The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4 + T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4 + T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer by T cells highlight unique aspects of cell-cell HIV transmission.

HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity, and 3D distribution of these compartments has remained controversial. Here, we report the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM, we show the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of ∼150–200 nm, and lengths of up to ∼5 µm that extend to the cell surface from vesicular compartments that contain assembling HIV-1 virions. These types of surface-connected tubular compartments are not observed in T cells infected with the 29/31 KE Gag-matrix mutant where the virus is targeted to multi-vesicular bodies and released into the extracellular medium. IA-SEM imaging also allows visualization of large sheet-like structures that extend outward from the surfaces of macrophages, which may bend and fold back to allow continual creation of viral compartments and virion-lined channels. This potential mechanism for efficient virus trafficking between the cell surface and interior may represent a subversion of pre-existing vesicular machinery for antigen capture, processing, sequestration, and presentation.

A quiet revolution is under way in technologies used for nanoscale cellular imaging. Focused ion beams, previously restricted to the materials sciences and semiconductor fields, are rapidly becoming powerful tools for ultrastructural imaging of biological samples. Cell and tissue architecture, as preserved in plastic-embedded resin or in plunge-frozen form, can be investigated in three dimensions by scanning electron microscopy imaging of freshly created surfaces that result from the progressive removal of material using a focused ion beam. The focused ion beam can also be used as a sculpting tool to create specific specimen shapes such as lamellae or needles that can be analyzed further by transmission electron microscopy or by methods that probe chemical composition. Here we provide an in-depth primer to the application of focused ion beams in biology, including a guide to the practical aspects of using the technology, as well as selected examples of its contribution to the generation of new insights into subcellular architecture and mechanisms underlying host-pathogen interactions.

The peptide editor HLA-DM (DM) mediates exchange of peptides bound to major histocompatibility (MHC) class II molecules during antigen processing; however, the mechanism by which DM displaces peptides remains unclear. Here we generated a soluble mutant HLA-DR1 with a histidine-to-asparagine substitution at position 81 of the β-chain (DR1βH81N) to perturb an important hydrogen bond between MHC class II and peptide. Peptide-DR1βH81N complexes dissociated at rates similar to the dissociation rates of DM-induced peptide-wild-type DR1, and DM did not enhance the dissociation of peptide-DR1βH81N complexes. Reintroduction of an appropriate hydrogen bond (DR1βH81N βV85H) restored DM-mediated peptide dissociation. Thus, DR1βH81N might represent a `post-DM effect' conformation. We suggest that DM may mediate peptide dissociation by a `hit-and-run' mechanism that results in conformational changes in MHC class II molecules and disruption of hydrogen bonds between βHis81 and bound peptide.Shortly after being synthesized in the antigen-presenting cell, major histocompatibility complex (MHC) class II αβ heterodimers form nonameric assemblies with invariant chain (Ii) in the endoplasmic reticulum and are then transported through the Golgi complex to the endocytic pathway 1, 2. During transport through the endocytic pathway, most Ii is removed from MHC class II molecules by low pH and acid proteases3, leaving a proteolytic fragment of Ii called `CLIP' bound to MHC class II molecule4. CLIP acts as a `placeholder' for the MHC class II groove, inhibiting conformational changes that render the groove closed5 -13 , and it must be removed to allow binding of exogenous peptides to nascent MHC class II complexes. Human HLA-DM (called `DM' here), or H2-M in mice, is a nonclassical HLA molecule that is critical in the displacement of CLIP 14-17. In addition to displacing CLIP, DM transiently interacts with empty MHC class II molecules to generate a peptide-receptive conformation and is active in the selection of specific peptide-MHC class II complexes during antigen processing18 -26. The two concurrent hypotheses for the recognition of certain peptide-MHC class II by DM relate to the intrinsic affinity between MHC class II © 2006 Nature Publishing Group Correspondence should be addressed to S.S-N (ssadegh@jhmi.edu).. 4 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Immunology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Immunol. Author manuscript; available in PMC 2011 January 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript and the peptide 22,27,28 or to subtle structural variations among different peptide-MHC complexes 25,[29][30][31][32] , whereby structurally flexible complexes are susceptible to DM-induced dissociation, and `rigid' complexes are resistant to DM 25 . Although those studies may have brought greater understanding of the crite...

Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB-SEM) to address this issue. We increase the speed, robustness and automation of the process, and achieve consistent z slice thickness of ~3 nm. We introduce “keyframe imaging” to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We apply these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a “target map” of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 109 in a single automated experimental run.

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA–SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ~10 to 20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA–SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.

The formation of the HIV-1 core is the final step in the viral maturation pathway, resulting in the formation of infectious virus. Most current models for HIV-1 core formation suggest that, upon proteolytic cleavage from the immature Gag, capsid (CA) dissociates into the viral interior before reforming into the core. Here we present evidence for an alternate view of core formation by taking advantage of our serendipitous observation of large membrane-enclosed structures in HIV-1 supernatants from infected cells. Cryo-electron tomographic studies show that these structures, which contain ordered arrays of what is likely the membrane-associated matrix protein, contain multiple cores that can be captured at different stages of maturation. Our studies suggest that HIV maturation involves a non-diffusional phase transition in which the detaching layer of the cleaved CA lattice is gradually converted into a roll that ultimately forms the surface of the mature conical core.

Chemotactic signals are relayed to neighboring cells through the secretion of additional chemoattractants. We previously showed in that the adenylyl cyclase A, which synthesizes the chemoattractant cyclic adenosine monophosphate (cAMP), is present in the intraluminal vesicles of multivesicular bodies (MVBs) that coalesce at the back of cells. Using ultrastructural reconstructions, we now show that ACA-containing MVBs release their contents to attract neighboring cells. We show that the released vesicles are capable of directing migration and streaming and are central to chemotactic signal relay. We demonstrate that the released vesicles not only contain cAMP but also can actively synthesize and release cAMP to promote chemotaxis. Through proteomic, pharmacological, and genetic approaches, we determined that the vesicular cAMP is released via the ABCC8 transporter. Together, our findings show that extracellular vesicles released by cells are functional entities that mediate signal relay during chemotaxis and streaming.