First comprehensive proteomic analysis of how proteins change as an animal develops
Carnegie Mellon University scientists have performed the first comprehensive proteome analysis of protein changes that occur in a developing animal, making surprising findings that could require scientists to revise standard thinking about how proteins orchestrate critical steps in embryonic development.
Their findings could one day provide a sensitive way to measure how drugs or environmental chemicals affect specific protein networks and harm development.
The research, reported online (http://dev.biologists.org/content/vol131/issue3/) and in the February 1 issue of Development, found that specific cells set to change shape during a key growth step are actually poised for their transformation far in advance and that many types of proteins are involved.
"Our findings counter long-held assumptions that a limited number of proteins are responsible for this step of development and that they become active right before the cells change shape," said Jonathan Minden, principal investigator on the study and associate professor of biological sciences at Carnegie Mellon University.
The researchers studied the complete proteome, or all the proteins, within embryos of the fruitfly, Drosophila melanogaster. They compared proteomes at three stages of fruitfly development to witness changes that occurred as some cells folded into the body to form structures including the nerves, immune system and muscles. This process, called gastrulation, is a critical growth step for virtually every animal, from insects to humans.
During gastrulation, in a process called ventral furrow formation, column-shaped cells along the underside of the fruitfly become cone-shaped, which drives them to the interior or the embryo.
In their proteomic analysis, the Carnegie Mellon scientists found changes in the abundance of many types of proteins well before gastrulation. These included proteins that control metabolism, protein breakdown, protein production and the formation of the cell's interior scaffolding, known as the cytoskeleton. Previous genetic studies have yielded limited information about the genes controlling the signaling pathway that specifies the ventral furrow cells. Moreover, these studies have failed to provide a coordinated framework for how the changes take place in concert.
To test that the protein changes they saw actually were critical to the formation of the ventral furrow, Minden and his colleagues used a technique called RNA interference to greatly decrease the expression of genes for altered proteins in the fruitfly embryos. They found that that the embryos failed to form ventral furrows.
"Our study demonstrates that the formation of the ventral furrow is a complex process that encompasses nearly all cellular processes," said Minden.
The scientists also found that only one protein was activated at the exact moment when cells changed shape. This protein is part of a complex cellular machine called the proteosome, which is responsible for breaking down proteins.
"This finding suggests that right before cells in the ventral furrow change shape, they break down proteins, perhaps the cytoskeletal proteins that preserve their columnar shape," said Minden.
To compare the abundance and kinds of proteins made at different stages of development, Minden and his colleagues used Difference Gel Electrophoresis, a tool created by Minden and commercialized by Amersham, plc. Using DIGE, scientists label two protein samples with different color fluorescent dyes and then run both samples on the same gel, which separates proteins by size and electrical charge. A computer program analyzes the gel to detect differences in the abundance and presence of all the proteins from the two samples and reports them back to the investigator.
"Our study is really a starting point. Comparing the proteomes at different stages in an animal's development, together with other experimental tools, can help uncover the network of functions and interactions required for a variety of developmental processes and disease states." Minden said.
Source: Eurekalert & othersLast reviewed: By John M. Grohol, Psy.D. on 21 Feb 2009
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