PIN2/3/4 auxin carriers mediate root growth inhibition under conditions of boron deprivation in Arabidopsis.

The mechanistic basis by which boron (B) deprivation inhibits root growth via the mediation of root apical auxin transport and distribution remains elusive. This study showed that B deprivation repressed root growth of wild-type Arabidopsis seedlings, which was related to higher auxin accumulation (observed with DII-VENUS and DR5-GFP lines) in B-deprived roots. Boron deprivation elevated the auxin content in the root apex, coinciding with upregulation of the expression levels of auxin biosynthesis-related genes (TAA1, YUC3, YUC9, and NIT1) in shoots, but not in root apices. Phenotyping experiments using auxin transport-related mutants revealed that the PIN2/3/4 carriers are involved in root growth inhibition caused by B deprivation. B deprivation not only upregulated the transcriptional levels of PIN2/3/4, but also restrained the endocytosis of PIN2/3/4 carriers (observed with PIN-Dendra2 lines), resulting in elevated protein levels of PIN2/3/4 in the plasma membrane. Overall, these results suggest that B deprivation not only enhances auxin biosynthesis in shoots by elevating the expression levels of auxin biosynthesis-related genes but also promotes the polar auxin transport from shoots to roots by upregulating the gene expression levels of PIN2/3/4, as well as restraining the endocytosis of PIN2/3/4 carriers, ultimately resulting in auxin accumulation in root apices and root growth inhibition.

Shining a light on autophagy in neurodegenerative diseases.

Small-molecule modulators of autophagy have been widely investigated as potential therapies for neurodegenerative diseases. In a recent issue of JBC, Safren et al. described a novel assay that uses a photoconvertible fusion protein to identify compounds that alter autophagic flux. Autophagy inducers identified using this assay were found to either alleviate or exacerbate neurotoxicity in different cellular models of amyotrophic lateral sclerosis, challenging the notion that autophagy stimulation can be used as a one-size-fits-all therapy for neurodegenerative disease.

Abscisic Acid Acts as a Regulator of Molecular Trafficking through Plasmodesmata in the Moss Physcomitrella patens.

In multi-cellular organisms, cell-to-cell communication is crucial for adapting to changes in the surrounding environment. In plants, plasmodesmata (PD) provide a unique pathway for cell-to-cell communication. PD interconnect most cells and generate a cytoplasmic continuum, allowing the trafficking of various micro- and macromolecules between cells. This molecular trafficking through PD is dynamically regulated by altering PD permeability dependent on environmental changes, thereby leading to an appropriate response to various stresses; however, how PD permeability is dynamically regulated is still largely unknown. Moreover, studies on the regulation of PD permeability have been conducted primarily in a limited number of angiosperms. Here, we studied the regulation of PD permeability in the moss Physcomitrella patens and report that molecular trafficking through PD is rapidly and reversibly restricted by abscisic acid (ABA). Since ABA plays a key role in various stress responses in the moss, PD permeability can be controlled by ABA to adapt to surrounding environmental changes. This ABA-dependent restriction of PD trafficking correlates with a reduction in PD pore size. Furthermore, we also found that the rate of macromolecular trafficking is higher in an ABA-synthesis defective mutant, suggesting that the endogenous level of ABA is also important for PD-mediated macromolecular trafficking. Thus, our study provides compelling evidence that P. patens exploits ABA as one of the key regulators of PD function.

PML-Nuclear Bodies Regulate the Stability of the Fusion Protein Dendra2-Nrf2 in the Nucleus.

BACKGROUND/AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine-zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. METHODS: Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2-Nrf2 in single live cells. RESULTS: Using this approach, we show that the half-life (t1/2) of Dendra2-Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG-132 or induction of oxidative stress with tert-butylhydroquinone (tBHQ) extended the half-life of Dendra2-Nrf2 by 6- and 28-fold, respectively. By inhibiting nuclear export using Leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML-NBs (Promyelocytic Leukemia-nuclear bodies). We further demonstrate that co-expression of Dendra2-Nrf2 and Crimson-PML-I lacking two PML-I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2-Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. CONCLUSION: Altogether, our findings provide direct evidence for degradation of Nrf2 in the nucleus and suggest that modification of Nrf2 in PML nuclear bodies contributes to its degradation in intact cells.

Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor.

Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5. Thus, photoconverted form of Dendra2 can be potentially used as a ratiometric pH-sensor in the physiological pH range. However, incomplete photoconversion makes ratiometric measurements irreproducible when using standard filter sets. Here, we describe the method to detect fluorescence of only photoconverted Dendra2 form, but not nonconverted green Dendra2. We show that the 350 nm excitation light induces solely the fluorescence of photoconverted protein. By measuring the red to green fluorescence ratio, we determined intracellular pH in live CHO and HEK 293 cells. Thus, Dendra2 can be used as a novel ratiometric genetically encoded pH sensor with emission maxima in the green-red spectral region, which is suitable for application in live cells.

Axially-confined in vivo single-cell labeling by primed conversion using blue and red lasers with conventional confocal microscopes.

Green-to-red photoconvertible fluorescent proteins have been found to undergo efficient photoconversion by a new method termed primed conversion that uses dual wave-length illumination with blue and red/near-infrared light. By modifying a confocal laser-scanning microscope (CLSM) such that two laser beams only meet at the focal plane, confined photoconversion at the axial dimension has been achieved. The necessity of this custom modification to the CLSM, however, has precluded the wide-spread use of this method. Here, we investigated whether spatially-restricted primed conversion could be achieved with CLSM without any hardware modification. We found that the primed conversion of Dendra2 using a conventional CLSM with two visible lasers (473 nm and 635 nm) and a high NA objective lens (NA, 1.30) resulted in dramatic restriction of photoconversion volume: half-width half-maximum for the axial dimension was below 5 µm, which is comparable to the outcome of the original method that used the microscope modification. As a proof of this method's effectiveness, we used this technique in living zebrafish embryos and succeeded in revealing the complex anatomy of individual neurons packed between neighboring cells. Because unmodified CLSMs are widely available, this method can be widely applicable for labeling cells with single-cell resolution.

Photo-convertible fluorescent proteins as tools for fresh insights on subcellular interactions in plants.

Optical highlighters comprise photo-activatable, photo-switchable and photo-convertible fluorescent proteins and are relatively recent additions to the toolbox utilized for live cell imaging research. Here, we provide an overview of four photo-convertible fluorescent proteins (pcFP) that are being used in plant cell research: Eos, Kaede, Maple and Dendra2. Each of these proteins has a significant advantage over other optical highlighters since their green fluorescent nonconverted forms and red fluorescent converted forms are generally clearly visible at expression levels that do not appear to interfere with subcellular dynamics and plant development. These proteins have become increasingly useful for understanding the role of transient and sustained interactions between similar organelles. Tracking of single organelles after green-to-red conversion has provided novel insights on plastids and their stroma-filled extensions and on the formation of mega-mitochondria. Similarly colour recovery after photo-conversion has permitted the estimation of nuclear endo-reduplication events and is being developed further to image protein trafficking within the lumen of the endoplasmic reticulum. We have also applied photo-convertible proteins to create colour-differentiation between similar cell types to follow their development. Both the green and red fluorescent forms of these proteins are compatible with other commonly used single coloured FPs. This has allowed us to develop simultaneous visualization schemes for up to five types of organelles and investigate organelle interactivity. The advantages and caveats associated with the use of photo-convertible fluorescent proteins are discussed.

Photo-convertible tagging for localization and dynamic analyses of low-expression proteins in filamentous fungi.

Photo-convertible fluorescent proteins (PCFPs) undergo a dramatic change in their excitation and emission spectra upon irradiation at specific wavelengths, thus rendering a different color. Dendra2 is a commercially available PCFP used to track the redistribution of proteins within cellular compartments, their life-time or interactions. Before photo-conversion Dendra2 exhibits green fluorescence, which becomes red after irradiation with either UV or blue lights. Multiple studies including Dendra2 as a molecular tool have been described in eukaryotes but not in filamentous fungi. Here we present a method to tag low-expression proteins from the filamentous fungus Aspergillus nidulans with Dendra2 and track their cellular dynamics. The regulator of asexual development FlbB was selected as control, a transcription factor that is expressed at low levels and can be used as a marker for the tip and nuclei of vegetative hyphae. This control provided us with a visual way to confirm the functionality of our genomic and plasmid constructs, since a non-functional FlbB protein renders a block in development and a characteristic aconidial phenotype. Our protocol combines standardized cloning and transformation procedures with the use of a mercury lamp microscope to convert and follow Dendra2 within cells. Hence, we present a rapid, simple and inexpensive method that makes tracking analysis of proteins that present technical difficulties to be followed feasible in filamentous fungi.

Tracking protein dynamics with photoconvertible Dendra2 on spinning disk confocal systems.

Understanding the dynamic properties of cellular proteins in live cells and in real time is essential to delineate their function. In this context, we introduce the Fluorescence Recovery After Photobleaching-Photoactivation unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk (Andor) confocal microscope as an advantageous and robust platform to exploit the properties of the Dendra2 photoconvertible fluorescent protein (Evrogen) and analyse protein subcellular trafficking in living cells. A major advantage of the spinning disk confocal is the rapid acquisition speed, enabling high temporal resolution of cellular processes. Furthermore, photoconversion and imaging are less invasive on the spinning disk confocal as the cell exposition to illumination power is reduced, thereby minimizing photobleaching and increasing cell viability. We have tested this commercially available platform using experimental settings adapted to track the migration of fast trafficking proteins such as UBC9, Fibrillarin and have successfully characterized their differential motion between subnuclear structures. We describe here step-by-step procedures, with emphasis on cellular imaging parameters, to successfully perform the dynamic imaging and photoconversion of Dendra2-fused proteins at high spatial and temporal resolutions necessary to characterize the trafficking pathways of proteins.

Transgenic Dendra2::tau expression allows in vivo monitoring of tau proteostasis in Caenorhabditis elegans.

Protein homeostasis is perturbed in aging-related neurodegenerative diseases called tauopathies, which are pathologically characterized by aggregation of the microtubule-associated protein tau (encoded by the human MAPT gene). Transgenic Caenorhabditis elegans serve as a powerful model organism to study tauopathy disease mechanisms, but moderating transgenic expression level has proven problematic. To study neuronal tau proteostasis, we generated a suite of transgenic strains expressing low, medium or high levels of Dendra2::tau fusion proteins by comparing integrated multicopy transgene arrays with single-copy safe-harbor locus strains generated by recombinase-mediated cassette exchange. Multicopy Dendra2::tau strains exhibited expression level-dependent neuronal dysfunction that was modifiable by known genetic suppressors or an enhancer of tauopathy. Single-copy Dendra2::tau strains lacked distinguishable phenotypes on their own but enabled detection of enhancer-driven neuronal dysfunction. We used multicopy Dendra2::tau strains in optical pulse-chase experiments measuring tau turnover in vivo and found that Dendra2::tau turned over faster than the relatively stable Dendra2. Furthermore, Dendra2::tau turnover was dependent on the protein expression level and independent of co-expression with human TDP-43 (officially known as TARDBP), an aggregating protein interacting with pathological tau. We present Dendra2::tau transgenic C. elegans as a novel tool for investigating molecular mechanisms of tau proteostasis.

A Lamb1Dendra2 mouse model identifies basement-membrane-producing origins and dynamics in PyMT breast tumors.

The basement membrane (BM) around tumor lobes forms a barrier to prevent cancer cells from invading the surrounding tissue. Although myoepithelial cells are key producers of the healthy mammary epithelium BM, they are nearly absent in mammary tumors. To study the origin and dynamics of the BM, we developed and imaged a laminin beta1-Dendra2 mouse model. We show that the turnover of laminin beta1 is faster in the BMs that surround the tumor lobes than in the BMs that surround the healthy epithelium. Moreover, we find that epithelial cancer cells and tumor-infiltrating endothelial cells synthesize laminin beta1 and that this production is temporarily and locally heterogeneous, leading to local discontinuity of the BM laminin beta1. Collectively, our data draw a new paradigm for tumor BM turnover in which the disassembly happens at a constant rate, and a local misbalance of compensating production leads to reduction or even complete disappearance of the BM.

CRISPR Based Programmable RNA Editing in Primary Hippocampal Neurons.

Investigating the RNA regulation landscape primarily relies on understanding how RNA-protein interactions are governed in various cell types, including neurons. Analysis of RNA-protein interactions in physiological environments warrants the development of new tools that rely on RNA manipulation. Recently, a CRISPR-based RNA-editing tool (dCas13b-ADAR2DD ) was developed to mitigate disease-associated point mutations in cell lines. Here, we explored the targeted sequence editing potential of the tool (dCas13b-ADAR2DD system) by adapting it to manipulate RNA function to visualize RNA editing in primary hippocampal neurons. This two-component system includes a programmable guide RNA (gRNA) complementary to the target RNA and a catalytically dead version of the Cas13b enzyme fused to ADAR. The RNA editing protocol outlined in this article relies on gRNA-dependent targeting of the dCas13b-ADAR fusion protein to the mutant form of the Dendra2 transcript. Dendra2 is not required for intrinsic cellular functioning. It was ectopically expressed for fluorescent detection as a proof-of-principle demonstration of targeted RNA editing. We first abrogated the fluorescence of Dendra2 by introducing a nonsense mutation that precludes the formation of the functional protein. To visualize the efficacy of the RNA editing in neurons, we used the dCas13b-ADAR2DD system to edit specific nucleotides within the Dendra2 mRNA to restore the amino acid codes critical for Dendra2 fluorescence. This method lays the foundation for future studies on the dynamics of activity-induced RNA-protein interactions in neurons and can be extended to manipulate the endogenous RNome in diverse neuronal subtypes. Furthermore, this methodology will enable investigators to visualize the spatial and temporal resolution of RNA-protein interactions without altering the genomes via conventional methods. © 2023 Wiley Periodicals LLC. Support Protocol: Preparation of mouse primary hippocampal culture Basic Protocol: Targeted editing of RNA.

Tracking Intercellular Movement of Fluorescent Proteins in Bryophytes.

An important approach to investigate intercellular connectivity via plasmodesmata is to visualize and track the movement of fluorescent proteins between cells. The intercellular connectivity is largely controlled by the size exclusion limit of the pores. Over the past few decades, the technique to observe and analyze intercellular movement of a fluorescent protein has been developed mainly in angiosperms such as Arabidopsis thaliana. We recently applied the corresponding system to track the intercellular movement of the fluorescent protein Dendra2 in the moss Physcomitrium (Physcomitrella) patens. The protonemal tissues are particularly suited for observation of the intercellular movement due to the simple organization. Here, we describe a protocol suitable for the analysis of Dendra2 movement between cells in P. patens.

The Olfactory Organ Is a Unique Site for Neutrophils in the Brain.

In the vertebrate olfactory tract new neurons are continuously produced throughout life. It is widely believed that neurogenesis contributes to learning and memory and can be regulated by immune signaling molecules. Proteins originally identified in the immune system have subsequently been localized to the developing and adult nervous system. Previously, we have shown that olfactory imprinting, a specific type of long-term memory, is correlated with a transcriptional response in the olfactory organs that include up-regulation of genes associated with the immune system. To better understand the immune architecture of the olfactory organs we made use of cell-specific fluorescent reporter lines in dissected, intact adult brains of zebrafish to examine the association of the olfactory sensory neurons with neutrophils and blood-lymphatic vasculature. Surprisingly, the olfactory organs contained the only neutrophil populations observed in the brain; these neutrophils were localized in the neural epithelia and were associated with the extensive blood vasculature of the olfactory organs. Damage to the olfactory epithelia resulted in a rapid increase of neutrophils both within the olfactory organs as well as the central nervous system. Analysis of cell division during and after damage showed an increase in BrdU labeling in the neural epithelia and a subset of the neutrophils. Our results reveal a unique population of neutrophils in the olfactory organs that are associated with both the olfactory epithelia and the lymphatic vasculature suggesting a dual olfactory-immune function for this unique sensory system.

Quantitative Analysis of the Cytoskeleton's Role in Inward Rectifier K IR 2.1 Forward and Backward Trafficking.

Alteration of the inward rectifier current I K1, carried by KIR2.1 channels, affects action potential duration, impacts resting membrane stability and associates with cardiac arrhythmias. Congenital and acquired KIR2.1 malfunction frequently associates with aberrant ion channel trafficking. Cellular processes underlying trafficking are intertwined with cytoskeletal function. The extent to which the cytoskeleton is involved in KIR2.1 trafficking processes is unknown. We aimed to quantify the dependence of KIR2.1 trafficking on cytoskeleton function. GFP or photoconvertible Dendra2 tagged KIR2.1 constructs were transfected in HEK293 or HeLa cells. Photoconversion of the Dendra2 probe at the plasma membrane and subsequent live imaging of trafficking processes was performed by confocal laser-scanning microscopy. Time constant of green fluorescent recovery (τg,s) represented recruitment of new KIR2.1 at the plasma membrane. Red fluorescent decay (τr,s) represented internalization of photoconverted KIR2.1. Patch clamp electrophysiology was used to quantify I KIR2.1. Biochemical methods were used for cytoskeleton isolation and detection of KIR2.1-cytoskeleton interactions. Cytochalasin B (20 µM), Nocodazole (30 µM) and Dyngo-4a (10 nM) were used to modify the cytoskeleton. Chloroquine (10 µM, 24 h) was used to impair KIR2.1 breakdown. Cytochalasin B and Nocodazole, inhibitors of actin and tubulin filament formation respectively, strongly inhibited the recovery of green fluorescence at the plasma membrane suggestive for inhibition of KIR2.1 forward trafficking [τg,s 13 ± 2 vs. 131 ± 31* and 160 ± 40* min, for control, Cytochalasin B and Nocodazole, respectively (*p < 0.05 vs. control)]. Dyngo-4a, an inhibitor of dynamin motor proteins, strongly slowed the rate of photoconverted channel internalization, whereas Nocodazole and Cytochalasin B had less effect [τr,s 20 ± 2 vs. 87 ± 14*, 60 ± 16 and 64 ± 20 min (*p < 0.05 vs. control)]. Cytochalasin B treatment (20 µM, 24 h) inhibited I KIR2.1. Chloroquine treatment (10 µM, 24 h) induced intracellular aggregation of KIR2.1 channels and enhanced interaction with the actin/intermediate filament system (103 ± 90 fold; p < 0.05 vs. control). Functional actin and tubulin cytoskeleton systems are essential for forward trafficking of KIR2.1 channels, whereas initial backward trafficking relies on a functional dynamin system. Chronic disturbance of the actin system inhibits KIR2.1 currents. Internalized KIR2.1 channels become recruited to the cytoskeleton, presumably in lysosomes.

Fate Mapping of Cancer Cells in Metastatic Lymph Nodes Using Photoconvertible Proteins.

The lymph node microenvironment is extremely dynamic and responds to immune stimuli in the host by reprogramming immune, stromal, and endothelial cells. In normal physiological conditions, the lymph node will initiate an appropriate immune response to clear external threats that the host may experience. However, in metastatic disease, cancer cells often colonize local lymph nodes, disrupt immune function, and even leave the lymph node to create additional metastases. Understanding how cancer cells enter, colonize, survive, proliferate, and interact with other cell types in the lymph node is challenging. Here, we describe the use of photoconvertible fluorescent proteins to label and trace the fate of cancer cells once they enter the lymph node.

CRISPR-Cas9-mediated labelling of the C-terminus of human laminin ß1 leads to secretion inhibition.

OBJECTIVES: The laminins (LM) are a family of basement membranes glycoproteins with essential roles in supporting epithelia, endothelia, nerves and muscle adhesion, and in regulating a range of processes including cell migration, stem cell maintenance and differentiation. However, surprisingly little is known about the mechanisms of turnover and remodelling of LM networks due to lack of appropriate tools to study these processes at the necessary resolution. Recently, the nematode C. elegans ortholog of human the LMß1 chain was labelled at the C-terminus with the photoconvertible fluorophore Dendra2. Here we used genome editing to establish a similar system in a mammalian cell line as proof of concept for future mammalian models. RESULTS: CRISPR-Cas9 was used to introduce the Dendra2 sequence at the C-terminus of LMß1 in the human lung adenocarcinoma cell line A549. Despite expression of the tagged protein within cells, no detectable LMß1-Dendra2 protein was deposited to the extracellular matrices or conditioned media of edited cells. Moreover, the edited cells displayed reduced proliferation rates. Together, these data suggest that, in humans, addition of C-terminal Dendra2 tag to LMß1 inhibits LM secretion, and is not a viable approach for use in animal models.

Measuring Exocytosis Rate in Arabidopsis Pollen Tubes Using Corrected Fluorescence Recovery After Photoconversion (cFRAPc) Technique.

Exocytosis is a fundamental process essential for many cellular functions by targeting signal peptides, proteins, and cell wall components to the plasma membrane (PM) or extracellular matrix. Conventional methods, such as FRAP, often underestimate the exocytosis rate of a specific molecule, because retrieval of the molecules from the PM by endocytosis can impact the measurement. To overcome this issue, we have previously established a novel method, corrected fluorescence recovery after photoconversion (cFRAPc), which allows us to accurately measure the exocytosis rate by monitoring both exocytosis-dependent and exocytosis-independent events. In this chapter, we provide detailed procedures for the cFRAPc method to measure the exocytosis rate of Arabidopsis receptor-like kinase PRK1 in pollen tubes. This method should be widely applicable to various cell types.

Relationship between relocation of phototropin to the chloroplast periphery and the initiation of chloroplast movement in Marchantia polymorpha.

The blue-light photoreceptor kinase phototropin (phot) mediates chloroplast movement in response to light and temperature. Phot predominantly localizes at the plasma membrane, but also resides in the cytosol and the chloroplast periphery. Although the phot localized to the chloroplast periphery is thought to mediate chloroplast movement, the localization mechanism is unknown. In this study, we found that chloroplast movement does not occur in 0-day-old gemma cells of the liverwort Marchantia polymorpha but that the movement is induced in 1-day-old gemmaling cells. Along with this physiological change, the subcellular localization of phot also changed: In 0-day-old gemma cells, phot localized at the plasma membrane and the cytosol, but in 1-day-old gemmaling cells, the phot disappeared from the cytosol and appeared at the chloroplast periphery. When the relocalization was tracked using a photoconvertible fluorescent protein, the cytosolic phot relocated to the plasma membrane, and the plasma membrane-resident phot relocated to the chloroplast periphery. The blue-light-dependent activation of phot kinase activity enhanced this relocalization. Mutated phot deficient in blue-light reception or kinase activity had a severely reduced ability to localize at the chloroplast periphery. These findings suggest that photoactivated phot localizes at the chloroplast periphery to initiate chloroplast movement.

The Photoconvertible Fluorescent Protein Dendra2 Tag as a Tool to Investigate Intracellular Protein Dynamics.

Fluorescence proteins changing spectral properties after exposure to light with a specific wavelength have recently become outstanding aids in the study of intracellular protein dynamics. Herein we show using Arabidopsis SYNAPTOTAGMIN 1 as a model protein that the Dendra2 green to red photoconvertible protein tag in combination with confocal scanning laser microscopy is a useful tool to study membrane protein intracellular dynamics.