Biology subjects

Chang, K. R.

Publications and source records attributed to Chang, K. R..

3 recordsLinked to original sources

bioRxiv2022

A genetic strategy to measure insulin signaling regulation and physiology in Drosophila

Insulin regulation is a hallmark of health, and impaired insulin signaling promotes metabolic diseases like diabetes mellitus. However, current assays for measuring insulin signaling in all animals remain semi-quantitative and lack the sensitivity, tissue-specificity or temporal resolution needed to quantify in vivo physiological signaling dynamics. Insulin signal transduction is remarkably conserved across metazoans, including insulin-dependent phosphorylation and regulation of Akt/Protein kinase B. Here, we generated transgenic fruit flies permitting tissue-specific expression of an immunoepitope-labelled Akt (AktHF). We developed enzyme-linked immunosorption assays (ELISA) to quantify picomolar levels of phosphorylated (pAktHF) and total AktHF in single flies, revealing dynamic tissue-specific physiological regulation of pAktHF in response to fasting and re-feeding, exogenous insulin, or targeted genetic suppression of established insulin signaling regulators. Genetic screening revealed Pp1-87B as an unrecognized regulator of Akt and insulin signaling. Tools and concepts here provide opportunities to discover tissue-specific regulators of in vivo insulin signaling responses. Author SummaryInsulin is an essential hormone that controls metabolism in all animals, by regulating energy use and growth of target tissues. Impaired insulin signaling ("resistance") in humans underlies development of type 2 diabetes, a pandemic disease causing significant morbidity and mortality. The genetic risk for insulin resistance is complex, and studies of diabetes are limited by a lack of tools to measure insulin resistance in a sensitive, quantitative, and tissue-specific way. Here, we describe a new technique to measure the strength of insulin signaling in fruit flies. By combining fruit fly genetics with antibody-based assays, we can quantify phosphorylated Akt, an evolutionarily conserved target of insulin signaling, in specific tissues of the adult fly. We show this technique can detect changes in insulin signaling after fasting and refeeding, addition of exogenous insulin, or genetic disruption of the insulin signaling pathway. We used this method to discover a new regulator of insulin signaling, a phosphatase enzyme encoded by Pp1-87B. This exciting new tool should advance our ability to study and discover additional regulators of insulin signaling and resistance.

genetics↗

bioRxiv2021

Transgenic Drosophila lines for LexA-dependent gene and growth regulation

Conditional expression of short hairpin RNAs (shRNAs) with binary genetic systems is an indispensable tool for studying gene function. Addressing mechanisms underlying cell-cell communication in vivo benefits from simultaneous use of two independent gene expression systems. To complement the abundance of existing Gal4/UAS-based resources in Drosophila, we and others have developed LexA/LexAop-based genetic tools. Here, we describe experimental and pedagogical advances that promote the efficient conversion of Drosophila Gal4 lines to LexA lines, and the generation of LexAop-shRNA lines to suppress gene function. We developed a CRISPR/Cas9-based knock-in system to replace Gal4 coding sequences with LexA, and a LexAop-based shRNA expression vector to achieve shRNA-mediated gene silencing. We demonstrate the use of these approaches to achieve targeted genetic loss-of-function in multiple tissues. We also detail our development of secondary school curricula that enable students to create transgenic flies, thereby magnifying the production of well-characterized LexA/LexAop lines for the scientific community. The genetic tools and teaching methods presented here provide LexA/LexAop resources that complement existing resources to study intercellular communication coordinating metazoan physiology and development.

genetics↗

bioRxiv2020

A toolkit to generate inducible and interconvertible Drosophila transgenes

The existence of three independent binary systems for conditional gene expression (Gal4/UAS; LexA/LexAop; QF/QUAST) has greatly expanded versatile genetic analyses in the fruit fly Drosophila melanogaster; however, the experimental application of these tools is limited by the need to generate multiple collections of non-interchangeable transgenic fly strains for each inducible gene expression system. To address this practical limitation, we developed a modular vector that contains the regulatory elements from all three binary systems, enabling Gal4-, LexA- or QF-dependent expression of transgenes. Our methods also incorporate DNA elements that facilitate independent site-specific recombination and elimination of regulatory UAS, LexAop or QUAST modules with spatial and temporal control, thus offering unprecedented possibilities and logistical advantages for in vivo genetic modulation and efficient interconversion of overexpression transgenic fly lines.

genetics↗