scholarly journals A Single Mutation N166D in Hemagglutinin Affects Antigenicity and Pathogenesis of H9N2 Avian Influenza Virus

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 709 ◽  
Author(s):  
Fang Jin ◽  
Xiaomei Dong ◽  
Zhimin Wan ◽  
Dan Ren ◽  
Min Liu ◽  
...  
Keyword(s):  
Immune Escape ◽  
Mdck Cells ◽  
Body Weight Loss ◽  
Escape Mutant ◽  
Single Mutation ◽  
H9n2 Avian Influenza Virus ◽  
Viral Shedding ◽  
Escape Mutants ◽  
The Impact ◽  
Darby Canine Kidney

Some immune escape mutants of H9N2 virus and the corresponding mutations in hemagglutinin (HA) have been documented, but little is known about the impact of a single mutation on the antigenicity and pathogenesis of H9N2. In this study, seven critical sites in HA associated with the antigenicity were identified and the effects of a HA mutation (N166D) derived from a H9N2 escape mutant (m3F2) were investigated. Although N166D did not significantly affect viral replication in Madin–Darby canine kidney (MDCK) cells and viral shedding in the larynx and cloaca of chicken, N166D attenuated the pathogenesis of the virus in mice. Compared to the rescued RgPR8-H9_166D, RgPR8-H9_166N caused greater body weight loss and higher viral titers in the lungs of the infected mice. Moreover, hemagglutination inhibition (HI) assay for the sera from the chickens infected with wild type H9N2 and mutant m3F2 showed that N166D mutation could result in weak antibody response in chickens. Considering the field strains of H9N2 with N166D mutation are frequently isolated in the countries with H9N2 vaccination, the findings that the single mutation in HA, N166D, affected both the antigenicity and pathogenesis of H9N2 highlight the significance of surveillance on such mutation that may contribute to the failure of H9N2 vaccination in the field.

scholarly journals Immune Escape Adaptive Mutations in the H7N9 Avian Influenza Hemagglutinin Protein Increase Virus Replication Fitness and Decrease Pandemic Potential

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Pengxiang Chang ◽  
Joshua E. Sealy ◽  
Jean-Remy Sadeyen ◽  
Sushant Bhat ◽  
Deimante Lukosaityte ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Avian Influenza ◽  
Virus Replication ◽  
Receptor Binding ◽  
Immune Escape ◽  
Influenza Viruses ◽  
Escape Mutant ◽  
Hemagglutinin Protein ◽  
Ha Protein ◽  
The Impact

ABSTRACT H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T) and 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in field isolates in 2019. To investigate the potential threat of serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability, and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo. They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor-binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in the HA protein may pose a reduced pandemic risk but remain a heightened threat for poultry. IMPORTANCE Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in virus-specific antiserum gives rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunized or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chicken embryos as well as (ii) increased acid and thermal stability but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing the indicated mutations remain a significant risk to poultry due to antigenic drift and improved fitness for poultry.

scholarly journals 37. Allogeneic, Off-the-Shelf, SARS-CoV-2-specific T Cells Demonstrate Reactivity Against Emerging Variant Strains

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S27-S27
Author(s):  
Spyridoula Vasileiou ◽  
Manik Kuvalekar ◽  
Aster Workineh ◽  
Ayumi Watanabe ◽  
Yovana Velazquez ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Function ◽  
Viral Infections ◽  
Immune Escape ◽  
Standard Of Care ◽  
Cytolytic Activity ◽  
Effector Memory ◽  
Severe Illness ◽  
Viral Shedding ◽  
The Impact

Abstract Background The impact of COVID-19 has been profound with >170,000,000 confirmed cases worldwide and emerging variants being a cause of global concern. Defects in T-cell function and trafficking have been described among those with severe illness, and immunodeficiency is a risk factor for persistent viral shedding and prolonged symptoms. Because of our prior clinical data demonstrating that allogeneic, off-the-shelf virus-specific T cells (VSTs) can safely and effectively treat viral infections, we investigated the feasibility of targeting COVID-19 using banked, SARS-CoV-2-specific VSTs. Methods We first screened PBMCs from convalescent individuals against 18 structural and non-structural/accessory (NSPs/APs) SARS-CoV-2 proteins and identified 5 [Spike (S), Membrane (M), Nucleoprotein (N), NSP4, and AP7a] as immunodominant which were then advanced to our VST production process. Results Using overlapping peptide libraries spanning these antigens as a stimulus, we achieved a mean 7.6±0.9 fold expansion (n=13) of VSTs (96±0.5%), with a mixture of cytotoxic (CD8+) and helper (CD4+) T cells that expressed activation and central/effector memory markers. These VSTs were potent, Th1-polarized and polyfunctional, producing IFNγ, TNFα, GM-CSF and Granzyme B. Moreover, the VSTs were able to kill pepmix-loaded autologous targets with no evidence of auto- or alloreactivity, attesting to their virus selectivity and safety for clinical use (Figure 1). Finally, though initially generated against the reference strain NC_045512.2 (Wuhan), these VSTs were able to recognize other clinically important variants including B1.1.7 (UK), B1.351 (South Africa) and P1 (Brazil). This demonstrates the cross-reactive potential of these polyclonal and diverse VSTs, which were developed to provide potent antiviral effects and minimize the risk of immune escape due to sequence variation. Figure 1: SARS-CoV-2 Specific T cells Have Demonstrated Selective Cytolytic Activity against SARS-CoV-2 While Leaving Non-Virus Infected Targets Intact. Conclusion In conclusion, it is feasible to generate polyclonal SARS-CoV-2 VSTs that provide coverage against variant strains using GMP-compliant manufacturing methodologies. We have advanced this product to the bedside for administration in a Phase I, randomized clinical trial [VSTs+ standard of care (SOC) vs SOC] in high-risk patients hospitalized with COVID-19 (NCT04401410). Disclosures Spyridoula Vasileiou, PhD, AlloVir (Consultant) Manik Kuvalekar, MSc, AlloVir (Consultant) Aster Workineh, MSc, AlloVir (Employee) Ayumi Watanabe, BSc, AlloVir (Consultant) Yovana Velazquez, BSc, AlloVir (Consultant) Helen Elisabeth Heslop, MD, AlloVir (Shareholder)Cell Medica (Grant/Research Support)Gilead Sciences (Consultant)Kiadis Pharma (Consultant)Marker Therapeutics (Consultant, Shareholder)Mesoblast (Consultant)Novartis (Consultant)PACT Pharma (Consultant)Tessa Therapeutics (Consultant, Research Grant or Support) Premal Lulla, MD, Johnson & Johnson (Shareholder) Ann Marie Leen, PhD, AlloVIr (Consultant, Shareholder)Marker Therapeutics (Consultant, Shareholder)

scholarly journals PO 8275 HEPATITIS B VIRUS IMMUNE ESCAPE MUTANTS AMONG APPARENTLY HEALTHY INHABITANTS IN IBADAN, SOUTHWESTERN NIGERIA

2019 ◽  
Vol 4 (Suppl 3) ◽  
pp. A25.3-A26
Author(s):  
Adeleye S Bakarey ◽  
Ijeoma M Ifeorah ◽  
Adegboyega Akere
Keyword(s):  
Immune Escape ◽  
Hbv Dna ◽  
Escape Mutant ◽  
Southwestern Nigeria ◽  
Significant Similarity ◽  
S Gene ◽  
Escape Mutants ◽  
Detectable Hbsag ◽  
Hbv Prevention ◽  
Genotype E

BackgroundThe documentation of circulation of immune escape mutants (IEMs) poses a risk on the continual success of HBV prevention and control. Therefore, this study aimed to determine the possible circulation of IEM among asymptomatic dwellers in southwestern Nigeria.MethodsBlood samples collected from consenting 133 males and 305 female participants in Ibadan were tested for HBsAg, HBeAg, HBcIgM, HBcTotal and HBsAb by ELISA technique. Samples positive for HBsAg were further analysed for HBV DNA by amplifying and sequencing the S gene. Isolates were genotyped and subtyped based on amino acid residues at position 122, 127, 134, 160 of the S gene.ResultsOf the 438 subjects tested 31 (7.1%) were positive for HBsAg, 2 (6.5%) of which were HBeAg positive. Ninety-nine (22.8%) had detectable HBsAb, 3 (0.7%) were positive for HBcIgM and 195 (44.5%) were HBcTotal positive. HBV DNA was amplified and sequenced in 27 out of 31 and 4 could not be amplified due to low titres. After sequencing, 9 (33.3%) were not exploitable due to the presence of multiple peaks. Of the 18 exploitable isolates, only 15 showed significant similarity to HBV S-gene. Eleven of the 15 isolates were subtyped as ayw4 while others could not due to substitution at s122p. Phylogram showed that the 11 isolates were genotype E. Two of the 4 isolates with R122Q/P substitutions also belonged to genotype E while the other 2 which were >11% divergent from the reference genotype E sequence clustered with an isolate previously described as an Immune Escape Mutant.ConclusionThis study identified high endemicity of HBV infection, presence of markers of infection even in non-detectable HBsAg levels and circulation of genotype E ayw4 and vaccine mutants in south-western Nigeria. It therefore emphasises the risk of development of an indigenous infected population that may not be protected by the current vaccine.

scholarly journals Cytotoxic T-Lymphocyte Escape Mutations Identified by HLA Association Favor Those Which Escape and Revert Rapidly

2012 ◽  
Vol 86 (16) ◽  
pp. 8568-8580 ◽  
Author(s):  
Helen R. Fryer ◽  
John Frater ◽  
Anna Duda ◽  
Duncan Palmer ◽  
Rodney E. Phillips ◽  
...  
Keyword(s):  
T Lymphocyte ◽  
Immune Escape ◽  
Cytotoxic T Lymphocyte ◽  
Population Level ◽  
Escape Mutation ◽  
Cross Sectional ◽  
Hla Association ◽  
Escape Mutants ◽  
Ctl Escape ◽  
The Impact

Identifying human immunodeficiency virus (HIV) immune escape mutations has implications for understanding the impact of host immunity on pathogen evolution and guiding the choice of vaccine antigens. One means of identifying cytotoxic-T-lymphocyte (CTL) escape mutations is to search for statistical associations between mutations and host human leukocyte antigen (HLA) class I alleles at the population level. The impact of evolutionary rates on the strength of such associations is not well defined. Here, we address this topic using a mathematical model of within-host evolution and between-host transmission of CTL escape mutants that predicts the prevalence of escape mutants at the population level. We ask how the rates at which an escape mutation emerges in a host who bears the restricting HLA and reverts when transmitted to a host who does not bear the HLA affect the strength of an association. We consider the impact of these factors when using a standard statistical method to test for an association and when using an adaptation of that method that corrects for phylogenetic relationships. We show that with both methods, the average sample size required to identify an escape mutation is smaller if the mutation escapes and reverts quickly. Thus, escape mutations identified as HLA associated systematically favor those that escape and revert rapidly. We also present expressions that can be used to infer escape and reversion rates from cross-sectional escape prevalence data.

scholarly journals Molecular surveillance of HIV, HBV, and HCV amongst blood donors in five Chinese regions

2020 ◽  
Author(s):  
Xiaoting Lv ◽  
Mary A Rodgers ◽  
Peng Yin ◽  
Ling Ke ◽  
Ping Fu ◽  
...  
Keyword(s):  
Blood Donors ◽  
Immune Escape ◽  
Scientific Basis ◽  
Blood Safety ◽  
Molecular Tests ◽  
Hbsag Detection ◽  
Immunodeficiency Virus ◽  
Escape Mutants ◽  
Different Strains ◽  
The Impact

AbstractHepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency virus (HIV) are transfusion transmissible infections (TTIs) agents that threaten the safety of the blood supply. Surveillance of the variance of those viruses is an important way to monitor their diversity and evolution to improve safety in blood transfusion. In this study, we characterized the specimens of blood donors from 13 blood centers located in 5 Chinese regions.Samples collected between 2014 and 2017 were screened with serological and molecular tests conducted on Abbott ARCHITECT and m2000 platforms. Sequencing was used to determine the classifications. The HBV immune escape mutations were also analyzed for assessing vaccine breakthrough risks and challenges for diagnostic tests. For HIV, 11 genotypes or recombinants were identified. The predominant genotype was C, which accounts for 42%. For HBV, the genotypes of B, C and D were identified, with B and C predominating. The major subgenotype was B2, comprising 84.1% of all infections. 79 out of 113 (69.9%) samples carried escape mutations in the “a” determinant region with 69 (87.3%) multiple mutants and 15 (19%) escape mutants which will affect HBsAg detection. For HCV, 7 genotypes or subtypes were identified. The major genotype was 1b (48%), followed by 6a (16.7%) and 2a & 3a (10%). This study provides the information of diversity of HBV, HCV and HIV strains circulating in blood centers from 5 regions in China. These data can also be scientific basis for development of detection assays that mitigate the impact of viral diversity on performance.ImportanceThe prevalence of TTIs in blood donations is important for evaluating blood safety and it can also reflect the burden of these disease among populations. Virus variance is threat to blood safety due to it may affect assays detection by nucleic acid, antigen and antibody-based methods in blood donors. HIV, HBV and HCV exhibit high degrees of genetic diversity, with different strains predominating in different geographic locations. The aim of this study is to assess the diversity of HBV, HCV and HIV among blood donors in China. In this study, 13 blood centers located in 5 Chinese regions were involved and the most informative phylogenetic regions of each virus had been sequenced. This will benefit for viral monitoring by subtype/genotype analyses to determine whether the distributions of variants are changing over time and geographically, and to speculate whether previously rare subtypes are becoming established in blood donors in China.

scholarly journals 1066. Immune Escape Mutant Detection Using Commercially Available Methods for Hepatitis B Surface Antigen Serological Testing

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S562-S562
Author(s):  
Robert Gish ◽  
Vincent Streva
Keyword(s):  
Hepatitis B ◽  
Immune Escape ◽  
Hepatitis B Surface Antigen ◽  
Panel Member ◽  
The United States ◽  
Hbv Infection ◽  
Hepatitis B Vaccination ◽  
Review Panel ◽  
B Virus ◽  
Escape Mutants

Abstract Background Although overall infection rates of Hepatitis B virus (HBV) in the United States (US) remain stable, as many as 2.2 million persons are still chronically infected with Hepatitis B Virus (HBV)1. Persons who inject drugs (PWID) are at a higher risk of HBV infection and since 2009 three states (KY, TN, WV) have reported up to a 114% increase in cases of acute HBV infection due to higher infection rates among a non-Hispanic white populations (30–39 years), and injection drug users2. Hepatitis B vaccination is recommended as primary prevention for adults who are at increased risk for HBV infection, including PWID. However, data from the National Health Interview Survey indicate that hepatitis B vaccination coverage is low among adults in the general population3, and it is likely to be lower among injection drug users. Hepatitis B Surface Antigen (HBsAg) is the first serological marker to appear after HBV exposure and infection; this marker is included in the recommended panel for acute hepatitis diagnosis and accurate detection is necessary for early and accurate diagnosis. Serological testing challenges exist for HBsAg due to the high degree of genetic variability which can further be exacerbated by endogenous and exogenous pressures. The immuno-dominant region may have one or more mutations described as immune escape mutations which can decrease or abrogate HBsAg binding to antibodies used in immunoassays. Although the prevalence of these mutations is not well documented in the United States, international studies have shown that up to 79% of HBV-reactivated patients (vs 3.1% of control patients; p< 0.001) carry HBsAg mutations localized in immune-active HBsAg regions4. Methods A study was conducted using a panel of 10 unique recombinant HBsAg immune escape mutants. Panel members were tested by commercially available HBsAg serological immunoassays. Results It was found that although commercially available HBsAg immunoassays are the primary diagnostic tool for HBV diagnosis, not all HBsAg immune escape mutants are detected, with some method detecting as few as 5 out of 10 of these mutant samples. Figure 1 Conclusion Improvement is needed in commercially available methods for the accurate detection of HBsAg. Disclosures Robert Gish, MD, Abbott (Consultant)AbbVie (Consultant, Advisor or Review Panel member, Speaker’s Bureau)Access Biologicals (Consultant)Antios (Consultant)Arrowhead (Consultant)Bayer (Consultant, Speaker’s Bureau)Bristol Myers (Consultant, Speaker’s Bureau)Dova (Consultant, Speaker’s Bureau)Dynavax (Consultant)Eiger (Consultant, Advisor or Review Panel member)Eisai (Consultant, Speaker’s Bureau)Enyo (Consultant)eStudySite (Consultant, Advisor or Review Panel member)Exelixis (Consultant)Fujifilm/Wako (Consultant)Genentech (Consultant)Genlantis (Consultant)Gilead (Consultant, Advisor or Review Panel member, Speaker’s Bureau)GLG (Consultant)HepaTX (Consultant, Advisor or Review Panel member)HepQuant (Consultant, Advisor or Review Panel member)Intercept (Consultant, Speaker’s Bureau)Ionis (Consultant)Janssen (Consultant)Laboratory for Advanced Medicine (Consultant)Lilly (Consultant)Merck (Consultant)Salix (Consultant, Speaker’s Bureau)Shionogi (Consultant, Speaker’s Bureau)Viking (Consultant)

Electrophysiological Measurements of a Toad Renal Epithelial Cell Line (A6) as an Assay for Predicting Ocular Eye Irritancy

1992 ◽  
Vol 20 (2) ◽  
pp. 218-221
Author(s):  
Henning F. Bjerregaard
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
South African ◽  
Mdck Cells ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Electrophysiological Measurements ◽  
Renal Epithelial Cell Line ◽  
Darby Canine Kidney

An established epithelial cell line (A6) from a South African clawed toad (Xenopus laevis) kidney was used as a model for the corneal epithelium of the eye in order to determine ocular irritancy. When grown on Millipore filter inserts, A6 cells form a monolayer epithelium of high electrical resistance and generate a trans-epithelial potential difference. These two easily-measured electrophysiological endpoints showed a dose-related decrease after exposure for 24 hours to seven selected chemicals of different ocular irritancy potential. It was demonstrated that both trans-epithelial resistance and potential ranked closely with in vivo eye irritancy data and correlated well (r = 0.96) with loss of trans-epithelial impermeability of Madin-Darby canine kidney (MDCK) cells, detected by use of a fluorescein leakage assay.

scholarly journals The Impact of Diet and Fibre Fractions on Plasma Adipocytokine Levels in Prediabetic Adults

Nutrients ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 487
Author(s):  
Margarita S. Dodevska ◽  
Sladjana S. Sobajic ◽  
Vesna D. Dragicevic ◽  
Ivan Stankovic ◽  
Nevena Dj. Ivanovic ◽  
...  
Keyword(s):  
Dietary Intervention ◽  
Polynomial Regression ◽  
Body Weight Loss ◽  
C Reactive Protein ◽  
Resistin Level ◽  
Reactive Protein ◽  
Obese Subjects ◽  
The Impact ◽  
Necrosis Factor ◽  
Recommended Level

The impact of diet and fibre fractions on adipocytokines in obese subjects with a risk of diabetes has not been investigated in detail yet. The purpose of the study is to evaluate the effects of a 12-month lifestyle intervention with different fibre profiles (resistant starch (RS)—rich fibre, or ordinary food fibre profiles) on adipocytokine levels. Fifty participants are divided into two groups (RS group and Fibre group). The groups differ only in the percentage of the recommended level of the RS consumed as a fraction of the same total fibre amount. The applied dietary intervention includes intake of 7531 KJ/daywith a total fibre portion of 25–35 g/dayfor both groups that includes 15 g/day of RS for the RS group only. The levels of leptin, adiponectin, apelin, resistin, tumor necrosis factor (TNF)-alpha and C-reactive protein (CRP) are measured, and their relationship to anthropometric and biochemical parameters is estimated. Along with significant body weight loss, only leptin is significantly reduced by 13% in the RS group while in the Fibre group, apelin levels are significant (−21%). Polynomial regression shows a negative correlation between RS intake and adiponectin (R2 = 0.145) and resistin level (R2 = 0.461) in the RS group. This study indicates the possibility that fibre fractions differently influence the outcome of lifestyle interventions, as well as their adipocytokine levels, in obese prediabetic adults.

scholarly journals SARS-CoV-2 Clinical Outcome in Domestic and Wild Cats: A Systematic Review

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 2056
Author(s):  
Sebastian Giraldo-Ramirez ◽  
Santiago Rendon-Marin ◽  
Javier A. Jaimes ◽  
Marlen Martinez-Gutierrez ◽  
Julian Ruiz-Saenz
Keyword(s):  
Systematic Review ◽  
Immune Escape ◽  
Case Reports ◽  
Clinical Signs ◽  
Press Releases ◽  
Animal Populations ◽  
Escape Mutants ◽  
The Subject ◽  
Wild Cats ◽  
Multiple Species

Recently, it has been proved that SARS-CoV-2 has the ability to infect multiple species. This work was aimed at identifying the clinical signs of SARS-CoV-2 infection in domestic and wild felids. A PRISMA-based systematic review was performed on case reports on domestic and wild cats, reports on experimental infections, case reports in databases, preprints and published press releases. Descriptive statistical analysis of the data was performed. A total of 256 articles, 63 detailed official reports and 2 press articles on SARS-CoV-2 infection in domestic and wild cats were analyzed, of which 19 articles and 65 reports were finally included. In domestic cats, most cats’ infections are likely to be asymptomatic, and 46% of the reported infected animals were symptomatic and predominantly presented respiratory signs such as sneezing and coughing. In wild felines, respiratory clinical signs were most frequent, and up to 96.5% of the reported affected animals presented coughing. It is noteworthy that, to date, symptomatic animals with SARS-CoV-2 infection have been reported to belong to two different subfamilies (Phanterinae and Felinae), with up to five different felid species affected within the Felidae family. Reported results evince that the signs developed in felids show similar progression to those occurring in humans, suggesting a relationship between the viral cycle and target tissues of the virus in different species. While viral transmission to humans in contact with animal populations has not been reported, spill-back could result in the emergence of immune-escape mutants that might pose a risk to public health. Despite the clear results in the identification of the typical clinical picture of SARS-CoV-2 infection in felines, the number of detailed academic reports and papers on the subject is scarce. Therefore, further description of these cases will allow for more accurate and statistically robust clinical approaches in the future.

scholarly journals Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells

2012 ◽  
Vol 23 (12) ◽  
pp. 2302-2318 ◽  
Author(s):  
Lynne A. Lapierre ◽  
Kenya M. Avant ◽  
Cathy M. Caldwell ◽  
Asli Oztan ◽  
Gerard Apodaca ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Mdck Cell ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Cellular Polarity ◽  
Interacting Protein ◽  
P120 Catenin ◽  
Myosin Vb ◽  
Darby Canine Kidney

The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin–Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)–expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells.

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