TIRF imaging of Fc gamma receptor microclusters dynamics and signaling on macrophages during frustrated phagocytosis
- 3.3k Downloads
Recent evidence indicates that in addition to the T-cell receptor, microclustering is an important mechanism for the activation of the B-cell receptor and the mast cell Fcε-receptor. In macrophages and neutrophils, particles opsonized with immunoglobulin G (IgG) antibodies activate the phagocytic Fcγ-receptor (FcγR) leading to rearrangements of the actin cytoskeleton. The purpose of this study was to establish a system for high-resolution imaging of FcγR microclustering dynamics and the recruitment of the downstream signaling machinery to these microclusters.
We developed a supported lipid bilayer platform with incorporated antibodies on its surface to study the formation and maturation of FcγR signaling complexes in macrophages. Time-lapse multicolor total internal reflection microscopy was used to capture the formation of FcγR-IgG microclusters and their assembly into signaling complexes on the plasma membrane of murine bone marrow derived macrophages.
Upon antibody binding, macrophages formed FcγR-IgG complexes at the leading edge of advancing pseudopods. These complexes then moved toward the center of the cell to form a structure reminiscent of the supramolecular complex observed in the T-cell/antigen presenting cell immune synapse. Colocalization of signaling protein Syk with nascent clusters of antibodies indicated that phosphorylated receptor complexes underwent maturation as they trafficked toward the center of the cell. Additionally, imaging of fluorescent BtkPH domains indicated that 3′-phosphoinositides propagated laterally away from the FcγR microclusters.
We demonstrate that surface-associated but mobile IgG induces the formation of FcγR microclusters at the pseudopod leading edge. These clusters recruit Syk and drive the production of diffusing PI(3,4,5)P3 that is coordinated with lamellar actin polymerization. Upon reaching maximal extension, FcγR microclusters depart from the leading edge and are transported to the center of the cellular contact region to form a synapse-like structure, analogous to the process observed for T-cell receptors.
KeywordsFcγ receptor IgG TIRF frustrated phagocytosis receptor synapses macrophage
Bruton’s tyrosine kinase PH domain
center of SMAC
fluorescent recovery after photobleaching
fragment crystalizable region
Guanine nucleotide exchange factor
immunoreceptor tyrosine-based activation motif
macrophage colony-stimulating factor
supported lipid bilayer
spleen tyrosine kinase
supramolecular activation cluster
- TIRF microscopy
total internal reflection microscopy
Of the immunoreceptors, microclustering of the T cell receptor (TCR) is the most studied for its role in forming an immunological synapse (IS) during interaction with antigen-presenting cells (APC) . Actin rearrangements downstream of the TCR drive the formation of the IS and it’s bull’s eye arrangement known as the supramolecular activation cluster (SMAC). After receptor ligation, ZAP70 or Syk are recruited to the TCR microclusters where they mediate the phosphorylation of downstream signaling molecules . Formation of the IS then mediated by F-actin rich protrusions that move around the distal SMAC (dSMAC, actin rich region) in a radial wave. TCR microclusters migrate inward along with downstream signaling molecules, such as Syk, Lyn, and VAV1, forming the central SMAC (cSMAC) [9, 20, 21, 22, 23, 24]. Similarly to TCRs, the B cell receptor (BCR) undergoes microclustering following binding with antigen triggers . Recently, FcεRs on mast cells have been observed to form microclusters upon contact with lipid bilayer presenting IgE. In all cases, these microclusters were directly transported to the center of the cell to form a patch [25, 26]. Together, these studies indicate that microclustering is a common theme for immunoreceptors.
Here, we captured the clustering behavior of FcγR-IgG complexes in macrophages using Total Internal Reflection Fluorescence (TIRF) Microscopy [27, 28, 29]. The ‘evanescent field’ generated by TIRF selectively excites fluorophores within 200 nm above the glass surface, thereby reducing out-of-focus fluorescence from remainder of the cell. By taking advantage of a supported lipid bilayer (SLB) to present IgG, TIRF microscopy can provide high-resolution imaging of the FcγR microcluster dynamics during initial macrophage interactions with the surface followed by frustrated phagocytosis. Furthermore, we applied this system to capture the dynamic recruitment of downstream signaling proteins to FcγR-IgG microclusters by expressing them as fluorescent protein fusions. These data provide a framework for understanding the transitions in signaling states of FcγRs during actin polymerization and phagocytosis in macrophages.
Alexa Fluor 594 IgG Fraction Monoclonal Mouse Anti-Biotin (Code: 200-582-211) was from Jackson ImmunoResearch Inc. l-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-oleoyl-2-(12-biotinyl(aminododecanoyl))-sn-glycero-3-phosphoethanolamine (Biotin-PE) were ordered from Avanti Polar Lipids. Sulfuric acid (H2SO4), hydrogen peroxide (H2O2), chloroform and glucose were from Sigma Aldrich. Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Cellgro (Manassas, VA). Phosphate buffered saline (PBS) and DiI were purchased from Thermo Fisher Scientific Inc. Fetal Bovine Serum (FBS) was from Atlanta Biologicals (Flowery Branch, GA). Anti-Biotin was purchased from Neomarkers (Fremont, CA). Platinum-E Retroviral Packaging Cell Line (Plat-E) were purchased from Cellbiolabs, San Diego, CA. FuGENE transfection reagent was obtained from Roche Applied Science (Germany). All supplementary materials were of the highest grade commercially available.
Glass supported lipid bilayer
The SLB was formed by spontaneous fusion of lipid vesicles. To achieve this, Biotin-PE and POPC were mixed at a molar ratio of 1:100 with total lipid concentration of 400 μg/ml. The lipid mixture was then dissolved in chloroform and dried under air for 10 min. The lipid film was re-suspended in PBS containing 2 mM Mg2+. The obtained solution was sonicated for 5 min using a probe sonicator (Branson Ultrasonics, Danbury, CT). Bilayer was formed on Piranha acid (H2SO4 (30 %, v/v):H2O2 (3:1, v/v)) cleaned coverslip by incubation in a water bath at 37 °C for 15 min. Excess liposomes were exchanged with imaging buffer (PBS + 5 mM glucose). The bilayer coated coverslip was kept in a buffer solution during washing and transferring to imaging chamber to protect SLB from drying out and to keep it uniform . Alexa Fluor 594 succidiminal ester was conjugated to anti-Biotin IgG for antibody fluorescent labeling (Jackson ImmunoResearch Inc.). The labeled antibody was incubated with SLB at 37 °C for 30 min. Excess IgG was washed with imaging buffer.
The mobility of SLB was confirmed by fluorescence recovery after photobleaching (FRAP) microscopy (Additional file 1). Briefly, the mobility of SLB labeled with Bodipy (5 μg/ml for 5 min) was observed by photobleaching the area of SLB and then imaging recovery of the fluorescent signal at the bleached location [25, 26].
Cell culture and retroviral transduction of signaling proteins
Murine bone marrow derived macrophages (BMM) were obtained as described in . Bone marrow was extruded from femurs and tibia of B57/BL6 mice (Charles River Laboratories, Wilmington, MA). The marrow was cultured in DMEM media containing 30 % L-cell supernatant as a source of MCSF (macrophage colony-stimulating factor), 20 % heat-inactive FBS. Cells were supplemented with fresh media to continue differentiation and proliferation . In general macrophages were fully differentiated by day 6.
Gene inserts of fluorescently tagged signaling protein of interest (Syk-mCitrine and BtkPH-mCitrine) were introduced into Murine leukemia virus (MLV)-based vectors. The assembled constructs were used to transfect Plat-E cells using FuGENE following the manufactures protocol. The retroviral supernatant was harvested 48 hours post transfection and used within one week after harvesting. BMMs were plated in the 6-well dish at a density of 1x106 per well. Retroviral supernatants (1x107 virus/mL) were added to the well in the presence of polybrene (10 μg/mL). BMMs were incubated with the virus for 24–48 hours, and then replaced with fresh bone marrow media. These transduced cells were used for following imaging experiments .
Image acquisition and data analysis
TIRF 360 was used to create uniform TIRF illumination by steering the laser at the back-focal plane. The microscope was custom-built based on iMIC system (TILL Photonics, Munich, Germany) with 60x 1.49 oil immersion objective lens (Olympus, Tokyo, Japan), previously described in . BMMs were lifted from culture dish, washed with PBS twice and then dropped onto the SLB surface in the imaging chamber. Cell samples were imaged 3 min after they were placed to the imaging chamber and images were acquired every 5 sec for a total duration of 6 min.
Cell images were processed in Matlab (The MathWorks, Inc., Natick, MA) with customized codes. Two channels were registered using the fiducial data registration method. Multiple-fluorophore beads (TetraSpeck, Invitrogen, CA) were employed for image registration . Individual protein complexes were analyzed with single particle tracking technique. Due to the dynamic movement, some complexes were moving out of the TIRF field. We only tracked molecules that moved within the TIRF field . We imaged at least 3 cells per each condition and performed the tracking and analysis from single cells as the behavior was consistent for all cells imaged under each condition.
Results and discussion
IgG-coated SLB for TIRF imaging of FcγR signaling on macrophages
Dynamic association of Syk with FcγR microclusters
Dynamics of actin and PI(3,4,5)P3 relative to IgG-FcγR complexes
In this work we demonstrated that like the TCR, BCR, and FcεR, FcγR on macrophages form microclusters that are transported into a synapse-like structure. In addition, this work provides a powerful system in which SLB presentation of IgG can be used to specifically activate FcγR on the surface of macrophages without incidental activation of other receptors by the glass surface. Using this system, we were able to observe FcγR microclustering at the pseudopod edge of macrophages engaged in frustrated phagocytosis, followed by the release of these receptors from the leading edge and their subsequent retrograde transport. By expressing fluorescent proteins in these cells and tracking the motions of FcγR-IgG microclusters, we were able to make two new observations. First, we found that as expected, Syk localized to the FcγR-IgG microcluster where it oscillated on and off, suggesting multiple rounds of phosphorylation of the FcγR. Second, we observed local PI(3,4,5)P3 was produced proximal to FcγR-IgG microclusters and this localization corresponded well with the localization of actin at the leading edge of the pseudopod and to a lesser extent on FcγR microclusters that have departed from the leading edge. In comparing this system to other imaging experiments that track the localization of downstream signaling components to IgG coated beads and erythrocytes, we note similar behaviors – Syk remains associated with the FcγR at all stages as observed for IgG bead phagosomes , and that PI(3,4,5)P3 is coordinated with actin on membrane that remains contiguous with the plasma membrane . Together, these observations indicate that there are many parallels across immunoreceptor signaling and that microclustering and actin-mediated transport of these receptors is a common theme. Furthermore, this approach can be used to address key questions regarding FcγR activation, deactivation and signal propagation.
This research does not involve human subjects, human materials. All animal research protocols for this work were reviewed and approved by the IACUC committee at South Dakota State University.
This material is based on work supported by the National Science Foundation under the National Science Foundation/EPSCoR Cooperative Agreement #IIA-1355423, the South Dakota Research and Innovation Center, BioSNTR, and by the State of South Dakota. Any opinions, findings, conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
FRAP of the Bodipy labeled bilayer. The mobility of SLB and photobleaching effect were checked with a fluorescence recovery after photobleaching (FRAP) microscopy. SLB was labeled with Bodipy (5 μg/ml) and a region was bleached and the recovery was recorded. Scale bar 10 μm. (AVI 857 kb)
DiI labeled BMM engage and spread on IgG-SLB. PC/PE-Biotin in the SLB was labeled with anti-Biotin IgG. Macrophages dropped onto this surface actively engaged in frustrated phagocytosis. Scale bar 10 μm. (AVI 310 kb)
DiI-labeled BMM do not interact with SLB in the absence of IgG and simply roll alone the surface. Scale bar 10 μm. (AVI 524 kb)
FcγR-IgG (magenta) microcluster formation and Syk recruitment (green). Syk associated with FcγR-IgG microclusters at the leading edge during spreading phase and to a lesser extent following accumulation of FcγR-IgG microclusters in the center of the cell. Oscillations were observed for Syk kinase as it was recruited to the FcγR-IgG microclusters. Scale bar 10 μm. (AVI 628 kb)
Dynamics of PI(3,4,5)P3 was tracked by imaging BtkPH (green) relative to FcγR-IgG (magenta) microclusters. BtkPH association with clustered receptors indicates lateral propagation of signals. Scale bar 10 μm. (AVI 1549 kb)
Dynamics of actin (cyan) in relation to FcγR-IgG (magenta) microclusters. These data are for the same cell as shown in Additional file 5. Fluorescent signal from Actin follow very similar patterns and dynamics as BtkPH. Scale bar 10 μm. (AVI 1617 kb)
- 14.Levin R, Grinstein S, Schlam D. Phosphoinositides in phagocytosis and macropinocytosis. BBA-Mol Cell Biol L. 2015;1851(6):805–23.Google Scholar
- 16.Sarantis H, Grinstein S. In: DiPaolo G, Wenk MR, editors. Monitoring phospholipid dynamics during phagocytosis: application of genetically-encoded fluorescent probes, in lipids, Vol 108. 2012. p. 429–44.Google Scholar
- 24.Grakoui A et al. The immunological synapse: a molecular machine controlling T cell activation. J Immunol. 2015;194(9):221–7.Google Scholar
- 32.Selvin PR, Selvin PR, Ha T. Q Rev Biol. 2008;83(4):406–6.Google Scholar
- 35.Freeman SA, et al. Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor. Nat Commun. 2015;6:6168. doi: 10.1038/ncomms7168.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.