulins to change dynamically from alterations in the number density of actins/tubulin, number density and type of binding agents, and electrolyte concentration is crucial for eukaryotic cells to regulate their cytoskeleton conformations in different cellular compartments. Conventional approaches on biopolymers solution break down for cytoskeleton filaments because they entail several approximations to treat their polyelectrolyte and mechanical properties. In this article, we introduce a novel density functional theory for polydisperse, semiflexible cytoskeleton filaments. The approach accounts for the equilibrium polymerization kinetics, length, and orientation filament distributions, as well as the electrostatic interaction between filaments and the electrolyte. This is essential for cytoskeleton polymerization in different cell compartments generating filaments of different lengths, sometimes long enough to become semiflexible. We characterized the thermodynamics properties of actin filaments in electrolyte aqueous solutions. We calculated the free energy, pressure, chemical potential, and second virial coefficient for each filament conformation. We also calculated the phase diagram of actin filaments solution and compared it with available experimental data.

Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples - so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample - a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

Mini Review: The Forensic Value of Heat Shock Proteins

Forensic pathologists are routinely confronted with unclear causes of death or related findings. In some instances, difficulties arise in relation to questions posed by criminal investigators or prosecutors. Such scenarios may include questions about wound vitality or cause of death where typical or landmark findings are difficult to ascertain. In addition to the usual examinations required to clarify unclear causes of death or address specific questions, immunohistochemistry and genetic analyses have become increasingly important techniques in this area since their establishment last century. Since then, many studies have determined the usefulness and significance of immunohistochemical and genetic investigations on cellular structures and proteins. For example, these proteins include heat shock proteins (Hsp), which were first described in 1962 and are so called based on their molecular weight. They predominantly act as molecular chaperones with cytoprotective functions that support cell survival under (sub) lethal conditions. They are expressed in specific cellular compartments and have many divergent functions. Central family members include, Hsp 27, 60, and 70. This mini review investigates recent research on the Hsp family, their application range, respective forensic importance, and current limitations and provides an outlook on possible applications within forensic science.

Specificity of H2O2 signaling in leaf senescence: is the ratio of H2O2 contents in different cellular compartments sensed in Arabidopsis plants?

Leaf senescence is an integral part of plant development and is driven by endogenous cues such as leaf or plant age. Developmental senescence aims to maximize the usage of carbon, nitrogen and mineral resources for growth and/or for the sake of the next generation. This requires efficient reallocation of the resources out of the senescing tissue into developing parts of the plant such as new leaves, fruits and seeds. However, premature senescence can be induced by severe and long-lasting biotic or abiotic stress conditions. It serves as an exit strategy to guarantee offspring in an unfavorable environment but is often combined with a trade-off in seed number and quality. In order to coordinate the very complex process of developmental senescence with environmental signals, highly organized networks and regulatory cues have to be in place. Reactive oxygen species, especially hydrogen peroxide (H2O2), are involved in senescence as well as in stress signaling. Here, we want to summarize the role of H2O2 as a signaling molecule in leaf senescence and shed more light on how specificity in signaling might be achieved. Altered hydrogen peroxide contents in specific compartments revealed a differential impact of H2O2 produced in different compartments. Arabidopsis lines with lower H2O2 levels in chloroplasts and cytoplasm point to the possibility that not the actual contents but the ratio between the two different compartments is sensed by the plant cells.

Liquid-Liquid Phase Separation Phenomenon on Protein Sorting Within Chloroplasts

In higher plants, chloroplasts are vital organelles possessing highly complex compartmentalization. As most chloroplast-located proteins are encoded in the nucleus and synthesized in the cytosol, the correct sorting of these proteins to appropriate compartments is critical for the proper functions of chloroplasts as well as plant survival. Nuclear-encoded chloroplast proteins are imported into stroma and further sorted to distinct compartments via different pathways. The proteins predicted to be sorted to the thylakoid lumen by the chloroplast twin arginine transport (cpTAT) pathway are shown to be facilitated by STT1/2 driven liquid-liquid phase separation (LLPS). Liquid-liquid phase separation is a novel mechanism to facilitate the formation of membrane-less sub-cellular compartments and accelerate biochemical reactions temporally and spatially. In this review, we introduce the sorting mechanisms within chloroplasts, and briefly summarize the properties and significance of LLPS, with an emphasis on the novel function of LLPS in the sorting of cpTAT substrate proteins. We conclude with perspectives for the future research on chloroplast protein sorting and targeting mechanisms.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immuno-staining.

Detection of nucleic acids within sub-cellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization and immuno-staining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels which escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in-situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.

The Signal Peptide and Chaperone UNC93B1 Both Influence TLR8 Ectodomain Intracellular Endosomal Localization

Toll-like receptor 8 (TLR8) is a single-stranded RNA sensing receptor and is localized in the cellular compartments, where it encounters foreign or self-nucleic acids and activates innate and adaptive immune responses. However, the mechanism controlling intracellular localization TLR8 is not completely resolved. We previously revealed the intracellular localization of TLR8 ectodomain (ECD), and in this study, we investigated the mechanism of the intracellular localization. Here we found that TLR8 ECDs from different species as well as ECDs from different TLRs are all intracellularly localized, similarly to the full-length porcine TLR8. Furthermore, porcine, bovine, and human TLR8 ECDs are all localized in cell endosomes, reflecting the cellular localization of TLR8. Intriguingly, none of post-translational modifications at single sites, including glycosylation, phosphorylation, ubiquitination, acetylation, and palmitoylation alter porcine TLR8-ECD endosomal localization. Nevertheless, the signal peptide of porcine TLR8-ECD determines its endosomal localization. On the other hand, signaling regulator UNC93B1 also decides the endosomal localization of porcine, bovine, and human TLR8 ECDs. The results from this study shed light on the mechanisms of not only TLR8 intracellular localization but also the TLR immune signaling.

Contour, a semi-automated segmentation and quantitation tool for cryo-soft-X-ray tomography

Cryo-soft-X-ray tomography is being increasingly used in biological research to study the morphology of cellular compartments and how they change in response to different stimuli, such as viral infections. Segmentation of these compartments is limited by time-consuming manual tools or machine learning algorithms that require extensive time and effort to train. Here we describe Contour, a new, easy-to-use, highly automated segmentation tool that enables accelerated segmentation of tomograms to delineate distinct cellular compartments. Using Contour, cellular structures can be segmented based on their projection intensity and geometrical width by applying a threshold range to the image and excluding noise smaller in width than the cellular compartments of interest. This method is less laborious and less prone to errors from human judgement than current tools that require features to be manually traced, and does not require training datasets as would machine-learning driven segmentation. We show that high-contrast compartments such as mitochondria, lipid droplets, and features at the cell surface can be easily segmented with this technique in the context of investigating herpes simplex virus 1 infection. Contour can extract geometric measurements from 3D segmented volumes, providing a new method to quantitate cryo-soft-X-ray tomography data. Contour can be freely downloaded at github.com/kamallouisnahas/Contour.Impact StatementMore research groups are using cryo-soft-X-ray tomography as a correlative imaging tool to study the ultrastructure of cells and tissues but very few tomograms are segmented with existing segmentation programs. Segmentation is usually a prerequisite for measuring the geometry of features in tomograms but the time- and labour-intensive nature of current segmentation techniques means that such measurements are rarely across a large number of tomograms, as is required for robust statistical analysis. Contour has been designed to facilitate the automation of segmentation and, as a result, reduce manual effort and increase the number of tomograms that can be segmented. Because it requires minimal manual intervention, Contour is not as prone to human error as programs that require the users to trace the edges of cellular features. Geometry measurements of the segmented volumes can be calculated using this program, providing a new platform to quantitate cryoSXT data. Contour also supports quantitation of volumes imported from other segmentation programs. The generation of a large sample of segmented volumes with Contour that can be used as a representative training dataset for machine learning applications is a long-term aspiration of this technique.

Ubiquitin Ligase Redundancy and Nuclear-Cytoplasmic Localization in Yeast Protein Quality Control

The diverse functions of proteins depend on their proper three-dimensional folding and assembly. Misfolded cellular proteins can potentially harm cells by forming aggregates in their resident compartments that can interfere with vital cellular processes or sequester important factors. Protein quality control (PQC) pathways are responsible for the repair or destruction of these abnormal proteins. Most commonly, the ubiquitin-proteasome system (UPS) is employed to recognize and degrade those proteins that cannot be refolded by molecular chaperones. Misfolded substrates are ubiquitylated by a subset of ubiquitin ligases (also called E3s) that operate in different cellular compartments. Recent research in Saccharomyces cerevisiae has shown that the most prominent ligases mediating cytoplasmic and nuclear PQC have overlapping yet distinct substrate specificities. Many substrates have been characterized that can be targeted by more than one ubiquitin ligase depending on their localization, and cytoplasmic PQC substrates can be directed to the nucleus for ubiquitylation and degradation. Here, we review some of the major yeast PQC ubiquitin ligases operating in the nucleus and cytoplasm, as well as current evidence indicating how these ligases can often function redundantly toward substrates in these compartments.

Understanding the Molecular Basis of Cardiomyopathy

Inherited cardiomyopathies are a major cause of mortality and morbidity worldwide and can be caused by mutations in a wide range of proteins located in different cellular compartments. The present review is based on Dr. Ju Chen's 2021 Robert M. Berne Distinguished Lectureship of the American Physiological Society Cardiovascular section, in which he provided an overview of the current knowledge on the cardiomyopathy-associated proteins that have been studied in his laboratory. The review provides a general summary of the proteins in different compartments of cardiomyocytes associated with cardiomyopathies with specific focus on the proteins that have been studied in Dr. Chen's laboratory.