A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays.

Protein-fragment complementation is a valuable tool for monitoring protein interactions. In complementation assays, the reporter fragments are directly fused to the interacting proteins, eliminating the possibility of monitoring native interactions. In principle, complementation could be achieved by placing the reporter fragments on antibodies which bind to the proteins of interest, enabling the monitoring of endogenous protein interactions or detection of a single protein in a homogeneous immunoassay. Previous reports have demonstrated proof-of-concept of this approach; however, current complementation systems have not met the practical requirements as suitable fusion partners for antibodies while providing the sensitivity needed for immunoassays. To surmount these challenges, we created a first-in-class, tri-part split luciferase consisting of two 11-residue peptides that are used as the antibody appendages. As an initial proof-of-concept, we used antibody-peptide fusions and found them to be capable of quantifying pg/mL concentrations of soluble or cell-bound HER2, proving this unique complementation system overcomes previous limitations and transforms this approach from merely possible to practical and useful. As shown herein, this dual-peptide system provides a rapid, simple, and sensitive "add-and-read" homogeneous immunoassay platform that can be broadly adapted as an alternative to traditional immunoassays, and in the future should enable complementation to be expanded to monitoring endogenous protein interactions.

Distinct requirements for beta-catenin in pancreatic epithelial growth and patterning.

Pancreatic exocrine and endocrine lineages arise from multipotent pancreatic progenitor cells (MPCs). Exploiting the mechanisms that govern expansion and differentiation of these cells could enhance efforts to generate ß-cells from stem cells. Although our prior work indicates that the canonical Wnt signaling component ß-catenin is required qualitatively for exocrine acinar but not endocrine development, precisely how this requirement plays out at the level of MPCs and their lineage-restricted progeny is unknown. In addition, the contribution of ß-catenin function to ß-cell development remains controversial. To resolve the potential roles of ß-catenin in development of MPCs and ß-cells, we generated pancreas- and pre-endocrine-specific ß-catenin knockout mice. Pancreas-specific loss of ß-catenin produced not only a dramatic reduction in acinar cell numbers, but also a significant reduction in ß-cell mass. The loss of ß-cells is due not to a defect in the differentiation of endocrine precursors, but instead correlates with an early and specific loss of MPCs. In turn, this reflects a novel role for ß-catenin in maintaining proximal-distal patterning of the early epithelium, such that distal MPCs resort to a proximal, endocrine-competent "trunk" fate when ß-catenin is deleted. Moreover, ß-catenin maintains proximal-distal patterning, in part, by inhibiting Notch signaling. Subsequently, ß-catenin is required for proliferation of both distal and proximal cells, driving overall organ growth. In distinguishing two distinct roles for ß-catenin along the route of ß-cell development, we suggest that temporally appropriate positive and negative manipulation of this molecule could enhance expansion and differentiation of stem cell-derived MPCs.

The V-ATPase B1-subunit promoter drives expression of Cre recombinase in intercalated cells of the kidney.

The collecting duct of the kidney is composed of two morphologically and physiologically distinct cell types, principal and intercalated cells. To better understand intercalated cell function we generated a transgenic mouse expressing Cre recombinase under the control of a cell type- specific promoter. We used 7 kb of the ATP6V1B1 5' untranslated region (B1 promoter), a gene found in the intercalated cells of the kidney and the male reproductive tract. We first crossed these B1-Cre transgenic mice with the ROSA26-loxP-stop-loxP-yellow fluorescent protein reporter mice to assess the specificity of Cre expression. Immunohistochemistry and confocal fluorescence microscopy showed that Cre is selectively active in all intercalated cells (type A, type B, and non-A/B cells) within the collecting duct and most cells of the connecting segment. About half of the principal cells of the connecting segment also expressed Cre, a pattern also seen in B1-driven enhanced green fluorescent protein transgenic mice. Cre was found to be active in the male reproductive tract and at a low level in limited non-ATP6V1B1 expressing tissues. The B1-Cre transgenic mice are healthy, breed normally, produce regular sized litters, and transmit the transgene in Mendelian fashion. This new cell-specific Cre expressing mouse should prove useful for the study of intercalated cell physiology and development.