Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

U-M scientists see ubiquitin-modified proteins in living cells

11.10.2004


New technology makes visualization possible



Researchers at the University of Michigan Medical School and Howard Hughes Medical Institute have found a way to see proteins in cells that have been tagged by a molecular "sticky note" called ubiquitin. "This technology allows us to see, under a microscope, proteins modified by ubiquitin inside the cell," says Tom K. Kerppola, Ph.D., an associate professor of biological chemistry in the Medical School and an HHMI associate investigator. "Visualization gives us a direct connection to cellular processes that we could only study in test tubes or indirectly before."

In a paper published online this week in the early edition of the Proceedings of the National Academy of Sciences, Kerppola and Deyu Fang, M.D., Ph.D., a U-M research investigator, describe the first use of a technology called ubiquitin-mediated fluorescence complementation to study a cell-signaling mechanism called ubiquitination.


In this process, a small peptide called ubiquitin is linked to a protein in ways that can change the protein’s function and location within the cell. Originally, scientists thought ubiquitin was simply a universal "destroy me" signal for unneeded or harmful proteins, but it has recently been found to be associated with many other cellular functions. "The same ubiquitin signal can cause one protein to be degraded, but another protein to be moved to a new location," Kerppola says. "We’re interested in learning how this works."

In their PNAS paper, Kerppola and Fang describe how ubiquitin latched onto Jun - a protein involved in cell growth and gene transcription – and moved Jun from its usual location in the cell’s nucleus into hollow spheres called lysosomes in the cytoplasm outside the nucleus. Filled with digestive enzymes, lysosomes break down unwanted proteins into amino acids the cell recycles to make new proteins. "Jun’s function in the nucleus is transient and time-dependent," Kerppola says. "If it’s turned on and doesn’t get turned off, that’s not normal. Prolonged signaling causes aberrant growth and other problems for the cell."

"When ubiquitin is attached to Jun, the complex is transported to the lysosome getting it away from DNA in the nucleus and preventing Jun from continuing its normal gene transcription function," he adds. "This is a new way for the cell to eliminate the function of a transcription factor."

U-M scientists also discovered that an E3 ligase binding enzyme called Itch was a key player in the process. "Itch is the adapter," Kerppola says. "It tags Jun with ubiquitin, and is necessary for the protein to be targeted to the lysosome."

If Itch doesn’t recognize Jun, Kerppola explains, the level of Jun builds up in the cell, which can alter the regulation of gene transcription and cell growth. "Applying ubiquitin-mediated fluorescence technology to Jun made it possible to discover new information on how Jun turnover is controlled in cells," Kerppola says.

The technology uses complementary fragments of a fluorescent protein, which are fused to ubiquitin and to the target protein being studied. When ubiquitin is linked to the target protein, the fragments of the fluorescent protein come together and produce a bright spot of glowing color, which can be seen with a fluorescence microscope. This allows scientists to determine the location of the ubiquitinated protein in the cell.

"Location is important," Kerppola adds, "because proteins must get to their sites of action in order to do their jobs. Each protein must fulfill many different functions in different cells and in response to different stimuli. It is the variety of modifications and interactions with partners that enable the same protein to accomplish different tasks. With this technology, we are able to see the subpopulation of a protein that is modified by ubiquitin or interacts with a particular partner."

Scientists in Kerppola’s laboratory have used bimolecular fluorescence complementation methods to study protein interactions and signaling pathways, in addition to ubiquitination. While the technology should be generally applicable to most interactions, it does have some limitations. "The assay requires the two fragments of the fluorescent protein to come together," Kerppola says. "If they can’t get together, the assay doesn’t work. It’s quite good at identifying the location where something happens in the cell, but the timing of the interaction is more difficult to study, since it takes about an hour for the fluorescent proteins to become visible."

Sally Pobojewski | EurekAlert!
Further information:
http://www.umich.edu

More articles from Life Sciences:

nachricht Could this protein protect people against coronary artery disease?
17.11.2017 | University of North Carolina Health Care

nachricht Microbial resident enables beetles to feed on a leafy diet
17.11.2017 | Max-Planck-Institut für chemische Ökologie

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: A “cosmic snake” reveals the structure of remote galaxies

The formation of stars in distant galaxies is still largely unexplored. For the first time, astron-omers at the University of Geneva have now been able to closely observe a star system six billion light-years away. In doing so, they are confirming earlier simulations made by the University of Zurich. One special effect is made possible by the multiple reflections of images that run through the cosmos like a snake.

Today, astronomers have a pretty accurate idea of how stars were formed in the recent cosmic past. But do these laws also apply to older galaxies? For around a...

Im Focus: Visual intelligence is not the same as IQ

Just because someone is smart and well-motivated doesn't mean he or she can learn the visual skills needed to excel at tasks like matching fingerprints, interpreting medical X-rays, keeping track of aircraft on radar displays or forensic face matching.

That is the implication of a new study which shows for the first time that there is a broad range of differences in people's visual ability and that these...

Im Focus: Novel Nano-CT device creates high-resolution 3D-X-rays of tiny velvet worm legs

Computer Tomography (CT) is a standard procedure in hospitals, but so far, the technology has not been suitable for imaging extremely small objects. In PNAS, a team from the Technical University of Munich (TUM) describes a Nano-CT device that creates three-dimensional x-ray images at resolutions up to 100 nanometers. The first test application: Together with colleagues from the University of Kassel and Helmholtz-Zentrum Geesthacht the researchers analyzed the locomotory system of a velvet worm.

During a CT analysis, the object under investigation is x-rayed and a detector measures the respective amount of radiation absorbed from various angles....

Im Focus: Researchers Develop Data Bus for Quantum Computer

The quantum world is fragile; error correction codes are needed to protect the information stored in a quantum object from the deteriorating effects of noise. Quantum physicists in Innsbruck have developed a protocol to pass quantum information between differently encoded building blocks of a future quantum computer, such as processors and memories. Scientists may use this protocol in the future to build a data bus for quantum computers. The researchers have published their work in the journal Nature Communications.

Future quantum computers will be able to solve problems where conventional computers fail today. We are still far away from any large-scale implementation,...

Im Focus: Wrinkles give heat a jolt in pillared graphene

Rice University researchers test 3-D carbon nanostructures' thermal transport abilities

Pillared graphene would transfer heat better if the theoretical material had a few asymmetric junctions that caused wrinkles, according to Rice University...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Ecology Across Borders: International conference brings together 1,500 ecologists

15.11.2017 | Event News

Road into laboratory: Users discuss biaxial fatigue-testing for car and truck wheel

15.11.2017 | Event News

#Berlin5GWeek: The right network for Industry 4.0

30.10.2017 | Event News

 
Latest News

NASA detects solar flare pulses at Sun and Earth

17.11.2017 | Physics and Astronomy

NIST scientists discover how to switch liver cancer cell growth from 2-D to 3-D structures

17.11.2017 | Health and Medicine

The importance of biodiversity in forests could increase due to climate change

17.11.2017 | Studies and Analyses

VideoLinks
B2B-VideoLinks
More VideoLinks >>>