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. Remarkably, talin knockdown specifically abrogates uptake through complement receptor 3 (CR3, CD11b/CD18, ␣ M ␤ 2 integrin) and not through the Fc ␥ receptor. We show that talin physically interacts with CR3/␣ M ␤ 2 and that this interaction involves the talin head domain and residues W747 and F754 in the ␤ 2 integrin cytoplasmic domain. The CR3/␣ M ␤ 2 -talin head interaction controls not only talin recruitment to forming phagosomes but also CR3/␣ M ␤ 2 binding activity, both in macrophages and transfected fibroblasts. However, the talin head domain alone cannot support phagocytosis. Our results establish for the first time at least two distinct roles for talin during CR3/␣ M ␤ 2 -mediated phagocytosis, most noticeably activation of the CR3/␣ M ␤ 2 receptor and phagocytic uptake. INTRODUCTIONPhagocytosis is an essential physiological function, common to most eukaryotic cell types. From serving a feeding role in amoebae, phagocytosis is observed in Metazoa as a homeostatic process that ensures the removal of microorganisms and apoptotic cells (Desjardins et al., 2005). Classically, phagocytosis is a multistep process that sequentially involves receptor-mediated particle recognition, actin-driven uptake, phagosome maturation and particle clearance. Numerous phagocytic receptors exist that can bind their target directly or indirectly through opsonins (Underhill and Ozinsky, 2002). Receptors for phagocytosis can show constitutive or inducible binding activities, as illustrated for the two bestcharacterized phagocytic receptors: the Fc ␥ receptor (Fc␥R) for complexed IgG and complement receptor 3 (CR3, CD11b/ CD18, ␣ M ␤ 2 integrin), respectively (Bianco et al., 1975). Ligand-bound receptors classically zipper around the phagocytic prey and induce intracellular signaling cascades that lead to the activation and recruitment of signaling and adaptor molecules at sites of particle binding. These locally assembled signaling complexes reorganize the actin cytoskeleton and regulate membrane dynamics underneath bound particles through the activation of Rho-and Arf-family GTPbinding proteins, respectively (Cougoule et al., 2004). According to the zipper model, phagocytosis of bound particles requires continual ligation of phagocytic receptors around the whole phagocytic object, at least for spherical particles Champion and Mitragotri, 2006). Several cytoskeletal proteins have been shown to be recruited to phagocytic cups, although their role is not always defined. Talin, a cytoskeletal protein of 2541 amino acids and 270 kDa has been repeatedly implicated in phagocytosis. Immunofluorescence studies of phagocytozing macrophages have shown that talin accumulates...

BackgroundPhagocytosis 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.ResultsHere, we propose a novel, three-dimensional and stochastic biophysical model of phagocytosis, and study the engulfment of particles of various sizes and shapes, including spiral and rod-shaped particles reminiscent of bacteria. Highly curved shapes are not taken up, in line with recent experimental results. Furthermore, we surprisingly find that even without actin-driven force generation, engulfment proceeds in a large regime of parameter values, albeit more slowly and with highly variable phagocytic cups. We experimentally confirm these predictions using fibroblasts, transfected with immunoreceptor FcγRIIa for engulfment of immunoglobulin G-opsonized particles. Specifically, we compare the wild-type receptor with a mutant receptor, unable to signal to the actin cytoskeleton. Based on the reconstruction of phagocytic cups from imaging data, we indeed show that cells are able to engulf small particles even without support from biological actin-driven processes.ConclusionsThis suggests that biochemical pathways render the evolutionary ancient process of phagocytic highly robust, allowing cells to engulf even very large particles. The particle-shape dependence of phagocytosis makes a systematic investigation of host-pathogen interactions and an efficient design of a vehicle for drug delivery possible.

ObjectivesThe 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.MethodsMonocytes/monocyte-derived macrophages from healthy volunteers homozygous for either wild type (WT) or 77H CD11b were studied. The genotype-specific expression of CD11b, and CD11b activation using conformation-specific antibodies were measured. Genotype-specific differences in iC3b-mediated phagocytosis, adhesion to a range of ligands and the secretion of cytokines following CR3 ligation were studied. The functionality of R77H was confirmed by replicating findings in COS7 cells expressing variant-specific CD11b.ResultsNo genotype-specific difference in CD11b expression or in the expression of CD11b activation epitopes was observed. A 31% reduction was observed in the phagocytosis of iC3b opsonised sheep erythrocytes (sRBCiC3b) by 77H cells (p=0.003) and reduced adhesion to a range of ligands: notably a 24% reduction in adhesion to iC3b (p=0.014). In transfected COS7 cells, a 42% reduction was observed in phagocytosis by CD11b (77H)-expressing cells (p=0.004). A significant inhibition was seen in the release of Toll-like receptor 7/8-induced pro-inflammatory cytokines from WT monocytes when CR3 was pre-engaged using sRBCiC3b, but no inhibition in 77H monocytes resulting in a significant difference between genotypes (interleukin (IL)-1β p=0.030; IL-6 p=0.029; tumour necrosis factor alpha p=0.027).ConclusionsThe R77H variant impairs a broad range of CR3 effector functions in human monocytes. This study discusses how perturbation of this pathway may predispose to SLE.

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. These data indicate that CD44 is the principal receptor mediating this response and that CD44 expression is not a general requirement for cell migration and gradient sensing, rather it elicits a ligand-specific response. However, expression of CD44 alone is not sufficient to drive chemotaxis towards hyaluronan, as NIH-3T3 fibroblasts were unable to respond to a hyaluronan gradient even when transfected with high levels of human CD44. For NIH-3T3 cells to bind exogenous hyaluronan, it was necessary to both increase the level of receptor expression and remove a hyaluronan pericellular matrix. Together, these studies reveal a direct mechanism for promoting cell invasion into the hyaluronan-rich matrix and predict that in the complex multicellular environment in vivo, multiple mechanisms exist to regulate the ability of a cell to respond to a chemotactic hyaluronan gradient.

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 Fcc receptors (FccRs) through the formation of an enveloping phagocytic cup. Using confocal microscopy, we precisely track the location of fluorescently tagged FccRs during cup growth. Surprisingly, we found that phagocytic cups growing around identical spherical particles showed great variability even within a single cell and exhibited two eventual fates: a cup either stalled before forming a half-cup or it proceeded until the particle was fully enveloped. We explain these observations in terms of a mechanical bottleneck using a simple mathematical model of the overall process of cup growth. The model predicts that reducing F-actin concentration levels, and hence the deforming force, does not necessarily lead to stalled cups, a prediction we verify experimentally. Our analysis gives a coherent explanation for the importance of geometry in phagocytic uptake and provides a unifying framework for integrating the key processes, both biochemical and mechanical, occurring during cup growth.