Understanding cellular ultrastructure is tightly bound to microscopic resolution and the ability to identify individual components at that resolution. Expansion microscopy has revolutionised this topic. Here we present and compare two protocols of ultrastructure expansion microscopy that allow for 4.5-fold mostly isotropic expansion and the use of antibodies, metabolic labelling, and DNA stains to demarcate individual regions such as the endoplasmic reticulum, the nuclei, the peripheral endocytic compartments as well as the ventral disc and the cytoskeleton in Giardia lamblia. We present an optimised, shortened, and modular protocol that can be swiftly adjusted to the investigators needs in this important protozoan model organism.

While Aβ and Tau cellular distribution has been largely studied, the comparative internalization and subcellular accumulation of Tau and Aβ isolated from human brain extracts in endothelial and neuronal cells has not yet been unveiled. We have previously demonstrated that controlled enrichment of Aβ from human brain extracts constitutes a valuable tool to monitor cellular internalization in vitro and in vivo. Herein, we establish an alternative method to strongly enrich Aβ and Tau aggregates from human AD brains, which has allowed us to study and compare the cellular internalization, distribution and toxicity of both proteins within brain barrier endothelial (bEnd.3) and neuronal (Neuro2A) cells. Our findings demonstrate the suitability of human enriched brain extracts to monitor the intracellular distribution of human Aβ and Tau, which, once internalized, show dissimilar sorting to different organelles within the cell and differential toxicity, exhibiting higher toxic effects on neuronal cells than on endothelial cells. While tau is strongly concentrated preferentially in mitochondria, Aβ is distributed predominantly within the endolysosomal system in endothelial cells, whereas the endoplasmic reticulum was its preferential location in neurons. Altogether, our findings display a picture of the interactions that human Aβ and Tau might establish in these cells.

Chemical synapses are essential for neuronal information storage and relay. The synaptic signal received or sent from spatially distinct subcellular compartments often generates different outcomes due to the distance or physical property difference. Therefore, the final output of postsynaptic neurons is determined not only by the type and intensity of synaptic inputs but also by the synaptic subcellular location. How synaptic subcellular specificity is determined has long been the focus of study in the neurodevelopment field. Genetic studies from invertebrates such as Caenorhabditis elegans (C. elegans) have uncovered important molecular and cellular mechanisms required for subcellular specificity. Interestingly, similar molecular mechanisms were found in the mammalian cerebellum, hippocampus, and cerebral cortex. This review summarizes the comprehensive advances in the cellular and molecular mechanisms underlying synaptic subcellular specificity, focusing on studies from C. elegans and rodents.

Determining how cell-scale processes lead to tissue-scale patterns is key to understanding how hormones and morphogens are distributed within biological tissues and control developmental processes. In this article, we use multiscale asymptotic analysis to derive a continuum approximation for hormone transport in a long file of cells to determine how subcellular compartments and cell growth and division affect tissue-scale hormone transport. Focusing our study on plant tissues, we begin by presenting a discrete multicellular ODE model tracking the hormone concentration in each cell\'s cytoplasm, subcellular vacuole, and surrounding apoplast, represented by separate compartments in the cell-file geometry. We allow the cells to grow at a rate that can depend both on space and time, accounting for both cytoplasmic and vacuolar expansion. Multiscale asymptotic analysis enables us to systematically derive the corresponding continuum model, obtaining an effective reaction-advection-diffusion equation and revealing how the effective diffusivity, effective advective velocity, and the effective sink term depend on the parameters in the cell-scale model. The continuum approximation reveals how subcellular compartments, such as vacuoles, can act as storage vessels, that significantly alter the effective properties of hormone transport, such as the effective diffusivity and the induced effective velocity. Furthermore, we show how cell growth and spatial variance across cell lengths affect the effective diffusivity and the induced effective velocity, and how these affect the tissue-scale hormone distribution. In particular, we find that cell growth naturally induces an effective velocity in the direction of growth, whereas spatial variance across cell lengths induces effective velocity due to the presence of an extra compartment, such as the apoplast and the vacuole, and variations in the relative sizes between the compartments across the file of cells. It is revealed that hormone transport is faster across cells of decreasing lengths than cells with increasing lengths. We also investigate the effect of cell division on transport dynamics, assuming that each cell divides as soon as it doubles in size, and find that increasing the time between successive cell divisions decreases the growth rate, which enhances the effect of cell division in slowing hormone transport. Motivated by recent experimental discoveries, we discuss particular applications for transport of gibberellic acid (GA), an important growth hormone, within the Arabidopsis root. The model reveals precisely how membrane proteins that mediate facilitated GA transport affect the effective tissue-scale transport. However, the results are general enough to be relevant to other plant hormones, or other substances that are transported in a similar way in any type of cells.

In eukaryotic organisms, subcellular protein location is critical in defining protein function and understanding sub-functionalization of gene families. Some proteins have defined locations, whereas others have low specificity targeting and complex accumulation patterns. There is no single approach that can be considered entirely adequate for defining the in vivo location of all proteins. By combining evidence from different approaches, the strengths and weaknesses of different technologies can be estimated, and a location consensus can be built. The Subcellular Location of Proteins in Arabidopsis database ( http://suba.live/ ) combines experimental data sets that have been reported in the literature and is analyzing these data to provide useful tools for biologists to interpret their own data. Foremost among these tools is a consensus classifier (SUBAcon) that computes a proposed location for all proteins based on balancing the experimental evidence and predictions. Further tools analyze sets of proteins to define the abundance of cellular structures. Extending these types of resources to plant crop species has been complex due to polyploidy, gene family expansion and contraction, and the movement of pathways and processes within cells across the plant kingdom. The Crop Proteins of Annotated Location database ( http://crop-pal.org/ ) has developed a range of subcellular location resources including a species-specific voting consensus for 12 plant crop species that offers collated evidence and filters for current crop proteomes akin to SUBA. Comprehensive cross-species comparison of these data shows that the sub-cellular proteomes (subcellulomes) depend only to some degree on phylogenetic relationship and are more conserved in major biosynthesis than in metabolic pathways. Together SUBA and cropPAL created reference subcellulomes for plants as well as species-specific subcellulomes for cross-species data mining. These data collections are increasingly used by the research community to provide a subcellular protein location layer, inform models of compartmented cell function and protein-protein interaction network, guide future molecular crop breeding strategies, or simply answer a specific question-where is my protein of interest inside the cell?

Calcium is a second messenger crucial to a myriad of cellular processes ranging from regulation of metabolism and cell survival to vesicle release and motility. Current strategies to directly manipulate endogenous calcium signals lack cellular and subcellular specificity. We introduce SpiCee, a versatile and genetically encoded chelator combining low- and high-affinity sites for calcium. This scavenger enables altering endogenous calcium signaling and functions in single cells in vitro and in vivo with biochemically controlled subcellular resolution. SpiCee paves the way to investigate local calcium signaling in vivo and directly manipulate this second messenger for therapeutic use.

Manganese (Mn) is an essential element for plant growth due to its participation in a series of physiological and metabolic processes. Mn is also considered a heavy metal that causes phytotoxicity when present in excess, disrupting photosynthesis and enzyme activity in plants. Thus, Mn toxicity is a major constraint limiting plant growth and production, especially in acid soils. To cope with Mn toxicity, plants have evolved a wide range of adaptive strategies to improve their growth under this stress. Mn tolerance mechanisms include activation of the antioxidant system, regulation of Mn uptake and homeostasis, and compartmentalization of Mn into subcellular compartments (e.g., vacuoles, endoplasmic reticulum, Golgi apparatus, and cell walls). In this regard, numerous genes are involved in specific pathways controlling Mn detoxification. Here, we summarize the recent advances in the mechanisms of Mn toxicity tolerance in plants and highlight the roles of genes responsible for Mn uptake, translocation, and distribution, contributing to Mn detoxification. We hope this review will provide a comprehensive understanding of the adaptive strategies of plants to Mn toxicity through gene regulation, which will aid in breeding crop varieties with Mn tolerance via genetic improvement approaches, enhancing the yield and quality of crops.

As an alternative to in vitro lipase dependent biotransformation and to traditional assembly of pathways in cytoplasm, the present study focused on targeting lipase dependent pathways to a subcellular compartment lipid body (LB), in combination with compartmentalization of associated pathways in other lipid relevant organelles including endoplasmic reticulum (ER) and peroxisome for efficient in vivo biosynthesis of fatty acid methyl esters (FAMEs) and hydrocarbons, in the context of improving Yarrowia lipolytica lipid pool. Through knock in and knock out of key genes involved in triacylglycerols (TAGs) biosynthesis and degradation, the TAGs content was increased to 51.5%, from 7.2% in parent strain. Targeting lipase dependent pathway to LB gave a 10-fold higher FAMEs titer (1028.0 mg/L) compared to cytosolic pathway (102.8 mg/L). Furthermore, simultaneously targeting lipase dependent pathway to LB, ER and peroxisome gave rise to the highest FAMEs titer (1644.8 mg/L). The subcellular compartment engineering strategy was extended to other lipase dependent pathways for fatty alkene and alkane biosynthesis, which resulted in a 14-fold titer enhancement compared to traditional cytosolic pathways. We developed yeast subcellular cell factories by directing lipase dependent pathways towards the TAGs storage organelle LB for efficient biosynthesis of TAG derived chemicals for the first time. The successful exploration of targeting metabolic pathways towards LB centered organelles is expected to promote subcellular compartment engineering for other lipid derived product biosynthesis.

cGMP is critical to a variety of cellular processes, but the available tools to interfere with endogenous cGMP lack cellular and subcellular specificity. We introduce SponGee, a genetically encoded chelator of this cyclic nucleotide that enables in vitro and in vivo manipulations in single cells and in biochemically defined subcellular compartments. SponGee buffers physiological changes in cGMP concentration in various model systems while not affecting cAMP signals. We provide proof-of-concept strategies by using this tool to highlight the role of cGMP signaling in vivo and in discrete subcellular domains. SponGee enables the investigation of local cGMP signals in vivo and paves the way for therapeutic strategies that prevent downstream signaling activation.

The gene family of serine acetyltransferases (SERATs) constitutes an interface between the plant pathways of serine and sulfur metabolism. SERATs provide the activated precursor, O-acetylserine for the fixation of reduced sulfur into cysteine by exchanging the serine hydroxyl moiety by a sulfhydryl moiety, and subsequently all organic compounds containing reduced sulfur moieties. We investigate here, how manipulation of the SERAT interface results in metabolic alterations upstream or downstream of this boundary and the extent to which the five SERAT isoforms exert an effect on the coupled system, respectively. Serine is synthesized through three distinct pathways while cysteine biosynthesis is distributed over the three compartments cytosol, mitochondria, and plastids. As the respective mutants are viable, all necessary metabolites can obviously cross various membrane systems to compensate what would otherwise constitute a lethal failure in cysteine biosynthesis. Furthermore, given that cysteine serves as precursor for multiple pathways, cysteine biosynthesis is highly regulated at both, the enzyme and the expression level. In this study, metabolite profiles of a mutant series of the SERAT gene family displayed that levels of the downstream metabolites in sulfur metabolism were affected in correlation with the reduction levels of SERAT activities and the growth phenotypes, while levels of the upstream metabolites in serine metabolism were unchanged in the serat mutants compared to wild-type plants. These results suggest that despite of the fact that the two metabolic pathways are directly connected, there seems to be no causal link in metabolic alterations. This might be caused by the difference of their pool sizes or the tight regulation by homeostatic mechanisms that control the metabolite concentration in plant cells. Additionally, growth conditions exerted an influence on metabolic compositions.