The rs1143679 (R77H) lupus associated variant of ITGAM (CD11b) impairs complement receptor 3 mediated functions in human monocytes.

Objectives: The rs1143679 variant of ITGAM, encoding the R77H variant of CD11b (part of complement receptor 3; CR3), is among the strongest genetic susceptibility effects in human systemic lupus erythematosus (SLE). The authors aimed to demonstrate R77H function in ex-vivo human cells.

Methods: Monocytes/monocyte-derived macrophages from healthy volunteers homozygous for either wild type (WT) or 77H CD11b were studied.

RhoG is required for both FcγR- and CR3-mediated phagocytosis.

Phagocytosis is a highly ordered process orchestrated by signalling through Rho GTPases to locally organise the actin cytoskeleton and drive particle uptake. Specific Rho family members that regulate phagocytosis are not known, as the majority of studies have relied on the use of dominant-negative mutants and/or toxins, which can inactivate multiple Rho GTPases. To identify the relevant GTPases for phagocytosis through the Fcγ receptor (FcγR) and complement receptor 3 (CR3), we depleted 20 Rho proteins individually in an RNA interference (RNAi) screen.

The zipper mechanism in phagocytosis: energetic requirements and variability in phagocytic cup shape.

Background: Phagocytosis is the fundamental cellular process by which eukaryotic cells bind and engulf particles by their cell membrane. Particle engulfment involves particle recognition by cell-surface receptors, signaling and remodeling of the actin cytoskeleton to guide the membrane around the particle in a zipper-like fashion. Despite the signaling complexity, phagocytosis also depends strongly on biophysical parameters, such as particle shape, and the need for actin-driven force generation remains poorly understood.

A mechanical bottleneck explains the variation in cup growth during FcgammaR phagocytosis.

Phagocytosis is the process by which cells internalize particulate material, and is of central importance to immunity, homeostasis and development. Here, we study the internalization of immunoglobulin G-coated particles in cells transfected with Fcgamma receptors (FcgammaRs) through the formation of an enveloping phagocytic cup. Using confocal microscopy, we precisely track the location of fluorescently tagged FcgammaRs during cup growth.

An essential role for talin during alpha(M)beta(2)-mediated phagocytosis.

The cytoskeletal, actin-binding protein talin has been previously implicated in phagocytosis in Dictyostelium discoideum and mammalian phagocytes. However, its mechanism of action during internalization is not understood. Our data confirm that endogenous talin can occasionally be found at phagosomes forming around IgG- and C3bi-opsonized red blood cells in macrophages.

Chemotaxis towards hyaluronan is dependent on CD44 expression and modulated by cell type variation in CD44-hyaluronan binding.

The accumulation of the extracellular matrix glycosaminoglycan hyaluronan by tumours and tumour-associated stroma promotes cancer cell invasion and metastasis. Using the Dunn chamber chemotaxis assay, we demonstrate for the first time that high molecular mass hyaluronan acts as a soluble chemoattractant promoting the directional migration of MDA-MB-468 and MDA-MB-231 breast cancer cells. Moreover, chemotaxis towards hyaluronan, but not foetal bovine serum, can be abrogated following treatment of the cells with siRNA oligonucleotides to downregulate CD44 expression.

A new spreadsheet method for the analysis of bivariate flow cytometric data.

Background: A useful application of flow cytometry is the investigation of cell receptor-ligand interactions. However such analyses are often compromised due to problems interpreting changes in ligand binding where the receptor expression is not constant. Commonly, problems are encountered due to cell treatments resulting in altered receptor expression levels, or when cell lines expressing a transfected receptor with variable expression are being compared.