GEN Exclusives

More »

Feature Articles

More »
Sep 1, 2012 (Vol. 32, No. 15)

Lighting Up Live Cells with Fluorescence

  • Click Image To Enlarge +
    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]

    Prior to the advent of the tools and technologies for live-cell imaging, biologists relied on single snapshots of cellular events to understand how cells function.

    Yet without the ability to observe dynamic cellular processes in real-time, it was difficult, if not impossible, to understand what was going on at the molecular level.

    “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.

    Over the past few decades, developments in two major research areas have made it possible for researchers to unveil more and more about how everyday cellular processes occur. Scientists have developed genetically encoded biosensors that allow cells to express fluorescent protein fusions that light up under a fluorescence microscope. On the imaging side, researchers have developed technologies that allow real-time visualization of the spatial arrangement of fluorescently labeled proteins inside live cells.

    These two research fronts converge into a field known as live-cell imaging. Despite the major strides made in the field in recent years, there is still much room for growth, and continual advancements will enable researchers to address fundamental biological questions about how cells function.

    Once we understand the molecular basis of normal cellular processes, we can begin to understand how alterations to these functions lead to disease, says Mark Rizzo, Ph.D., assistant professor of physiology at the University of Maryland. Dr. Rizzo uses live-cell imaging techniques to understand the molecular and cellular details underlying the progression of diabetes.

    Dr. Rizzo’s lab studies mouse pancreatic β cells, which are responsible for secreting insulin in response to a rise in glucose levels. The process of insulin secretion is tightly regulated by glucokinase and several cellular enzymes. Glucokinase is known to play a key role in regulating blood sugar levels, but what remains unclear is whether the mechanism of glucokinase regulation breaks down during the progression of type 2 diabetes, Dr. Rizzo says. If so, does it promote disease progression, or is it simply correlated?

    In the field of diabetes research, these and many other questions remain, but Dr. Rizzo says one thing is clear: “There’s a lot of research out there now that says that early intervention of diabetes produces the best prognosis.” With a better understanding of how the disease develops, researchers will be better positioned to develop therapeutic interventions.

    Live-cell imaging is essential to this work, Dr. Rizzo says, because it allows his research team to observe what is happening on the time scale on which glucokinase is regulated. Researchers in his lab have developed a cyan fluorescent protein (CFP)-glucokinase fusion protein that can be expressed in pancreatic β cells isolated from normal mice. Using live-cell imaging, they have demonstrated for the first time that glucokinase is activated on the surface of insulin secretory granules.

    Dr. Rizzo is currently working on developing better and brighter reagents that will enable faster data acquisition and will require less reagent, and plans to continue fundamental research on diabetes as well.

  • Endogenous-Level Protein Studies

    A common approach to genetically engineering cells to express a protein fused to GFP involves introducing a recombinant DNA molecule into the cell. The DNA molecule contains both the gene encoding the protein hybrid and a promoter region that is used to trick the cell into expressing both the endogenous gene and the introduced hybrid gene.

    The problem is that this method often causes the protein of interest to be expressed in the cell at levels that are much higher than normal, says Dr. Drubin. Overexpressing a protein inside a cell can drastically alter cellular processes and can lead researchers to incorrectly conclude that an observed behavior is representative of normal cellular function when, in fact, it is simply an artifact of the experiment.

    To minimize the likelihood of experimental artifacts in the investigation of clathrin-mediated endocytosis, Dr. Drubin teamed up with Fyodor D. Urnov from Sangamo BioSciences to perform targeted genome editing on a mammalian cell line. Instead of introducing the genes encoding the fluorescent protein fusions through an ectopic DNA molecule, they used zinc finger nucleases (ZFN) to introduce the GFP genes directly into the genome of the cell at the locus of the endogenous gene of interest. In this way, the cell would express the protein fusion at endogenous levels.

    The team investigated the role of both clathrin light chain A and dynamin-2 in clathrin-mediated endocytosis, and found that genome-edited cells exhibited increased endocytic function compared to previous literature reports that relied on protein overexpression. This study raises questions about the reliability of data presented in previous reports where the proteins were overexpressed. Dr. Drubin is now working on extending this ZFN approach to study other genes and cell lines.

    The primary challenge when working with fluorescent protein fusions at endogenous levels is sensitivity.

    “One advantage of overexpressing a fluorescent protein fusion is you get more light and can see it better,” Dr. Drubin says. “When expressing at endogenous levels, if they’re not highly expressed, it’s harder to see them.”

    For this reason, Dr. Drubin says the development of both brighter fluorescent probes and more sensitive instrumentation are important research areas.


Add a comment

  • You must be signed in to perform this action.
    Click here to Login or Register for free.
    You will be taken back to your selected item after Login/Registration.

Related content

Jobs

GEN Jobs powered by HireLifeScience.com connects you directly to employers in pharma, biotech, and the life sciences. View 40 to 50 fresh job postings daily or search for employment opportunities including those in R&D, clinical research, QA/QC, biomanufacturing, and regulatory affairs.
 Searching...

Unable to get Jobs Listings.

More »

GEN Poll

More » Poll Results »

Lab-Grown Vaginas

Which body part do you think will be successfully engineered in the lab next?