A method using a virus to screen for chemical or genetically-encoded inhibitors of ion channels is published online this week in Nature Methods.
Ion channels are cell membrane-spanning proteins that, when activated, allow the influx of ions. They encompass a large family of over 400 proteins and play key roles in maintaining cellular function. While blocking their activity has proven useful for certain medical applications, these drugs usually elicit severe side effects due to their lack of specificity.
Joseph Glorioso and colleagues wanted to find more specific channel modulators and set up a virus-based screen in which the virus level is an indicator for inhibitor efficacy. They reasoned that overexpression of an ion channel in cells via a virus would be detrimental to the cell and consequently impede viral replication while the presence of an inhibitor of channel function would keep the cells healthy and allow viral replication. The advance over other screens for channel inhibitors is that the assay can easily be adapted to screen for genetically-encoded inhibitors by coinfection of a second virus that encodes the inhibitor. The inhibitor’s DNA can then be easily retrieved from the viral genome.
This method can easily be scaled up to screen whole cDNA libraries for channel modulators. These genetically encoded inhibitors will elucidate the biology behind channel regulation and are likely to lead to more specific inhibitors.
A sponge to soak up regulatory RNAs
Nature Methods
Genetically encoded inhibitors for small regulatory RNAs are presented online this week in Nature Methods.
MicroRNAs, unlike longer messenger RNAs (mRNAs), do not contain information for making a protein, but their short, 21 nucleotide, sequences specifically regulate mRNA expression. MicroRNAs bind to partly complementary sequences on their target mRNAs and as a consequence the mRNA is either marked for decay or protein translation is inhibited.
Overexpression of certain microRNAs has been linked to cancer and other diseases and a better understanding of their action is therefore important. Philip Sharp and colleagues have now developed a tool to specifically inhibit microRNAs. The principle of their system is based on "soaking up" all the microRNAs with a complementary decoy sequence, so the mRNA can express its protein unhindered. In contrast to previously described chemically synthesized microRNA inhibitors, Sharp’s inhibitors, or microRNA sponges as he calls them, are genetically encoded. They are provided to the cell either in form of a plasmid or they are stably integrated into the genome of a cell. This provides a much higher level of control over the amount of microRNA sponges a cell produces and allows microRNA action in a specific cell or tissue type to be studied.
CONTACT
Philip A Sharp (Massachusetts Institute of Technology 77 Massachusetts Avenue, Cambridge, MA 02139-4307 USA)
Tel: (617) 253-6421; E-mail: sharppa@mit.edu
Observing worms
Nature Methods
A pair of microfluidic devices for monitoring nerve cell activity in live worms is presented online this week Nature Methods. This will help meet one of the major goals of neuroscience: to correlate the activity of nerve cells with behavior in the living organism.
The microscopic worm C. elegans has a very simple nervous system, with only 302 neurons. However, it is difficult to monitor neuronal activity in worms without either using invasive methods or restraining them in an unphysiological way.
Nikos Chronis and colleagues present devices which make it possible to use a microscope to monitor nerve activity in intact, live worms. The key is that the worms are enclosed in microfluidic channels only slightly bigger than the animals, so that they are free to move to some extent, but restrained sufficiently to allow for imaging of the neurons. The trapped worms can also be stimulated in a very controlled fashion by substances delivered at the tip of the animal’s nose, so that their effect on nerve activity can be recorded.
The researchers used these devices to uncover new information about particular neurons involved in movement and sensation in the worm. This may very well pave the way to ever more sophisticated devices, as well as to devices for monitoring other small organisms in physiological and precisely controlled environments.
CONTACT
Nikos Chronis (University of Michigan, Ann Arbor, MI 48109 USA)
Tel: (734) 763-0154; E-mail: chronis@umich.edu
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