Archive for February, 2013

Senior Scientist Rudolf Oldenbourg and other MBL-affiliated biologists and physicists revealed their collaborative process to create informative, beautiful images of cell structure and behavior at the American Association for the Advancement of Science (AAAS) meeting last weekend in Boston, Mass.

The symposium “Innovations in Imaging: Seeing is Believing” was organized by Amy Gladfelter of Dartmouth College, an MBL Whitman Investigator.

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Fluorescence image of a living cell (MDCK) expressing septin molecules linked to green fluorescent protein (GFP). The image was recorded with the Fluorescence LC-PolScope and shows fluorescent septin fibers in color, indicating that the fluorescence is polarized and the septin molecules are aligned in the fibers. Credit: Rudolf Oldenbourg/MBL

“We are beginning to understand the basis for cell organization at unprecedented spatial and temporal resolution through the creative application of fundamental physics to microscopy,” Gladfelter stated. “This symposium will help motivate the next phase of interdisciplinary approaches to advance the visualization of life, from the scale of a single molecule to the whole organism.”

The data collected in biological images, Gladfelter noted, not only illuminates basic cellular processes, but is useful for medical purposes: to diagnose a metastasizing cancer or microbial infection, for example, or to screen chemical libraries for new pharmaceuticals.

“These images bring us to a beautiful world beyond the grasp of our normal senses,” Gladfelter stated. “In this way microscopes give us beauty and [biological or medical] application, often in the same image.”

The capacity of microscopes to reach beyond the senses is well appreciated by Oldenbourg, who spoke on New Frontiers in Polarized Light Microscopy for Live Cell Imaging.
(Oldenbourg’s MBL co-authors are Michael Shribak, Tomomi Tani, and Shinya Inoué.)

“Polarization is a basic property of light that is often overlooked, because the human eye is not sensitive to polarization. Therefore, we don’t have an intuitive understanding of it and optical phenomena that are based on polarization either elude us or we find them difficult to comprehend,” Oldenbourg stated.

“Like most scientific instruments, the polarized light microscope translates polarization effects so they can be perceived by our senses, in this case by our eyes, and makes them amenable to quantitative and analytical analysis. At the MBL, we are developing polarized light imaging techniques, including fluorescence polarization … for generating time-lapse images that clearly reveal the otherwise invisible dynamics of single molecules and molecular assemblies in organelles, cells, and tissues.”


The events of cell division during meiosis I in a living insect spermatocyte, beginning at diakinesis through telophase to the near completion of cytokinesis. Testes from the Crane fly Nephrotoma suturalis were observed with time-lapse liquid crystal polarized light microscopy (LC-PolScope, MBL, Woods Hole MA, and PerkinElmer, Hopkinton MA). Movie images display the naturally occurring birefringence of cell organelles and structures that are made up of aligned molecules, such as the meiotic spindle and mitochondria. Horizontal image width is 56 µm. Credits: James LaFountain and Rudolf Oldenbourg/MBL

Other talks in the symposium included:

Navigating the Dynamic Cell
Jennifer Lippincott-Swartz (National Institutes of Heath/MBL Physiology Course)

Imaging Three-Dimensional Dynamics in Cells and Embryos
Eric Betzig (Howard Hughes Medical Institute/MBL Physiology Course and MBL Neurobiology Course)

Structured Illumination and the Analysis of Single Molecules in Cells
Rainer Heintzmann (King’s College, London)

Imaging Single Cells in the Breast Tumor Microenvironment
John Condeelis (Albert Einstein College of Medicine)

Single Molecule Imaging in Live Cells
Amy S. Gladfelter (Dartmouth College/MBL Whitman Investigator)

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Among the animals that are appealing “cover models” for scientific journals, lancelets don’t spring readily to mind. Slender, limbless, primitive blobs that look pretty much the same end to end, lancelets “are extremely boring. I wouldn’t recommend them for a home aquarium,” says Enrico Nasi, adjunct senior scientist in the MBL’s Cellular Dynamics Program. Yet Nasi and his collaborators managed to land a lancelet on the cover of The Journal of Neuroscience last December. These simple chordates, they discovered, offer insight into our own biological clocks.

The head of the marine invertebrate amphioxus (Branchiostoma floridae), magnified 15 times. Amphioxus are the most ancient of the chordates (animals whose features include a nerve cord), according to molecular analysis. They are important to the study of the origin of vertebrates. Photo by Maria del Pilar Gomez. Click for larger version.

Nasi and his wife, MBL adjunct scientist Maria del Pilar Gomez, are interested in photo-transduction, the conversion of light by light-sensitive cells into electrical signals that are sent to the brain. The lancelet, also called amphioxus, doesn’t have eyes or a true brain. But what it does have in surprising abundance is melanopsin, a photopigment that is also produced by the third class of light-sensitive cells in the mammalian retina, besides the rods and cones. This third class of cells, called “intrinsically photosensitive retinal ganglion cells” (ipRGCs), were discovered in 2002 by Brown University’s David Berson and colleagues. Now sometimes called “circadian receptors,” they are involved in non-visual, light-dependent functions, such as adjustment of the animal’s circadian rhythms.

“It seemed like colossal overkill that amphioxus have melanopsin-producing cells,” Nasi says. “These animals do nothing. If you switch on a light, they dance and float to the top of the tank, and then they drop back down to the bottom. That’s it for the day.” But that mystery aside, Gomez and Nasi realized that studying amphioxus could help reveal the evolutionary history of the circadian receptors.

Amphioxus can grow as long as 2.5 inches and are typically found half-burrowed in sand. Photo by Hans Hillewaert.

As so it has. In 2009, Gomez and Nasi isolated the animal’s melanopsin-producing cells and described how they transduce light. In their recent paper, they tackled the puzzling question of why the light response of these amphioxus cells is several orders of magnitude higher than that of their more sophisticated, presumed descendents, the ipRGCs. (In mammals, the ipRGCs relay information on light and dark to the biological clock in the hypothalamus, where it is crucial for the regulation of circadian rhythms and associated control of hormonal secretion.)

By detailing how the large light response occurs in the amphioxus cells, Gomez and Nasi could relate their observations to the functional changes that may have occurred as the circadian receptors evolved and “eventually tailored their performance to the requirements of a reporter of day and night, rather than to a light sensor meant to mediate spatial vision.” The light-sensing cells of amphioxus, they discovered, may be the ”missing link“ between the visual cells of invertebrates and the circadian receptors in our own eyes.

Citations

Ferrer C., Melagón G., del Pilar Gomez M., and Nasi E. (2012) Dissecting the Determinants of Light Sensitivity in Amphioxus Microvillar Photoreceptors: Possible Evolutionary Implications for Melanopsin Signaling. J. Neurosci. 32: 17977-17987.

del Pilar Gomez M., Angueyra J.M, and Nasi E. (2009) Light-transduction in melanopsin-expressing photoreceptors of Amphioxus. PNAS 16: 9081-9086.

Enrico Nasi and Maria Gomez (second and third from left) with some of their students from the Universidad Nacional, de Colombia, where they hold faculty appointments. Nasi and Gomez regularly bring students to the MBL, where they are affiliated with the Program in Sensory Physiology and Behavior in addition to Cellular Dynamics.