Mechanically induced localisation of SECONDARY WALL INTERACTING bZIP is associated with thigmomorphogenic and secondary cell wall gene expression.

Plant growth requires the integration of internal and external cues, perceived and transduced into a developmental programme of cell division, elongation and wall thickening. Mechanical forces contribute to this regulation, and thigmomorphogenesis typically includes reducing stem height, increasing stem diameter, and a canonical transcriptomic response. We present data on a bZIP transcription factor involved in this process in grasses. Brachypodium distachyon SECONDARY WALL INTERACTING bZIP (SWIZ) protein translocated into the nucleus following mechanostimulation. Classical touch-responsive genes were upregulated in B. distachyon roots following touch, including significant induction of the glycoside hydrolase 17 family, which may be unique to grass thigmomorphogenesis. SWIZ protein binding to an E-box variant in exons and introns was associated with immediate activation followed by repression of gene expression. SWIZ overexpression resulted in plants with reduced stem and root elongation. These data further define plant touch-responsive transcriptomics and physiology, offering insights into grass mechanotranduction dynamics.

Harmonizing the Generation and Pre-publication Stewardship of FAIR Image data.

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges, and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.

Micro-Meta App: an interactive tool for collecting microscopy metadata based on community specifications.

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.

QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy.

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.

Arc self-association and formation of virus-like capsids are mediated by an N-terminal helical coil motif.

Activity-regulated cytoskeleton-associated protein (Arc) is a protein interaction hub with diverse roles in intracellular neuronal signaling, and important functions in neuronal synaptic plasticity, memory, and postnatal cortical development. Arc has homology to retroviral Gag protein and is capable of self-assembly into virus-like capsids implicated in the intercellular transfer of RNA. However, the molecular basis of Arc self-association and capsid formation is largely unknown. Here, we identified a 28-amino-acid stretch in the mammalian Arc N-terminal (NT) domain that is necessary and sufficient for self-association. Within this region, we identified a 7-residue oligomerization motif, critical for the formation of virus-like capsids. Purified wild-type Arc formed capsids as shown by transmission and cryo-electron microscopy, whereas mutant Arc with disruption of the oligomerization motif formed homogenous dimers. An atomic-resolution crystal structure of the oligomerization region peptide demonstrated an antiparallel coiled-coil interface, strongly supporting NT-NT domain interactions in Arc oligomerization. The NT coil-coil interaction was also validated in live neurons using fluorescence lifetime FRET imaging, and mutation of the oligomerization motif disrupted Arc-facilitated endocytosis. Furthermore, using single-molecule photobleaching, we show that Arc mRNA greatly enhances higher-order oligomerization in a manner dependent on the oligomerization motif. In conclusion, a helical coil in the Arc NT domain supports self-association above the dimer stage, mRNA-induced oligomerization, and formation of virus-like capsids. DATABASE: The coordinates and structure factors for crystallographic analysis of the oligomerization region were deposited at the Protein Data Bank with the entry code 6YTU.

Live-cell FLIM-FRET using a commercially available system.

Förster resonance energy transfer (FRET)-based sensors have been powerful tools in cell biologists' toolkit for decades. Informed by fundamental understanding of fluorescent proteins, protein-protein interactions, and the structural biology of reporter components, researchers have been able to employ creative design approaches to build sensors that are uniquely capable of probing a wide range of phenomena in living cells including visualization of localized calcium signaling, sub-cellular activity gradients, and tension generation to name but a few. While FRET sensors have significantly impacted many fields, one must also be cognizant of the limitations to conventional, intensity-based FRET measurements stemming from variation in probe concentration, sensitivity to photobleaching, and bleed-through between the FRET fluorophores. Fluorescence lifetime imaging microscopy (FLIM) largely overcomes the limitations of intensity-based FRET measurements. In general terms, FLIM measures the time, which for the reporters described in this chapter is nanoseconds (ns), between photon absorption and emission by a fluorophore. When FLIM is applied to FRET sensors (FLIM-FRET), measurement of the donor fluorophore lifetime provides valuable information such as FRET efficiency and the percentage of reporters engaged in FRET. This chapter introduces fundamental principles of FLIM-FRET toward informing the practical application of the technique and, using two established FRET reporters as proofs of concept, outlines how to use a commercially available FLIM system.

Anionic Polymers Promote Mitochondrial Targeting of Delocalized Lipophilic Cations.

Mitochondria are therapeutic targets in many diseases including cancer, metabolic disorders, and neurodegenerative diseases. Therefore, strategies to deliver therapeutics of interest to mitochondria are important for therapeutic development. As delocalized lipophilic cations (DLCs) preferentially accumulate in mitochondria, DLC-conjugation has been utilized to facilitate therapeutic delivery systems with mitochondrial targeting capability. Here we report that upon DLC-conjugation, anionic polymers exhibit significantly improved mitochondrial targeting when compared to cationic polymers and charge-neutral polymers. Considering that the cell membrane generally bears a net negative charge, the observed phenomenon is unexpected. Notably, the DLC-conjugated anionic polymers circumvent endosomal entrapment. The rapid mitochondrial accumulation of DLC-conjugated anionic polymers is likely a membrane-potential-driven process, along with the involvement of the mitochondrial pyruvate carrier. Moreover, the structural variations on the side chain of DLC-conjugated anionic polymers do not compromise the overall mitochondrial targeting capability, widely extending the applicability of anionic macromolecules in therapeutic delivery systems.

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk.

According to the prevailing 'clock' model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the 'ruler' model. Here we found that Cdk1 remains active during anaphase due to ongoing APC/CCdc20- and APC/CCdh1-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular 'rulers' and 'clocks' licenses mitotic exit only after proper chromosome separation.

DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth.

In many model organisms, diffuse patterning of cell wall peptidoglycan synthesis by the actin homolog MreB enables the bacteria to maintain their characteristic rod shape. In Caulobacter crescentus and Escherichia coli, MreB is also required to sculpt this morphology de novo. Mycobacteria are rod-shaped but expand their cell wall from discrete polar or subpolar zones. In this genus, the tropomyosin-like protein DivIVA is required for the maintenance of cell morphology. DivIVA has also been proposed to direct peptidoglycan synthesis to the tips of the mycobacterial cell. The precise nature of this regulation is unclear, as is its role in creating rod shape from scratch. We find that DivIVA localizes nascent cell wall and covalently associated mycomembrane but is dispensable for the assembly process itself. Mycobacterium smegmatis rendered spherical by peptidoglycan digestion or by DivIVA depletion are able to regain rod shape at the population level in the presence of DivIVA. At the single cell level, there is a close spatiotemporal correlation between DivIVA foci, rod extrusion and concentrated cell wall synthesis. Thus, although the precise mechanistic details differ from other organisms, M. smegmatis also establish and propagate rod shape by cytoskeleton-controlled patterning of peptidoglycan. Our data further support the emerging notion that morphology is a hardwired trait of bacterial cells.

Modular chemical probes for visualizing and tracking endogenous ion channels.

Fluorescently labeled, small molecule ligands designed for the labeling and tracking of neuronal receptors have become an increasingly popular tool in neurobiology. The small size of these probes allows for subcellular imaging of proteins in their native state with minimal perturbation of the system. Several factors such as the selectivity of the pharmacophore, the size and composition of linkers used, and the fluorescence stability of the fluorophore can all influence the effectiveness of the small molecule probe. Here we discuss a few key molecular targets of this technology including the NMDA receptor, serotonin transporter, dopamine transporter, and adenosine receptor due to their involvement in numerous neurodegenerative diseases. Future iterations of these probes will allow for a better understanding of many important neurological proteins as well as the development of new and potent therapeutic drugs. This review will cover probe design considerations and discuss examples of specific small molecule fluorescent ligands that have been used to study a multitude of neuronal receptors through fluorescent imaging. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.

Ligand-directed delivery of fluorophores to track native calcium-permeable AMPA receptors in neuronal cultures.

Subcellular trafficking of neuronal receptors is known to play a key role in synaptic development, homeostasis, and plasticity. We have developed a ligand-targeted and photo-cleavable probe for delivering a synthetic fluorophore to AMPA receptors natively expressed in neurons. After a receptor is bound to the ligand portion of the probe molecule, a proteinaceous nucleophile reacts with an electrophile on the probe, covalently bonding the two species. The ligand may then be removed by photolysis, returning the receptor to its non-liganded state while leaving intact the new covalent bond between the receptor and the fluorophore. This strategy was used to label polyamine-sensitive receptors, including calcium-permeable AMPA receptors, in live hippocampal neurons from rats. Here, we describe experiments where we examined specificity, competition, and concentration on labeling efficacy as well as quantified receptor trafficking. Pharmacological competition during the labeling step with either a competitive or non-competitive glutamate receptor antagonist prevented the majority of labeling observed without a blocker. In other experiments, labeled receptors were observed to alter their locations and we were able to track and quantify their movements. We used a small molecule, ligand-directed probe to deliver synthetic fluorophores to endogenously expressed glutamate receptors for the purpose of tracking these receptors on live, hippocampal neurons. We found that clusters of receptors appear to move at similar rates to previous studies. We also found that the polyamine toxin pharmacophore likely binds to receptors in addition to calcium-permeable AMPA receptors.

Methods to locate and track ion channels and receptors expressed in live neurons.

Brain cells use electrical and chemical signaling to communicate with each other and to send and receive information from the body. These neurons also encode information such as memories and are constantly adapting to changes as a result of positive alterations, such as learning, or negative events, such as neurological insults or neurodegeneration. In the last two decades, it has become clear that the placement of minute branches of neurons and, more importantly for the topic of this review, the placement of individual protein molecules, are the key events that enable neuronal network building and pruning. Advances in both electrophysiology and light-based imaging have allowed neuroscientists to answer fundamental questions about the key proteins involved in memory formation, maintenance, and loss. These findings have been enabled often through the clever use of chemical biology, biotechnology, and genetic engineering. In this review, we highlight numerous examples where chemical biology was used to provide new tools to answer difficult and near impossible questions in neurobiology.

Engineering a light-regulated GABAA receptor for optical control of neural inhibition.

Optogenetics has become an emerging technique for neuroscience investigations owing to the great spatiotemporal precision and the target selectivity it provides. Here we extend the optogenetic strategy to GABAA receptors (GABAARs), the major mediators of inhibitory neurotransmission in the brain. We generated a light-regulated GABAA receptor (LiGABAR) by conjugating a photoswitchable tethered ligand (PTL) onto a mutant receptor containing the cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism. In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents. Moreover, it allowed reversible photocontrol over neuron excitation in response to presynaptic stimulation. LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.

A photoinduced, benzyne click reaction.

The [3 + 2] cycloaddition of azides and alkynes has proven invaluable across numerous scientific disciplines for imaging, cross-linking, and site-specific labeling among many other applications. We have developed a photoinitiated, benzyne-based [3 + 2] cycloaddition that is tolerant of a variety of functional groups as well as polar, protic solvents. The reaction is complete on the minute time scale using a single equivalent of partner azide, and the benzyne photoprecursor is stable for months under ambient light at room tempurature. Herein we report the optimization and scope of the photoinitiated reaction as well as characterization of the cycloaddition products.

Photoinitiated release of an aziridinium ion precursor for the temporally controlled alkylation of nucleophiles.

A photo-activatable aziridinium precursor has been developed to investigate the possibility of a photo-initiated traditional nucleophilic reaction. The photolysis of a quaternary amine yields a tertiary amine and has allowed us to temporally control aziridinium formation and subsequent alkylation of a colorimetric nucleophilic reporter molecule. We have also used this photo-initiated reaction to alkylate a sulfhydryl group. This new photo-initiated alkylation strategy is water-soluble and expands the toolkit of photo-activated crosslinkers for protein labeling research.