Researchers with the U.S. Department of Energy's Lawrence
Berkeley National Laboratory (Berkeley Lab) and the University of
California (UC), Berkeley, have successfully attached imaging
probes to glycans - the sugar molecules that are abundant on the
surfaces of living cells – in the embryos of zebrafish less
than seven hours after fertilization. Glycans are key regulators of
the processes that guide cell development, and zebrafish are a top
vertebrate model organism of embryogenesis. This new technique
enables scientists to study the physiological changes cells undergo
during embryogenesis without invading and doing damage to the
embryos.
The team of researchers led by Carolyn Bertozzi, a Berkeley
Lab-UC Berkeley chemical biologist and leading authority on
glycobiology, used a combination of glycan metabolic labeling and
copper-free click chemistry to record the earliest images ever of
glycan activity on embryonic cells. The images were obtained during
a stage of development in which many of the cells were still in the
"multipotent stem cell" state, meaning they had yet to
differentiate into specific tissue types.
"We know from earlier studies of developmental biology that
glycan structures can change a lot during the early stages of
embryogenesis," Bertozzi says. "With this new technology, we hope
to witness some of those changes in the glycome in real time and to
understand better how cell surface glycans might contribute to the
decisions that stem cells make about their destiny."
Bertozzi is the director of Berkeley Lab's Molecular Foundry, a
faculty scientist with Berkeley Lab's Materials Sciences and
Physical Biosciences Divisions, and the T.Z. and Irmgard Chu
Distinguished Professor of Chemistry as well as a professor of
Molecular and Cell Biology at UC Berkeley, and an investigator with
the Howard Hughes Medical Institute (HHMI).
She is also the principal investigator and corresponding author
on a paper published in the Proceedings of the National Academy
of Science (PNAS) that describes this latest glycan imaging
research. The paper is titled "Visualizing enveloping layer glycans
during zebrafish early embryogenesis." Co-authoring the paper with
Bertozzi were graduate students Jeremy Baskin, Karen Dehnert and
Scott Laughlin, and Sharon Amacher, Associate Professor of
Molecular and Cell Biology at UC Berkeley.
Embryogenesis is the process by which a fertilized egg develops
into a fetus. It starts with a single zygote cell that multiplies
through rapid division (mitosis) into stem cells that subsequently
differentiate into the specific types of cells that make up organs
and tissues. Biologists need non-invasive imaging techniques to
capture in detail such developments at the molecular as well as the
cellular levels. Glycans play a central role in the signaling that
takes place between cells during embryogenesis, which makes them
appealing targets for molecular imaging. However, prior to
Bertozzi's research, glycans were difficult to visualize using the
standard tools of molecular imaging.
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In 2007, Bertozzi and her research group announced the first
copper-free variant of a chemical reaction known as "click
chemistry," one of the most proficient methods for attaching probes
to biological molecules. Conventional click chemistry requires a
copper catalyst to accelerate the reaction of alkynes with azides -
functional groups featuring three nitrogen atoms that can be
integrated into biomolecules by metabolic labeling. However,
because of copper's toxicity, the original click chemistry
reactions could only be used on fixed cells or cells in a
test-tube. Bertozzi and her group extended click chemistry to
living cells and organisms by developing a difluorinated
cyclooctyne or DIFO reagent that reacts with azides at
physiological temperatures, eliminating the need for the toxic
catalyst.
"It is a two-step chemical strategy for labeling glycans with
imaging agents in vivo," Bertozzi says. "It entails metabolic
labeling with synthetic azido sugars that hijack glycan
biosynthesis, followed by covalent chemical tagging of the
azide-labeled glycans with a compound bearing both an
azide-reactive group, such as DIFO, and an imaging probe."
Two years ago, Bertozzi and her group used their copper-free
click chemistry technique to image cells in live zebrafish embryos,
which by virtue of being transparent, are popular for scientists
studying embryogenesis. Those studies revealed dramatic differences
in cell-surface expression, intracellular trafficking patterns, and
tissue distributions of glycans at different stages of zebrafish
larval development.
"However, we were unable to detect labeled glycans in zebrafish
embryos earlier than 24 hours post-fertilization," Bertozzi says.
"Because many important developmental events including cell
migration, tissue morphogenesis, and cell differentiation occur in
the first 24 hours of zebrafish embryogenesis, and glycan
biosynthesis is also known to occur within that 24 hour period, we
sought to develop methods that would enable us to image the glycans
early during embryogenesis."
For this study, Bertozzi and her co-authors microinjected
embryos with azido sugars at the one-cell stage, allowed the
zebrafish to develop, and then detected the metabolically labeled
glycans with the copper-free click chemistry technique. This
enabled them to image certain types of glycans as early as seven
hours post-fertilization. In addition, they used a complementary,
non-metabolic labeling technique to target a class of glycans that
carry sialic acid, giving them simultaneous but independent ways to
image two distinct classes of glycans.
Time-lapse and multicolor imaging experiments highlighted
differences between the O-glycans and sialylated glycans in the
cells during the gastrulation and segmentation periods of
embryogenesis. The results revealed a dramatic re-organization of
cell-surface glycans during mitosis, highlighted by their rapid
migration to the cleavage furrow of mitotic cells. A cleavage
furrow is the indentation that forms in a mother cell, marking the
position where it will divide into two daughter cells.
"As the saying goes, a picture is worth a thousand words," says
Jeremy Baskin, the lead author on this new PNAS paper who is now
doing post-doctoral research at Yale University. "In our case, the
glycan imaging technology led directly to the observation that
membrane-associated glycans traffic laterally within the plasma
membrane to the cleavage furrow during cell division. Down the
road, we hope to learn the precise molecular structures of the
glycans involved in this process and what roles they play."
Baskin says the ability of this click chemistry technique to
provide visualization of specific molecules in cells is an
important first step toward an eventual understanding of the
function of these molecules.
"By perturbing the system using genetic or pharmacological means
and then observing any changes in glycan localization and behavior
through imaging," he says, "we may be able to understand the many
functions of glycans in embryogenesis and many other physiological
processes, including diseases."
Bertozzi says she and her colleagues are following up their
glycan imaging studies of zebrafish embryos with mechanistic
studies that will help determine what role the labeled glycans
might play in the process of cell division.
"There are other applications of glycan imaging in mice that we
also are aggressively pursuing," she says, "such as tumor imaging,
in addition to studies of how glycans change during
embryogenesis."
SOURCE