Ultrasensitive optical imaging with lanthanide lumiphores.

In principle, the millisecond emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. In practice, however, lanthanide imaging techniques have provided no better sensitivity than conventional fluorescence microscopy. Here, we identified three fundamental problems that have impeded lanthanide microscopy: low photon flux, inefficient excitation, and optics-derived background luminescence. We overcame these limitations with a new lanthanide imaging modality, transreflected illumination with luminescence resonance energy transfer (trLRET), which increases the time-integrated signal intensities of lanthanide lumiphores by 170-fold and the signal-to-background ratios by 75-fold. We demonstrate that trLRET provides at least an order-of-magnitude increase in detection sensitivity over that of conventional epifluorescence microscopy when used to visualize endogenous protein expression in zebrafish embryos. We also show that trLRET can be used to optically detect molecular interactions in vivo. trLRET promises to unlock the full potential of lanthanide lumiphores for ultrasensitive, autofluorescence-free biological imaging.

Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal.

Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial. HSCs are rare and few can be found in thin tissue sections or upon live imaging, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as α-catulin(GFP)), we discover that α-catulin(GFP) is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of α-catulin-GFP(+)c-kit(+) cells give long-term multilineage reconstitution of irradiated mice, indicating that α-catulin-GFP(+)c-kit(+) cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of α-catulin-GFP(+)c-kit(+) cells and to digitally reconstruct large segments of bone marrow. The distribution of α-catulin-GFP(+)c-kit(+) cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr(+)) and Cxcl12(high) niche cells, and approximately 85% of HSCs were within 10 µm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67(+)) and non-dividing (Ki-67(-)), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr(+)Cxcl12(high) cells throughout the bone marrow.

The immunoglobulin superfamily member Hbs functions redundantly with Sns in interactions between founder and fusion-competent myoblasts.

The body wall muscle of a Drosophila larva is generated by fusion between founder cells and fusion-competent myoblasts (FCMs). Initially, a founder cell recognizes and fuses with one or two FCMs to form a muscle precursor, then the developing syncitia fuses with additional FCMs to form a muscle fiber. These interactions require members of the immunoglobulin superfamily (IgSF), with Kin-of-IrreC (Kirre) and Roughest (Rst) functioning redundantly in the founder cell and Sticks-and-stones (Sns) serving as their ligand in the FCMs. Previous studies have not resolved the role of Hibris (Hbs), a paralog of Sns, suggesting that it functions as a positive regulator of myoblast fusion and as a negative regulator that antagonizes the activity of Sns. The results herein resolve this issue, demonstrating that sns and hbs function redundantly in the formation of several muscle precursors, and that loss of one copy of sns enhances the myoblast fusion phenotype of hbs mutants. We further show that excess Hbs rescues some fusion in sns mutant embryos beyond precursor formation, consistent with its ability to drive myoblast fusion, but show using chimeric molecules that Hbs functions less efficiently than Sns. In conjunction with a physical association between Hbs and SNS in cis, these data account for the previously observed UAS-hbs overexpression phenotypes. Lastly, we demonstrate that either an Hbs or Sns cytodomain is essential for muscle precursor formation, and signaling from IgSF members found exclusively in the founder cells is not sufficient to direct precursor formation.

Analysis of the cell adhesion molecule sticks-and-stones reveals multiple redundant functional domains, protein-interaction motifs and phosphorylated tyrosines that direct myoblast fusion in Drosophila melanogaster.

The larval body wall muscles of Drosophila melanogaster arise by fusion of founder myoblasts (FMs) and fusion-competent myoblasts (FCMs). Sticks-and-Stones (SNS) is expressed on the surface of all FCMs and mediates adhesion with FMs and developing syncytia. Intracellular components essential for myoblast fusion are then recruited to these adhesive contacts. In the studies herein, a functional analysis of the SNS cytodomain using the GAL4/UAS system identified sequences that direct myoblast fusion, presumably through recruitment of these intracellular components. An extensive series of deletion and site-directed mutations were evaluated for their ability to rescue the myoblast fusion defects of sns mutant embryos. Deletion studies revealed redundant functional domains within SNS. Surprisingly, highly conserved consensus sites for binding post-synaptic density-95/discs large/zonula occludens-1-domain-containing (PDZ) proteins and serines with a high probability of phosphorylation play no significant role in myoblast fusion. Biochemical studies establish that the SNS cytodomain is phosphorylated at multiple tyrosines and their site-directed mutagenesis compromises the ability of the corresponding transgenes to rescue myoblast fusion. Similar mutagenesis revealed a requirement for conserved proline-rich regions. This complexity and redundancy of multiple critical sequences within the SNS cytodomain suggest that it functions through a complex array of interactions that likely includes both phosphotyrosine-binding and SH3-domain-containing proteins.