September 1, 2012 (Vol. 32, No. 15)

Christine Herman

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.

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]

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.

“Smart” Biosensors

Alan Waggoner, Ph.D., professor and PI of the Technology Center for Networks and Pathways (TCNP) at Carnegie Mellon University, is part of a team that has developed a new technology that allows scientists to tune the fluorescent signal of a new class of genetically encoded biosensors. This biosensor technology can enable researchers to obtain new information about pathway regulation, and has the potential to lower background fluorescence and improve overall sensitivity compared to traditional fluorescent protein biosensors.

The approach involves single-chain antibodies, known as FAPs, or fluorescence activating proteins. FAPs are able to bind to nonfluorescent fluorogen dyes and switch them “on,” displaying a large increase in fluorescence. Researchers can use membrane-permeant or impermeant fluorogens and both spatially and temporally control the fluorescent signal. These “smart” biosensors are amenable to high-content imaging, in addition to high-throughput analyses such as flow cytometry and plate reader assays.

In cells, the process of placing and removing a membrane protein from the cell surface is complex and tightly regulated, and is important for cell function, Dr. Waggoner says. Dr. Waggoner’s research has focused on the study of GPCRs and other cell-surface molecules that can be tagged with a FAP.

Diseased cells often exhibit altered levels of cell surface proteins that may play a role in disease progression. For example, researchers have found that the transport of a surface protein, CFTR, is altered in cells from cystic fibrosis patients compared to healthy cells.

Dr. Waggoner and colleagues in the Pittsburgh TCNP and the University of New Mexico have attached a FAP to the CFTR protein and are now performing high-throughput screening assays to identify compounds that help restore normal protein transport of the CFTR protein to the cell surface. These compounds could serve as potential drug candidates to treat the disease.

Dr. Waggoner says he and his TCNP colleagues are always interested in speaking with researchers in both academia and industry to learn what the key questions are in the field that the FAP technology may be useful for addressing.

“It’s going to take years and years to figure out all the assays that people can use so that they can in detail understand the kinetics of different pathways,” Dr. Waggoner says. This detailed understanding of how cells are regulated is the first key step to being able “to understand and change disease states.”

Enabling Technologies

At PerkinElmer, Louise Armstrong, Ph.D., market development director, is part of a team that is working on the development of improved imaging technologies to meet researchers’ live-cell imaging needs.

Photobleaching has long been a problem for the field of fluorescence imaging. During image acquisition, the same light that is used to cause fluorescence also causes fluorophores to become bleached and no longer emit a signal.

There are two primary directions that researchers have turned in an effort to minimize photobleaching, Dr. Armstrong says.

Some choose to forgo the higher quality images obtained with laser scanning confocal microscopy, and instead acquire images and movies with a standard fluorescence microscope, which uses a less intense light source and reduces the rate of photobleaching.

Others have looked to spinning disk confocal microscopy, which combines a confocal microscope with a pair of spinning disks that splits the laser beam into many points across the specimen. By splitting the beam, researchers can obtain a sharp image across the entire field of view, instead of building up the image point-by-point as with traditional confocal microscopy.

The spinning disk approach significantly reduces and can even eliminate photobleaching, Dr. Armstrong explains. It also reduces the time to acquire images, which makes it possible to perform imaging at a fast rate over short time scales, or have longer time intervals between images over a time scale of multiple days. Prior to multipoint confocal microscopy, imaging over multiple days was not feasible because the fluorophores would become entirely bleached.

The key to performing live-cell imaging is optimizing experimental conditions such that the minimum amount of light is used, Dr. Armstrong says. “It’s all about getting as much signal as possible without photobleaching.”

While real-time two-dimensional live-cell imaging is a step up over simple snapshots of cellular events, Dr. Armstrong says that three-dimensional live-cell imaging will be the way of the future.

“In the last few years, technology has advanced enough that it’s now possible to generate 3D images and to manage the large amounts of data that this creates,” Dr. Armstrong notes. The cameras are fast enough, photobleaching is managed, and with the right software, two-dimensional image slices can be reconstructed to give quantifiable three-dimensional models of cellular processes changing over time.

These advancements, in conjunction with new developments in the field of three-dimensional cell culture, make 3D live-cell imaging an expanding field with lots of room for growth, Dr. Armstrong says.

Technological advancements have made it possible for researchers to perform fluorescence imaging of live cells in three dimensions. The image represents a 3D reconstruction of a polarized mammary epithelial acinus. [Professor Streuli and Dr. Akhtar, University of Manchester, U.K./PerkinElmer]

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