Use of nanobodies to localize endogenous cytoskeletal proteins and to determine their contribution to cancer cell invasion by using an ecm degradation assay

Abstract

There are numerous ways to study actin cytoskeletal structures, and thereby identify the underlying mechanisms of organization and their regulating proteins. Traditional approaches make use of protein overexpression or siRNA. However to study or modulate resident endogenous proteins, complementary methods are required. Since the discovery of nanobodies in 1993, they have proven to represent interesting tools in a variety of applications due to their high affi nity, solubility, and stability. Especially their intracellular functionality makes them ideally suited for the study of actin cytoskeletal regulation. Here we provide a protocol to clone nanobody cDNAs in frame with an EGFP or mCherry fl uorescent tag. We explain how to transfect this fusion protein in eukaryotic (cancer) cells and how to perform immunofl uorescence. This allows microscopic analysis of endogenous (cytoskeletal) proteins and gives insight into their endogenous localization. Moreover, we outline an extracellular matrix (ECM) degradation assay as an application of the general protocol. By seeding cells onto a fl uorescently labeled gelatin matrix, degradation can be quantifi ed by means of a matrix degradation index. This assay demonstrates the contribution of a protein during cancer cell invasiveness in vitro and the potential of a nanobody to inhibit this degradation through modulation of its target.