Jolanta Vidugiriene is a Group Leader in R&D at Promega leading development of new technologies for mechanistic studies of cell survival and adaptation, particularly those involved in regulation of cellular energy metabolism.
Prior to joining Promega, Jolanta was a Principal Investigator of a Howard Hughes International Grant, where she conducted collaborative research between the Department of Biochemistry, University of Wisconsin-Madison and Vilnius University, Lithuania. Jolanta received her Ph.D. in biochemistry from Vilnius University, completed post-doctoral studies at Boston Biomedical Research Institute and Laboratory of Molecular Parasitology, Rockefeller University.
Topics & Abstracts
Intracellular signaling and cell function are largely mediated through protein dynamics, including changes in protein abundance, interactions, location, or post-translational modification. HiBiT is an 11-amino-acid quantitative peptide tag that is well suited for such studies. High-affinity complementation of HiBiT with the 18kDa polypeptide, known as large BiT (LgBiT), generates a bright luminescent enzyme, enabling sensitive and linear quantitation of HiBiT-tagged proteins over seven orders of magnitude. Changes in protein abundance are monitored in either a lytic endpoint assay using purified LgBiT protein or in a live-cell kinetic assay by expressing the LgBiT subunit in cells. HiBiT can also be used to measure translocation of proteins to the cell surface in a live-cell assay as LgBiT is membrane impermeable. Additionally, HiBiT-tagged proteins can be detected at sub-picogram levels on membranes following SDS-PAGE and transfer, using a simple protocol that eliminates multiple blocking, binding, and washing steps of traditional blotting techniques.
In this presentation we demonstrate applications of HiBiT technology for studying diverse biological responses including time-dependent protein secretion, PROTAC-induced degradation and insulin-induced translocation of proteins to the cell surface. We also show how the small size and brightness of HiBiT tag facilitates rapid knock-in at endogenous loci using a plasmid-free protocol with Cas9:gRNA ribonucleoprotein complex and a single-stranded oligonucleotide donor DNA, and demonstrate the advantages of studying protein dynamics under endogenous expression conditions as opposed to more common over-expression methods.
Better understanding the role of cell metabolism in cancer, immunology, obesity, diabetes, and neurodegenerative disease presents specific research challenges, which drive the need for more rapid and reliable methods for measuring changes in metabolic pathways. Metabolites produced by the major metabolic pathways serve as signaling molecules and link the metabolic state of a cell to transcriptional control, epigenetics and cell signaling. Therefore, the ability to measure changes in key metabolites rapidly and robustly in higher throughput formats (e.g., 96-, 384-well plates) should provide a powerful approach for establishing the links between cellular metabolism and cell function under normal and disease conditions.
Here we introduce a series of luminescent metabolite detection assays (i.e., for glucose uptake, glucose, lactate, glutamate, glutamine, and NAD(P)/NAD(P)H detection). We discuss how the assays provide valuable information about metabolic reprograming of cancer cells, allow monitoring the activation of T cells or measuring insulin sensitivity in primary adipocytes and muscle cells.