Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

University of Delaware

We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process. 

An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy.

Inter-organellar communication is vital for successful innate immune responses that confer defense against pathogens. However, little is known about how chloroplasts, which are a major production site of pro-defense molecules, communicate and coordinate with other organelles during defense. Here we show that chloroplasts send out dynamic tubular extensions called stromules during innate immunity or exogenous application of the pro-defense signals, hydrogen peroxide (H2O2) and salicylic acid. Interestingly, numerous stromules surround nuclei during defense response, and these connections correlate with an accumulation of chloroplast-localized NRIP1 defense protein and H2O2 in the nucleus. Furthermore, silencing and knockout of chloroplast unusual positioning 1 (CHUP1) that encodes a chloroplast outer envelope protein constitutively induces stromules in the absence of pathogen infection and enhances programmed cell death. These results support a model in which stromules aid in the amplification and/or transport of pro-defense signals into the nucleus and other subcellular compartments during immunity.

Chloroplast Stromules Function during Innate Immunity.

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA-mRNA interactions combined with a fluorophore-binding sequence 'Spinach', a GFP-like RNA aptamer for which the RNA-fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo.

FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs.

Alleles that confer multiple disease resistance (MDR) are valuable in crop improvement, although the molecular mechanisms underlying their functions remain largely unknown. A quantitative trait locus, qMdr, associated with resistance to three important foliar maize diseases-southern leaf blight, gray leaf spot and northern leaf blight-has been identified on maize chromosome 9. Through fine-mapping, association analysis, expression analysis, insertional mutagenesis and transgenic validation, we demonstrate that ZmCCoAOMT2, which encodes a caffeoyl-CoA O-methyltransferase associated with the phenylpropanoid pathway and lignin production, is the gene within qMdr conferring quantitative resistance to both southern leaf blight and gray leaf spot. We suggest that resistance might be caused by allelic variation at the level of both gene expression and amino acid sequence, thus resulting in differences in levels of lignin and other metabolites of the phenylpropanoid pathway and regulation of programmed cell death.

A gene encoding maize caffeoyl-CoA O-methyltransferase confers quantitative resistance to multiple pathogens.

Contents Summary 1012 I. Introduction 1012 II. The endomembrane system in plant-microbe interactions 1013 III. The cytoskeleton in plant-microbe interactions 1017 IV. Organelles in plant-microbe interactions 1019 V. Inter-organellar communication in plant-microbe interactions 1022 VI. Conclusions and prospects 1023 Acknowledgements 1024 References 1024 SUMMARY: Plants have evolved a multilayered immune system with well-orchestrated defense strategies against pathogen attack. Multiple immune signaling pathways, coordinated by several subcellular compartments and interactions between these compartments, play important roles in a successful immune response. Pathogens use various strategies to either directly attack the plant's immune system or to indirectly manipulate the physiological status of the plant to inhibit an immune response. Microscopy-based approaches have allowed the direct visualization of membrane trafficking events, cytoskeleton reorganization, subcellular dynamics and inter-organellar communication during the immune response. Here, we discuss the contributions of organelles and the cytoskeleton to the plant's defense response against microbial pathogens, as well as the mechanisms used by pathogens to target these compartments to overcome the plant's defense barrier.

Plant-microbe interactions: organelles and the cytoskeleton in action.

Dynamic tubular extensions from chloroplasts called stromules have recently been shown to connect with nuclei and function during innate immunity. We demonstrate that stromules extend along microtubules (MTs) and MT organization directly affects stromule dynamics since stabilization of MTs chemically or genetically increases stromule numbers and length. Although actin filaments (AFs) are not required for stromule extension, they provide anchor points for stromules. Interestingly, there is a strong correlation between the direction of stromules from chloroplasts and the direction of chloroplast movement. Stromule-directed chloroplast movement was observed in steady-state conditions without immune induction, suggesting it is a general function of stromules in epidermal cells. Our results show that MTs and AFs may facilitate perinuclear clustering of chloroplasts during an innate immune response. We propose a model in which stromules extend along MTs and connect to AF anchor points surrounding nuclei, facilitating stromule-directed movement of chloroplasts to nuclei during innate immunity.

Stromule extension along microtubules coordinated with actin-mediated anchoring guides perinuclear chloroplast movement during innate immunity.

The development of genetically encoded fluorescent probes for analyte-specific imaging has revolutionized our understanding of intracellular processes. Current classes of intracellular probes depend on the selection of binding domains that either undergo conformational changes on analyte binding or can be linked to thiol redox chemistry. Here we have designed novel probes by fusing a flavoenzyme, whose fluorescence is quenched on reduction by the analyte of interest, with a GFP domain to allow for rapid and specific ratiometric sensing. Two flavoproteins, Escherichia coli thioredoxin reductase and Saccharomyces cerevisiae lipoamide dehydrogenase, were successfully developed into thioredoxin and NAD/NADH specific probes, respectively, and their performance was evaluated in vitro and in vivo. A flow cell format, which allowed dynamic measurements, was utilized in both bacterial and mammalian systems. In E. coli the first reported intracellular steady-state of the cytoplasmic thioredoxin pool was measured. In HEK293T mammalian cells, the steady-state cytosolic ratio of NAD/NADH induced by glucose was determined. These genetically encoded fluorescent constructs represent a modular approach to intracellular probe design that should extend the range of metabolites that can be quantitated in live cells.

Designing Flavoprotein-GFP Fusion Probes for Analyte-Specific Ratiometric Fluorescence Imaging.

Considering their sessile life, plants must efficiently coordinate their resources and energy for maintaining their presence in normal living conditions and for defending themselves against environmental threats. Collaboration between multiple subcellular compartments is a common strategy in several biological processes to modify cells' architecture for their growth and development and to respond to acute changes in the environment. When plants defend themselves against microbial pathogens, chloroplasts generate tubular structures - so-called stromules- to facilitate chloroplast movement towards nuclei during innate immunity. Morphological changes and movements of stromules are directed by interactions with microtubule and actin cytoskeleton. Microtubules provide a direction for the stromule extension, while actin filaments restrict stromule retraction which provides a driving force for repositioning of chloroplast near nucleus during plant immune responses. These findings implicated a critical role for stromules in signal transduction from chloroplast to the nucleus in plant defense.

Dynamic coordination of plastid morphological change by cytoskeleton for chloroplast-nucleus communication during plant immune responses.

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA-FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co-localization of miR2275 and a 24-nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi-photon fluorescence excitation microscopy can be used to separate the target sRNA-FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA-FISH signals can be imaged using super-resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super-resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step-by-step sRNA-FISH protocol for studying sRNAs at the cellular and even subcellular level.

sRNA-FISH: versatile fluorescent in situ detection of small RNAs in plants.

Upon phagocytosis into macrophages, the intracellular bacterial pathogen  secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the  effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein-lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P)  We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the -containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of  within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of , RavD is a part of the molecular mechanism that steers the containing vacuole away from endolysosomal maturation pathways.

The  effector RavD binds phosphatidylinositol-3-phosphate and helps suppress endolysosomal maturation of the -containing vacuole.

The dopamine transporter (DAT) is a plasma membrane protein responsible for the uptake of released dopamine back to the presynaptic terminal and ending dopamine neurotransmission. The DAT is the molecular target for cocaine and amphetamine as well as a number of pathological conditions including autism spectrum disorders, attention-deficit hyperactivity disorder (ADHD), dopamine transporter deficiency syndrome (DTDS), and Parkinson's disease. The DAT uptake capacity is dependent on its level in the plasma membrane.  studies show that DAT functional expression is regulated by a balance of endocytosis, recycling, and lysosomal degradation. However, recent reports suggest that DAT regulation by endocytosis in neurons is less significant than previously reported. Therefore, additional mechanisms appear to determine DAT steady-state level and functional expression in the neuronal plasma membrane. Here, we hypothesize that the ubiquitin-like protein small ubiquitin-like modifier 1 (SUMO1) increases the DAT steady-state level in the plasma membrane. In confocal microscopy, fluorescent resonance energy transfer (FRET), and Western blot analyses, we demonstrate that DAT is associated with SUMO1 in the rat dopaminergic N27 and DAT overexpressing Human Embryonic Kidney cells (HEK)-293 cells. The overexpression of SUMO1 and the Ubc9 SUMO-conjugase induces DAT SUMOylation, reduces DAT ubiquitination and degradation, enhancing DAT steady-state level. In addition, the Ubc9 knock-down by interference RNA (RNAi) increases DAT degradation and reduces DAT steady-state level. Remarkably, the Ubc9-mediated SUMOylation increases the expression of DAT in the plasma membrane and dopamine uptake capacity. Our results strongly suggest that SUMOylation is a novel mechanism that plays a central role in regulating DAT proteostasis, dopamine uptake, and dopamine signaling in neurons. For that reason, the SUMO pathway including SUMO1, SUMO2, Ubc9, and DAT SUMOylation, can be critical therapeutic targets in regulating DAT stability and dopamine clearance in health and pathological states.

The SUMO-Conjugase Ubc9 Prevents the Degradation of the Dopamine Transporter, Enhancing Its Cell Surface Level and Dopamine Uptake.

Mitochondrial Ca uniporter (MCU)-mediated Ca uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCU) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca (Ca) uptake, delays cytosolic Ca (Ca) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCU were a direct result of extramitochondrial Ca-dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCU hepatocytes. Conversely, gain-of-function MCU promotes rapid Ca uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca dynamics to hepatic lipid metabolism.

Jeffrey Caplan

Plant and Soil Sciences,
Delaware Biotechnology Institute,
Plant and Soil Sciences, Delaware Biotechnology Institute

University of Delaware

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Jeffrey Caplan

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University of Delaware

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Plant and Soil Sciences,
Delaware Biotechnology Institute,
Plant and Soil Sciences, Delaware Biotechnology Institute