Leo M. Carlin

Nr4a1-Dependent Ly6C(low) Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal

Summary The functions of Nr4a1-dependent Ly6Clow monocytes remain enigmatic. We show that they are enriched within capillaries and scavenge microparticles from their lumenal side in a steady state. In the kidney cortex, perturbation of homeostasis by a TLR7-dependent nucleic acid “danger” signal, which may signify viral infection or local cell death, triggers Gαi-dependent intravascular retention of Ly6Clow monocytes by the endothelium. Then, monocytes recruit neutrophils in a TLR7-dependent manner to mediate focal necrosis of endothelial cells, whereas the monocytes remove cellular debris. Prevention of Ly6Clow monocyte development, crawling, or retention in Nr4a1−/−, Itgal−/−, and Tlr7host−/−BM+/+ and Cx3cr1−/− mice, respectively, abolished neutrophil recruitment and endothelial killing. Prevention of neutrophil recruitment in Tlr7host+/+BM−/− mice or by neutrophil depletion also abolished endothelial cell necrosis. Therefore, Ly6Clow monocytes are intravascular housekeepers that orchestrate the necrosis by neutrophils of endothelial cells that signal a local threat sensed via TLR7 followed by the in situ phagocytosis of cellular debris.

Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis

Förster resonance energy transfer (FRET) detected via fluorescence lifetime imaging microscopy (FLIM) and global analysis provide a way in which protein–protein interactions may be spatially localized and quantified within biological cells. The FRET efficiency and proportion of interacting molecules have been determined using bi-exponential fitting to time-domain FLIM data from a multiphoton time-correlated single-photon counting microscope system. The analysis has been made more robust to noise and significantly faster using global fitting, allowing higher spatial resolutions and/or lower acquisition times. Data have been simulated, as well as acquired from cell experiments, and the accuracy of a modified Levenberg–Marquardt fitting technique has been explored. Multi-image global analysis has been used to follow the epidermal growth factor-induced activation of Cdc42 in a short-image-interval time-lapse FLIM/FRET experiment. Our implementation offers practical analysis and time-resolved-image manipulation, which have been targeted towards providing fast execution, robustness to low photon counts, quantitative results and amenability to automation and batch processing.

The size of the synaptic cleft and distinct distributions of filamentous actin, ezrin, CD43, and CD45 at activating and inhibitory human NK cell immune synapses.

In this study, we report the organization of cytoskeletal and large transmembrane proteins at the inhibitory and activating NK cell immunological or immune synapse (IS). Filamentous actin accumulates at the activating, but not the inhibitory, NK cell IS. However, surprisingly, ezrin and the associated protein CD43 are excluded from the inhibitory, but not the activating, NK cell IS. This distribution of ezrin and CD43 at the inhibitory NK cell IS is similar to that previously seen at the activating T cell IS. CD45 is also excluded from the inhibitory, but not activating, NK cell IS. In addition, electron microscopy reveals wide and narrow domains across the synaptic cleft. Target cell HLA-C, located by immunogold labeling, clusters where the synaptic cleft spans the size of HLA-C bound to the inhibitory killer Ig-like receptor. These data are consistent with assembly of the NK cell IS involving a combination of cytoskeletal-driven mechanisms and thermodynamics favoring the organization of receptor/ligand pairs according to the size of their extracellular domains.

Essential Role of hIST1 in Cytokinesis

The last steps of multivesicular body (MVB) formation, human immunodeficiency virus (HIV)-1 budding and cytokinesis require a functional endosomal sorting complex required for transport (ESCRT) machinery to facilitate topologically equivalent membrane fission events. Increased sodium tolerance (IST) 1, a new positive modulator of the ESCRT pathway, has been described recently, but an essential function of this highly conserved protein has not been identified. Here, we describe the previously uncharacterized KIAA0174 as the human homologue of IST1 (hIST1), and we report its conserved interaction with VPS4, CHMP1A/B, and LIP5. We also identify a microtubule interacting and transport (MIT) domain interacting motif (MIM) in hIST1 that is necessary for its interaction with VPS4, LIP5 and other MIT domain-containing proteins, namely, MITD1, AMSH, UBPY, and Spastin. Importantly, hIST1 is essential for cytokinesis in mammalian cells but not for HIV-1 budding, thus providing a novel mechanism of functional diversification of the ESCRT machinery. Last, we show that the hIST1 MIM activity is essential for cytokinesis, suggesting possible mechanisms to explain the role of hIST1 in the last step of mammalian cell division.

Biomedical Imaging: From Nano to Macro

Studying cellular protein-protein interactions in situ requires a technique such as fluorescence resonance energy transfer (FRET) which is sensitive on the nanometer scale. Observing FRET is significantly simplified if the fluorescence lifetime of the donor can be monitored. Results from live cells and tissue micro arrays are presented from an automated microscope incorporating time-domain TCSPC fluorescence lifetime imaging (FLIM). Novel hardware and software with a modular approach and scripting abilities allow us to work towards speed-optimized acquisition and ease of use to bring FLIM into the high-throughput regime.

The Actin Cytoskeleton Controls the Efficiency of Killer Ig-Like Receptor Accumulation at Inhibitory NK Cell Immune Synapses

Killer cell Ig-like receptors (KIRs) are MHC class I-specific receptors expressed in NK and T lymphocytes. KIR antagonism of activation signals occurs at the immune synapse between the effector and target cells. The processes that regulate clustering of KIR are not well defined. We have expressed KIR-GFP receptor chimeras in two human NK-like lines, YTS and NK92. In this study, we show that the frequency of KIR enrichment at the synapse was decreased for a KIR that lacks a portion of the cytoplasmic tail. Strikingly, blocking actin polymerization with a high dose of cytochalasin D also substantially decreased clustering of KIR as well as KIR-induced clustering of HLA-C-GFP in target cells. However, the effect of inhibiting actin polymerization was only clearly evident at the earlier time points after cell mixing, and eventually clustering of KIR and HLA-C occurred independently of actin remodeling. Although treatment with anti-LFA-1 also decreased conjugate formation, the frequency of KIR clustering remained normal within the population of conjugates that did form, suggesting that the effect of cytochalasin D is not solely through LFA-1. Collectively, these data suggest that the actin cytoskeleton and the cytoplasmic tail of KIR regulate the efficiency by which KIR accumulates at inhibitory NK cell synapses.

Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8+ T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c+ dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c+ MHCIIhi CD8αneg epithelial cell adhesion molecule (EpCAMneg) CD11b+ langerin (Lang; CD207)neg DCs, but neither Langerhans cells nor Lang+ DCs were required for CD8+ T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8+ T-cell priming by live rAdHu5 MAs.

A targeted siRNA screen identifies regulators of Cdc42 activity at the natural killer cell immunological synapse.

The oscillating activity of a cytoskeletal regulator enables natural killer cells to effectively perform their surveillance functions and polarize cytotoxic vesicles. Oscillatory Behavior at the Immunological Synapse Natural killer (NK) cells are required for effective immune responses against virally infected cells and tumor cells. The activity of NK cells is controlled by coordinated signals from stimulatory and inhibitory receptors at the cell surface, which are engaged when the NK cell forms conjugates with target cells. Effective cell killing by NK cells is dependent on changes in the actin cytoskeleton that require Rho family GTPases, such as Cdc42. Carlin et al. used fluorescence-based live-cell imaging to show that after an initial “spike,” the activity of Cdc42 at the NK cell–target cell interface oscillated over time. Bioinformatics analysis and a short inhibitory RNA (siRNA)–based screen identified Akt and a subunit of PI3K as required for the stimulation and oscillation of Cdc42 activity, as well as for the polarization of cytotoxic vesicles, a critical step in NK cell cytotoxicity. Natural killer (NK) cells kill tumor cells and virally infected cells, and an effective NK cell response requires processes, such as motility, recognition, and directional secretion, that rely on cytoskeletal rearrangement. The Rho guanosine triphosphatase (GTPase) Cdc42 coordinates cytoskeletal reorganization downstream of many receptors. The Rho-related GTPase from plants 1 (ROP1) exhibits oscillatory activation behavior at the apical plasma membrane of growing pollen tubes; however, a similar oscillation in Rho GTPase activity has so far not been demonstrated in mammalian cells. We hypothesized that oscillations in Cdc42 activity might occur within NK cells as they interact with target cells. Through fluorescence lifetime imaging of a Cdc42 biosensor, we observed that in live NK cells forming immunological synapses with target cells, Cdc42 activity oscillated after exhibiting an initial increase. We used protein-protein interaction networks and structural databases to identify candidate proteins that controlled Cdc42 activity, leading to the design of a targeted short interfering RNA screen. The guanine nucleotide exchange factors RhoGEF6 and RhoGEF7 were necessary for Cdc42 activation within the NK cell immunological synapse. In addition, the kinase Akt and the p85α subunit of phosphoinositide 3-kinase (PI3K) were required for Cdc42 activation, the periodicity of the oscillation in Cdc42 activity, and the subsequent polarization of cytotoxic vesicles toward target cells. Given that PI3Ks are targets of tumor therapies, our findings suggest the need to monitor innate immune function during the course of targeted therapy against these enzymes.

Integrating Receptor Signal Inputs That Influence Small Rho GTPase Activation Dynamics at the Immunological Synapse

ABSTRACT The Rho GTPase Cdc42 regulates cytoskeletal changes at the immunological synapse (IS) that are critical to T-cell activation. By imaging fluorescent activity biosensors (Raichu) using fluorescence lifetime imaging microscopy, Cdc42 activation was shown to display kinetics that are conditional on the specific receptor input (through two IS-associated receptors, CD3 and β1 integrin). CD3-triggered Cdc42 activity is dependent on the cyto-2 (NPIY) motif of the β1 integrin cytoplasmic domain. Perturbations of the ezrin-radixin-moesin (ERM) function blocked CD3- and β1-dependent increases in Cdc42 activity. Both IS-associated receptors probably lie on a serial molecular pathway and transduce signals through the ERM-dependent machinery that is responsible for the remodeling and stabilization of the synapse. Cdc42 activity is impaired in β1 integrin-deficient T cells that form conjugates with antigen-presenting cells but is partially restored in the context of an antigen-specific synapse. This restoration of Cdc42 activity is due, at least in part, to the recruitment and activation of β2 integrin.

Imaging immune surveillance by T cells and NK cells

Summary: As T cells and natural killer (NK) cells survey the surface of other cells, cognate receptors and ligands are commonly organized into distinct micrometer‐scale domains at the intercellular contact, creating an immune or immunological synapse (IS). We aim to address the still unanswered questions of how this organization of proteins aids immune surveillance and how these domains are biophysically constructed. Molecular mechanisms for the formation of the IS include a role for the cytoskeleton, segregation of proteins according to the size of their extracellular domains, and association of proteins with lipid rafts. Towards understanding the function of the IS, it is instructive to compare and contrast the supramolecular organization of proteins at the inhibitory and activating NK cell IS with that at the activating T cell IS. Finally, it is essential to develop new technologies for probing molecular recognition at cell surfaces. Imaging parameters other than fluorescence intensity, such as the lifetime of the fluorophores excited state, could be used to report on protein environments.