Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands.

Despite recent advances in single-cell genomic, transcriptional, and mass-cytometric profiling, it remains a challenge to collect highly multiplexed measurements of secreted proteins from single cells for comprehensive analysis of functional states. Herein, we combine spatial and spectral encoding with polydimethylsiloxane (PDMS) microchambers for codetection of 42 immune effector proteins secreted from single cells, representing the highest multiplexing recorded to date for a single-cell secretion assay. Using this platform to profile differentiated macrophages stimulated with lipopolysaccharide (LPS), the ligand of Toll-like receptor 4 (TLR4), reveals previously unobserved deep functional heterogeneity and varying levels of pathogenic activation. Uniquely protein profiling on the same single cells before and after LPS stimulation identified a role for macrophage inhibitory factor (MIF) to potentiate the activation of LPS-induced cytokine production. Advanced clustering analysis identified functional subsets including quiescent, polyfunctional fully activated, partially activated populations with different cytokine profiles. This population architecture is conserved throughout the cell activation process and prevails as it is extended to other TLR ligands and to primary macrophages derived from a healthy donor. This work demonstrates that the phenotypically similar cell population still exhibits a large degree of intrinsic heterogeneity at the functional and cell behavior level. This technology enables full-spectrum dissection of immune functional states in response to pathogenic or environmental stimulation, and opens opportunities to quantify deep functional heterogeneity for more comprehensive and accurate immune monitoring.

Lysophospholipid micelles sustain the stability and catalytic activity of diacylglycerol kinase in the absence of lipids.

There has been a renewal of interest in interactions of membrane proteins with detergents and lipids, sparked both by recent results that illuminate the structural details of these interactions and also by the realization that some experimental membrane protein structures are distorted by detergent-protein interactions. The integral membrane enzyme diacylglycerol kinase (DAGK) has long been thought to require the presence of lipid as an obligate "cofactor" in order to be catalytically viable in micelles. Here, we report that near-optimal catalytic properties are observed for DAGK in micelles composed of lysomyristoylphosphatidylcholine (LMPC), with significant activity also being observed in micelles composed of lysomyristoylphosphatidylglycerol and tetradecylphosphocholine. All three of these detergents were also sustained high stability of the enzyme. NMR measurements revealed significant differences in DAGK-detergent interactions involving LMPC micelles versus micelles composed of dodecylphosphocholine. These results highlight the fact that some integral membrane proteins can maintain native-like properties in lipid-free detergent micelles and also suggest that C(14)-based detergents may be worthy of more widespread use in studies of membrane proteins.

Structural basis for dimerization of the BNIP3 transmembrane domain.

Mutagenesis data suggest that BNIP3 transmembrane domain dimerization depends critically on hydrogen bonding between His 173 and Ser 172, but a recent structural analysis indicates that these residues adopt multiple conformations and are not always hydrogen bonded. We show that in dodecylphosphocholine micelles the structure of the BNIP3 transmembrane domain is modulated by phospholipids and that appropriate reconstitution and lipid titration yield a single set of peptide resonances. NMR structure determination reveals a symmetric dimer in which all interfacial residues, including His 173 and Ser 172, are well-defined. Small residues Ala 176, Gly 180, and Gly 184 allow close approach of essentially ideal helices in a geometry that supports intermonomer hydrogen bond formation between the side chains of His 173 and Ser 172. Bulky residues Ile 177 and Ile 181 pack against small residues of the opposite monomer, and favorable polar backbone-backbone contacts at the interface likely include noncanonical Calpha-H.O=C hydrogen bonds from Gly 180 to Ile 177. Modeling mutations into the structure shows that most deleterious hydrophobic substitutions eliminate the His-Ser hydrogen bond or introduce an intermonomer clash, indicating critical roles for sterics and hydrogen bonding in the sequence dependence of dimerization. Substitutions at most noninterfacial positions do not alter dimerization, but the disruptive effects of substitutions at Ile 183 cannot be rationalized in terms of peptide-peptide contacts and therefore may indicate a role for peptide-detergent or peptide-lipid interactions at this position.

Sequence dependence of BNIP3 transmembrane domain dimerization implicates side-chain hydrogen bonding and a tandem GxxxG motif in specific helix-helix interactions.

The transmembrane domain of the pro-apoptotic protein BNIP3 self-associates strongly in membranes and in detergents. We have used site-directed mutagenesis to analyze the sequence dependence of BNIP3 transmembrane domain dimerization, from which we infer the physical basis for strong and specific helix-helix interactions in this system. Hydrophobic substitutions identify six residues as critical to dimerization, and the pattern of sensitive residues suggests that the BNIP3 helices interact at a right-handed crossing angle. Based on the dimerization propensities of single point mutants, we propose that: polar residues His173 and Ser172 make inter-monomer hydrogen bonds to one another through their side-chains; Ala176, Gly180, and Gly184 form a tandem GxxxG motif that allows close approach of the helices; and Ile183 makes inter-monomer van der Waals contacts. Since neither the tandem GxxxG motif nor the hydrogen bonding pair is sufficient to drive dimerization, our results demonstrate the importance of sequence context for either hydrogen bonding or GxxxG motif involvement in BNIP3 transmembrane helix-helix interactions. In this study, hydrophobic substitutions away from the six interfacial positions have almost no effect on dimerization, confirming the expectation that hydrophobic replacements affect helix-helix interactions only if they interfere with packing or hydrogen bonding by interfacial residues. However, changes to slightly polar residues are somewhat disruptive even when located away from the interface, and the degree of disruption correlates with the decrease in hydrophobicity. Changing the hydrophobicity of the BNIP3 transmembrane domain alters its helicity and protection of its backbone amides. We suggest that polar substitutions decrease the fraction of dimer by stabilizing an unfolded monomeric state of the transmembrane span, rather than by affecting helix-helix interactions. This result has broad implications for interpreting the sequence dependence of membrane protein stability in detergents.

Systems analysis of latent HIV reversal reveals altered stress kinase signaling and increased cell death in infected T cells.

Viral latency remains the most significant obstacle to HIV eradication. Clinical strategies aim to purge the latent CD4+ T cell reservoir by activating viral expression to induce death, but are undercut by the inability to target latently infected cells. Here we explored the acute signaling response of latent HIV-infected CD4+ T cells to identify dynamic phosphorylation signatures that could be targeted for therapy. Stimulation with CD3/CD28, PMA/ionomycin, or latency reversing agents prostratin and SAHA, yielded increased phosphorylation of IκBα, ERK, p38, and JNK in HIV-infected cells across two in vitro latency models. Both latent infection and viral protein expression contributed to changes in perturbation-induced signaling. Data-driven statistical models calculated from the phosphorylation signatures successfully classified infected and uninfected cells and further identified signals that were functionally important for regulating cell death. Specifically, the stress kinase pathways p38 and JNK were modified in latently infected cells, and activation of p38 and JNK signaling by anisomycin resulted in increased cell death independent of HIV reactivation. Our findings suggest that altered phosphorylation signatures in infected T cells provide a novel strategy to more selectively target the latent reservoir to enhance eradication efforts.

Analysis of single-cell cytokine secretion reveals a role for paracrine signaling in coordinating macrophage responses to TLR4 stimulation.

Macrophages not only produce multiple cytokines but also respond to multiple cytokines, which likely shapes the ultimate response of the population. To determine the role of paracrine signaling in shaping the profile of inflammatory cytokines secreted by macrophages in response to stimulation of Toll-like receptor 4 (TLR4) with lipopolysaccharide (LPS), we combined multiplexed, microwell-based measurements of cytokine secretion by single cells with analysis of cytokine secretion by cell populations. Loss of paracrine signaling as a result of cell isolation reduced the secretion by macrophage-like U937 cells and human monocyte-derived macrophages (MDMs) of a subset of LPS-stimulated cytokines, including interleukin-6 (IL-6) and IL-10. Graphical Gaussian modeling (GGM) of the single-cell data defined a regulatory network of paracrine signals, which was validated experimentally in the population through antibody-mediated neutralization of individual cytokines. Tumor necrosis factor-α (TNF-α) was the most influential cytokine in the GGM network. Paracrine signaling by TNF-α secreted from a small subpopulation of "high-secreting" cells was necessary, but not sufficient, for the secretion of large amounts of IL-6 and IL-10 by the cell population. Decreased relative IL-10 secretion by isolated MDMs was linked to increased TNF-α secretion, suggesting that inhibition of the inflammatory response also depends on paracrine signaling. Our results reveal a previously uncharacterized role for cell-to-cell communication within a population in coordinating a rapid innate immune response despite underlying cell-to-cell heterogeneity.

Intermonomer hydrogen bonds enhance GxxxG-driven dimerization of the BNIP3 transmembrane domain: roles for sequence context in helix-helix association in membranes.

We determined the sequence dependence of human BNIP3 transmembrane domain dimerization using the biological assay TOXCAT. Mutants in which intermonomer hydrogen bonds between Ser172 and His173 are abolished show moderate interaction, indicating that side-chain hydrogen bonds contribute to dimer stability but are not essential to dimerization. Mutants in which a GxxxG motif composed of Gly180 and Gly184 has been abolished show little or no interaction, demonstrating the critical nature of the GxxxG motif to BNIP3 dimerization. These findings show that side-chain hydrogen bonds can enhance the intrinsic dimerization of a GxxxG motif and that sequence context can control how hydrogen bonds influence helix-helix interactions in membranes. The dimer interface mapped by TOXCAT mutagenesis agrees closely with the interfaces observed in the NMR structure and inferred from mutational analysis of dimerization on SDS-PAGE, showing that the native dimer structure is retained in detergents. We show that TOXCAT and SDS-PAGE give complementary and consistent information about BNIP3 transmembrane domain dimerization: TOXCAT is insensitive to mutations that have modest effects on self-association in detergents but readily discriminates among mutations that completely disrupt detergent-resistant dimerization. The close agreement between conclusions reached from TOXCAT and SDS-PAGE data for BNIP3 suggests that accurate estimates of the relative effects of mutations on native-state protein-protein interactions can be obtained even when the detergent environment is strongly disruptive.

Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase.

Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer with the use of solution nuclear magnetic resonance. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK's three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK's lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis.

Sequence-specific dimerization of the transmembrane domain of the "BH3-only" protein BNIP3 in membranes and detergent.

Mitochondria-mediated apoptosis is regulated by proteins of the Bcl-2 superfamily, most of which contain a C-terminal hydrophobic domain that plays a role in membrane targeting. Experiments with BNIP3 have implicated the transmembrane (TM) domain in its proapoptotic function, homodimerization, and interactions with Bcl-2 and Bcl-xL. We show that the BNIP3 TM domain self-associates strongly in Escherichia coli cell membranes and causes reversible dimerization of a soluble protein in the detergent SDS when expressed as an in-frame fusion. Limited mutational analysis identifies specific residues that are critical for BNIP3 TM self-association in membranes, and these residues are also important for dimerization in SDS micelles, suggesting that the self-association observed in membranes is preserved in detergent. The effects of sequence changes at positions Ala176 and Gly180 suggest that the BNIP3 TM domain associates using a variant of the GXXXG motif previously shown to be important in the dimerization of glycophorin A. The importance of residue His173 in BNIP3 TM domain dimerization indicates that polar residues, which have been implicated in self-association of model TM peptides, can act in concert with the AXXXG motif to stabilize TM domain interactions. Our results demonstrate that the hydrophobic C-terminal TM domain of the pro-apoptotic BNIP3 protein dimerizes tightly in lipidic environments, and that this association has a strong sequence dependence but is independent of the identity of flanking regions. Thus, the transmembrane domain represents another region of the Bcl-2 superfamily of proteins that is capable of mediating strong and specific protein-protein interactions.