Aug. 19, 2006 -
Cryoelectron tomography pries into the secrets of the cilia.
Daniela Nicastro, Cindi Schwartz, Jason Pierson, Richard Gaudette, Mary E. Porter, J. Richard McIntosh (2006), The molecular architecture of axonemes revealed by cryoelectron tomography. Science 331:944-948.
The cellular whip structures known as cilia and flagella are attached to molecular motors constructed of proteins and powered by ATP. These hair-like appendages on single-celled organisms are used for movement and feeding while in small multicellular organisms the cilia whip up water currents that move food into the mouth. Even humans have cilia. Ciliated cells in the lung and airway move mucus along as it traps dust, bacteria, pollen, etc., ultimately bringing it to the throat where it can be swallowed and disposed of.
But how do these marvelous little motors work? Researchers working at the Universities of Colorado and Minnesota have been studying cilia at the molecular level for some time and they just published some great electron microscope photos of the axonemes, the technical name for the ciliary structure, in this week’s Science.
The axonemes are constructed of bundles of long fibrous proteins called microtubules (MTs). The MTs are tethered to the motor molecules which are known as dyneins and which convert chemical energy into a mechanical oscillatory motion that moves the MTs back and forth. Exactly how this is accomplished on the molecular level has remained a mystery.
The scientists reasoned that if they could freeze the motion of the cilia very quickly and then examine the structure using the electron microscope, they might be able to catch the motor in action. So that is exactly what they did—freeze the cells and keep them frozen.
They chose to study two types of cell. One was the sperm of the sea urchin which is a good example of the flagellar structure and the other was the alga, Chlamydomonas, which has a pair of cilia that it uses for movement.
Electron microscopy as employed in labs today has come a long way since its invention in the late 1940’s. Combined improvements in optics, electronics and computer software produce far better image control and signal-to-noise ratios than were possible even just a few years ago. The authors of this study used a highly sophisticated technique called cryoelectron tomography and obtained wonderful photos and even movies of the motors that can be seen in this article and in supplementary material on the Science magazine website.
The dynein proteins are attached to the base at one end and have arms that can be seen in the micrographs attaching to the MTs. As the array of dyneins is stimulated to retract their arms they pull the MT fibers back and forth creating the whiplike motion.
The researchers emphasize that the complexity of the cilia and flagella means that there are numerous points at which defects can occur. Indeed several human diseases are known to be caused by cilia that don’t work the way they should. One of these, known as polycystic kidney disease, affects about 500,000 people in this country alone and is incurable. It is hoped that important new research findings such as these can help us to develop treatments to reverse the harmful effects of ciliary disease.
July 20, 2006 -
Cells organize silica home.
The July 21st issue of Science has a research paper describing a method for encapsulating cells in phospholipids, the fatty molecules that make up the cell’s membrane envelope, and attaching these cells-in-overcoats to a silica support. The goal is to make a bio-machine, linking living cells to non-living hardware in such a way that the cell’s unique capabilities can be utilized. This is hard to do because most cells are fragile and do not take kindly to interfacing with the metals and ceramics that engineers would like to combine them with.
In this study, silica (silicon dioxide, the same compound that makes sand and quartz crystals) was used as the matrix. A 3-D silica structure was created by a technique called evaporation-induced self-assembly (EISA) in which the controlled evaporation of a solvent from a silica solution produces a hard, porous structure. The authors needed to use a solvent/surfactant that was gentle enough to cells to preserve them from breakage yet still possessed of the right chemical properties. They came up with a modified phosphatidylcholine (PC) which is one of the lipids that naturally occurs in cell membranes and is both fat-soluble and water-soluble.
When the silica matrix was made with PC, it formed channels and pores in which the authors figured their cells would feel right at home. So next, they added cells of yeast—the kind used in bread- and beer-making, scientific name Saccharomyces cerevisiae to the silica/PC solution and subjected the mix to EISA. To their surprise, the yeast took over the process and appropriated the PC to form a protective layer around itself. By increasing the concentration of PC, the researchers produced a silica chip of many layers containing yeast cells that were protected from drying and remained alive and active for at least a month. The cells were even able to withstand the high vacuum of the electron microscope in which they were placed to be photographed!
So, what can we do with live cells on a silica chip? The purpose of using cells is to identify or ‘sense’ specific molecules and to register this recognition in a physical way such as emission of light or an electric current. The combination of cells and a solid matrix is called a biosensor. The research on biosensors is still in its exploratory phase but potential applications include sniffing out pathogens such as anthrax and sounding an alarm, monitoring pollutant levels or indicating levels of specific molecules in the human body such as glucose in diabetics or cholesterol in heart patients.
The whole area of combining non-organic materials with cells or indeed with organs in the human body is, I think, set to explode in the next decade into a wonderful array of new gadgets for helping people monitor disease states or prevent them, for providing mechanical eyes to the blind, electronic nerve replacements for paralyzed persons, and even for machine-brain connections that allow direct data transfer between our neurons and the mechanical brain of a computer. Don’t ever say that some idea is too far-fetched to ever be put into use, because there’s an army of smart and highly driven creative people out there working hard to take that idea and turn it into a usable machine.
|July 15, 2006|
Protein modification helps cells move.
Just when you think you have mastered all the basics of molecular biology, along comes another new item to add to the list. In the latest issue of Science (14 July 06) I noticed an article entitled “Arginylation of Beta-Actin Regulates Actin Cytoskeleton and Cell Motility”. Arginylation? That was something I’d missed in my reading. I know proteins can be modified by the body which can add sugar groups, phosphates or fatty acids but I didn’t remember arginine being one of the additions. The report is a collaboration among labs from the Department of Animal Biology, University of Pennsylvania, Philadelphia, PA, The Scripps Research Institute, La Jolla, CA, University of California, Davis, CA, and Rockefeller University, New York, NY, USA.
If you remember biology class, you know that proteins are made of a string of 20 or so different amino acids that fold up into a tight little tangle. The protein modifications happen after the folding and change the function of the protein in some way. Fat, for example, added to a protein as a fatty acid may cause the protein to stick in the cell’s membrane because it has a fatty acid core. Such proteins poking out of a cell’s membrane act as antennas to pick up signals from the bloodstream. Phosphates are another favorite addition to a protein and their negative charge causes them to repel other negative charges such as those on DNA.
The author of the paper in Science states that ‘arginylation’—the addition of the amino acid arginine (Arg) to a protein has been known for forty years, but the purpose and details of the modification are only now becoming clear. One of the proteins that is known to be arginylated is actin. Actin forms the ‘bones’ of a cell, an internal scaffolding that gives the cell support and structure, and also is used in cell movement. The arginylation of actin allows the scaffold to form properly. Mutant cells that have lost the ability to add Arg to actin are smaller and move poorly. Mice with this mutation usually die in the embryonic stage or have developmental problems later on. The authors do not mention any diseases in humans caused by a defect in actin arginylation but it is certainly possible.
A PubMed search for ‘arginylation’ came up with 50 hits, the oldest being 1971. Reading some of the abstracts, I found that arginylation of proteins is also a way of causing them to be degraded. The replacement of an amino acid at the amino-terminal (‘N’ terminus) end of a protein by Arg tells the cells waste removal team to target that protein and get rid of it. This is called the ‘N-end Rule’ because it is the amino acid at the N-terminus of the protein that determines its fate. In the case of actin, however, the experimenters in the Science article tested the beta-actin protein with and without Arg addition, and they found that arginylation does not target the protein for degradation. How beta-actin escapes from the enzyme police is not explained.
Whether or not you believe that everything can be explained in terms of molecules, the actions and reactions of our proteins make for interesting reading. Molecular biology is kind of like mathematics. It is daunting at first until you understand the basic ideas, then it becomes entertaining and finally totally amazing. The human mind is such a great tool for good, if only we could teach people how to get along and stop killing each other over real estate, minerals and ideologies.
July 8, 2006|
Apple juice for Alzheimer's?
Alzheimer's disease is a devastating illness that affects one person in ten over the age of 65. Antioxidants in fresh fruits and vegetables may help to slow down the disease.
In a paper published in the December issue of the Journal of Alzheimer's Disease, the protective effects of apple juice concentrate were examined in mice. The researchers were from the Center for Cellular Neurobiology and Neurodegeneration Research, Department of Biological Sciences, University of Massachusetts at Lowell where, under the leadership of Thomas Shea, PhD, they have been studying the role of oxidants in Alzheimer’s disease for several years. It has been known that the brains of Alzheimer's disease patients show greater amounts of oxidative damage than normal brains. If they were given antioxidants in sufficient amounts then perhaps the disease could be stopped or at least slowed down.
The mice that were used in these studies had a defect in the gene for apolipoprotein E (ApoE), a protein that transports cholesterol in the bloodstream, and this causes them to be prone to Alzheimer’s disease. To mimic the condition in humans, mice that were young (9-10 weeks) and others that were elderly (2-2.5 years) were included in the experiments. They were fed either a normal diet or an oxidative stress diet that was deficient in the vitamins, folic acid and E, and contained extra iron to cause more oxidation damage.
Oxygen radicals and other oxygen species can damage neurons directly and may be one of the agents that cause harm in Alzheimer’s disease. These reactive chemicals are part of the body’s natural defense against bacteria and viruses but if they are made in the wrong place or in too great of quantities then they can cause problems. Cells have their own antioxidants, but sometimes they aren’t enough to cancel out all the bad oxygen radicals and they need a little help. This is where antioxidants in the diet may act to restore the balance of oxidant-antioxidant that is necessary for us to live healthy and long lives.
The researchers divided the mice into two groups—those that got the normal diet and those that got the oxidative stress diet. The question that they were asking was, Does feeding animals antioxidants prevent them from getting Alzheimer’s disease? The source of antioxidants they used was something you might not think of—just plain old apple juice from concentrate. Some of the mice in each group got apple juice and the others got only sugar water for comparison.
So, how can you tell that a mouse has Alzheimer’s disease? Humans are given special tests that measure their ability to remember things and the results are compared to an average value for normal, healthy persons. Mice that are placed in a maze with food as a reward will eventually find there way to the center where the food is, and healthy mice are able to remember the route so the next time they are put in the maze they get to the food more quickly. Alzheimer’s disease interferes with the brain’s ability to remember things and mice that have the disease don’t run the maze as quickly as those who were protected from the disease.
After the experiments were done, the results were all in favor of apple juice—at least for the elderly mice. The young mice apparently were not affected by the stress diet. In contrast, the older mice who got the oxidative stress diet forgot how to run the maze. The group who got apple juice along with the stress diet, however, did just as well as those on the normal, non-stress diet. When their brains were examined, it could be seen that the apple juice drinkers had less damage which correlates with their better mental ability.
There are other diseases that are made worse by oxidants, such as asthma and arthritis, and I wonder if eating apples will reduce the symptoms of those as well? Eating apples every day is not a problem for me because I love them. You should do your brain--and the rest of your body--a favor and keep the oxidants in check by eating lots of fruit and vegies every day.