IntAct: a non-disruptive internal tagging strategy to study actin isoform organization and function

Actin plays a central role in many biological processes such as cell division, motility and contractility. In birds and mammals, actin has six, highly conserved isoforms, four of which are primarily present in muscles and two that are ubiquitously expressed across tissues. While each isoform has non-redundant biological functions, we currently lack the tools to investigate the molecular basis for isoform specificity due to their high similarity and the limited possibilities to manipulate actin. To solve this technical challenge, we developed IntAct, an internally tagged actin system to study actin isoform organization in fixed and living cells. For this, we performed a microscopy-based screen for 11 internal actin positions and identified one residue pair that allows for non-disruptive epitope tag integration. Using knockin cell lines with tags into the ubiquitously expressed β-actin, we demonstrate that IntAct actins are properly expressed and that their incorporation into filaments is indistinguishable from wildtype. We further show that IntAct actins can be visualized in living cells by exploiting the nanobody-targeted ALFA tag and that they keep their ability to interact with the actin binding proteins profilin and cofilin. Lastly, we also introduced the tag in the ubiquitously expressed γ-actin and demonstrate that the differential localization observed for actin isoforms remains unaltered for the IntAct actins. Together, our data demonstrate that IntAct is a versatile tool to study actin isoform localization, dynamics and molecular interactions to finally enable the molecular characterization of actin isoforms in biological processes.

Design and validation of plasmid vectors for characterizing protein-protein interactions in Spodoptera frugiperda insect cells

There are limited molecular biology resources for interrogating protein-protein interactions (PPI) in insect cells. To address this deficiency, we developed plasmid vectors for localization, bi-molecular fluorescence complementation (BiFC), and co-immunoprecipitation (co-IP) assays in Sf9 insect cells. Plasmids were designed to express a protein of interest as a fusion with epitope tags and autofluorescent proteins using the Gateway cloning system. Two robust interactors were utilized to validate this system, the nucleoprotein (N) and the phosphoprotein (P) of maize mosaic virus. The viral N was fused with the carboxy-terminal portion of eYFP and a FLAG epitope tag, and P was fused with the amino-terminal portion of eYFP and a c-myc epitope tag. The two expression plasmids were co-transfected into Sf9 cells, and fluorescence microscopy was used to visualize BiFC and co-IP was performed to confirm that this system was sensitive enough to detect PPI between the two proteins. BiFC was seen in cells co-transfected with N and P and co-IP validated the interaction. This plasmid-based system can be used to investigate a variety of PPI that occur in insects. We validated viral protein interactions that occur in the insect vector which provides further insights into the biology of rhabdoviruses that are transmitted by insects. The ability to express viral and insect proteins in insect cells for studying PPI with this streamlined system represents an advancement for protein research in insects. Future work will focus on identifying interacting viral and host proteins and discovery of targets for control of viruses and insect vectors.

Workflow for proteomic analysis of purified lysosomes with or without damage v2

Lysosomes are a major degradative organelle within eukaryotic cells. Previous work has developed a method wherein the TMEM192 protein is tagged on its C-terminus with an epitope tag in order to immunopurify (IP) lysosomes from cell extracts.1 This process is referred to as Lyso-IP. Such lysosomes can be used for proteomic analysis or for metabolomic analysis. The Lyso-IP is adapted from a previous reported method (Wyant et al., 2018). Here we also describe processing steps using proteomics after lysosome purification in the context of lysosomal damaging agents. Agents such as L-Leucyl-L-Leucine methyl ester (hydrochloride) (LLoMe) and Gly-Phe-β-naphthylamide (GPN) induce lysosomal damage, leading to the degradation of damaged lysosomes by lysophagy. This adaptation of Lyso-IP provides a route to identify proteins that are recruited to damaged lysosomes using quantitative proteomics.

HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants

G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues. In an effort to resolve this problem, we have developed a novel mouse model by fusing the hemagglutinin (HA)-epitope tag sequence to the amino-terminus of the µ-opioid receptor (MOP). Although HA-MOP knock-in mice exhibit reduced receptor expression, we found that this approach allowed for highly efficient immunodetection of low abundant GPCR targets. We also show that the HA-tag facilitates both high-resolution imaging and immunoisolation of MOP. Mass spectrometry (MS) confirmed post-translational modifications, most notably agonist-selective phosphorylation of carboxyl-terminal serine and threonine residues. MS also unequivocally identified the carboxyl-terminal 387LENLEAETAPLP398 motif, which is part of the canonical MOP sequence. Unexpectedly, MS analysis of brain lysates failed to detect any of the 15 MOP isoforms that have been proposed to arise from alternative splicing of the MOP carboxyl-terminus. For quantitative analysis, we performed multiple successive rounds of immunodepletion using the well-characterized rabbit monoclonal antibody UMB-3 that selectively detects the 387LENLEAETAPLP398 motif. We found that >98% of HA-tagged MOP contain the UMB-3 epitope indicating that virtually all MOP expressed in the mouse brain exhibit the canonical amino acid sequence.

Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein–protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein–protein interactions.

Workflow for proteomic analysis of purified lysosomes with or without damage v1

Lysosomes are a major degradative organelle within eukaryotic cells. Previous work has developed a method wherein the TMEM192 protein is tagged on its C-terminus with an epitope tag in order to immunopurify (IP) lysosomes from cell extracts.1 This process is referred to as Lyso-IP. Such lysosomes can be used for proteomic analysis or for metabolomic analysis. A detailed protocol has been described by Dong et al (2021)2 for the isolation of lysosomes (https://protocols.io/view/sample-preparation-protocol-for-lipidomics-harvest-br9ym97w), with an emphasis on downstream analysis by metabolomics. Here we describe processing steps using proteomics after lysosome purification in the context of lysosomal damaging agents. Agents such as L-Leucyl-L-Leucine methyl ester (hydrochloride) (LLoMe) and Gly-Phe-β-naphthylamide (GPN) induce lysosomal damage, leading to the degradation of damaged lysosomes by lysophagy. This adaptation of Lyso-IP provides a route to identify proteins that are recruited to damaged lysosomes using quantitative proteomics.

Mutation E48K in PB1 Polymerase Subunit Improves Stability of a Candidate Live Attenuated Influenza B Virus Vaccine

Influenza B virus (IBV) is a major respiratory pathogen of humans, particularly in the elderly and children, and vaccines are the most effective way to control it. In previous work, incorporation of two mutations (E580G, S660A) along with the addition of an HA epitope tag in the PB1 segment of B/Brisbane/60/2008 (B/Bris) resulted in an attenuated strain that was safe and effective as a live attenuated vaccine. A third attempted mutation (K391E) in PB1 was not always stable. Interestingly, viruses that maintained the K391E mutation were associated with the mutation E48K. To explore the contribution of the E48K mutation to stability of the K391E mutation, a vaccine candidate was generated by inserting both mutations, along with attenuating mutations E580G and S660A, in PB1 of B/Bris (B/Bris PB1att 4M). Serial passages of the B/Bris PB1att 4M vaccine candidate in eggs and MDCK indicated high stability. In silico structural analysis revealed a potential interaction between amino acids at positions 48 and 391. In mice, B/Bris PB1att 4M was safe and provided complete protection against homologous challenge. These results confirm the compensatory effect of mutation E48K to stabilize the K391E mutation, resulting in a safer, yet still protective, IBV LAIV vaccine.

Mutation E48K in PB1 Polymerase Subunit Improves Stability of a Candidate Live Attenuated Influenza B Virus Vaccine

Influenza B virus (IBV) is a major respiratory pathogen of humans, particularly in the elderly and children and vaccines are the most effective way to control it. In previous work, incorporation of two mutations (E580G, S660A) along with the addition of a HA epitope tag in the PB1 segment of B/Brisbane/60/2008 (B/Bris) resulted in an attenuated strain that was safe and effective as a live attenuated vaccine. A third attempted mutation (K391E) in PB1 was not always stable. Interestingly, viruses that maintained the K391E mutation were associated with the mutation E48K. To explore the contribution of the E48K mutation for stability of the K391E mutation, a vaccine candidate was generated by inserting both mutations along with attenuating mutations E580G and S660A in PB1 of B/Bris (B/Bris PB1att 4M). Serial passage of the B/Bris PB1att 4M vaccine candidate in eggs and MDCK indicated high stability. In silico structural analysis revealed a potential interaction between amino acids at positions 48 and 391. In mice, B/Bris PB1att 4M was safe and provided complete protection against homologous challenge. These results confirm the compensatory effect of mutation E48K to stabilize the K391E mutation, resulting in a safer, yet still protective, IBV LAIV vaccine.

A Drosophila Toolkit for Imaging of HA-tagged Proteins Unveiled a Block in Autophagy Flux in the Last Instar Larval Fat Body

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with POI harboring different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: HA Frankenbody. This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Autophagy was dispensable for the swelling of lysosomes, indicating that lysosomal activity is downregulated at this stage. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.