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 fullspectrum 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.single-cell analysis | cytokine | immune effector function | cellular heterogeneity | Toll-like receptor activation E merging evidence indicates that cell-to-cell variability can give rise to phenotypic differences within a genetically identical cell population (1, 2). Nongenetic heterogeneity is also emerging as a potential barrier to effective therapeutic intervention (3, 4). Recent advances in single-cell molecular profiling are beginning to address these questions. Single-cell RNA sequencing revealed dynamic and bimodal gene expression (5). Single-cell multicolor flow cytometry (6) and mass cytometry (7) can quantify phenotypic diversity and differential drug response even across the hematopoietic continuum. Although a limited number of signaling proteins can be measured using intracellular staining, most of these technologies measure transcriptional or phenotypic marker expression in single cells. It remains an unmet need to directly measure cellular functional outcomes in a highly multiplexed manner and in single cells. In the immune system, the immune effector functions are largely mediated by a panel of effector proteins (e.g., cytokines and chemokines) secreted from single cells. Due to phenotypic plasticity and functional diversity, immune cells purified for a well-defined phenot...

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 using solution NMR. 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.

Cellular responses are mediated by heterogeneous intermediate signals that are secreted and sensed by the same cells. Cell-to-cell communication through these intermediate signals likely affects the collective response of cells within a population. We combined multiplexed, microwell-based measurements of cytokine secretion by single cells with data from the analysis of cell populations to determine the role of paracrine signaling in shaping the profile of inflammatory cytokines secreted by macrophages in response to the stimulation of Toll-like receptor 4 (TLR4) with lipopolysaccharide (LPS). Loss of paracrine signaling as a result of cell isolation substantially reduced the secretion of a subset of LPS-stimulated cytokines, including interleukin-6 (IL-6) and IL-10, by macrophage-like U937 cells and human monocyte-derived macrophages (MDMs). 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 identified as the most influential cytokine in the GGM network, and our data suggest that paracrine signaling from a small subpopulation of “high-secreting” cells, which generated most of the TNF-α produced, was necessary but not sufficient to achieve high secretion of IL-6 and IL-10 in the cell population. Decreased IL-10 secretion in 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 and reliable innate immune response in spite of underlying cell-to-cell heterogeneity.

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-x L . 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 selfassociation 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 Ala 176 and Gly 180 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 His 173 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.The Bcl-2 superfamily of proteins plays a central role in regulating mitochondria-mediated apoptosis; the Bax subfamily promotes apoptosis, whereas the Bcl-2 subfamily protects against apoptosis (reviewed in Ref. 1). Protein-protein interactions between Bax and Bcl-2 subfamily members help determine cell fate (2, 3) and have accordingly been the subject of intensive study. Four regions of sequence homology, the BH1 through BH4 domains, contribute to the structure and function of these proteins, and the BH3 domain is particularly implicated in heterodimerization events (4 -9). Peptide and small molecule inhibitors of these protein-protein interactions can modulate apoptosis, further demonstrating the functional and pharmaceutical importance of these contacts (10 -13).Differentiation and development in metazoans requires celland signal-specific inputs to a functional Bcl-2/Bax checkpoint. The pro-apoptotic "BH3-only" proteins, which show homology to the Bcl-2 superfamily through the BH3 domain alone (14), are expressed or activated in specific tissues and in response to certain stimuli, making them candidates for connecting diverse signaling pathways to the ubiquitous apoptosis effector machinery (15, 16). BNIP3 (Bcl-2/19-kDa interacting protein 3) is a BH3-only protein whose expression and pro-apoptotic activity is induced following hyp...

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