Abscisic Acid Increases Hydrogen Peroxide in Multiple Subcellular Compartments to Drive Stomatal Closure

Stomatal closure regulates transpiration and gas exchange in response to environmental cues. Drought upregulates ABA signaling, which elevates levels of reactive oxygen species (ROS). However, the subcellular location and identity of these ROS has received limited study. We found that in guard cells, ABA increased fluorescence of the general redox sensor, dichlorofluorescein (DCF), in distinct subcellular locations including chloroplasts, cytosol, nuclei, and cytosolic puncta. These changes were lost in ABA-insensitive quintuple receptor mutant and accentuated in an ABA-hypersensitive mutant. ABA induced ROS accumulation in these subcellular compartments was lost in mutants with defects in genes encoding hydrogen peroxide synthesizing respiratory burst oxidase homolog (RBOH) enzymes and guard cells treated with the RBOH inhibitor VAS2870, while exogenous hydrogen peroxide treatment is sufficient to close guard cells. The hydrogen peroxide-selective probe, peroxy orange1, also showed ABA-dependent increases in chloroplasts and cytosolic puncta. Using the more sensitive genetically-encoded hydrogen peroxide reporter roGFP-Orp1, we also detected significant hydrogen peroxide increases in the cytosol and nucleus. These cytosolic puncta accumulate ROS after ABA treatment show colocalization with Mitotracker and with a mitochondrial targeted mt-roGFP2-Orp1, which also revealed ABA-increased ROS in mitochondria. These results indicate that elevated hydrogen peroxide after ABA treatment in these subcellular compartments is necessary and sufficient to drive stomatal closure.

Massively parallel identification of zipcodes in primary cortical neurons

Cells adopt highly polarized shapes and form distinct subcellular compartments largely due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called "zipcodes." Their recognition by RNA-binding proteins (RBPs) leads to the integration of the mRNAs into macromolecular complexes and their localization. While there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here, we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3'UTRs. This approach combines a method of separating the principal subcellular compartments of neurons – cell bodies and neurites - with a massively parallel reporter assay. Our analysis identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons and suggests a strategy for detecting many more.

SubcellulaRVis: an app to visualize subcellular compartment enrichment

High-throughput 'omics methods result in lists of differentially regulated or expressed genes or proteins, whose function is generally studied through statistical methods such as enrichment analyses. One aspect of protein regulation is subcellular localization, which is crucial for their correct processing and function and can change in response to various cellular stimuli. Enrichment of proteins for subcellular compartments is often based on Gene Ontology Cellular Compartment annotations. Results of enrichment are typically visualized using bar-charts, however enrichment analyses can result in a long list of significant annotations which are highly specific, preventing researchers from gaining a broad understanding of the subcellular compartments their proteins of interest may be located in. Schematic visualization of known subcellular locations has become increasingly available for single proteins via the UniProt and COMPARTMENTS platforms. However, it is not currently available for a list of proteins (e.g. from the same experiment) or for visualizing the results of enrichment analyses. To generate an easy-to-interpret visualization of protein subcellular localization after enrichment we developed the SubcellulaRVis web app, which visualizes the enrichment of subcellular locations of gene lists in an easy and impactful manner. SubcellulaRVis projects the results of enrichment analysis on a graphical representation of a eukaryotic cell. Implemented as a web app and an R package, this tool is user-friendly, provides exportable results in different formats, and can be used for gene lists derived from multiple organisms. Here, we show the power of SubcellulaRVis to assign proteins to the correct subcellular compartment using gene list enriched in previously published spatial proteomics datasets. We envision SubcellulaRVis will be useful for cell biologists with limited bioinformatics expertise wanting to perform precise and quick enrichment analysis and immediate visualization of gene lists.

Spezifische Membrankontaktstellen bei Pflanzen: wer mit wem, warum und wie

Cellular membranes can serve as barriers between subcellular compartments, but they can also interact to form dynamically regulated membrane contact sites between a specific pair of organelles. Focussing on plants, this article discusses local redox environments and the current knowledge on membrane contact sites as examples for the dividing and connecting functions of membranes, respectively.

Massively parallel identification of zipcodes in primary cortical neurons

Cells adopt highly polarized shapes and form distinct subcellular compartments largely due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called "zipcodes." Their recognition by RNA-binding proteins (RBPs) leads to the integration of the mRNAs into macromolecular complexes and their localization. While there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here, we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3'UTRs. This approach combines a method of separating the principal subcellular compartments of neurons - cell bodies and neurites - with a massively parallel reporter assay. Our analysis identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons and suggests a strategy for detecting many more.

Wdr47, Camsaps, and Katanin cooperate to generate ciliary central microtubules

The axonemal central pair (CP) are non-centrosomal microtubules critical for planar ciliary beat. How they form, however, is poorly understood. Here, we show that mammalian CP formation requires Wdr47, Camsaps, and microtubule-severing activity of Katanin. Katanin severs peripheral microtubules to produce central microtubule seeds in nascent cilia. Camsaps stabilize minus ends of the seeds to facilitate microtubule outgrowth, whereas Wdr47 concentrates Camsaps into the axonemal central lumen to properly position central microtubules. Wdr47 deficiency in mouse multicilia results in complete loss of CP, rotatory beat, and primary ciliary dyskinesia. Overexpression of Camsaps or their microtubule-binding regions induces central microtubules in Wdr47−/− ependymal cells but at the expense of low efficiency, abnormal numbers, and wrong location. Katanin levels and activity also impact the central microtubule number. We propose that Wdr47, Camsaps, and Katanin function together for the generation of non-centrosomal microtubule arrays in polarized subcellular compartments.

A Functional Dissection of the mRNA and Locally Synthesized Protein Population in Neuronal Dendrites and Axons

Neurons are characterized by a complex morphology that enables the generation of subcellular compartments with unique biochemical and biophysical properties, such as dendrites, axons, and synapses. To sustain these different compartments and carry a wide array of elaborate operations, neurons express a diverse repertoire of gene products. Extensive regulation at both the messenger RNA (mRNA) and protein levels allows for the differentiation of subcellular compartments as well as numerous forms of plasticity in response to variable stimuli. Among the multiple mechanisms that control cellular functions, mRNA translation is manipulated by neurons to regulate where and when a protein emerges. Interestingly, transcriptomic and translatomic profiles of both dendrites and axons have revealed that the mRNA population only partially predicts the local protein population and that this relation significantly varies between different gene groups. Here, we describe the space that local translation occupies within the large molecular and regulatory complexity of neurons, in contrast to other modes of regulation. We then discuss the specialized organization of mRNAs within different neuronal compartments, as revealed by profiles of the local transcriptome. Finally, we discuss the features and functional implications of both locally correlated—and anticorrelated—mRNA-protein relations both under baseline conditions and during synaptic plasticity. Expected final online publication date for the Annual Review of Genetics, Volume 55 is November 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

An Algorithm to Quantify Inducible Protein Condensates In Eukaryotic Cells

Reorganization of cellular proteins into subcellular compartments, such as the rearrangement of RNA-binding proteins into cytoplasmic stress granules and P-bodies, is a well-recognized, widely studied physiological process currently under intense investigation. Using the assembly of a novel, inducible, nuclear granule formed from the east RNA-binding transcription termination factors Nab3 and Nrd1, we present a freely-accessible, high-throughput and unbiased algorithm written in MATLAB that detects and measures protein distribution, partitioning, and sequestration into subcellular compartments captured by fluorescence microscopy; an invaluable advancement to current image analysis methods which utilize experiment-specific custom scripts or subjective manual counting. Employing our algorithm, we quantified thousands of cells, ensuring rigorous examination of Nab3 granule formation across strains with reproducible statistical analyses. We document strain differences in Nab3 granule formation and an associated growth defect. Additionally, we applied our algorithm to immunofluorescent images of the inducible polymerization into filaments of an enzyme in human cells, demonstrating the algorithms versatility and adaptability.

DYT-TOR1A Subcellular Proteomics Reveals Selective Vulnerability of the Nuclear Proteome to Cell Stress

TorsinA is a AAA+ ATPase that shuttles between the ER lumen and outer nuclear envelope in an ATP-dependent manner and is functionally implicated in nucleocytoplasmic transport. We hypothesized that the DYT-TOR1A dystonia disease-causing variant, ΔE TorsinA, may therefore disrupt the normal subcellular distribution of proteins between the nuclear and cytosolic compartments. To test this hypothesis, we performed proteomic analysis on nuclear and cytosolic subcellular fractions from DYT-TOR1A and wildtype mouse embryonic fibroblasts (MEFs). We further examined the compartmental proteomes following exposure to thapsigargin (Tg), an endoplasmic reticulum (ER) stressor, because DYT-TOR1A dystonia models have previously shown abnormalities in cellular stress responses. Across both subcellular compartments, proteomes of DYT-TOR1A cells showed basal state disruptions consistent with an activated stress response, and in response to thapsigargin, a blunted stress response. However, the DYT-TOR1A nuclear proteome under Tg cell stress showed the most pronounced and disproportionate degree of protein disruptions - 3-fold greater than all other conditions. The affected proteins extended beyond those typically associated with stress responses, including enrichments for processes critical for neuronal synaptic function. These findings highlight the advantage of subcellular proteomics to reveal events that localize to discrete subcellular compartments and refine thinking about the mechanisms and significance of cell stress in DYT-TOR1A pathogenesis.

CamelliA-based simultaneous imaging of Ca2+ dynamics in subcellular compartments

As a universal second messenger, calcium (Ca2+) transmits specific cellular signals via a spatiotemporal signature generated from its extracellular source and internal stores. Our knowledge of the mechanisms underlying generation of a Ca2+ signature is hampered by limited tools enabling simultaneous monitoring of the dynamics of Ca2+ levels in multiple subcellular compartments. To overcome the limitation and to further improve spatiotemporal resolutions, here we have assembled a molecular toolset (the CamelliA lines) in Arabidopsis that enables simultaneous and high-resolution monitoring of Ca2+ dynamics in multiple subcellular compartments through imaging analyses of different single-colored GECIs (Genetically Encoded Calcium Indicators). Indeed, the uncovering of the previously unrecognized Ca2+ signatures in three types of Arabidopsis cells in response to internal and external cues is a testimony to the wide applicability of the newly generated toolset for elucidating the subcellular sources contributing to the Ca2+signatures in plants.