Can Single-Molecule Structures be Defined in Vivo?

Monday, September 22, 2014 — Poster Session II
4:00 p.m. – 6:00 p.m.	FAES Academic Center	NHLBI	MOLBIO-18

* FARE Award Winner

Authors

• JM Solis
• A Nagy
• F Buss
• JR Sellers

Abstract

Genetic mutations encoding myosin-7a have been associated with Usher Syndrome, the most common cause of deaf-blindness in humans. However, the precise molecular structure and mechanism by which myosin-7a functions remains unclear. In vitro expressed full-length Drosophila myosin-7a is monomeric, while in vivo studies demonstrate dimeric phenotypes when co-expressed with its binding partner, M7BP. Characterizing the structure of myosins elucidates movement capabilities and intracellular roles, such as transporting cargo along actin microfilaments. We evaluated the oligomeric state of myosin-7a in vivo using nanobody pull-down assays. Nanobodies are single-domain monovalent antibodies, which circumvent the potential complication of false multimeric results ensuing to the bivalent nature of conventional IgG antibody structure. GFP-tagged myosin-7a, with and without its binding partner, was transfected into S2 cells. Cell lysates were exposed to a flow chamber surface coated with anti-GFP nanobodies to capture the expressed protein. Bound myosin was visualized and photobleached using Total Internal Reflection Fluorescence (TIRF) microscopy. Photobleaching kinetics were analyzed and correlated to oligomeric structure. To test the validity of the system, we used known monomeric and dimeric GFP-tagged constructs as positive controls. Our results demonstrate that nanobody pull-down is a novel approach to ascertain single-molecule oligomeric structure in vivo.

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