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Release of Cholesterol-Rich Particles from the Macrophage Plasma RESEARCH ARTICLE Release of cholesterol-rich particles from the macrophage plasma membrane during movement of filopodia and lamellipodia Xuchen Hu1, Thomas A Weston1, Cuiwen He1, Rachel S Jung1, Patrick J Heizer1, Brian D Young2, Yiping Tu1, Peter Tontonoz3, James A Wohlschlegel2, Haibo Jiang1,4*, Stephen G Young1,5*, Loren G Fong1* 1Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States; 2Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, United States; 3Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, United States; 4School of Molecular Sciences, University of Western Australia, Perth, Australia; 5Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States Abstract Cultured mouse peritoneal macrophages release large numbers of ~30-nm cholesterol- rich particles. Here, we show that those particles represent fragments of the plasma membrane that are pulled away and left behind during the projection and retraction of filopodia and lamellipodia. Consistent with this finding, the particles are enriched in proteins found in focal adhesions, which attach macrophages to the substrate. The release of particles is abolished by blocking cell movement (either by depolymerizing actin with latrunculin A or by inhibiting myosin II with blebbistatin). Confocal microscopy and NanoSIMS imaging studies revealed that the plasma membrane–derived particles are enriched in ‘accessible cholesterol’ (a mobile pool of cholesterol detectable with the modified cytolysin ALO-D4) but not in sphingolipid-sequestered cholesterol [a *For correspondence: pool detectable with ostreolysin A (OlyA)]. The discovery that macrophages release cholesterol-rich [email protected] (HJ); particles during cellular locomotion is likely relevant to cholesterol efflux and could contribute to [email protected] (SGY); extracellular cholesterol deposition in atherosclerotic plaques. [email protected] (LGF) DOI: https://doi.org/10.7554/eLife.50231.001 Competing interest: See page 23 Funding: See page 23 Introduction Received: 16 July 2019 A key function of macrophages is to engulf and digest cellular debris. The cholesterol in the debris Accepted: 04 September 2019 can be esterified and stored in cytosolic lipid droplets (Brown et al., 1980), thereby avoiding toxicity Published: 05 September 2019 associated with an accumulation of free cholesterol, but macrophages ultimately must dispose of the surplus cholesterol, a process generally referred to as ‘cholesterol efflux’ (Rosenson et al., 2012; Reviewing editor: Fredric B Kraemer, Stanford University, Tall et al., 2002; Rothblat and Phillips, 2010; Adorni et al., 2007). One mechanism for cholesterol United States efflux involves transferring free cholesterol to high density lipoproteins (HDL), a process that is facili- tated by ABC transporters (Rosenson et al., 2012; Tall et al., 2002; Rothblat and Phillips, 2010; Copyright Hu et al. This article Yvan-Charvet et al., 2010; Westerterp et al., 2014; Duong et al., 2006). A deficiency of ABCA1 is distributed under the terms of interferes with cholesterol efflux by macrophages, leading to an accumulation of foam cells (macro- the Creative Commons Attribution License, which phages containing numerous cholesterol ester droplets) in tissues (Brooks-Wilson et al., 1999; permits unrestricted use and Bodzioch et al., 1999; Clee et al., 2001; Rust et al., 1999). Another potential mechanism for cho- redistribution provided that the lesterol efflux by macrophages is the release of cholesterol-rich particles from the plasma mem- original author and source are brane. Using a cholesterol-specific monoclonal antibody and immunocytochemical approaches, the credited. laboratory of Howard Kruth reported that cultured human monocyte–derived macrophages release Hu et al. eLife 2019;8:e50231. DOI: https://doi.org/10.7554/eLife.50231 1 of 26 Research article Biochemistry and Chemical Biology Cell Biology ‘cholesterol microdomains’ (varying in size but as large as several hundred nm) onto the surrounding substrate (Jin et al., 2016; Freeman et al., 2014; Ong et al., 2010; Jin et al., 2015; Jin et al., 2018). The release of these microdomains was reduced by decreasing the expression of ABC trans- porters (Jin et al., 2016; Freeman et al., 2014; Jin et al., 2015; Jin et al., 2018). Recently, we dem- onstrated, by scanning electron microscopy (SEM), that large numbers of ~30-nm vesicular particles were released from the plasma membrane of mouse peritoneal macrophages and a mouse macro- phage cell line by a process that morphologically resembles ‘budding.’ The plasma membrane– derived particles were enriched in ‘accessible cholesterol’ (He et al., 2018), a mobile pool of choles- terol that can be detected by ALO-D4, a modified cholesterol-binding cytolysin (Gay et al., 2015). The cholesterol content of the particles could be increased by loading macrophages with cholesterol or by treating the cells with a liver X receptor (LXR) agonist, and the cholesterol content of the par- ticles could be depleted with HDL (He et al., 2018). The SEM studies by He and coworkers (He et al., 2018) demonstrated that the particles released by macrophages are derived from the plasma membrane, but the mechanism was unclear. One pos- sibility is that the budding of particles from the plasma membrane was driven by shuttling choles- terol into plasma membrane microdomains, causing outward ballooning of a localized segment of the plasma membrane and ultimately to the release of a vesicular particle. A second possibility, men- tioned by He and coworkers (He et al., 2018) but not pursued, was that the particles represented segments of the macrophage plasma membrane that had been affixed to the substrate but then ‘torn away and left behind’ during movement of filopodia and lamellipodia. In the current study, we used live-cell microscopy and SEM, along with proteomic studies, to explore the mechanism for the release of particles from the plasma membrane of macrophages. We also used super-resolution fluorescence microscopy and NanoSIMS imaging, in combination with two different cholesterol-binding proteins (one specific for ‘accessible cholesterol’ and the other for sphingomyelin-sequestered cholesterol), to examine the cholesterol pools within the plasma mem- brane–derived particles released by macrophages. Results Macrophages release plasma membrane–derived particles during the movement of filopodia and lamellipodia In the current studies, we again found that particles are released from macrophage filopodia and lamellipodia onto the surrounding substrate by a process that resembles budding (Figure 1—figure supplement 1). Because the released particles remain adherent to the substrate, we suspected that the particles might represent fragments of the plasma membrane that were affixed to the underlying substrate but then pulled away and left behind during movement of filopodia/lamellipodia. To explore this idea, we plated mouse peritoneal macrophages onto gridded glass-bottom dishes and recorded images of cells by live-cell microscopy (Figure 1—videos 1–2), making it possible to visual- ize the projection and retraction of filopodia/lamellipodia. The same cells were then fixed and imaged by SEM. Lawns of ~30-nm particles were present on the substrate surrounding macro- phages, generally at the lagging pole of the cell and invariably where we had observed, by live-cell imaging, the extension and retraction of filopodia/lamellipodia (Figure 1, Figure 1—videos 1–2). To determine whether the extension and retraction of filopodia/lamellipodia are required for par- ticle release, cell movement was blocked by treating macrophages with an actin-depolymerizing agent (latrunculin A) or a myosin II inhibitor (blebbistatin). Live-cell imaging revealed that the drug- treated macrophages were unable to project and retract filopodia/lamellipodia (Figure 2—videos 1–6). Macrophages were incubated with latrunculin A or blebbistatin in suspension for 1 hr (‘pre- treatment’) and then plated onto poly-D-lysine–coated silicon wafers and incubated with the drugs overnight (Figure 2). Both latrunculin A and blebbistatin abolished particle release from ‘pre-treat- ment’ macrophages (Figure 2A–B). Other macrophages were allowed to adhere to the substrate for 1 hr before initiating drug treatment (‘post-adherence’) (Figure 2A–B). In latrunculin A–treated ‘post-adherence’ macrophages, a circumferential ring of particles was left behind on the substrate as the cell retracted with actin depolymerization (Figure 2A). Blebbistatin eliminated particle release in post-adherence cells (Figure 2B). Macrophages treated with vehicle alone (DMSO) released large numbers of particles onto the surrounding substrate (Figure 2A–B). As an additional control, Hu et al. eLife 2019;8:e50231. DOI: https://doi.org/10.7554/eLife.50231 2 of 26 Research article Biochemistry and Chemical Biology Cell Biology Figure 1. Macrophages release plasma membrane–derived particles onto the substrate during extension and retraction of filopodia and lamellipodia, as judged by correlative live-cell imaging and SEM. Cells were plated onto poly-D-lysine–coated gridded glass-bottom Petri dishes, and videos were recorded for 24 hr at 5 min intervals (see Figure 1—videos 1–2). The ‘Live cell’ images show the final frame of the videos. The imaging of cells by SEM
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