A phosphatidylinositol-3-kinase-dependent signal transition regulates ARF1 and ARF6 during Fcgamma receptor-mediated phagocytosis.

Molecular cloning and DNA manipulation.

Plasmids incorporating monomeric versions

(A207K) of the GFP spectral variants ECFP and Citrine (an EYFP variant) were used for all constructs [ 44 , 45 ]. Human ARF1 and ARF6 from the UMR cDNA Resource Center ( http://www.cdna.org ) were PCR-amplified for subcloning into pECFP-N1 and pEYFP-N1 (Clontech, Mountain View, California, United States) on EcoRI-KpnI fragments producing a fusion to the fluorescent protein at the C-terminus of the GTPase. To generate YFP-NGAT, the base pairs encoding amino acid residues 165–210 from transcript variant 1 of human GGA1, a gift from J. Bonifacino, were PCR-amplified for insertion into the XhoI-EcoRI site of pEYFP-C1. YFP-NGAT was mutated to NGAT(A193T, N194Y) in the YFP-NGAT expression construct using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California, United States). ARF1(T31N), ARF1(Q71L), ARF6(T27N), and ARF6(Q67L) were generated from the appropriate ARF-CFP expression constructs using the Stratagene QuikChange mutagenesis kit. The sequences of constructs were confirmed by DNA sequencing at the University of Michigan DNA Sequencing Core. Tissue culture and transfection. RAW264.7 macrophage-like cells were obtained from the American Type Culture Collection (Manassas, Virginia, United States) and maintained at 37 °C under 5% CO 2 . RAW264.7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% HIFBS, 100 U/ml penicillin, and 100 μg/ml streptomycin, or Advanced-DMEM supplemented with 2% HIFBS, 4 mM L-glutamine, 20 U/ml penicillin, and 20 μg/ml streptomycin. Cell culture reagents were products of Invitrogen (Carlsbad, California, United States). Cells under microscopic observation were maintained in Leiden chambers (Harvard Apparatus, Holliston, Massachusetts, United States) at 37 °C in Ringer's buffer (155 mM NaCl, 5mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 2 mM NaH 2 PO 4 , 10 mM glucose, 10 mM HEPES [pH 7.2]). The opsonization of sheep erythrocytes with rabbit IgG (ICN Biochemicals, Aurora, Ohio, United States) has been described previously [ 46 ]. To prepare RAW264.7 cells for FRET or ratiometric microscopy, ˜2.5 × 10 5 cells per coverslip were plated the day before imaging. Transfection of plasmids encoding fluorescent chimeras was performed ˜18 h prior to the start of imaging using FuGene-6 according to the manufacturer's recommended protocol (Roche Diagnostics, Indianapolis, Indiana, United States). Pharmacological treatments. BFA and LY294002 were purchased from EMD Biosciences (San Diego, California, United States). Stocks were prepared by dilution of the lyophilized solids into DMSO to a concentration of 1 mM for BFA and 10 mM for LY294002. Aliquoted stocks were stored at −20 °C until needed. BFA was added to the cells in the Leiden chamber to a final concentration of 5 μM, and fluorescence images were recorded as described below. LY294002 was added to cells at a final concentration of 50 μM 30 min prior to the addition of opsonized erythrocytes. FRET microscopy. FRET component fluorescence images were acquired using a Nikon Eclipse TE-300 inverted microscope with a 60×, numerical aperture 1.4, oil-immersion PlanApo objective lens (Nikon, Tokyo, Japan) and Lambda LS xenon arc lamp for epifluorescence illumination (Sutter Instruments, Novato, California, United States). Image acquisition and processing were performed using Metamorph 6.2r6 (Universal Imaging, Malvern, Pennsylvania, United States). Fluorescence excitation and emission wavelengths were selected using a JP4v2 filter set (Chroma Technology, Rockingham, Vermont, United States) and a Lambda 10–2 filter wheel controller (Sutter Instruments) equipped with a shutter for epifluorescence illumination control. Phase contrast illumination was controlled using a Uniblitz VMM-D1 shutter driver (Vincent Associates, Rochester, New York, United States). Images were recorded with a Photometrics CoolSnap HQ cooled CCD camera (Roper Scientific, Tucson, Arizona, United States). Images were acquired by positioning excitation and emission filters to visualize CFP (I D ): excitation at 430 ± 12.5 nm, emission at 470 ± 15 nm; YFP (I A ): excitation at 500 ± 10 nm, emission at 535 ± 15 nm; or FRET (I F ): excitation at 430 ± 12.5 nm, emission at 535 ± 15 nm. Images were collected with exposure times of 100–400 ms. To account for variable exposure lengths, image scaling was performed following shading and bias correction, e.g., to correct for I D :I F :I A exposure lengths of 200 ms : 200 ms : 100 ms, the shade/bias corrected I A image was multiplied by two prior to further image processing. Shading correction images for I D , I A , and I F were collected from a mixture of CFP and YFP between two coverglasses. Bias-correction images to correct for camera dark noise were collected with the excitation light blocked. The FRET parameters α and β were measured from RAW264.7 cells expressing YFP or CFP, respectively. The parameters γ and ξ were determined using cells expressing a covalently linked CFP–YFP molecule, whose FRET efficiency (E C ) has been measured by fluorescence lifetime spectroscopy, and back-calculating using the expression for f A and an updated expression for f D (see Protocol S1 ), respectively [ 24 ]. The E A , E D , and R M images were calculated from the corrected fluorescence images and FRET parameters as described here in Protocol S1 (for E D and R M ) and as described in previous work (E A ) [ 24 ]. To observe phagocytosis, RAW264.7 cells expressing fluorescent chimeras were located, and phagocytosis initiated by delivery of ˜2 × 10 5 IgG-opsonized erythrocytes to the chamber. Collection of I A , I D , I F , and phase contrast images began as erythrocytes landed on cells expressing fluorescent chimeras. Image sets were recorded within 2 s at 30-s intervals. Following acquisition of complete time series, shade/bias-corrected I A , I D , and I F images were used to calculate the FRET stoichiometry images E A , E D , and R M . The calculated images represent: the fraction of YFP chimera (f A ) in complex times the characteristic FRET efficiency (E C ) of the complex (E A ), the fraction of CFP chimera in complex (f D ) times E C (E D ), or the molar ratio of YFP chimera to CFP chimera (R M ) at each pixel in the image. Note that the value of E C for the ARF–CFP–YFP–NGAT interaction is not required for calculation of E A , E D , and R M . To measure the BFA-mediated disruption of ARF1 activation at the Golgi complex, I A , I D , I F , and phase contrast images of RAW264.7 macrophages expressing both ARF1-CFP and YFP-NGAT were collected before the addition of BFA (final concentration of 5 μM). Following the addition of toxin, fluorescence and phase contrast images were collected at 30-s intervals. Image processing to obtain the E A , E D , and R M images was performed as described above. To define the Golgi compartment, a manual threshold was applied to the high intensity region in the shade/bias-corrected I D image. This threshold was used to create a binary mask; the mask was applied to the E A , E D , and R M images to define the region of measurement. Metamorph's Integrated Morphometry Analysis was used to measure the area and average gray value of the resulting region in the masked E A , E D , and R M images at each time point. As BFA reduced the Golgi network, the region of high intensity used to make measurements decreased in size. After ˜15–20 min in 50 μM BFA, the measurement region could no longer be defined due to redistribution of ARF1-CFP. Cells expressed uniform E D values of ˜0 at these time points. To compare FRET values measured in cells transfected with different ARF-CFP and YFP-NGAT chimeras, the average fluorescence intensities of the masked, shade-corrected, and bias-corrected I A , I D , and I F component images for each cell were recorded into a spreadsheet. The average fluorescence values were then used to calculate E A , E D , and R M using the FRET stoichiometry equations and E AVG , the arithmetic mean of E A and E D . Because E A and E D are sensitive to the amount of acceptor and donor available for incorporation into FRET complexes, these values are dependent on the value of R M ; while E A is suppressed in cells that express a large excess of YFP relative to CFP, E D is increased in cells with an overabundance of acceptor. E AVG serves as a weighted average that adjusts for varying levels of donor and acceptor in a population of cells, and was therefore used to compare FRET values in cells that express variable relative amounts of different CFP and YFP chimeras. The relationships between E A , E D , E AVG , and R M can be seen in Figure S1 .

Statistical analysis to compare

FRET in cells expressing the wild-type and mutant chimeras was performed using Prism 3.0 (GraphPad Software, San Diego, California, United States). Ratiometric microscopy. During phagocytosis by cells expressing YFP chimeras, quantification of the association of the YFP chimera with the phagosome is complicated by the changes in optical path length induced by morphological rearrangements at the cell periphery near the phagosome. Ratiometric imaging can be used to distinguish specific association of a marker with the phagosome from intensity changes due to the increase in bulk cellular material at the site of phagocytosis [ 47 , 48 ]. The application of FRET stoichiometry to ratiometric imaging analysis allows the quantification of the molar ratio of YFP chimera to soluble CFP marker [ 30 ]. In the absence of CFP–YFP energy transfer, the molar ratio can be calculated as: The factors α and γ are defined as described previously, ξ is described in Protocol S1 , and we have replaced the notation R with R M [ 24 ]. To make measurements of protein localization that are independent of the relative transfection efficiency of the YFP chimera and CFP in a particular cell, a recruitment index has been developed [ 30 ]. This recruitment index is calculated as R M for the phagosome region divided by R M for the entire cell: Image collection for ratiometric microscopy was performed as for FRET microscopy except that only the I A , I D , and phase contrast images were collected. Particle-tracking and phagosome analysis.

Methods for measuring phagosome-associated signals using the centroid-tracking algorithm

TRACKOBJ in Metamorph have been described [ 30 , 48 ]. Briefly, the TRACKOBJ algorithm was used to identify the center of the erythrocyte in the phase contrast image and to position a 5-μm circular region encompassing the erythrocyte in the phase contrast, E A , E D , and R M images. The average E A , E D , and R M values of the phagosome measurement region, excluding the non-cell portion, were then recorded for each image in the series. Phagocytic progress was monitored using the phase-bright to phase-dark transition that accompanies phagocytosis of opsonized erythrocytes [ 49 ]. Phagosomes were aligned at the time point corresponding to the midpoint of the phase-bright to phase-dark transition; this corresponds to 7.5 min. For phagocytic cups formed in the presence of LY294002, particle-tracking measurements were temporally aligned at the onset of pseudopod extension observed in the fluorescence images. Measurement of phagocytic efficiencies. To quantify the effects of the ARF GTPase mutants on phagocytosis, phagocytic efficiencies were measured for cells expressing the GTPases as CFP fusions. ˜5 × 10 4 RAW264.7 cells were plated in triplicate onto 13-mm coverslips. The cells were transfected the same day with CFP-expression plasmid or plasmids encoding the CFP fusions of ARF1, ARF1(T31N), ARF1(Q71L), ARF6, ARF6(T27N), or ARF6(Q67L). The following day, ˜10 6 IgG-opsonized sheep erythrocytes were delivered to each coverslip in 50 μL of warm, serum-free DMEM. Erythrocytes were allowed to sediment and phagocytosis to proceed for 30 min in a 37 °C–CO 2 incubator. Phagocytosis was arrested and unbound erythrocytes removed by rinsing the coverslips three times with PBS followed by fixation of cells in 20 mM HEPES, 0.2 M sucrose, 4% PFA, 0.01% glutaraldehyde [pH 7.4] for 30 min; fixation and all subsequent steps were performed at room temperature. Following three rinses with PBS, cells were treated with sodium borohydride (Sigma Aldrich, St. Louis, Missouri, United States) to reduce primary fluorescence induced by glutaraldehyde. Coverslips were incubated with a 1 mg/mL solution of borohydride in PBS for 5 min. After four incubations in the borohydride solution, cells were rinsed six times with PBS and twice with PBS supplemented with 2% goat serum (PBS-GS); coverslips were left in the second PBS-GS rinse for 15 min to block nonspecific binding sites. To mark uninternalized erythrocytes, coverslips were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, California, United States) at a concentration of 1 μg/mL in PBS-GS for 30 min. Cells were rinsed with PBS-GS followed by PBS and mounted on slides using Prolong Gold antifade reagent (Invitrogen, Carlsbad, California, United States). Visualization of CFP and Alexa Fluor 488 was accomplished on the inverted Nikon Eclipse TE300 FRET microscope using the JP4v2 filter set. Alexa Fluor 488 was imaged through the YFP channel of this filter set. Cells transfected with the CFP expression construct were located on coverslips, and then the internalized erythrocytes (Alexa488 − ) and bound, uninternalized erythrocytes (Alexa488 + ) counted. The percent phagocytosis was calculated from at least 25 CFP-positive macrophages on each coverslip as: The binding index was calculated as the sum of the uninternalized and internalized erythrocytes per macrophage. Statistical analysis of the phagocytic efficiencies was performed using SigmaPlot 8.0 (Systat Software, Point Richmond, California, United States).

Supporting Information Figure S1 FRET Values (E A , E D , and E AVG ) Measured from Cells Expressing ARF1-CFP and YFP-NGAT and YFP-Rac2V12 and PBD-CFP, Graphed as Functions of R M The average FRET values were calculated from ˜40 cells expressing ARF1-CFP and YFP-NGAT (A) or ˜125 cells expressing YFP-Rac2V12 and PBD-CFP (B). The construction of the Rac-PBD FRET pair is described elsewhere [ 30 ]. The values of E A , E D , and E AVG for each cell were then plotted against the corresponding measured R M values. For R M values below 1, the CFP donor is in excess, while for R M values above 1, the YFP acceptor is the excess binding partner. For low R M values, more acceptors are able to form complex with the excess donors, leading to relatively higher E A values; however, the lack of sufficient acceptors reduces the fraction of donors that can enter into FRET complexes and reduces E D . In the case of excess acceptors, the impact on E A and E D values is reversed, with E A being suppressed. E AVG , though, does not demonstrate this sharp, asymmetric dependence on R M . When graphed as a function of R M , E AVG is essentially independent of R M , particularly in the range R M = 0.1 – 10. Because E A and E D are distinctly dependent on R M , E AVG is preferred for comparing FRET values for multiple samples where average R M values differ between some samples. (204 KB PDF) Click here for additional data file. Protocol S1 Explanation of the Changes to the E D and R M Expressions (25 KB DOC) Click here for additional data file. Table S1 Table of E A , E D , E AVG , and R M Values from Cells Expressing the ARF-CFP and YFP-NGAT Molecules Measured for Figure 1 C (40 KB DOC) Click here for additional data file. Video S1 Movie of ARF6 Activation during Phagocytosis A phase contrast movie of an RAW264.7 macrophage engulfing an IgG-coated RBC is shown in the panel to the left. I A , I D , and I F images were used to calculate the time-lapse R M images (center panel), E D images (right-hand panel) and E A images (unpublished data). Activated ARF6 can be seen at a site of ruffling (upper left of the macrophage, near the center of the panels). Phagocytosis of an erythrocyte, initiated below the ruffle, induced the activation of ARF6 at the leading edge of the phagosome pseudopod during the extension phase. ARF6 was deactivated as the phagosome sealed. All videos seen here play at 6 frames/s. Frames were collected at 30-s intervals. (2.9 MB MOV) Click here for additional data file. Video S2 Movie of ARF1 Activation during Phagocytosis Images shown left to right: phase contrast, R M and E D time-lapse images of a macrophage expressing ARF1-CFP and YFP-NGAT. The activation of ARF1-CFP at the phagosome is revealed in the E D image. ARF1 activation at the phagosome increased during the extension phase and was resolved during closure. The juxtanuclear Golgi compartment is visible in the E D image as well, to the lower right of the forming phagosome. Levels of activated ARF1 at the Golgi did not vary during phagocytosis. (2.2 MB MOV) Click here for additional data file. Video S3 Movie of ARF1 Activation during Phagocytosis by a BFA-Treated Macrophage Panels in the video, left to right: phase contrast, R M and E D images from a macrophage expressing ARF1-CFP and YFP-NGAT, and treated with 5 μM BFA for 30 min prior to microscopic observation. Exposure to BFA prevented the activation of ARF1 at the Golgi—no regions of high FRET are visible near the nucleus in the E D image at the start of the movie. However, the two phagocytic events (lower right, followed by upper left) led to the activation of ARF1, indicating that ARF1 activation at the phagosome is mediated by a BFA-resistant ARNO/cytohesin-family GEF. (2.7 MB MOV) Click here for additional data file. Video S4 Movie of ARF6 Activation in the Presence of LY294002 RAW264.7 macrophages co-transfected with ARF6-CFP and YFP-NGAT were incubated in 50 μM LY294002 for 30 min before the addition of IgG-opsonized erythrocytes. The presented images, left to right, are: phase contrast, R M and E D time-lapse images. Following binding of a particle at the right, ARF6-CFP was activated and pseudopod extension commenced. GTP-bound ARF6-CFP continued to accumulate in the phagocytic cup and was not deactivated during observation. The accumulation of ARF6-CFP at the arrested phagocytic cup can be seen in the R M image series (the blue band at the phagocytic cup represents a molar excess of ARF6-CFP relative to YFP-NGAT). The generation of ARF6-GTP in the absence of PI-3K function indicates ARF6 is activated at the phagosome by a PI-3K-independent GEF activity. (2.8 MB MOV) Click here for additional data file. Video S5 Movie of ARF1 Activity with PI-3K Activity Blocked by LY294002 RAW264.7 macrophages co-transfected with ARF1-CFP and YFP-NGAT demonstrated high FRET signals in the area of their Golgi network in the presence of LY294002: the nucleus is visible in the phase contrast image shown at the left; next to the nucleus, the crescent-shaped region of high E D (far-right panel) corresponds to the Golgi apparatus. The Golgi is also visible in the R M image (center panel) where ARF1-CFP is present in excess over YFP-NGAT. Cells treated with LY294002 could bind opsonized particles and form phagocytic cups but could not complete phagocytosis. At the upper left and right of the macrophage, binding of opsonized erythrocytes was not accompanied by the activation of ARF1-CFP. This result indicates that amplification of FcγR-signaling by PI-3K is required for the activation of ARF1 at the phagosome. (2.9 MB MOV) Click here for additional data file. Accession Numbers Genbank ( http://www.ncbi.nlm.nih.gov/Genbank ) accession numbers are: ARF1 cDNA used to produce ARF1-CFP ( AF493881 ), ARF6 source cDNA ( AF493885 ), GGA1 transcript variant used to create NGAT ( NM_013365 ). All plasmids used to produce the data for this manuscript can be obtained from the Addgene plasmid repository ( http://www.addgene.org/Joel_Swanson , and the Addgene plasmid ID numbers are 11381–11390.