Transcriptional and functional motifs defining renal function revealed by single-nucleus RNA sequencing.

Recent advances in single-cell sequencing provide a unique opportunity to gain novel insights into the diversity, lineage, and functions of cell types constituting a tissue/organ. Here, we performed a single-nucleus study of the adult Drosophila renal system, consisting of Malpighian tubules and nephrocytes, which shares similarities with the mammalian kidney. We identified 11 distinct clusters representing renal stem cells, stellate cells, regionally specific principal cells, garland nephrocyte cells, and pericardial nephrocytes. Characterization of the transcription factors specific to each cluster identified fruitless (fru) as playing a role in stem cell regeneration and Hepatocyte nuclear factor 4 (Hnf4) in regulating glycogen and triglyceride metabolism. In addition, we identified a number of genes, including Rho guanine nucleotide exchange factor at 64C (RhoGEF64c), Frequenin 2 (Frq2), Prip, and CG1093 that are involved in regulating the unusual star shape of stellate cells. Importantly, the single-nucleus dataset allows visualization of the expression at the organ level of genes involved in ion transport and junctional permeability, providing a systems-level view of the organization and physiological roles of the tubules. Finally, a cross-species analysis allowed us to match the fly kidney cell types to mouse kidney cell types and planarian protonephridia, knowledge that will help the generation of kidney disease models. Altogether, our study provides a comprehensive resource for studying the fly kidney.

Enhanced Production of the Mical Redox Domain for Enzymology and F-actin Disassembly Assays.

To change their behaviors, cells require actin proteins to assemble together into long polymers/filaments-and so a critical goal is to understand the factors that control this actin filament (F-actin) assembly and stability. We have identified a family of unusual actin regulators, the MICALs, which are flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in Redox reactions. F-actin is a specific substrate for these MICAL Redox enzymes, which oxidize specific amino acids within actin to destabilize actin filaments. Furthermore, this MICAL-catalyzed reaction is reversed by another family of Redox enzymes (SelR/MsrB enzymes)-thereby revealing a reversible Redox signaling process and biochemical mechanism regulating actin dynamics. Interestingly, in addition to the MICALs' Redox enzymatic portion through which MICALs covalently modify and affect actin, MICALs have multiple other domains. Less is known about the roles of these other MICAL domains. Here we provide approaches for obtaining high levels of recombinant protein for the Redox only portion of Mical and demonstrate its catalytic and F-actin disassembly activity. These results provide a ground state for future work aimed at defining the role of the other domains of Mical - including characterizing their effects on Mical's Redox enzymatic and F-actin disassembly activity.

Defining cell types and lineage in the Drosophila midgut using single cell transcriptomics.

The Drosophila midgut has emerged in recent years as a model system to study stem cell renewal and differentiation and tissue homeostasis. Histological, genetic and gene expression studies have provided a wealth of information on gut cell types, regionalization, genes and pathways involved in cell proliferation and differentiation, stem cell renewal, and responses to changes in environmental factors such as the microbiota and nutrients. Here, we review the contribution of single cell transcriptomic methods to our understanding of gut cell type diversity, lineage and behavior.

A single-cell survey of Drosophila blood.

Drosophila blood cells, called hemocytes, are classified into plasmatocytes, crystal cells, and lamellocytes based on the expression of a few marker genes and cell morphologies, which are inadequate to classify the complete hemocyte repertoire. Here, we used single-cell RNA sequencing (scRNA-seq) to map hemocytes across different inflammatory conditions in larvae. We resolved plasmatocytes into different states based on the expression of genes involved in cell cycle, antimicrobial response, and metabolism together with the identification of intermediate states. Further, we discovered rare subsets within crystal cells and lamellocytes that express fibroblast growth factor (FGF) ligand branchless and receptor breathless, respectively. We demonstrate that these FGF components are required for mediating effective immune responses against parasitoid wasp eggs, highlighting a novel role for FGF signaling in inter-hemocyte crosstalk. Our scRNA-seq analysis reveals the diversity of hemocytes and provides a rich resource of gene expression profiles for a systems-level understanding of their functions.

A cell atlas of the adult Drosophila midgut.

Studies of the adult Drosophila midgut have led to many insights in our understanding of cell-type diversity, stem cell regeneration, tissue homeostasis, and cell fate decision. Advances in single-cell RNA sequencing provide opportunities to identify new cell types and molecular features. We used single-cell RNA sequencing to characterize the transcriptome of midgut epithelial cells and identified 22 distinct clusters representing intestinal stem cells, enteroblasts, enteroendocrine cells (EEs), and enterocytes. This unbiased approach recovered most of the known intestinal stem cells/enteroblast and EE markers, highlighting the high quality of the dataset, and led to insights on intestinal stem cell biology, cell type-specific organelle features, the roles of new transcription factors in progenitors and regional variation along the gut, 5 additional EE gut hormones, EE hormonal expression diversity, and paracrine function of EEs. To facilitate mining of this rich dataset, we provide a web-based resource for visualization of gene expression in single cells. Altogether, our study provides a comprehensive resource for addressing functions of genes in the midgut epithelium.

The Septate Junction Protein Tsp2A Restricts Intestinal Stem Cell Activity via Endocytic Regulation of aPKC and Hippo Signaling.

Hippo signaling and the activity of its transcriptional coactivator, Yorkie (Yki), are conserved and crucial regulators of tissue homeostasis. In the Drosophila midgut, after tissue damage, Yki activity increases to stimulate stem cell proliferation, but how Yki activity is turned off once the tissue is repaired is unknown. From an RNAi screen, we identified the septate junction (SJ) protein tetraspanin 2A (Tsp2A) as a tumor suppressor. Tsp2A undergoes internalization to facilitate the endocytic degradation of atypical protein kinase C (aPKC), a negative regulator of Hippo signaling. In the Drosophila midgut epithelium, adherens junctions (AJs) and SJs are prominent in intestinal stem cells or enteroblasts (ISCs or EBs) and enterocytes (ECs), respectively. We show that when ISCs differentiate toward ECs, Tsp2A is produced, participates in SJ assembly, and turns off aPKC and Yki-JAK-Stat activity. Altogether, our study uncovers a mechanism allowing the midgut to restore Hippo signaling and restrict proliferation once tissue repair is accomplished.

miR-263a Regulates ENaC to Maintain Osmotic and Intestinal Stem Cell Homeostasis in Drosophila.

Proper regulation of osmotic balance and response to tissue damage is crucial in maintaining intestinal stem cell (ISC) homeostasis. We found that Drosophila miR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes (ECs) to maintain osmotic and ISC homeostasis. In the absence of miR-263a, the intraluminal surface of the intestine displays dehydration-like phenotypes, Na+ levels are increased in ECs, stress pathways are activated in ECs, and ISCs overproliferate. Furthermore, miR-263a mutants have increased bacterial load and expression of antimicrobial peptides. Strikingly, these phenotypes are reminiscent of the pathophysiology of cystic fibrosis (CF) in which loss-of-function mutations in the chloride channel CF transmembrane conductance regulator can elevate the activity of ENaC, suggesting that Drosophila could be used as a model for CF. Finally, we provide evidence that overexpression of miR-183, the human ortholog of miR-263a, can also directly target the expressions of all three subunits of human ENaC.

Characterizing F-actin Disassembly Induced by the Semaphorin-Signaling Component MICAL.

The MICALs are a family of phylogenetically conserved cytoplasmic proteins that modulate numerous cellular behaviors and play critical roles in semaphorin-plexin signaling. Our recent results have revealed that the MICALs are an unusual family of actin regulatory proteins that use actin filaments (F-actin) as a direct substrate-controlling F-actin dynamics via stereospecific oxidation of conserved methionine (Met44 and Met47) residues within actin. In particular, the MICALs have a highly conserved flavoprotein monooxygenase (redox) enzymatic domain in their N-terminus that directly oxidizes and destabilizes F-actin. Here, we describe methods to characterize MICAL-mediated F-actin disassembly using in vitro assays with purified proteins.

F-actin dismantling through a redox-driven synergy between Mical and cofilin.

Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF (actin-depolymerizing factor)/cofilins (encoded by the twinstar gene in Drosophila), sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. Here we uncover a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. We find that Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared with either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodelling, axon guidance and Semaphorin-Plexin repulsion. Mical and cofilin, therefore, form a redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties.

A simple and efficient method for generating high-quality recombinant Mical enzyme for in vitro assays.

We have recently identified a new family of multidomain oxidoreductase (redox) enzymes, the MICALs, that directly regulate the actin cytoskeletal elements necessary for the morphology, motility, and trajectory of cells. Our genetic assays reveal that Mical is both necessary and sufficient for actin organization and cellular effects in vivo and our biochemical assays with purified Mical protein reveal that Mical utilizes its redox activity to directly disassemble actin filaments. These results identify Mical proteins as novel actin disassembly factors and uncover a redox signaling mechanism that directly regulates the actin cytoskeleton. These results have also set the stage for in-depth characterization of the Mical enzyme. However, it has been difficult to obtain sufficient amounts of highly-pure Mical protein to conduct further biochemical, structural, imaging, catalytic, and other high-precision studies. Herein, we describe a means for expressing high levels of soluble recombinant Mical protein in bacteria. Likewise, we have designed a new purification strategy that enables the rapid and efficient purification of milligram quantities of highly-pure and >99% active Mical protein. This new strategy for generating large amounts of highly-pure and active Mical protein will aid research objectives designed to characterize the biochemical, enzymology, and structural biology of Mical and its effects on actin filament dynamics.

SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics.

Actin's polymerization properties are markedly altered by oxidation of its conserved Met 44 residue. Mediating this effect is a specific oxidation-reduction (redox) enzyme, Mical, that works with Semaphorin repulsive guidance cues and selectively oxidizes Met 44. We now find that this actin-regulatory process is reversible. Employing a genetic approach, we identified a specific methionine sulfoxide reductase (MsrB) enzyme SelR that opposes Mical redox activity and Semaphorin-Plexin repulsion to direct multiple actin-dependent cellular behaviours in vivo. SelR specifically catalyses the reduction of the R isomer of methionine sulfoxide (methionine-R-sulfoxide) to methionine, and we found that SelR directly reduced Mical-oxidized actin, restoring its normal polymerization properties. These results indicate that Mical oxidizes actin stereospecifically to generate actin Met-44-R-sulfoxide (actin(Met(R)O-44)), and also implicate the interconversion of specific Met/Met(R)O residues as a precise means to modulate protein function. Our results therefore uncover a specific reversible redox actin regulatory system that controls cell and developmental biology.

Direct redox regulation of F-actin assembly and disassembly by Mical.

Different types of cell behavior, including growth, motility, and navigation, require actin proteins to assemble into filaments. Here, we describe a biochemical process that was able to disassemble actin filaments and limit their reassembly. Actin was a specific substrate of the multidomain oxidation-reduction enzyme, Mical, a poorly understood actin disassembly factor that directly responds to Semaphorin/Plexin extracellular repulsive cues. Actin filament subunits were directly modified by Mical on their conserved pointed-end, which is critical for filament assembly. Mical posttranslationally oxidized the methionine 44 residue within the D-loop of actin, simultaneously severing filaments and decreasing polymerization. This mechanism underlying actin cytoskeletal collapse may have broad physiological and pathological ramifications.

Extracellular inhibitors, repellents, and semaphorin/plexin/MICAL-mediated actin filament disassembly.

Multiple extracellular signals have been identified that regulate actin dynamics within motile cells, but how these instructive cues present on the cell surface exert their precise effects on the internal actin cytoskeleton is still poorly understood. One particularly interesting class of these cues is a group of extracellular proteins that negatively alter the movement of cells and their processes. Over the years, these types of events have been described using a variety of terms and herein we provide an overview of inhibitory/repulsive cellular phenomena and highlight the largest known protein family of repulsive extracellular cues, the Semaphorins. Specifically, the Semaphorins (Semas) utilize Plexin cell-surface receptors to dramatically collapse the actin cytoskeleton and we summarize what is known of the direct molecular and biochemical mechanisms of Sema-triggered actin filament (F-actin) disassembly. We also discuss new observations from our lab that reveal that the multidomain oxidoreductase (Redox) enzyme Molecule Interacting with CasL (MICAL), an important mediator of Sema/Plexin repulsion, is a novel F-actin disassembly factor. Our results indicate that MICAL triggers Sema/Plexin-mediated reorganization of the F-actin cytoskeleton and suggest a role for specific Redox signaling events in regulating actin dynamics.

Tumor-induced endothelial cell apoptosis: roles of NAD(P)H oxidase-derived reactive oxygen species.

Many tumor cells are capable of migrating through endothelial cell (EC) junctions and disintegrating sub-endothelial extracellular matrix to achieve extravasation. We demonstrate in this study that certain solid tumor cells can induce EC apoptosis to facilitate their escape from the circulation. The EC apoptosis is triggered by elevated intracellular reactive oxygen species (ROS) levels and direct contacts with tumor cells are required. Treating ECs with antioxidants, such as ascorbate and N-acetyl-L-cysteine (NAC), and a glutathione precursor can rescue the ECs from tumor-induced apoptosis and reduce the number of tumor cells migrating across endothelial barriers. NAD(P)H oxidase was identified as the major ROS producer in the event since inhibitors and small interference RNA specific to the enzyme could abrogate the tumor-induced ROS production and hence EC death. This study also provides evidence showing that the interaction between tumor and EC increases intracellular Ca(2+) concentration and activates protein kinase C (PKC) activity, which leads to NAD(P)H oxidase activation through the serine-phosphorylation of p47(phox) subunit. These findings suggest that blocking the tumor-induced EC apoptosis is a potential way to prevent tumor metastasis.

Mical links semaphorins to F-actin disassembly.

How instructive cues present on the cell surface have their precise effects on the actin cytoskeleton is poorly understood. Semaphorins are one of the largest families of these instructive cues and are widely studied for their effects on cell movement, navigation, angiogenesis, immunology and cancer. Semaphorins/collapsins were characterized in part on the basis of their ability to drastically alter actin cytoskeletal dynamics in neuronal processes, but despite considerable progress in the identification of semaphorin receptors and their signalling pathways, the molecules linking them to the precise control of cytoskeletal elements remain unknown. Recently, highly unusual proteins of the Mical family of enzymes have been found to associate with the cytoplasmic portion of plexins, which are large cell-surface semaphorin receptors, and to mediate axon guidance, synaptogenesis, dendritic pruning and other cell morphological changes. Mical enzymes perform reduction-oxidation (redox) enzymatic reactions and also contain domains found in proteins that regulate cell morphology. However, nothing is known of the role of Mical or its redox activity in mediating morphological changes. Here we report that Mical directly links semaphorins and their plexin receptors to the precise control of actin filament (F-actin) dynamics. We found that Mical is both necessary and sufficient for semaphorin-plexin-mediated F-actin reorganization in vivo. Likewise, we purified Mical protein and found that it directly binds F-actin and disassembles both individual and bundled actin filaments. We also found that Mical utilizes its redox activity to alter F-actin dynamics in vivo and in vitro, indicating a previously unknown role for specific redox signalling events in actin cytoskeletal regulation. Mical therefore is a novel F-actin-disassembly factor that provides a molecular conduit through which actin reorganization-a hallmark of cell morphological changes including axon navigation-can be precisely achieved spatiotemporally in response to semaphorins.

Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1.

UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD(+)-dependent oxidation of UDP-glucose to produce UDP-glucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP-glucose as a substrate to generate UDP-glucuronic acid. The K(m) values of PA2022 and PA3559 for UDP-glucose are approximately 0.1 and 0.4 mM, whereas the K(m) values for NAD(+) are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP-glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg(2+), which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.

Assembly of adherens junctions is required for sphingosine 1-phosphate-induced matriptase accumulation and activation at mammary epithelial cell-cell contacts.

Sphingosine 1-phosphate (S1P), a bioactive phospholipid, simultaneously induces actin cytoskeletal rearrangements and activation of matriptase, a membrane-associated serine protease in human mammary epithelial cells. In this study, we used a monoclonal antibody selective for activated, two-chain matriptase to examine the functional relationship between these two S1P-induced events. Ten minutes after exposure of 184 A1N4 mammary epithelial cells to S1P, matriptase was observed to accumulate at cell-cell contacts. Activated matriptase first began to appear as small spots at cell-cell contacts, and then its deposits elongated along cell-cell contacts. Concomitantly, S1P induced assembly of adherens junctions and subcortical actin belts. Matriptase localization was observed to be coincident with markers of adherens junctions at cell-cell contacts but likely not to be incorporated into the tightly bound adhesion plaque. Disruption of subcortical actin belt formation and prevention of adherens junction assembly led to prevention of accumulation and activation of the protease at cell-cell contacts. These data suggest that S1P-induced accumulation and activation of matriptase depend on the S1P-induced adherens junction assembly. Although MAb M32, directed against one of the low-density lipoprotein receptor class A domains of matriptase, blocked S1P-induced activation of the enzyme, the antibody had no effect on S1P-induced actin cytoskeletal rearrangement. Together, these data indicate that actin cytoskeletal rearrangement is necessary but not sufficient for S1P-induced activation of matriptase at cell-cell contacts. The coupling of matriptase activation to adherens junction assembly and actin cytoskeletal rearrangement may serve to ensure tight control of matriptase activity, restricted to cell-cell junctions of mammary epithelial cells.