Membrane topography and the overestimation of protein clustering in single molecule localisation microscopy - identification and correction.

According to single-molecule localisation microscopy almost all plasma membrane proteins are clustered. We demonstrate that clusters can arise from variations in membrane topography where the local density of a randomly distributed membrane molecule to a degree matches the variations in the local amount of membrane. Further, we demonstrate that this false clustering can be differentiated from genuine clustering by using a membrane marker to report on local variations in the amount of membrane. In dual colour live cell single molecule localisation microscopy using the membrane probe DiI alongside either the transferrin receptor or the GPI-anchored protein CD59, we found that pair correlation analysis reported both proteins and DiI as being clustered, as did its derivative pair correlation-photoactivation localisation microscopy and nearest neighbour analyses. After converting the localisations into images and using the DiI image to factor out topography variations, no CD59 clusters were visible, suggesting that the clustering reported by the other methods is an artefact. However, the TfR clusters persisted after topography variations were factored out. We demonstrate that membrane topography variations can make membrane molecules appear clustered and present a straightforward remedy suitable as the first step in the cluster analysis pipeline.

Downregulation of miRNA-26a by HIV-1 Enhances CD59 Expression and Packaging, Impacting Virus Susceptibility to Antibody-Dependent Complement-Mediated Lysis.

MicroRNAs (miRNAs) play important roles in the control of HIV-1 infection. Here, we performed RNA-seq profiling of miRNAs and mRNAs expressed in CD4+ T lymphocytes upon HIV-1 infection. Our results reveal significant alterations in miRNA and mRNA expression profiles in infected relative to uninfected cells. One of the miRNAs markedly downregulated in infected cells is miRNA-26a. Among the putative targets of miRNA-26a are CD59 receptor transcripts, which are significantly upregulated in infected CD4+ T cells. The addition of miRNA-26a mimics to CD4+ T cells reduces CD59 at both the mRNA and surface protein levels, validating CD59 as a miRNA-26a target. Consistent with the reported inhibitory role of CD59 in complement-mediated lysis (CML), knocking out CD59 in CD4+ T cells renders both HIV-1-infected cells and progeny virions more prone to antibody-dependent CML (ADCML). The addition of miRNA-26a mimics to infected cells leads to enhanced sensitivity of progeny virions to ADCML, a condition linked to a reduction in CD59 packaging into released virions. Lastly, HIV-1-mediated downregulation of miRNA-26a expression is shown to be dependent on integrated HIV-1 expression but does not involve viral accessory proteins. Overall, these results highlight a novel mechanism by which HIV-1 limits ADCML by upregulating CD59 expression via miRNA-26a downmodulation.

A Novel Marker for Screening Paroxysmal Nocturnal Hemoglobinuria Using Routine Complete Blood Count and Cell Population Data

BACKGROUND: Final diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) may take years demanding a quick diagnosis measure. We used the facts that PNH cells are damaged in acid, and reagents for measuring reticulocytes in Coulter DxH800 (Beckman Coulter, USA) are weakly acidic and hypotonic, to create a new PNH screening marker. METHODS: We analyzed 979 complete blood counts (CBC) data from 963 patients including 57 data from 44 PNH patients. Standard criteria for PNH assay for population selection were followed: flow cytometry for CD55 and CD59 on red blood cells (RBCs) to a detection level of 1%; and fluorescent aerolysin, CD24 and CD15 in granulocytes to 0.1%. Twenty-four PNH minor clone-positive samples (minor-PNH+) were taken, in which the clone population was <5% of RBCs and/or granulocytes. Excluding PNH and minor-PNH+ patients, the population was divided into anemia, malignancy, infection, and normal groups. Parameters exhibiting a distinct demarcation between PNH and non-PNH groups were identified, and each parameter cutoff value was sought that includes the maximum [minimum] number of PNH [non-PNH] patients. RESULTS: Cutoff values for 5 selected CBC parameters (MRV, RDWR, MSCV, MN-AL2-NRET, and IRF) were determined. Positive rates were: PNH (86.0%), minor-PNH+ (33.3%), others (5.0%), anemia (13.4%), malignancy (5.3%), infection (3.7%), normal (0.0%); within anemia group, aplastic anemia (40.0%), immune hemolytic anemia (11.1%), iron deficiency anemia (1.6%). Sensitivity (86.0%), specificity (95.0%), PPV (52.1%), and NPV (99.1%) were achieved in PNH screening. CONCLUSION: A new PNH screening marker is proposed with 95% specificity and 86% sensitivity. The flag identifies PNH patients, reducing time to final diagnosis by flow cytometry.