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Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3ζ, ZAP70, LAT, SLP-76, Itk, and PLCγ15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.
Fluorescent dyes used in this protocol are effective for binding to proteins, are sufficiently photo-stabile for microscopy imaging, and match the excitation wavelengths of the lasers available with our microscope. These three criteria need to be met when selecting fluorescent molecules for tagging proteins.
The general procedure for flow cell and bilayer preparation has been described in detail previously14. Here we outline specifically the protocol to prepare bilayers containing His6-tagged gp120 DH12 on a Bioptech flow cell. For studies involving infectious viruses or infected cells and when small volumes are necessary due to limited reagents, a disposable Ibidi flow chamber (sticky Slide I 0.2 Luer) may be used and followed with the same bi-layer preparation procedure in Steps 2.4-2.9. Information for Ibidi flow chambers can be obtained from the company website, http://www.ibidi.com/service/display_material/CA_8p_EN_150dpi.pdf.
Proteins in bilayers must be laterally diffusible. Before adding cells onto bilayers, it is important to assure the mobility of proteins in the prepared bilayer. Here we describe a Fluorescent Recovery After Photobleaching (FRAP) method3 that may be performed manually on most objective illuminated TIRF, wide field epifluorescence, or laser scanning confocal microscopes. The goal is to image fully uniform fields of fluorescence in the lipid bilayer, bleach a spot, and to monitor the return of unquenched molecules to the bleached area. A recovery of 50% in 2 minutes may be acceptable. All the major microscope manufacturers (Leica, Nikon, Olympus, Zeiss) sell objective illuminated TIRF systems that work well for this application. While each company provides variations of TIRF illumination and other operational features, the image quality is essentially the same.
Note: In our current microscope, we can deliver 0.9 mW of 641 nm light to a circular spot with an area of 240 μm2 which bleaches fluorescent ICAM at typical concentrations in less than 4 seconds. Readers should find that critical alignment of an Hg or Xe lamp with bleaching times less than 30 seconds is more than sufficient to perform this assay in a repeatable manner. We would also refer you to reference3 for additional details.
TIRF microscopy allows excitation of fluorescent signals limited to a 250 nm or thinner plane at the glass substrate and cell interface. This guarantees image acquisition of only signals from the lipid bilayer and from the immediately apposed cell membranes. Therefore, only molecules at the synapse are imaged. Because TIRF images only the membrane-proximal area at the bottom of the cell, proteins that are internalized or redistributed into the dorsal membrane will not be detected. It may then be important to additionally acquire images by standard widefield or confocal microscopy to determine accumulation or distribution of the proteins at the synaptic area as compared to the other volumes of the cell.
All the major microscope manufacturers (Leica, Nikon, Olympus, Zeiss) sell objective illuminated TIRF systems that work well for this application. While each company provides variations of TIRF illumination and other operational features, the image quality is essentially the same. Each TIRF microscope has its own details for operation, but the following general rules apply to obtain high-resolution imaging.
To study the intracellular signals activated as a result of CD4+ T cell interaction with gp120 at the VS, quantitative analyses of TIRF images are done to determine whether or not intracellular signaling molecules such as Lck and Fyn are activated and recruited to the synapse 15. Here we describe a simple method applicable for most image analysis application packages such as ImageJ and Metamorph. We have used ImageJ, which runs on Windows, Macintosh, and Linux, for our method here 12.
In the Analyze > Set Measurements tab, check the boxes for Area and Mean gray value. When you make a region of interest on the image that is a polygon or a freehand traced closed shape and do Analyze > Measure, the software will put the data from this measurement in a results table. The Area will be in number of pixels or in μm2. The Mean is the average intensity in arbitrary units. It must have background intensity subtracted from it. Multiplying the mean minus background by area returns the integrated intensity of the protein in arbitrary units.
To measure activation and recruitment of the initial membrane-proximal signaling molecules Lck and Fyn as a result of interaction with gp120 at the VS, primary human CD4+ T cells were introduced onto bilayers bearing gp120 and ICAM-1 15. Cells introduced to bilayers with only ICAM-1 served as a control to define the basal levels of signaling. Specific fluorescence intensity was measured within the gp120 contact area for cells on gp120 and ICAM-1 containing bilayers and within the whole contact area for cells interacting with the ICAM-1 bilayer. An increase of average fluorescence intensity is a sign of augmented recruitment and activation of the signaling molecule to the VS. This will be further demonstrated by a comparable increase in the integrated fluorescence intensity. If however, there is no change in the integrated fluorescence intensity, this indicates a redistribution of the signaling molecule.
After the cells interacted with bilayers containing gp120 and ICAM-1, total Lck and pLck(Y394) were recruited to the VS interface and colocalized with gp120 (Figs. 1 & 2). The average intensity of total Lck (Fig. 1) was higher on the bilayer containing both gp120 and ICAM-1 than with ICAM-1 alone, but the integrated intensity levels (Fig. 1) were similar, suggesting that Lck is redistributed into a central cluster upon CD4+ T cell binding to gp120. However, quantification of pLck(Y394) (Fig. 2) showed that the average intensity on gp120 and ICAM-1 containing bilayers was higher than that on ICAM-1 alone bilayers, and the integrated intensity was higher on bilayers with both gp120 and ICAM-1 than on ICAM-1 alone bilayers. This indicates that while the levels of total Lck at the interface are similar in cells adhering onto gp120 and ICAM-1 and ICAM-1 alone bilayers, gp120 binding increased phosphorylation of residue Y394 at the Lck activation loop. In contrast, Fyn was not recruited to the VS (Fig. 3), as more Fyn was present in the contact area of cells on ICAM-1 alone bilayers than on bilayers containing both gp120 and ICAM-1. Consequently, we conclude that Lck, not Fyn, is the active kinase in the HIV-1 gp120-induced VS.
Figures: Membrane-proximal signaling at HIV-1 gp120-induced VS. Images of representative cells on the gp120+ICAM-1 bilayer (top panels) and the ICAM-1 bilayer (bottom panels) are shown. Fluorescence intensities of the individual cells were quantified within manually traced areas of the cell footprints as illustrated by the region marked with the yellow line in Figure 1. Quantification of average and integrated intensities detected by TIRF microscopy are presented in the left and right graphs, respectively. A total of 30 to 350 cells were quantified for each condition. Bars = 5 μm. Data from one of three repeat experiments are shown.
Figure 1. CD4+ T cells were introduced onto bilayers containing gp120 and ICAM-1 or ICAM-1 alone for 45 min and then fixed and stained for total Lck.
Figure 2. CD4+ T cells were introduced onto bilayers containing gp120 and ICAM-1 or ICAM-1 alone for 45 min and then fixed and stained for pLck(Y394).
Figure 3. CD4+ T cells were introduced onto bilayers containing gp120 and ICAM-1 or ICAM-1 alone for 45 min and then fixed and stained for total Fyn.
Previous studies have visualized VS in the cell-cell conjugate system; however these studies did not provide images of high enough resolution to visualize the supramolecular structures at the synapse. In our lab, we utilized the glass-supported planar bilayer system to represent the surface of infected cells expressing the virus envelope gp120 and the cellular adhesion molecule ICAM-1. In conjunction with TIRF microscopy, which detects fluorescence signals within 100-200 nm from the bilayer surface with a high signal-to-noise ratio, we were able to detect supramolecular segregation of gp120 from ICAM-1 at the VS. Moreover, the standard immunostaining method can be applied to the bilayer system and was used here to detect and quantify the specific recruitment of active Lck, but not Fyn, to the gp120-contact area at VS 15. Hence, the planar bilayer offers an experimental system for high-resolution imaging of synapse interface in a 2D plane by TIRF microscopy, as well as wide-field or confocal illumination methods. However, the system also has limitations as adjusting ligand mobility, out-of-plane bending, and fluctuations of biological membranes are not reproduced by planar bilayers. Furthermore, this is an in-vitro system and therefore has other limitations, such as lack of other membrane molecules that would be present on an infected cell and the cytoskeleton machinery that regulates molecular mobility and cellular motility. Also, the dynamics and distribution of the molecules, such as trimers versus monomers of gp120, may not be represented physiologically on the bilayer. Nevertheless, even with these limitations, this system is still very valuable for studying virus-cell or cell-cell interactions, and these methods can serve as a useful guide to researchers who are seeking high-resolution images to detect supramolecular organization that is not discernible in the conventional cell-cell conjugate system.
The authors declare no conflict of interests.
This work was supported by NIH grants AI071815 (C.E.H.) and the Roadmap Nanomedicine Development Center award PN2EY016586 (M.L.D.).