|Molecular Switch Discovery Helps Explain Development of Cell Polarity|
|SciMed - Biology|
|TS-Si News Service|
|Tuesday, 21 February 2012 09:00|
Kansas City, MO, USA. Research into cell polarity in yeast has found a switch that moves molecules called phospholipids from the outer to the inner layer of the cell membrane, an intricate mechanism found in mammalian cells as well.
Upon casual inspection, cells tend to look more or less the same from all sides, but in real life they have a structured orientation (front and back, top and bottom), which explains why yeast cells prferentially bud at one end and not the other.
Over the last few years, Rong Li, PhD, and her team at the Stowers Institute for Medical Research have figured out many important details of the basic biochemical mechanism that establishes cell polarity in yeast. Now, applying microscopy and advanced mathematics, the team has discovered that an enzyme known as a lipid flippase literally flips lipids from one side of yeast cell membranes to the other. New findings from predoctoral researcher Arupratan Das, Rong Li, and five colleagues appear in the journal Nature Cell Biology.
Rong Li, PhD, is Professor of Physiology and Investigator at the Stowers Institute for Medical Research.
Her laboratory investigates the fundamental laws governing the behavior and interactions of cellular systems. Li's current work focuses on questions pertaining to the molecular and physical basis of self-organization during morphogenesis, and the evolutionary dynamics of cell division pathways.
"What the [current] study really highlights is the future of cell biology the ability to make these very accurate biochemical and biophysical measurements in single live cells and reconstituted systems, rather than using traditional bulk biochemical assays," says Li.
"Our finding could lead to a lot of clues mechanistically."
Li and her colleagues hope that others will pick up on the discovery and apply it to the cells they study.All of the molecules involved are found in mammalian cells, opening up new avenues for exploring how cell polarity is created in, say, liver cells, and whether or not glitches in the mechanism can contribute to disease. "Cell polarity is critical for the specialized function of the vast majority of cells," explains Li, who is also a professor in the Department of Molecular and Integrative Physiology at the University of Kansas Medical Center (KUMC).
Several decades ago, scientists thought that cell polarity might result from external cues such as spatially localized chemical signals outside cells that tell the cells how to move and behave. However, researchers have also long noticed that without such localized signals cells can still polarize suggesting that the ability to polarize and bud is innate in the cell.
Li's team uncovered an important clue to the underlying process. Embedded in the cell membrane is a molecule, named Cdc42, that acts as a key regulator. In a non-polarized cell, Cdc42 is randomly distributed around the membrane, like stones randomly set in a brick wall. It also floats around within the cell.
When activated, Cdc42 stimulates the formation of a skeleton of microfilaments that guide free-floating Cdc42 towards the Cdc42 already in the membrane. Some parts of the membrane start out with a bit more Cdc42 than others and the process guided by the microfilaments concentrates the molecule in those areas. Eventually, cells ends up with a high concentration of Cdc42 in one spot the spot where the bud will emerge.
Of course it isn't that simple. Membrane-bound Cdc42 can diffuse away but can also be pulled out of the membrane by another molecule named Rdi1 and then recycled back to the same spot on the membrane. The speed of the recycling determines how big the eventual spot will be, which directly affects the shape of the bud that grows out.
Li says we should imagine ants in a ditch. The ants keep running out. To keep them there, you pick them up and put them back. If your hands are fast, you can contain them in a very small area. If your hands are slow, the ants spread out more. It's the same in the yeast cell. Faster recycling of Cdc42 results in a smaller spot on the cell; slower recycling causes a bigger spot. In a 2009 paper, Li's team showed that the recycling process can be fast or slow.
So what controls the speed of Cdc42 dissociation and recycling? Li and her colleagues addressed the question by looking for genes that, when knocked out, affect the speed. They found one that slowed things way down. It was a big surprise. "It wasn't something we were envisioning," says Li. In fact, she wasn't even sure if her team would pursue it. The reason: The gene was for a type of an enzyme known as a lipid flippase, which literally flips lipids from one side of cell membranes to the other. And lipids are "really messy and hard to work with," she says. Predoctoral researcher Arupratan Das, however, was willing to take it on. "I have to give Das a lot of credit he's fearless," says Li.
Using cutting-edge fluorescence fluctuation spectroscopy, Das and his colleagues were able to chart the movement of molecules in live yeast cells, and figure out the role of the flippase. This is what they found:
Case closed? Not quite. The researchers were hopeful that they'd uncovered the mechanism based upon their observations with living cells. But in a living cell "it's hard to rule out the effects of something else," says Li. So Das painstakingly created small bits of man-made, reconstituted membrane to test the mechanism. "It was a great challenge and required much troubleshooting before we performed it successfully," he recalls.
After a year of effort, it worked. Using an imaging technique called total internal reflection fluorescence microscopy, the team was able to show that the charge of the membrane does indeed determine how fast Cdc42 can be pulled out by Rdi1.
The next challenge will be figuring out what controls the flippase. "There are some hints that it gets even messier that the flippase may be regulated by another kind of lipid in the membrane and perhaps other enzymes," says Li. But the work in the new paper is already opening the door to a better understanding of the development of cell polarity in all types of cells. And it points to the power of new experimental tools and mathematical analysis that can help track and count molecules in cells, says Li.
FundingThe work was part of a project funded by the National Institute of General Medical Sciences (NIGMS).
ParticipationResearchers who also contributed to the study include Brian D. Slaughter, Jay R. Unruh, William D. Bradford, Richard Alexander and Boris Rubinstein at the Stowers Institute for Medical Research.
CitationFlippase-mediated phospholipid asymmetry promotes fast Cdc42 recycling in dynamic maintenance of cell polarity. Arupratan Das, Brian D. Slaughter, Jay R. Unruh, William D. Bradford, Richard Alexander, Boris Rubinstein, Rong Li. Nature Cell Biology 2012. doi:10.1038/ncb2444
Lipid asymmetry at the plasma membrane is essential for such processes as cell polarity, cytokinesis and phagocytosis. Here we find that a lipid flippase complex, composed of Lem3, Dnf1 or Dnf2, has a role in the dynamic recycling of the Cdc42 GTPase, a key regulator of cell polarity, in yeast. By using quantitative microscopy methods, we show that the flippase complex is required for fast dissociation of Cdc42 from the polar cortex by the guanine nucleotide dissociation inhibitor. A loss of flippase activity, or pharmacological blockage of the inward flipping of phosphatidylethanolamine, a phospholipid with a neutral head group, disrupts Cdc42 polarity maintained by guanine nucleotide dissociation inhibitor-mediated recycling. Phosphatidylethanolamine flipping may reduce the charge interaction between a Cdc42 carboxy-terminal cationic region with the plasma membrane inner leaflet, enriched for the negatively charged lipid phosphatidylserine. Using a reconstituted system with supported lipid bilayers, we show that the relative composition of phosphatidylethanolamine versus phosphatidylserine directly modulates Cdc42 extraction from the membrane by guanine nucleotide dissociation inhibitor.
|Last Updated on Tuesday, 21 February 2012 09:06|