Cell Surface Distribution Research Articles

Novel Transmission Electron Microscopic Method to Quantify Protein Clustering on the Cell Surface.

The cell surface distribution patterns (clustering) of membrane proteins have been widely investigated in cell biology. Here we describe a novel transmission electron microscopic (TEM) protocol designed to improve the quality of information obtained about the protein distribution patterns detected. This novel method makes it possible to study the clustering of all transmembrane proteins on one half of the cytoplasmic membrane of a whole cell. To achieve better imaging, we combine various methods, including critical-point drying, fixation of gold beads with a carbon layer, and a newly developed chemical thinning method. In addition, in our image-processing algorithm, we implemented pair correlation and pair cross-correlation functions, providing more details and better quantitative accuracy in characterizing the size and numbers of possible protein clusters. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and transmission electron micrography Alternate Protocol: Direct cell labeling for transmission electron micrography Basic Protocol 2: Analysis of TEM images to detect immunogold-labeled proteins.

HTLV-1 biofilm polarization maintained by tetraspanin CD82 is required for efficient viral transmission.

In the early stages of infection, human T-lymphotropic virus type 1 (HTLV-1) dissemination within its host is believed to rely mostly on cell-to-cell contacts. Past studies unveiled a novel mechanism of HTLV-1 intercellular transmission based on the remodeling of the host-cell extracellular matrix and the generation of cell-surface viral assemblies whose structure, composition, and function resemble bacterial biofilms. These polarized aggregates of infectious virions, identified as viral biofilms, allow the bulk delivery of viruses to target cells and may help to protect virions from immune attacks. However, viral biofilms' molecular and functional description is still in its infancy, although it is crucial to fully decipher retrovirus pathogenesis. Here, we explore the function of cellular tetraspanins (CD9, CD81, CD82) that we detect inside HTLV-1 particles within biofilms. Our results demonstrate specific roles for CD82 in the cell-surface distribution and intercellular transmission of HTLV-1 biofilms, which we document as two essential parameters for efficient viral transmission. At last, our findings indicate that N-glycosylation of cell-surface molecules, including CD82, is required for the polarization of HTLV-1 biofilms and for the efficient transmission of HTLV-1 between T-lymphocytes.

Downregulation of EphA2 stability by RNF5 limits its tumor-suppressive function in HER2-negative breast cancers

Ephrin receptor A2 (EphA2) plays dual functions in tumorigenesis through ligand-independent tumor promotion or ligand-dependent tumor suppression. However, the regulation of EphA2 tumor-suppressive function remains unclear. Here, we showed that RNF5 interacts with EphA2 and induces its ubiquitination and degradation, decreases the stability and cell surface distribution of EphA2 and alters the balance of its phosphorylation at S897 and Y772. In turn, RNF5 inhibition decreases ERK phosphorylation and increases p53 expression through an increase in the EphA2 level in HER2-negative breast cancer cells. Consequently, RNF5 inhibition increases the adhesion and decreases the migration of HER2-negative breast cancer cells, and RNF5 silencing suppresses the growth of xenograft tumors derived from ER-positive, HER2-negative breast cancer cells with increased EphA2 expression and altered phosphorylation. RNF5 expression is inversely correlated with EphA2 expression in breast cancers, and a high EphA2 level accompanied by a low RNF5 level is related to better survival in patients with ER-positive, HER2-negative breast cancers. These studies revealed that RNF5 negatively regulates EphA2 properties and suppresses its tumor-suppressive function in HER2-negative breast cancers.

HLA DQ protein changes the cell surface distribution pattern of HLA proteins as monitored by Förster resonance energy transfer and high-resolution electron microscopy.

Peptide presentation by MHC class I and MHC class II molecules plays important roles in the regulation of the immune response. One factor in these displays is the density of antigen, which must exceed a critical threshold for the effective activation of T cells. Nonrandom distribution of MHC class I and class II has already been detected at the nanometer level and at higher hierarchical levels. It is not clear how the absence and reappearance of some protein molecules can influence the nonrandom distribution. Therefore, we performed experiments on HLA II-deficient bare lymphocyte syndrome (BLS1) cells: we created a stable transfected cell line, tDQ6-BLS-1, and were able to detect the effect of the appearance of HLA-DQ6 molecules on the homo and heteroassociation of different cell surface molecules by comparing Förster resonance energy transfer (FRET) efficiency on transfected cells to that on nontransfected BLS-1 and JY human B-cell lines. Our FRET results show a decrease in homoassociation FRET between HLA I chains in HLA-DQ6-transfected tDQ6-BLS-1 cells compared with the parent BLS-1 cell line and an increase in heteroassociation FRET between HLA I and HLA II (compared with JY cells), suggesting a similar pattern of antigen presentation by the HLA-DQ6 allele. Transmission electron microscopy (TEM) revealed that both HLA class I and class II molecules formed clusters at higher hierarchical levels on the tDQ6-BLS-1 cells, and the de novo synthesized HLA DQ molecules did not intersperse with HLA class I islands. These observations could be important in understanding the fine tuning of the immune response.

Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds toGM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

Distribution of Glycolipids in the Plasma Membrane Monitored by Specific Probes in Combination with Sodium Dodecyl Sulfate-Digested Freeze-Fracture Replica Labeling (SDS-FRL).

Glycolipids are mainly distributed in the outer leaflet of the plasma membrane and are involved in cellular signaling by modulating the activity of cell surface receptor proteins. Glycolipids themselves also work as cell surface receptors of bacterial toxins. Anti-glycolipid antibodies are associated with various pathological conditions. The cellular distribution of glycolipids has been studied using specific toxins or antibodies. However, these proteins are multivalent and thus potentially induce the artificial aggregation of glycolipids. Since chemical fixative such as paraformaldehyde does not fix glycolipids, an alternative methodology is required to localize glycolipids with multivalent probes. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) physically fixes glycolipids on the cast after quick freezing. Thus, SDS-FRL provides the opportunity to observe the natural distribution of glycolipids using multivalent probes. Here, we describe the application of SDS-FRL on the cell surface distribution of phosphatidylglucoside.

Egg White Protein Ovotransferrin-Derived IRW (Ile-Arg-Trp) Inhibits LPS-Induced Barrier Integrity Dysfunction and Inflammation in Caco-2 Cells.

Tripeptide IRW derived from egg ovotransferrin was initially identified to be an inhibitor of angiotensin-converting enzyme. Later, IRW has been shown to possess various bioactivities, including anti-inflammatory activity and the ability to suppress colitis development. Nevertheless, its role in protecting intestinal barrier integrity has not been reported. This study aims to investigate the effect of IRW on inhibiting intestinal barrier dysfunction and inflammation in lipopolysaccharide (LPS)-treated Caco-2 cells. Pretreatment with IRW could mitigate the LPS-induced reduction of transepithelial electronic resistance values and decrease the paracellular permeation of differentiated Caco-2 cell monolayers. Meanwhile, IRW restored the expression level and cell surface distribution of the tight junction protein occludin. Furthermore, IRW showed LPS-neutralizing activity and could significantly inhibit LPS-induced activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. In conclusion, our study demonstrated the ability of IRW to prevent LPS-induced intestinal barrier dysfunction and prohibit inflammatory responses.

Cutaneous manifestations in Costello syndrome: HRAS p.Gly12Ser affects RIN1-mediated integrin trafficking in immortalized epidermal keratinocytes.

Heterozygous germline missense variants in the HRAS gene underlie Costello syndrome (CS). The molecular basis for cutaneous manifestations in CS is largely unknown. We used an immortalized human cell line, HaCaT keratinocytes, stably expressing wild-type or CS-associated (p.Gly12Ser) HRAS and defined RIN1 as quantitatively most prominent, high-affinity effector of active HRAS in these cells. As an exchange factor for RAB5 GTPases, RIN1 is involved in endosomal sorting of cell-adhesion integrins. RIN1-dependent RAB5A activation was strongly increased by HRASGly12Ser, and HRAS-RIN1-ABL1/2 signaling was induced in HRASWT- and HRASGly12Ser-expressing cells. Along with that, HRASGly12Ser expression decreased total integrin levels and enriched β1 integrin in RAB5- and EEA1-positive early endosomes. The intracellular level of active β1 integrin was increased in HRASGly12Ser HaCaT keratinocytes due to impaired recycling, whereas RIN1 disruption raised β1 integrin cell surface distribution. HRASGly12Ser induced co-localization of β1 integrin with SNX17 and RAB7 in early/sorting and late endosomes, respectively. Thus, by retaining β1 integrin in intracellular endosomal compartments, HRAS-RIN1 signaling affects the subcellular availability of β1 integrin. This may interfere with integrin-dependent processes as we detected for HRASGly12Ser cells spreading on fibronectin. We conclude that dysregulation of receptor trafficking and integrin-dependent processes such as cell adhesion are relevant in the pathobiology of CS.

Characterization of alternatively spliced transcript variants of glycophorin A and glycophorin B genes in Chinese blood donors.

The molecular basis of MNS blood group variants is not fully clear yet. In this study, we have characterized mRNA variants of GYPA and GYPB genes to reveal whether alternative RNA splicing may cause antigenic diversity of the MNS system. Total RNA was extracted from peripheral blood of Chinese blood donors and full-length cDNA products were generated. A nested polymerase chain reaction (PCR)-based method was established for fragment amplification and Sanger sequencing. Resulted full-length mRNA sequences were aligned with GYPA or GYPB genomic sequences respectively for exon identification. Amino acid (AA) sequences of GPA and GPB proteins were extrapolated and GYPA-EGFP, GYPB-EGFP fusion genes were generated to monitor subcellular distribution of the encoded glycophorin (GP) proteins. Totally 10 blood samples were analysed. GYPB mRNAs of all the subjects demonstrated frequent exon insertion or deletion whereas this kind of variation was only observed in 3 of 10 GYPA mRNA samples. None of the reported Miltenberger hybrids was detected in any of the mRNA samples. The alternative splicing resulted in changes of AA sequences in N-terminal domains where the MNS antigenic motifs resided; however, subcellular localizations of GP-EGFP fusion proteins showed that the above-mentioned AA changes did not affect cell surface distribution of the encoded GP proteins. Alternative RNA splicing may influence the antigenic features of GP proteins but not their cell surface distribution. Therefore, GYPA and GYPB mRNA characterization might be an invaluable supplement to serological phenotyping and DNA-based genotyping in MNS blood grouping.

Siglec Signaling in the Tumor Microenvironment.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of receptors that recognize sialoglycans – sialic acid containing glycans that are abundantly present on cell membranes. Siglecs are expressed on most immune cells and can modulate their activity and function. The majority of Siglecs contains immune inhibitory motifs comparable to the immune checkpoint receptor PD-1. In the tumor microenvironment (TME), signaling through the Siglec-sialoglycan axis appears to be enhanced through multiple mechanisms favoring tumor immune evasion similar to the PD-1/PD-L1 signaling pathway. Siglec expression on tumor-infiltrating immune cells appears increased in the immune suppressive microenvironment. At the same time, enhanced Siglec ligand expression has been reported for several tumor types as a result of aberrant glycosylation, glycan modifications, and the increased expression of sialoglycans on proteins and lipids. Siglec signaling has been identified as important regulator of anti-tumor immunity in the TME, but the key factors contributing to Siglec activation by tumor-associated sialoglycans are diverse and poorly defined. Among others, Siglec activation and signaling are co-determined by their expression levels, cell surface distribution, and their binding preferences for cis- and trans-ligands in the TME. Siglec binding preference are co-determined by the nature of the proteins/lipids to which the sialoglycans are attached and the multivalency of the interaction. Here, we review the current understanding and emerging conditions and factors involved in Siglec signaling in the TME and identify current knowledge gaps that exist in the field.

An alternate covalent form of platelet αIIbβ3 integrin that resides in focal adhesions and has altered function

AbstractThe αIIbβ3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbβ3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbβ3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Gene silencing-mediated immune checkpoint blockade for tumor therapy boosted by dendrimer-entrapped gold nanoparticles

Immune checkpoint blockade (ICB) has been regarded as one promising approach for tumor immunotherapy. Here, we report a functional nanoplatform based on generation 5 (G5) poly(amidoamine) (PAMAM) dendrimer-entrapped gold nanoparticles (Au DENPs) as a nonviral vector to deliver programmed death-ligand 1 (PD-L1) small interfering RNA (siPD-L1) for subsequent PD-L1 gene silencing-mediated tumor immunotherapy. In this work, G5 dendrimers with amine termini were partially decorated with methoxy polyethylene glycol ( m PEG) on their periphery, entrapped Au NPs within their interiors, and were eventually labeled with fluorescamine. The generated functional Au DENPs possess desired dispersibility in water and colloidal stability, satisfactory cytocompatibility after complexation with siPD-L1, and efficient gene delivery performance. Strikingly, the functional Au DENPs enabled the delivery of siPD-L1 to cancer cells to efficiently knock down the PD-L1 protein expression, thus boosting the ICB-based immunotherapy of a xenografted melanoma mouse tumor model with a tumor inhibition efficiency much higher than the PD-L1 antibody. The immune responses were also well demonstrated by downregulation of PD-L1 protein on the tumor cell surface and abundant distribution of CD8+ and CD4+ T cells in the infiltrating tumor tissue and spleen organ. The developed functional dendrimer-based nanoplatform may be promising to boost ICB-based immunotherapy of other tumor types.

Post-translational suppression of the high affinity IgE receptor expression on mast cells by an intestinal bacterium

Mast cells, which express the high-affinity IgE receptor (FcεRI) on their surface, play a crucial role in inducing allergic inflammation. Since mast cells are activated by crosslinking of FcεRI with IgE and allergens, the cell surface expression level of FcεRI is an important factor in determining the sensitivity to allergens. Recently, the involvement of gut microbiota in the prevalence and regulation of allergy has attracted attention but the precise underlying mechanisms are not fully understood. In this study, the effect of intestinal bacteria on cell surface expression of FcεRI was examined. Bacteroides acidifaciens type A 43 specifically suppressed cell surface expression of FcεRI on mouse bone marrow-derived mast cells (BMMCs) without reduction in FcεRI α and β-chain mRNA and total protein expression. The suppressive effect required sustained exposure to this bacterium, with a corresponding reduction in Erk activation. Inhibition of Erk decreased cell surface distribution of FcεRI in BMMCs, at least in part, through facilitated endocytosis of FcεRI. These results indicate that B. acidifaciens type A 43 suppresses cell surface expression of FcεRI on mast cells in a post-translational manner via inhibition of Erk. The suppression of FcεRI expression on mast cells by specific bacteria might be the underlying mechanism involved in the regulation of allergy by gut microbiota.

Porphyromonas gingivalis laboratory strains and clinical isolates exhibit different distribution of cell surface and secreted gingipains

ABSTRACT Background: The cell-surface cysteine proteinases RgpA, RgpB (Arg-gingipain), and Kgp (Lys-gingipain) are major virulence factors of P. gingivalis, a keystone pathogen in the development of destructive periodontal disease. The gingipains function as proteinases and transpeptidases utilising small peptides such as glycylglycine as acceptor molecules. However, the characteristics of the gingipains from most P. gingivalis strains have not been determined. Methods: We determined the phenotypes of a panel of P. gingivalis laboratory strains and global clinical isolates with respect to growth on blood agar plus whole-cell and vesicle-free culture supernatant (VFSN) Arg- and Lys-specific proteinase activities. Results: The P. gingivalis isolates exhibited different growth characteristics and hydrolysis of haemoglobin in solid media. Whole-cell Arg-gingipain Vmax varied 5.8-fold and the whole cell Lys-gingipain Vmax varied 2.1-fold across the strains. Furthermore, the P. gingivalis strains showed more than 107-fold variance in soluble Arg-gingipain activity in VFSN and more than 371-fold variance in soluble Lys-gingipain activity in VFSN. Glycylglycine and cysteine stimulated Arg- and Lys-specific cleavage activities of all strains. The stimulation by cysteine was in addition to its redox effect consistent with both glycylglycine and cysteine promoting transpeptidation. Conclusion: The global P. gingivalis clinical isolates exhibit different Arg- and Lys‑gingipain activities with substantial variability in the level of soluble proteinases released into the environment.

Insights into Cell Surface Expression, Supramolecular Organization, and Functions of Aquaporin 4 Isoforms in Astrocytes.

Aquaporin 4 (AQP4) is the most abundant water channel in the central nervous system (CNS). Its expression is confined to non-neuronal glial cells, predominantly to astrocytes that represent a heterogeneous glial cell type in the CNS. The membrane of astrocyte processes, which align brain capillaries and pia, is particularly rich in AQP4. Several isoforms of AQP4 have been described; however, only some (AQP4a (M1), AQP4 c (M23), AQP4e, and AQP4ex) have been identified in the plasma membrane assemblies of astrocytes termed orthogonal arrays of particles (OAPs). Intracellular splicing isoforms (AQP4b, AQP4d, AQP4f, AQP4-Δ4) have been documented, and most of them are postulated to have a role in the cell surface distribution of the plasma membrane isoforms and in the formation of OAPs in murine and human astrocytes. Although OAPs have been proposed to play various roles in the functioning of astrocytes and CNS tissue as a whole, many of these still need to be described. OAPs are studied primarily from the perspective of understanding water permeability regulation through the plasma membrane and of their involvement in cell adhesion and in the dynamics of astrocytic processes. This review describes the cellular distribution of various AQP4 isoforms and their implications in OAP assembly, which is regulated by several intracellular and extracellular proteins.

Radiation doses from 161Tb and 177Lu in single tumour cells and micrometastases

BackgroundTargeted radionuclide therapy (TRT) is gaining importance. For TRT to be also used as adjuvant therapy or for treating minimal residual disease, there is a need to increase the radiation dose to small tumours. The aim of this in silico study was to compare the performances of 161Tb (a medium-energy β− emitter with additional Auger and conversion electron emissions) and 177Lu for irradiating single tumour cells and micrometastases, with various distributions of the radionuclide.MethodsWe used the Monte Carlo track-structure (MCTS) code CELLDOSE to compute the radiation doses delivered by 161Tb and 177Lu to single cells (14 μm cell diameter with 10 μm nucleus diameter) and to a tumour cluster consisting of a central cell surrounded by two layers of cells (18 neighbours). We focused the analysis on the absorbed dose to the nucleus of the single tumoral cell and to the nuclei of the cells in the cluster. For both radionuclides, the simulations were run assuming that 1 MeV was released per μm3 (1436 MeV/cell). We considered various distributions of the radionuclides: either at the cell surface, intracytoplasmic or intranuclear.ResultsFor the single cell, the dose to the nucleus was substantially higher with 161Tb compared to 177Lu, regardless of the radionuclide distribution: 5.0 Gy vs. 1.9 Gy in the case of cell surface distribution; 8.3 Gy vs. 3.0 Gy for intracytoplasmic distribution; and 38.6 Gy vs. 10.7 Gy for intranuclear location. With the addition of the neighbouring cells, the radiation doses increased, but remained consistently higher for 161Tb compared to 177Lu. For example, the dose to the nucleus of the central cell of the cluster was 15.1 Gy for 161Tb and 7.2 Gy for 177Lu in the case of cell surface distribution of the radionuclide, 17.9 Gy for 161Tb and 8.3 Gy for 177Lu for intracytoplasmic distribution and 47.8 Gy for 161Tb and 15.7 Gy for 177Lu in the case of intranuclear location.Conclusion161Tb should be a better candidate than 177Lu for irradiating single tumour cells and micrometastases, regardless of the radionuclide distribution.

The deubiquitinase USP6 affects memory and synaptic plasticity through modulating NMDA receptor stability.

Ubiquitin-specific protease (USP) 6 is a hominoid deubiquitinating enzyme previously implicated in intellectual disability and autism spectrum disorder. Although these findings link USP6 to higher brain function, potential roles for USP6 in cognition have not been investigated. Here, we report that USP6 is highly expressed in induced human neurons and that neuron-specific expression of USP6 enhances learning and memory in a transgenic mouse model. Similarly, USP6 expression regulates N-methyl-D-aspartate-type glutamate receptor (NMDAR)-dependent long-term potentiation and long-term depression in USP6 transgenic mouse hippocampi. Proteomic characterization of transgenic USP6 mouse cortex reveals attenuated NMDAR ubiquitination, with concomitant elevation in NMDAR expression, stability, and cell surface distribution with USP6 overexpression. USP6 positively modulates GluN1 expression in transfected cells, and USP6 down-regulation impedes focal GluN1 distribution at postsynaptic densities and impairs synaptic function in neurons derived from human embryonic stem cells. Together, these results indicate that USP6 enhances NMDAR stability to promote synaptic function and cognition.

Impaired dopamine- and adenosine-mediated signaling and plasticity in a novel rodent model for DYT25 dystonia

Dystonia is a neurological movement disorder characterized by sustained or intermittent involuntary muscle contractions. Loss-of-function mutations in the GNAL gene have been identified to be the cause of “isolated” dystonia DYT25. The GNAL gene encodes for the guanine nucleotide-binding protein G(olf) subunit alpha (Gαolf), which is mainly expressed in the olfactory bulb and the striatum and functions as a modulator during neurotransmission coupling with D1R and A2AR. Previously, heterozygous Gαolf -deficient mice (Gnal+/−) have been generated and showed a mild phenotype at basal condition. In contrast, homozygous deletion of Gnal in mice (Gnal−/−) resulted in a significantly reduced survival rate. In this study, using the CRISPR-Cas9 system we generated and characterized heterozygous Gnal knockout rats (Gnal+/−) with a 13 base pair deletion in the first exon of the rat Gnal splicing variant 2, a major isoform in both human and rat striatum. Gnal+/− rats showed early-onset phenotypes associated with impaired dopamine transmission, including reduction in locomotor activity, deficits in rotarod performance and an abnormal motor skill learning ability. At cellular and molecular level, we found down-regulated Arc expression, increased cell surface distribution of AMPA receptors, and the loss of D2R-dependent corticostriatal long-term depression (LTD) in Gnal+/− rats. Based on the evidence that D2R activity is normally inhibited by adenosine A2ARs, co-localized on the same population of striatal neurons, we show that blockade of A2ARs restores physiological LTD. This animal model may be a valuable tool for investigating Gαolf function and finding a suitable treatment for dystonia associated with deficient dopamine transmission.

Madin-Darby canine kidney cell sialic acid receptor modulation induced by culture medium conditions: Implications for the isolation of influenza A virus.

BackgroundThe influenza A virus (IAV) binds to α‐2,3‐ and α‐2,6‐linked sialic acid (SA) receptors expressed by Madin‐Darby canine kidney (MDCK) cells. The receptor distribution may therefore be important in regulating IAV propagation. Serum‐free medium (SFM) avoids variability in conventional culture medium containing fetal bovine serum (FBS), which can have variable composition and may contain endotoxins. However, little is known about the distribution of SA receptors on cells maintained in SFM.ObjectivesWe assessed the influence of culture media on MDCK cell SA receptor distribution along with the effect of SA receptor distribution on IAV recovery. We hypothesized that SFM would increase the proportion of α‐2,6‐linked SA receptors present and alter isolate recovery.MethodsMadin‐Darby canine kidney cells were cultured in medium containing FBS and two SFMs. Cell surface distribution of α‐2,6‐ and α‐2,3‐linked receptors was determined using flow cytometry. Recovery of swine‐ and avian‐lineage IAVs from MDCK cells maintained in each medium was quantified as TCID50.ResultsMadin‐Darby canine kidney cells cultured in UltraMDCK SFM expressed both SA receptors and supported the growth of both swine‐ and avian‐lineage IAVs. Cells maintained in other medium inconsistently expressed each receptor and the avian IAV grew to lower titers in cells cultured with FBS.ConclusionsMedium conditions altered the distribution of SA receptors present on MDCK cells and affected IAV recovery. Culture in UltraMDCK SFM resulted in cells expressing both receptors and IAVs grew to higher titers than in the other culture condition, indicating that this medium may be useful for culturing IAV from multiple species.

A novel motif in the proximal C-terminus of Pannexin 1 regulates cell surface localization

The Pannexin 1 (Panx1) ion and metabolite channel is expressed in a wide variety of cells where it regulates a number of cell behaviours including proliferation and differentiation. Panx1 is expressed on the cell surface as well as intracellular membranes. Previous work suggests that a region within the proximal Panx1 C-terminus (Panx1CT) regulates cell surface localization. Here we report the discovery of a putative leucine-rich repeat (LRR) motif in the proximal Panx1CT necessary for Panx1 cell surface expression in HEK293T cells. Deletion of the putative LRR motif results in significant loss of Panx1 cell surface distribution. Outcomes of complementary cell surface oligomerization and glycosylation state analyses were consistent with reduced cell surface expression of Panx1 LRR deletion mutants. Of note, the oligomerization analysis revealed the presence of putative dimers and trimers of Panx1 at the cell surface. Expression of Panx1 increased HEK293T cell growth and reduced doubling time, while expression of a Panx1 LRR deletion mutant (highly conserved segment) did not reproduce this effect. In summary, here we discovered the presence of a putative LRR motif in the Panx1CT that impacts on Panx1 cell surface localization. Overall these findings provide new insights into the molecular mechanisms underlying C-terminal regulation of Panx1 trafficking and raise potential new lines of investigation with respect to Panx1 oligomerization and glycosylation.