“The difference between taking snapshots of the process and watching a movie is just night and day,” says David Drubin, Ph.D., professor of cell and developmental biology at the University of California, Berkeley, whose lab uses fluorescence to understand the intricate details underlying clathrin-mediated endocytosis.
Researchers in David Drubin’s lab at the University of California, Berkeley genetically engineered a human cell line to express endogenous levels of RFP-tagged clathrin light chain A (red) and GFP-tagged dynamin 2 (green) for studying clathrin-mediated endocytosis. The above 3D kymograph of the cell surface, with the time dimension in the z-axis, shows the full lifetime of hundreds of clathrin patches on the membrane, which terminate upon recruitment of dynamin. [Aaron T. Cheng]
The May 2012 edition of Biowire, a publication of Sigma-Aldrich, includes an interview with David Drubin about the projects in our lab looking at clathrin-mediated endocytosis (CME) in mammalian cells using zing finger nuclease (ZFN) technology to undertake targeted genome modification. Traditionally, CME has been studied in cells in which fluorescently-tagged components of endocytic machinery are overexpressed using exogenous constructs. Data obtained in many labs using these methods suggested that CME was highly variable. Using ZFN technology, in collaboration with Sangamo Biosciences, our lab recently showed that CME is robust and efficient in mammalian cells. The new results highlight the technical advantages of tagging genes at their endogenous loci, an approach that has been historically limited to genetically tractable organisms, such as the Drubin/Barnes Lab favorite Saccharomycescerevisiae (budding yeast). Emerging technologies, such as ZFNs and TALENs, however, are now making this sort of precise genomic manipulation possible in animal cells, including human cells, giving us new and powerful ways of studying cellular biology.
Cellular processes should be studied as close to their natural states as possible. I suspect that, as we see more uses of zinc finger nucleases [for tagging endogenous genes], people will find that they have been inadvertently perturbing the processes that they have been studying.