Hemagglutinin Tag Research Articles

Development of a sensor to detect methylmercury toxicity

Methylmercury (MeHg) is a well-known neurotoxicant that induces various cellular functions depending on cellular- and developmental-specific vulnerabilities. MeHg has a high affinity for selenol and thiol groups, thus impairing the antioxidant system. Such affinity characteristics of MeHg led us to develop sensor vectors to assess MeHg toxicity. In this study, MeHg-mediated defects in selenocysteine (Sec) incorporation were demonstrated using thioredoxin reductase 1 cDNA fused with the hemagglutinin tag sequence at the C-terminus. Taking advantage of such MeHg-mediated defects in Sec incorporation, a cDNA encoding luciferase with a Sec substituted for cysteine-491 was constructed. This construct showed MeHg-induced decreases in signaling in a dose-dependent manner. To directly detect truncated luciferase under MeHg exposure, we further constructed a new sensor vector fused with a target for proteasomal degradation. However, this construct was inadequate because of the low rate of Sec insertion, even in the absence of MeHg. Finally, a Krab transcriptional suppressor fused with Sec was constructed and assessed to demonstrate MeHg-dependent increases in signal intensity. We confirmed that the vector responded specifically and in a dose-dependent manner to MeHg in cultured cerebellar granule cells. This vector is expected to allow monitoring of MeHg-specific toxicity via spatial and temporal imaging.

Development of a Biosafety Level 1 Cellular Assay for Identifying Small-Molecule Antivirals Targeting the Main Protease of SARS-CoV-2: Evaluation of Cellular Activity of GC376, Boceprevir, Carmofur, Ebselen, and Selenoneine.

While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S-transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases.

A cell based assay using virus-like particles to screen AM type mimics for SARS-CoV-2 neutralisation

A number of small molecule and protein therapeutic candidates have been developed in the last four years against SARS-CoV-2 spike. However, there are hardly a few molecules that have advanced through the subsequent discovery steps to eventually work as a therapeutic agent. This is majorly because of the hurdles in determining the affinity of potential therapeutics with live SARS-CoV-2 virus. Furthermore, affinity determined for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, at times, fails to mimic physiological conditions of the host-virus interaction. To bridge this gap between in vitro and in vivo methods of therapeutic agent screening, we report an improved screening protocol for therapeutic candidates using SARS-CoV-2 virus like particles (VLPs). To minimise the interference from the bulkier reporters like GPF in the affinity studies, a smaller hemagglutinin (HA) tag has been fused to one of the proteins of VLP. This HA tag serves as readout, when probed with fluorescent anti-HA antibodies. Outcome of this study sheds light on the lesser known virus neutralisation capabilities of AM type miniprotein mimics. Further, to assess the stability of SARS-CoV-2 spike - miniprotein complex, we have performed molecular dynamic simulations on the membrane embedded protein complex. Simulation results reveal extremely stable intermolecular interactions between RBD and one of the AM type miniproteins, AM1. Furthermore, we discovered a robust network of intramolecular interactions that help stabilise AM1. Findings from our in vitro and in silico experiments concurrently highlight advantages and capabilities of mimic based miniprotein therapeutics.

SERPINC1 c.1247dupC: a novel SERPINC1 gene mutation associated with familial thrombosis results in a secretion defect and quantitative antithrombin deficiency

BackgroundAntithrombin (AT) is an important anticoagulant in hemostasis. We describe here the characterization of a novel AT mutation associated with clinically relevant thrombosis. A pair of sisters with confirmed type I AT protein deficiency was genetically analyzed on suspicion of an inherited SERPINC1 mutation. A frameshift mutation, c.1247dupC, was identified and the effect of this mutation was examined on the cellular and molecular level.MethodsPlasmids for the expression of wild-type (WT) and mutated SERPINC1 coding sequence (CDS) fused to green fluorescent protein (GFP) or hemagglutinin (HA) tag were transfected into HEK293T cells. Subcellular localization and secretion of the respective fusion proteins were analyzed by confocal laser scanning microscopy and Western blot.ResultsThe c.1247dupC mutation results in a frameshift in the CDS of the SERPINC1 gene and a subsequently altered amino acid sequence (p.Ser417LysfsTer48). This alteration affects the C-terminus of the AT antigen and results in impaired secretion as confirmed by GFP- and HA-tagged mutant AT analyzed in HEK293T cells.ConclusionThe p.Ser417LysfsTer48 mutation leads to impaired secretion, thus resulting in a quantitative AT deficiency. This is in line with the type I AT deficiency observed in the patients.

A versatile 2A peptide-based strategy for ectopic expression and endogenous gene tagging in Trypanosoma cruzi

Nearly all expression vectors currently available for Trypanosoma cruzi were conceived to produce a single primary transcript containing the genes of interest along with those that confer antibiotic resistance. However, since each messenger RNA (mRNA) matures separately, drug selection will only guarantee the expression of those derived from the selectable marker. Therefore, commonly a considerable fraction of the cells recovered after selection with these expression vectors, although resistant do not express the protein of interest. Consequently, in order to counteract this disadvantage, we developed vectors with an alternative arrangement in which the gene of interest and antibiotic resistance are fused sharing the same mRNA. To test this configuration, we included the coding sequence for the green fluorescent protein (mEGFP) linked to the one conferring neomycin resistance (Neo). Additionally, to allow for the production of two independent proteins the sequence for a Thosea asigna virus self-cleaving 2A peptide (T2A) was inserted in-between. Cells obtained with these vectors displayed higher mEGFP expression levels with more homogeneous transgenic parasite populations than those transfected with more conventional independent mRNA-based alternatives. Moreover, as determined by Western blot, 2A mediated fusion protein dissociation occurred with high efficiency in all parasite stages. In addition, these vectors could easily be transformed into endogenous tagging constructs that allowed the insertion, by ends-in homologous recombination, of a hemagglutinin tag (HA) fused to the actin gene. The use of 2A self-cleaving peptides in the context of single mRNA vectors represents an interesting strategy capable of improving ectopic transgene expression in T. cruzi as well as providing a simple alternative to more sophisticated methods, such as the one based on CRISPR/Cas9, for the endogenous labeling of genes.

An Atypical Somatic Calr Variant Acquired in Cis to a Germline Calr variant Induces Constitutive Signaling of the Thrombopoietin Receptor and Results in Essential Thrombocythemia

Acquired mutations in the gene encoding the endoplasmic reticulum (ER) chaperone calreticulin (CALR) are responsible for ~ 30% of essential thrombocythemia (ET) and myelofibrosis (MF) cases. Two CALR mutations, a deletion of 52 base pairs (del52) and an insertion of 5 base pairs (ins5) are the most frequent in ET and MF patients. All CALR mutations result in a +1-frameshift leading to 1) the replacement of the negatively-charged wild-type (WT) C-terminus that binds calcium in the ER by a positively-charged mutant tail including 44 residues that are common to all CALR mutant proteins and 2) the loss of the ER retention KDEL; together leading to the deregulation of calcium homeostasis. The CALR mutants are also secreted and bind to MPL in the ER but induce its constitutive dimerization and downstream signaling pathways at the cell surface. A 16-year-old girl presented with a platelet count of 797 x 10 3/mL rising to 1,228 x 10 3/mL within 2 years. After Informed Consent, we detected two atypical CALR variants in her blood cells by NGS analysis: V1 (c.1122-1125del) at 47% variant allele frequency (VAF) and V2 (c.1227-1231del) in cis of the V1 leading to V1-V2 (c.1122-1125del ; c.1227-1231del) at 5% VAF. The presence of V1 in blood and nails from the propositus and her healthy mother confirmed its germline origin. The V1-V2 was only found in the propositus at 5% in isolated monocytes and at very low levels in NK and B cells, confirming its somatic acquisition. The germline V1 induces a frameshift resulting in the replacement of the CALR WT C-terminus by the positively charged 44 residue-long C-terminal mutant tail present in del52 and ins5 and the loss of KDEL together with an insertion of 10 amino acids not present in CALR del52 nor ins5. In contrast, the V1-V2 generates a similar but shortened C-terminus containing the Arg-rich MPL binding site and KDEL (Fig. 1A). To investigate the functional impact of these CALR variants, we cloned V1 and V1-V2 with a N-terminal hemagglutinin (HA) tag as well as CALR WT and del52 as controls in a retroviral plasmid. Viral particles were produced to transduce Ba/F3 cell lines expressing MPL. Structural modeling using AlphaFold was used to investigate the role of KDEL restoration in V1-V2 in producing an oncogenic protein leading to ET. Both CALR WT and V1-V2 variants that contain the KDEL motif were detected to a similar extent in Ba/F3-MPL cell lysates. In contrast, del52 and V1, that lost the ER-retention KDEL motif, were barely detected in cell lysates. Unexpectedly, despite the presence of the KDEL motif, V1-V2 was found at a similar level than del52 at the Ba/F3-MPL cell surface. In contrast, V1 was barely detectable at the cell surface, like CALR WT. In order to investigate the role of the KDEL in V1-V2 cell surface expression, we generated a CALR WT lacking the KDEL and added the KDEL motif to del52. The presence of the KDEL motif stabilized the intracellular expression of del52 without affecting cell surface localization while the removal of the KDEL motif in CALR WT appeared to destabilize the protein and induce a slight leak to the cell surface. Structural modeling using AlphaFold predicted that the KDEL in V1-V2 structure is accessible to KDEL receptors, suggesting that the motif may be masked by the interaction of V1-V2 with MPL while trafficking to the cell surface. Indeed, cell-surface expression of V1-V2 was decreased in Ba/F3 that do not express MPL. We show that V1-V2, but not V1, constitutively activates the phosphorylation of STATs, ERK and AKT in Ba/F3-MPL. TPO further increases activation of STATs but not of AKT and ERK by V1-V2 and del52. Coherently, V1-V2, but not V1, sustained oncogenic MPL stimulation able to sustain cell autonomous Ba/F3-MPL cell proliferation (Fig. 1B). Both del52 and V1-V2 up-regulated BCL-2 mRNA. Dose-response cytotoxicity assays with the BCL-2 inhibitor venetoclax showed that these two mutants are sensitive to lower doses compared to CALR WT- or V1-expressing Ba/F3-MPL cells. This is the first report of a germline loss-of-function CALR variant (V1) expressed as an unstable protein and that might predispose to the acquisition of a somatic gain-of-function CALR variant (V1-V2) responsible for ET. The presence of KDEL in V1-V2 leads to stabilized CALR expression that does not prevent cell surface localization. Moreover, it leads to induction of MPL signaling and cytokine-independent cell growth.

Analysis of PERV-C superinfection resistance using HA-tagged viruses

BackgroundUsing pigs as organ donors has advanced xenotransplantation to the point that it is almost ready for clinical use. However, there is still a zoonotic risk associated with xenotransplantation, and the potential transmission of porcine endogenous retroviruses needs to be surveyed. Despite significant attempts to eliminate this risk, by the selection of PERV-C free pigs with low expression of PERV-A, -B, and by the genome-wide inactivation of PERV using CRISPR/Cas9, the impact of superinfection resistance (SIR) was not investigated. SIR is a viral trait that prevents reinfection (superinfection). For PERV, the underlying mechanism is unclear, whether and how cells, that harbor functional PERV, are protected. Using PERV-C(5683) as a reference virus, we investigated SIR in a newly developed in vitro model to pursue the mechanism and confirm its protective effect.ResultsWe developed three PERV-C constructs on the basis of PERV-C(5683), each of which carries a hemagglutinin tag (HA-tag) at a different position of the envelope gene (SP-HA, HA-VRA, and RPep-HA), to distinguish between primary infection and superinfection. The newly generated PERV-C(5683)-HA viruses were characterized while quantifying the viral RNA, reverse transcriptase activity, protein expression analysis, and infection studies. It was demonstrated that SP-HA and RPep-HA were comparable to PERV-C(5683), whereas HA-VRA was not replication competent. SP-HA and RPep-HA were chosen to challenge PERV-C(5683)-positive ST-IOWA cells demonstrating that PERV-C-HA viruses are not able to superinfect those cells. They do not integrate into the genome and are not expressed.ConclusionsThe mechanism of SIR applies to PERV-C. The production of PERV-C particles serves as a defense mechanism from superinfection with exogenous PERV-C. It was demonstrated by newly generated PERV-C(5683)-HA clones that might be used as a cutting-edge tool. The HA-tagging of PERV-C is novel, providing a blueprint for the tagging of other human tropic PERV viruses. The tagged viruses are suitable for additional in vitro and in vivo infection studies and will contribute, to basic research on viral invasion and pathogenesis. It will maintain the virus safety of XTx.

NAP1L1 is a novel microtubule-associated protein.

Microtubule-associated proteins (MAPs) regulate assembly and stability of microtubules (MTs) during cell cytokinesis, cell migration, neuronal growth, axon guidance, and synapse formation. Using data mining of the Human Protein Atlas database and experimental screening, we identified nucleosome assembly protein 1 like 1 (NAP1L1) as a new MAP. The Human Protein Atlas and PubMed database screening identified 99 potential new MAPs. Twenty candidate proteins that highly co-localized with MTs were exogenously expressed with green fluorescent protein (GFP) or hemagglutinin (HA) tags in tissue culture cells and MTs were co-stained for immunofluorescent microscopy. We found that NAP1L1 is mainly localized in the cytosol with MTs during interphase. Using bacterially expressed recombinant NAP1L1 fragments and purified MTs, we biochemically mapped the MT-binding site on the N-terminal region (1-72aa) and the central region (164-269aa) of NAP1L1. NAP1L1 dimerizes through the long helix region (73-163aa), and full-length NAP1L1 induces the formation of thick MTs, indicating that NAP1L1 has the ability to bundle MTs in cells. Analysis of publicly available RNA-seq data of NAP1L1 depleted cells suggested that NAP1L1 is involved in cell adhesion and migration in agreement with the function of NAP1L1 as a MAP.

Methods to Quantify the Dynamic Recycling of Plasma Membrane Channels.

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling modality mediated by Orai Ca2+ channels at the plasma membrane (PM) and the endoplasmic reticulum (ER) Ca2+ sensors STIM1/2. At steady state, Orai1 constitutively cycles between an intracellular compartment and the PM. Orai1 PM residency is modulated by its endocytosis and exocytosis rates. Therefore, Orai1 trafficking represents an important regulatory mechanism to define the levels of Ca2+ influx. Here, we present a protocol using the dually tagged YFP-HA-Orai1 with a cytosolic YFP and extracellular hemagglutinin (HA) tag to quantify Orai1 cycling rates. For measuring Orai1 endocytosis, cells expressing YFP-HA-Orai1 are incubated with mouse anti-HA antibody for various periods of time before being fixed and stained for surface Orai1 with Cy5-labeled anti-mouse IgG. The cells are fixed again, permeabilized, and stained with Cy3-labeled anti-mouse IgG to reveal anti-HA that has been internalized. To quantify Orai1 exocytosis rate, cells are incubated with anti-HA antibody for various incubation periods before being fixed, permeabilized, and then stained with Cy5-labeled anti-mouse IgG. The Cy5/YFP ratio is plotted over time and fitted with a mono-exponential growth curve to determine exocytosis rate. Although the described assays were developed to measure Orai1 trafficking, they are readily adaptable to other PM channels. Key features Detailed protocols to quantify endocytosis and exocytosis rates of Orai1 at the plasma membrane that can be used in various cell lines. The endocytosis and exocytosis assays are readily adaptable to study the trafficking of other plasma membrane channels.

Nerve injury increases native Ca V 2.2 trafficking in dorsal root ganglion mechanoreceptors.

Neuronal N-type (Ca V 2.2) voltage-gated calcium channels are essential for neurotransmission from primary afferent terminals in the dorsal horn. In this study, we have used a knockin mouse containing Ca V 2.2 with an inserted extracellular hemagglutinin tag (Ca V 2.2_HA), to visualise the pattern of expression of endogenous Ca V 2.2 in dorsal root ganglion (DRG) neurons and their primary afferents in the dorsal horn. We examined the effect of partial sciatic nerve ligation (PSNL) and found an increase in Ca V 2.2_HA only in large and medium dorsal root ganglion neurons and also in deep dorsal horn synaptic terminals. Furthermore, there is a parallel increase in coexpression with GFRα1, present in a population of low threshold mechanoreceptors, both in large DRG neurons and in their terminals. The increased expression of Ca V 2.2_HA in these DRG neurons and their terminals is dependent on the presence of the auxiliary subunit α 2 δ-1, which is required for channel trafficking to the cell surface and to synaptic terminals, and it likely contributes to enhanced synaptic transmission at these synapses following PSNL. By contrast, the increase in GFRα1 is not altered in α 2 δ-1-knockout mice. We also found that following PSNL, there is patchy loss of glomerular synapses immunoreactive for Ca V 2.2_HA and CGRP or IB4, restricted to the superficial layers of the dorsal horn. This reduction is not dependent on α 2 δ-1 and likely reflects partial deafferentation of C-nociceptor presynaptic terminals. Therefore, in this pain model, we can distinguish 2 different events affecting specific DRG terminals, with opposite consequences for Ca V 2.2_HA expression and function in the dorsal horn.

Direct in situ protein tagging in Chlamydomonas reinhardtii utilizing TIM, a method for CRISPR/Cas9-based targeted insertional mutagenesis.

Chlamydomonas reinhardtii is an important model organism for the study of many cellular processes, and protein tagging is an increasingly indispensable tool for these studies. To circumvent the disadvantages of conventional approaches in creating a tagged cell line, which involve transforming either a wild-type or null-mutant cell line with an exogenous DNA construct that inserts randomly into the genome, we developed a strategy to tag the endogenous gene in situ. The strategy utilizes TIM, a CRISPR/Cas9-based method for targeted insertional mutagenesis in C. reinhardtii. We have tested the strategy on two genes: LF5/CDKL5, lack of which causes a long-flagella phenotype, and Cre09.g416350/NAP1L1, which has not been studied previously in C. reinhardtii. We successfully tagged the C-terminus of wild-type LF5 with the hemagglutinin (HA) tag with an efficiency of 7.4%. Sequencing confirmed that these strains are correctly edited. Western blotting confirmed the expression of HA-tagged LF5, and immunofluorescence microscopy showed that LF5-HA is localized normally. These strains have normal length flagella and appear wild type. We successfully tagged the N-terminus of Cre09.g416350 with mNeonGreen-3xFLAG with an efficiency of 9%. Sequencing showed that the tag region in these strains is as expected. Western blotting confirmed the expression of tagged protein of the expected size in these strains, which appeared to have normal cell size, growth rate, and swimming speed. This is the first time that C. reinhardtii endogenous genes have been edited in situ to express a wild-type tagged protein. This effective, efficient, and convenient TIM-tagging strategy promises to be a useful tool for the study of nuclear genes, including essential genes, in C. reinhardtii.

Astrocyte heterogeneity within white matter tracts and a unique subpopulation of optic nerve head astrocytes.

Much of what we know about astrocyte form and function is derived from the study of gray matter protoplasmic astrocytes, whereas white matter fibrous astrocytes remain relatively unexplored. Here, we used the ribotag approach to isolate ribosome-associated mRNA and investigated the transcriptome of uninjured fibrous astrocytes from three regions: unmyelinated optic nerve head, myelinated optic nerve proper, and corpus callosum. Astrocytes from each region were transcriptionally distinct and we identified region-specific astrocyte genes and pathways. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as key differentiators of astrocyte populations. Optic nerve astrocytes expressed higher levels of neuroinflammatory pathways than corpus callosum astrocytes and we further identified CARTPT as a new marker of optic nerve head astrocytes. These previously uncharacterized transcriptional profiles of white matter astrocyte types reveal their functional diversity and a greater heterogeneity than previously appreciated.

Functional Properties of Two Distinct PTH1R Mutants Associated With Either Skeletal Defects or Pseudohypoparathyroidism.

ABSTRACTConsistent with a vital role of parathyroid hormone (PTH) receptor type 1 (PTH1R) in skeletal development, homozygous loss‐of‐function PTH1R mutations in humans results in neonatal lethality (Blomstrand chondrodysplasia), whereas such heterozygous mutations cause a primary failure of tooth eruption (PFE). Despite a key role of PTH1R in calcium and phosphate homeostasis, blood mineral ion levels are not altered in such cases of PFE. Recently, two nonlethal homozygous PTH1R mutations were identified in two unrelated families in which affected members exhibit either dental and skeletal abnormalities (PTH1R‐V204E) or hypocalcemia and hyperphosphatemia (PTH1R‐R186H). Arg186 and Val204 map to the first transmembrane helix of the PTH1R, and thus to a critical region of this class B G protein‐coupled receptor. We used cell‐based assays and PTH and PTH‐related protein (PTHrP) ligand analogs to assess the impact of the R186H and V204E mutations on PTH1R function in vitro. In transiently transfected HEK293 cells, PTH1R‐R186H mediated cyclic adenosine monophosphate (cAMP) responses to PTH(1‐34) and PTHrP(1‐36) that were of comparable potency to those observed on wild‐type PTH1R (PTH1R‐WT) (half maximal effective concentrations [EC50s] = 0.4nM to 1.2nM), whereas the response‐maxima were significantly reduced for the PTH1R‐V204E mutant (maximum effect [Emax] = 81%–77% of PTH1R‐WT, p ≤ 0.004). Antibody binding to an extracellular hemagglutinin (HA) tag was comparable for PTH1R‐R186H and PTH1R‐WT, but was significantly reduced for PTH1R‐V204E (maximum binding level [Bmax] = 44% ± 11% of PTH1R‐WT, p = 0.002). The potency of cAMP signaling induced by a PTH(1‐11) analog was reduced by ninefold and threefold, respectively, for PTH1R‐R186H and PTH1R‐V204E, relative to PTH1R‐WT, and a PTH(1‐15) radioligand analog that bound adequately to PTH1R‐WT exhibited little or no specific binding to either mutant receptor. The data support a general decrease in PTH1R surface expression and/or function as a mechanism for PFE and a selective impairment in PTH ligand affinity as a potential PTH1R‐mutation‐based mechanism for pseudohypoparathyroidism. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Terahertz immunosensing assisted by functionalized Au NPs based on all-dielectric metasurface

Terahertz (THz) sensors have attracted significant attention in the biological field due to their non-destructive and harmless interaction with biochemical samples, but how to achieve efficiently specific sensing for the small molecular weight samples is still challenging. In this work, we incorporated an all-dielectric metasurface with the functionalized gold nanoparticles (Au NPs) to realize an immunosensing of the target biomolecule at nanomole-level concentration. These Au NPs are modified by the Anti-HA tag antibody, which realizes the specific immunobinding of the human influenza hemagglutinin tag protein (HA antigen). By comparing with and without the functionalized Au NPs, the experimental results show that this sensor can quantitatively detect the concentration of HA antigen, and its sensitivity can be greatly improved by the functionalized Au NPs. The maximum sensitivity can be achieved to 2.96 GHz∙ml/nmol, which is 2.66 times higher than that without the Au NPs, and the highest detection accuracy reaches 1.05 nmol/ml. Furthermore, by comparing three other nonspecific proteins, only the HA antigen can be sensitively distinguished, which proves the specificity of this sensing method. Our results illustrate a reliable platform for THz immunosensing applications, and it is expected that the proposed sensing strategy opens new avenues for developing high-efficient, low-cost, and real-time THz all-dielectric sensors for biomolecular detection.

The Chlamydia trachomatis inclusion membrane protein CT006 associates with lipid droplets in eukaryotic cells.

Chlamydia trachomatis causes genital and ocular infections in humans. This bacterial pathogen multiplies exclusively within host cells in a characteristic vacuole (inclusion) and delivers proteins such as inclusion membrane proteins (Incs) into the host cell. Here, we identified CT006 as a novel C. trachomatis protein that when expressed ectopically eukaryotic cells can associate with lipid droplets (LDs). A screen using Saccharomyces cerevisiae identified two Incs causing vacuolar protein sorting defects and seven Incs showing tropism for eukaryotic organelles. Ectopic expression in yeast and mammalian cells of genes encoding different fragments of CT006 revealed tropism for the endoplasmic reticulum and LDs. We identified a LD-targeting region within the first 88 amino acid residues of CT006, and positively charged residues important for this targeting. Comparing with the parental wild-type strain, cells infected by a newly generated C. trachomatis strain overproducing CT006 with a double hemagglutinin tag showed a slight increase in the area occupied by LDs within the inclusion region. However, we could not correlate this effect with the LD-targeting regions within CT006. We further showed that both the amino and carboxy-terminal regions of CT006, flanking the Inc-characteristic bilobed hydrophobic domain, are exposed to the host cell cytosol during C. trachomatis infection, supporting their availability to interact with host cell targets. Altogether, our data suggest that CT006 might participate in the interaction of LDs with C. trachomatis inclusions.

In Vivo Epitope Tagging of Plant Mitochondria.

Mitochondria play a key role in cellular metabolism. Analyses of the genome, the proteome, metabolic, physiological, and biochemical functions of mitochondria frequently require the isolation of intact and functional mitochondria from various plant tissues with sufficient yield. For this purpose, we generated a transgenic Arabidopsis thaliana (Arabidopsis) line which presents a triple hemagglutinin tag on the surface of the outer mitochondrial membrane. The affinity tag enables immunocapture of the organelles in a single step. This chapter gives detailed instructions on how to generate transgenic Arabidopsis lines harboring a ubiquitously expressed 3xHA-sGFP-TOM5 mitochondrial fusion protein that is targeted to the outer mitochondrial membrane and enables purification of the organelles in a single step.

Application of a RiboTag‐based approach to generate and analyze mRNA from enteric neural cells

Transcriptional profiling of specific intestinal cell populations under health and disease is generally based on traditional sorting approaches followed by gene expression analysis. Therein, specific cell populations are identified either by expressing reporter genes under a cell type-specific promotor or by specific surface antigens. This method provides adequate results for blood-derived and tissue-resident immune cells. However, in stromal cell analysis, cellular stress due to digestion often results in degraded RNA. Particularly, ramified cells integrated into the tissue, such as enteric neurons and glial cells, suffer from these procedures. These cell types are involved in various intestinal processes, including a prominent immune-regulatory role, which requires suitable approaches to generate cell-specific transcriptional profiles. Sox10iCreERT2 and choline acetyltransferase (ChATCre ) mice were crossed with mice labeling the ribosomal Rpl22 protein upon Cre activity with a hemagglutinin tag (Rpl22-HA, termed RiboTag). This approach enabled cellular targeting of enteric glia and neurons and the immediate isolation of cell-specific mRNA from tissue lysates without the need for cell sorting. We verified the specific expression of Rpl22-HA in enteric glia and neurons and provided gene expression data demonstrating a successful enrichment of either Sox-10+ glial or ChAT+ neuronal mRNAs by the RiboTag-mRNA procedure using qPCR and RNA-Seq analysis. We present a robust and selective protocol that allows the generation of cell type-specific transcriptional in vivo snapshots of distinct enteric cell populations that will be especially useful for various intestinal disease models involving peripheral neural cells.

Elucidating the Importance and Regulation of Key Enhancers for Human MEIS1 Expression

Myeloid ecotropic virus insertion site 1 (MEIS1) is essential for normal hematopoiesis and is deregulated in a large subset of acute myeloid leukemia (AML) by yet unknown mechanisms. We previously identified 3 candidate enhancer regions: enhancer region 1 (E1) at -2 kb upstream; enhancer region 2 (E2) at +10.6 kb downstream inside intron 6; and enhancer region 3 (E3) +140 kb downstream of the translation start site. In the current study, we utilized CRISPR-Cas9 genome editing to further characterize these enhancers in a human AML cell line and identify the key transcription factors (TFs) associated with their function.To efficiently track MEIS1 expression levels, a GFP reporter, a P2A self-cleaving peptide tag and a hemagglutinin tag at its translation start site was introduced in a MEIS1 high expressing human AML cell line, U937. Then we introduced random mutations (Indels) along the MEIS1 locus utilizing a CRISPR-Cas9 mediated genome editing vector system in mono-allelic MEIS1-GFP-tagged U937 cells with special focus on the previously identified enhancer regions to find the key sequences important to the function of the MEIS1 enhancer regions. Two targeted regions yielding the highest proportion of GFP - cells corresponded to the E2 enhancer region within intron 6 and were referred to as E2.1 and E2.2. Using chromosome conformation capture (3C) assay, we detected a significantly decreased interaction (p=0.0022) between the promoter and the intron 6 region surrounding the E2 region in E2.2 targeted cells compared to the parental cells. Moreover, our data indicated that the DNA sequence within E2.2 is highly critical to this region's enhancer function which is further influenced by the larger genomic region surrounding the E2.1 gRNA targeted site.To identify TFs binding to the E2 region, we further scrutinized the E2.2 indel region for loss of TF binding sites. We performed TF prediction analysis and performed a protein pull down-mass spectrometry experiment to identify TF candidates. The overlap yielded a list of 7 TFs, each of which we targeted via CRISPR/Cas9. Reduction in GFP levels was only observed for FLI1 locus targeting but not for the other 6 TFs. Concordant reduction in MEIS1 and FLI1 levels were confirmed by immunoblotting. Additionally, chromatin immunoprecipitation (ChIP) followed by quantitative PCR revealed significant FLI1 enrichment at the promoter and at 3 sites surrounding the E2.2 region (p=0.0004) compared to 4 control regions scattered along the MEIS1 locus. Given a previous study indicating MEIS1 upregulation of FLI1 in normal hematopoiesis, we hypothesised that a positive feedback loop may exist between FLI1 and MEIS1 in AML. Since MEIS1 levels are frequently elevated in normal karyotype AML (CN-AML), we used the murine Hoxa9/Meis1 AML model as a surrogate for CN-AML and performed Meis1 ChIP-seq analysis. We detected direct Meis1 binding to the intronic region of the mouse Fli1 gene as well as other ETS factor loci, in Hoxa9/Meis1 cells.To better understand the clinical relevance of FLI1 in AML, we analyzed the Beat AML dataset. High FLI1 transcript levels correlated with adverse overall survival in CN-AML (p=0.044). Additionally, we observed a trend towards worse outcome with high FLI1 in the NPM1-mutated CN-AML subtype (p=0.069). We also observed a similar correlation in CN-AML for another ETS factor, ELF1, which we had previously shown to bind and upregulate MEIS1 expression in AML, suggesting a broader unrecognized role for ETS factors in AML.In summary, we have developed a rapid flow cytometry-based readout for the fine dissection and characterization of the cis-regulatory elements and associated TFs critical for MEIS1 transcription via CRISPR-Cas9 genetic manipulation. Our study revealed FLI1 as the candidate key regulator of MEIS1 expression and a positive correlation between FLI1 mRNA levels and worse overall survival in MEIS1-high AML subgroups. DisclosuresNo relevant conflicts of interest to declare.

Assembly Apparatus of Light-Harvesting Complexes: Identification of Alb3.1–cpSRP–LHCP Complexes in the Green Alga Chlamydomonas reinhardtii

The unicellular green alga, Chlamydomonas reinhardtii, contains many light-harvesting complexes (LHCs) associating chlorophylls a/b and carotenoids; the major LHCIIs (types I, II, III and IV) and minor light-harvesting complexes, CP26 and CP29, for photosystem II, as well as nine LHCIs (LHCA1-9), for photosystem I. A pale green mutant BF4 exhibited impaired accumulation of LHCs due to deficiency in the Alb3.1 gene, which encodes the insertase involved in insertion, folding and assembly of LHC proteins in the thylakoid membranes. To elucidate the molecular mechanism by which ALB3.1 assists LHC assembly, we complemented BF4 to express ALB3.1 fused with no, single or triple Human influenza hemagglutinin (HA) tag at its C-terminus (cAlb3.1, cAlb3.1-HA or cAlb3.1-3HA). The resulting complemented strains accumulated most LHC proteins comparable to wild-type (WT) levels. The affinity purification of Alb3.1-HA and Alb3.1-3HA preparations showed that ALB3.1 interacts with cpSRP43 and cpSRP54 proteins of the chloroplast signal recognition particle (cpSRP) and several LHC proteins; two major LHCII proteins (types I and III), two minor LHCII proteins (CP26 and CP29) and eight LHCI proteins (LHCA1, 2, 3, 4, 5, 6, 8 and 9). Pulse-chase labeling experiments revealed that the newly synthesized major LHCII proteins were transiently bound to the Alb3.1 complex. We propose that Alb3.1 interacts with cpSRP43 and cpSRP54 to form an assembly apparatus for most LHCs in the thylakoid membranes. Interestingly, photosystem I (PSI) proteins were also detected in the Alb3.1 preparations, suggesting that the integration of LHCIs to a PSI core complex to form a PSI-LHCI subcomplex occurs before assembled LHCIs dissociate from the Alb3.1-cpSRP complex.

Variable Distribution of DOCK-D Proteins between Cytosol and Nucleoplasm in Cell Lines, Effect of Interleukin-4 on DOCK10 in B-Cell Lymphoid Neoplasms, and Validation of a New DOCK10 Antiserum for Immunofluorescence Studies.

Dedicator-of-cytokinesis (DOCK), a family of guanine-nucleotide exchange factors (GEFs), comprises four subfamilies, named from A to D. DOCK-D comprises DOCK9, DOCK10, and DOCK11. The GEF activity involves translocation from the cytoplasm to the plasma membrane (PM), as assessed by the transfection of tagged proteins. However, the cellular localization of endogenous DOCK proteins is poorly understood. In this paper, to gain a better understanding of the role of the DOCK-D proteins, we studied their distribution between cytosol and nucleoplasm in 11 cell lines. DOCK-D proteins were distributed with variable cytosolic or nuclear predominance, although the latter was common for DOCK9 and DOCK11. These results suggest that the DOCK-D proteins may perform new nuclear functions, which remain to be discovered. Furthermore, we found that DOCK10 levels are increased by interleukin-4 (IL-4) in B-cell lymphoid neoplasms other than chronic lymphocytic leukemia (CLL) such as mantle cell lymphoma and diffuse large B-cell lymphoma. We also found evidence for an induction of the cytosolic levels of DOCK10 by IL-4 in CLL. Finally, we obtained a valid DOCK10 antiserum for immunofluorescence (IF) microscopy that, as an antibody against the hemagglutinin (HA) tag, marked PM ruffles and filopodia in HeLa cells with inducible expression of HA-DOCK10.