Drosophila cell culture is used as a model system with multiple applications including the identification of new therapeutic targets in screens, the study of conserved signal transduction pathway mechanisms, and as an expression system for recombinant proteins. However, in vitro methods for Drosophila cell and organ culture are relatively undeveloped. To characterize the minimal requirements for long-term maintenance of Drosophila cell lines, we developed an inverse screening strategy to identify small molecules and synergies stimulating proliferation in a chemically defined medium. In this chemical-genetics approach, a compound-protein interaction database is used to systematically score genetic targets on a screen-wide scale to extract further information about cell growth. In the pilot screen, we focused on two well-characterized cell lines, Clone 8 (Cl.8) and Schneider 2 (S2). Validated factors were investigated for their ability to maintain cell growth over multiple passages in the chemically defined medium (CDM). The polyamine spermidine proved to be the critical component that enables the CDM to support the long-term maintenance of Cl.8 cells. Spermidine supplementation upregulates DNA synthesis for Cl.8 and S2 cells and increases MAPK signaling for Cl.8 cells. The CDM also supports the long-term growth of Kc167 cells. Our target scoring approach validated the importance of polyamines, with enrichment for multiple polyamine ontologies found for both cell lines. Future iterations of the screen will enable the identification of compound combinations optimized for specific applications—maintenance and generation of new cell lines or the production and purification of recombinant proteins— thus increasing the versatility of Drosophila cell culture as both a genetic and biochemical model system. Our cumulative target scoring approach improves on traditional chemical-genetics methods and is extensible to biological processes in other species.

A technological revolution in both light and electron microscopy imaging now allows unprecedented views of clotting, especially in animal models of hemostasis and thrombosis. However, our understanding of three-dimensional high-resolution clot structure remains incomplete since most of our recent knowledge has come from studies of relatively small clots or thrombi, due to the optical impenetrability of clots beyond a few cell layers in depth. Here, we developed an optimized optical clearing method termed cCLOT that renders large whole blood clots transparent and allows confocal imaging as deep as one millimeter inside the clot. We have tested this method by investigating the 3D structure of clots made from reconstituted pre-labeled blood components yielding new information about the effects of clot contraction on erythrocytes. Although it has been shown recently that erythrocytes are compressed to form polyhedrocytes during clot contraction, observations of this phenomenon have been impeded by the inability to easily image inside clots. As an efficient and nondestructive method, cCLOT represents a powerful research tool in studying blood clot structure and mechanisms controlling clot morphology. Additionally, cCLOT optical clearing has the potential to facilitate imaging of ex vivo clots and thrombi derived from healthy or pathological conditions. W. Weisel, I. N. Chernysh, C. Nagaswami, P. R. Williams, and K. Hawkins, "Effects of unidirectional flow shear stresses on the formation, fractal microstructure and rigidity of incipient whole blood clots and fibrin gels," Clin. Hemorheol. Microcirc. 60(4), 451-464 (2015).

Virulent strains of Streptococcus pyogenes (GAS) recruit host single-chain human plasminogen (hPg) to the cell surface - where in the case of Pattern D strains of GAS - hPg binds directly to the cells through a surface receptor, plasminogen-binding group A streptococcal M-protein (PAM).  The coinherited Pattern D GAS-secreted streptokinase (SK2b) then accelerates cleavage of hPg at the R561-V562 peptide bond, resulting in the disulfide-linked two-chain protease, plasmin (hPm).  hPm localizes on the bacterial surface, assisting bacterial dissemination via proteolysis of host defense proteins.  Studies using isolated domains from PAM and hPg revealed that the A-domain of PAM binds to the hPg kringle-2 module (K2hPg), but how this relates to the function of the full-length proteins is unclear.  Herein, we use intact proteins to show that the lysine binding site (LBS) of K2hPg is a major determinant of the activation-resistant T-conformation of hPg.  The binding of PAM to the LBS of K2hPg relaxes the conformation of hPg, leading to a greatly enhanced activation rate of hPg by SK2b.  Domain swapping between hPg and mPg emphasizes the importance of the Pg latent heavy chain (residues 1-561) in PAM binding and shows that while SK2b binds to both hPg and mPg, the activation properties of SK are strictly attributed to the serine protease domain (residues 562-791) of hPg.  Overall, these data show that native hPg is locked in an activation-resistant conformation that is relaxed upon its direct binding to PAM, allowing hPm to form and provide GAS cells with a proteolytic surface.

Recently, organ-scale intercellular Ca 2+ transients (ICTs) were reported in the Drosophila wing disc. However, the functional in vivo significance of ICTs remains largely unknown. Here we demonstrate the in vivo relevance of intercellular Ca 2+ signaling and its impact on wing development. We report that Ca 2+ signaling in vivo decreases as wing discs mature. Ca 2+ signaling ex vivo responds to fly extract in a dose-dependent manner. This suggests ICTs occur in vivo due to chemical stimulus that varies in concentration during development. RNAi mediated inhibition of genes required for ICTs results in defects in the size, shape, and vein patterning of adult wings. It also leads to reduction or elimination of in vivo Ca 2+ transients. Further, perturbations to the extracellular matrix along the basal side of the wing disc stimulates intercellular Ca 2+ waves. This is the first identified chemically defined, non-wounding stimulus of ICTs. Together, these results point toward specific in vivo functions of intercellular Ca 2+ signaling to mediate mechanical stress dissipation and ensure robust patterning during development.

We report a novel approach that generates a high resolution, three-dimensional (3D) fluorescent staining atlas of tissue microbiopsies in a microfluidic device without destroying the tissue. We demonstrate that this method preserves tissue architecture for multiple murine organs by comparing traditional 2D slices to an optically sectioned 3D H&E-mimic. The H&E-mimic slices show a close qualitative match to traditional H&E. The 3D spatial and molecular information obtainable from this method significantly increases the amount of data available for evaluating both tissue morphology and specific biomarkers in a wide range of both research and clinically driven applications and is amenable to automation.