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MEETING NEWS Rajendrani Mukhopadhyay reports from the
51st Annual Meeting of the Biophysical Society—Baltimore, Md.
Multifocal plane imaging reveals exocytosis dynamics Sometimes you just need to tinker with an existing technique to see things in a whole new way. Investigators at the University of Texas Southwestern and the University of Texas Dallas modified a conventional inverted optical microscope so that they could image several planes simultaneously to get 3D images. In doing so, they were able to observe exocytotic events in cells that haven’t been described previously.
And with a technique such as confocal microscopy, Ram says, “you can always miss events in the other planes when you’re imaging one plane.” So, the investigators tinkered with a conventional inverted microscope and came up with a new simultaneous multifocal plane imaging method. They split the light coming out of the objective lens into two paths. In one path, they placed a camera where it would normal-
Intermediate plane 0.00
0.51
1.70
2.38
2.55
2.89
3.23
4.25
5.95
8.33
Membrane plane
Cell Endosome
Tubule extending
Tubule approaching Exocytosis membrane
Tubule retracting
Tubule extending
Tubule detaching
Multifocal plane imaging shows a tubule extending from a sorting endosome for exocyotosis and retracting over several seconds. The neonatal Fc receptor is shown in green, and the events of interest are highlighted in red (resulting in a yellow-orange overlay). Scale bar = 1 µm. (Adapted with permission. Copyright 2007 National Academy of Sciences, U.S.A.)
Sripad Ram—a graduate student under the direction of E. Sally Ward and Raimund Ober— along with fellow students Prashant Prabhat, Zhuo Gan, and Jerry Chao were studying how a protein called the neonatal Fc receptor moved inside cells. They had also caught glimpses of the protein undergoing exocytosis at the plasma membrane. So the investigators naturally wanted to know how the protein moved from the depths of the cell to the plasma membrane. But Ram and colleagues lacked a way to image a cell through its entire thickness in one shot. Epifluorescence microscopy could track the protein inside the cell but couldn’t image exocytotic events at the plasma membrane with sufficient sensitivity. Total internal reflection fluorescence (TIRF) microscopy is great for imaging events at the plasma membrane but can’t penetrate deep inside the cell. 3958
ly be positioned. In the other path, they shifted the camera along the optical axis. “If you do that and work out the simple optics, it turns out you can image a distinct plane,” says Ram. The investigators demonstrated that they could simultaneously image two or more focal planes, one at the plasma membrane by TIRF and the others inside the cell by epifluorescence (IEEE Trans. Nanobiosci. 2004, 3, 237–242). Ram notes that there is no “movement related to drift or vibration problems,” he says. “Everything is stationary, so whatever moves is essentially inside the cell.” Armed with the new technique, the investigators returned to the question of how their protein of interest got to the plasma membrane for exocytosis. Exocytosis from sorting endosomes— tiny lipid-coated recycling bags for pro-
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teins—is poorly understood despite being extensively studied. But now, the investigators could get an idea of the dynamics of the process because they could visualize the rapid movements of intracellular compartments in 3D. When the researchers looked inside the cells, “we identified some new pathways which have not been reported before,” says Ram. “There is more than one simple route that the receptor takes. We call them the direct and indirect pathways” (Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5889–5894). The protein, along with its ligand, definitely took the classical exocytosis route in which small vesicles pinch off endosomes and move down to the plasma membrane to release their contents. However, Ram and colleagues discovered something that hadn’t been previously described in the literature. “We also see a tubular structure that extends all the way from the endosome and touches down on the membrane. That tubular connection leads to a release event,” says Ram. Once the tubule released its contents, it retracted from the plasma membrane and then detached from the endosome. Exocytosis also occurred via other means. For example, vesicles that were nowhere near sorting endosomes traveled significant distances inside the cell before standing still above the plasma membrane. The vesicles hovered in these “holding zones” for several seconds before undergoing exocytosis at the plasma membrane. The new imaging method also has allowed the investigators to tackle 3D imaging of single molecules. So far, they’ve looked at the exocytosis of immunoglobulin G molecules labeled with quantum dots. “With a multifocal plane setup, you can get very accurate information about object depth,” says Ram. “As a result, now people can do 3D tracking with high accuracy.”
