Profilin-Mediated Actin Allocation Regulates the Growth of Epithelial Microvilli.

Transporting epithelial cells, like those that line the intestinal tract, are specialized for solute processing and uptake. One defining feature is the brush border, an array of microvilli that serves to amplify apical membrane surface area and increase functional capacity. During differentiation, upon exit from stem-cell-containing crypts, enterocytes build thousands of microvilli, each supported by a parallel bundle of actin filaments several microns in length. Given the high concentration of actin residing in mature brush borders, we sought to determine whether enterocytes were resource (i.e., actin monomer) limited in assembling this domain. To examine this possibility, we inhibited Arp2/3, the ubiquitous branched actin nucleator, to increase G-actin availability during brush border assembly. In native intestinal tissues, Arp2/3 inhibition led to increased microvilli length on the surface of crypt, but not villus, enterocytes. In a cell culture model of brush border assembly, Arp2/3 inhibition accelerated the growth and increased the length of microvilli; it also led to a redistribution of F-actin from cortical lateral networks into the brush border. Effects on brush border growth were rescued by treatment with the G-actin sequestering drug, latrunculin A. G-actin binding protein, profilin-1, colocalized in the terminal web with G-actin, and knockdown of this factor compromised brush border growth in a concentration-dependent manner. Finally, the acceleration in brush border assembly induced by Arp2/3 inhibition was abrogated by profilin-1 knockdown. Thus, brush border assembly is limited by G-actin availability, and profilin-1 directs unallocated actin monomers into microvillar core bundles during enterocyte differentiation.

An actin-based protrusion originating from a podosome-enriched region initiates macrophage fusion.

Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating this process is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase-contrast video microscopy demonstrated that in the majority of events, short protrusions (∼3 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (∼12 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data suggest that an actin-based protrusion formed at the leading edge initiates macrophage fusion.

Interaction between the integrin Mac-1 and signal regulatory protein α (SIRPα) mediates fusion in heterologous cells.

Macrophage fusion leading to the formation of multinucleated giant cells is a hallmark of chronic inflammation. Several membrane proteins have been implicated in mediating cell-cell attachment during fusion, but their binding partners remain unknown. Recently, we demonstrated that interleukin-4 (IL-4)-induced fusion of mouse macrophages depends on the integrin macrophage antigen 1 (Mac-1). Surprisingly, the genetic deficiency of intercellular adhesion molecule 1 (ICAM-1), an established ligand of Mac-1, did not impair macrophage fusion, suggesting the involvement of other counter-receptors. Here, using various approaches, including signal regulatory protein α (SIRPα) knockdown, recombinant proteins, adhesion and fusion assays, biolayer interferometry, and peptide libraries, we show that SIRPα, which, similar to ICAM-1, belongs to the Ig superfamily and has previously been implicated in cell fusion, interacts with Mac-1. The following results support the conclusion that SIRPα is a ligand of Mac-1: (a) recombinant ectodomain of SIRPα supports adhesion of Mac-1-expressing cells; (b) Mac-1-SIRPα interaction is mediated through the ligand-binding αMI-domain of Mac-1; (c) recognition of SIRPα by the αMI-domain conforms to general principles governing binding of Mac-1 to many of its ligands; (d) SIRPα reportedly binds CD47; however, anti-CD47 function-blocking mAb produced only a limited inhibition of macrophage adhesion to SIRPα; and (e) co-culturing of SIRPα- and Mac-1-expressing HEK293 cells resulted in the formation of multinucleated cells. Taken together, these results identify SIRPα as a counter-receptor for Mac-1 and suggest that the Mac-1-SIRPα interaction may be involved in macrophage fusion.

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion.

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

Development of fusogenic glass surfaces that impart spatiotemporal control over macrophage fusion: Direct visualization of multinucleated giant cell formation.

Implantation of synthetic material, including vascular grafts, pacemakers, etc. results in the foreign body reaction and the formation of multinucleated giant cells (MGCs) at the exterior surface of the implant. Despite the long-standing premise that fusion of mononucleated macrophages results in the formation of MGCs, to date, no published study has shown fusion in context with living specimens. This is due to the fact that optical-quality glass, which is required for the majority of live imaging techniques, does not promote macrophage fusion. Consequently, the morphological changes that macrophages undergo during fusion as well as the mechanisms that govern this process remain ill-defined. In this study, we serendipitously identified a highly fusogenic glass surface and discovered that the capacity to promote fusion was due to oleamide contamination. When adsorbed on glass, oleamide and other molecules that contain long-chain hydrocarbons promoted high levels of macrophage fusion. Adhesion, an essential step for macrophage fusion, was apparently mediated by Mac-1 integrin (CD11b/CD18, αMß2) as determined by single cell force spectroscopy and adhesion assays. Micropatterned glass further increased fusion and enabled a remarkable degree of spatiotemporal control over MGC formation. Using these surfaces, we reveal the kinetics that govern MGC formation in vitro. We anticipate that the spatiotemporal control afforded by these surfaces will expedite studies designed to identify the mechanism(s) of macrophage fusion and MGC formation with implication for the design of novel biomaterials.

The Role of Integrins αMß2 (Mac-1, CD11b/CD18) and αDß2 (CD11d/CD18) in Macrophage Fusion.

The subfamily of ß2 integrins is implicated in macrophage fusion, a hallmark of chronic inflammation. Among ß2 family members, integrin Mac-1 (αMß2, CD11b/CD18) is abundantly expressed on monocyte/macrophages and mediates critical adhesive reactions of these cells. However, the role of Mac-1 in macrophage fusion leading to the formation of multinucleated giant cells remains unclear. Moreover, the role of integrin αDß2 (CD11d/CD18), a receptor with recognition specificity overlapping that of Mac-1, is unknown. We found that multinucleated giant cells are formed in the inflamed mouse peritoneum during the resolution phase of inflammation, and their numbers were approximately twofold higher in wild-type mice than in Mac-1(-/-) mice. Analyses of isolated inflammatory peritoneal macrophages showed that IL-4-induced fusion of Mac-1-deficient cells was strongly reduced compared with wild-type counterparts. The examination of adhesive reactions known to be required for fusion showed that spreading, but not adhesion and migration, was reduced in Mac-1-deficient macrophages. Fusion of αDß2-deficient macrophages was also significantly decreased, albeit to a smaller degree. Deficiency of intercellular adhesion molecule 1, a counter-receptor for Mac-1 and αDß2, did not alter the fusion rate. The results indicate that both Mac-1 and αDß2 support macrophage fusion with Mac-1 playing a dominant role and suggest that Mac-1 may mediate cell-cell interactions with a previously unrecognized counter-receptor(s).

Food grade titanium dioxide disrupts intestinal brush border microvilli in vitro independent of sedimentation.

Bulk- and nano-scale titanium dioxide (TiO2) has found use in human food products for controlling color, texture, and moisture. Once ingested, and because of their small size, nano-scale TiO2 can interact with a number of epithelia that line the human gastrointestinal tract. One such epithelium responsible for nutrient absorption is the small intestine, whose constituent cells contain microvilli to increase the total surface area of the gut. Using a combination of scanning and transmission electron microscopy it was found that food grade TiO2 (E171 food additive coded) included ∼25% of the TiO2 as nanoparticles (NPs; <100 nm), and disrupted the normal organization of the microvilli as a consequence of TiO2 sedimentation. It was found that TiO2 isolated from the candy coating of chewing gum and a commercially available TiO2 food grade additive samples were of the anatase crystal structure. Exposure to food grade TiO2 additives, containing nanoparticles, at the lowest concentration tested within this experimental paradigm to date at 350 ng/mL (i.e., 100 ng/cm(2) cell surface area) resulted in disruption of the brush border. Through the use of two independent techniques to remove the effects of gravity, and subsequent TiO2 sedimentation, it was found that disruption of the microvilli was independent of sedimentation. These data indicate that food grade TiO2 exposure resulted in the loss of microvilli from the Caco-2BBe1 cell system due to a biological response, and not simply a physical artifact of in vitro exposure.