Adventures in Nontranslational Research

Over the last ten years administrators, governmental officials, and clinical researchers have increasingly called for a greater emphasis on translational research , i.e., research that translates findings in the laboratory into new treatments for medical conditions, over basic research in the biological sciences. Although the application of biological research to human disease (as well as toward improvements in agriculture and development of biotechnology) is important, I feel that an increased emphasis is not needed and is actually detrimental. Ultimately, this emphasis is self-defeating because one needs the fruits of basic research to fuel these applications. I will give examples from my own research developing green fluorescent protein (GFP) as a biological marker and uncovering the molecular basis of the sense of touch to argue that basic research into fundamental problems in biology is important for its own sake and, not surprisingly, for the development of various applications

The 2009 Lindau Nobel Laureate Meeting: Martin Chalfie, Chemistry 2008

American Biologist Martin Chalfie shared the 2008 Nobel Prize in Chemistry with Roger Tsien and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP)

Regulation of Caenorhabditis elegans degenerin proteins by a putative extracellular domain

AbstractBackground: Rare, dominant mutations in the degenerin genes of Caenorhabditis elegans (deg-1, mec-4 and mec-10) cause neuronal degeneration. The extensive sequence similarity between degenerins and mammalian genes that encode subunits of the amiloride-sensitive sodium channel from kidney, colon and lung suggests that the C. elegans degenerins form ion channels. As mec-4 and mec-10 are needed for the reception of gentle touch stimuli, they may contribute to a mechanosensory ion channel. All the dominant degeneration-causing mutations in the C. elegans degenerin genes affect equivalent residues in a hydrophobic region that is structurally similar to the H5 domain of several ion channels, and so could form the channel lining. Increased channel activity may underlie the resulting degeneration, in which the affected cells vacuolate and swell.Results We now demonstrate that a missense change in a predicted extracellular region of the proteins encoded by deg-1 and mec-4 causes cell death similar to that caused by the dominant mutations. The missense mutation lies within a 22 amino-acid region found in all the C. elegans degenerins for which the sequences have been published, but not in the similar mammalian proteins. Deletion of nine amino acids surrounding the mutation site in mec-4 also causes neuronal degeneration. The degeneration-causing mutations in either the predicted pore-lining or the predicted extracellular regions of deg-1 are suppressed by additional, dominantly acting mutations that substitute larger for smaller residues within the channel lining.Conclusion Our data suggest that the putative extracellular domain negatively regulates degenerin activity, perhaps by gating the channel. As this region is only found in the C. elegans proteins, it may allow more rapid regulation of the nematode channels, which may be needed for them to function in mechanosensation. The suppressor mutations, by adding larger amino acids to the putative pore lining, could prevent degeneration by blocking the pore of a multisubunit channel

protaTETHER - A Method for the Incorporation of Variable Linkers in Protein Fusions Reveals Impacts of Linker Flexibility in a PKAc-GFP Fusion Protein

Protein fusions are of fundamental importance in the study of cellular biology and the elucidation of cell signaling pathways, and the importance of linkers for the proper function of protein fusions is well documented in the literature. However, there are few convenient methods available to experimentalists for the systematic implementation of linkers in protein fusions. In this work, we describe a universal approach to the creation and insertion of focused linker libraries into protein fusions. This process, deemed protaTETHER, utilizes reiterative oligomer design, PCR-mediated linker library generation, and restriction enzyme-free cloning methods in a straightforward, three-step cloning process. We utilize a fusion between the catalytic subunit of cAMP-dependent protein kinase A (PKAc) and green fluorescent protein (GFP) for the development of the protaTETHER method, implementing small linker libraries that vary by length, sequence, and predicted secondary structural elements. We analyze the impact of linker length and sequence on the expression, activity, and subcellular localization of the PKAc-GFP fusions, and use these results to select a PKAc-GFP fusion construct with robust expression and enzymatic activity. Based upon the results of both biochemical experiments and molecular modeling, we determine that linker flexibility is more important than linker length for optimal kinase activity and expression

A scalable optical detection scheme for matter wave interferometry

Imaging of surface adsorbed molecules is investigated as a novel detection method for matter wave interferometry with fluorescent particles. Mechanically magnified fluorescence imaging turns out to be an excellent tool for recording quantum interference patterns. It has a good sensitivity and yields patterns of high visibility. The spatial resolution of this technique is only determined by the Talbot gratings and can exceed the optical resolution limit by an order of magnitude. A unique advantage of this approach is its scalability: for certain classes of nano-sized objects, the detection sensitivity will even increase significantly with increasing size of the particle.Comment: 10 pages, 4 figure

NOMPC, a Member of the TRP Channel Family, Localizes to the Tubular Body and Distal Cilium of Drosophila Campaniform and Chordotonal Receptor Cells

Mechanoreception underlies the senses of touch, hearing and balance. An early event in mechanoreception is the opening of ion channels in response to mechanical force impinging on the cell. Here, we report antibody localization of NOMPC, a member of the transient receptor potential (TRP) ion channel family, to the tubular body of campaniform receptors in the halteres and to the distal regions of the cilia of chordotonal neurons in Johnston's organ, the sound-sensing organ of flies. Because NOMPC has been shown to be associated with the mechanotransduction process, our studies suggest that the transduction apparatus in both types of sensory cells is located in regions where a specialized microtubule-based cytoskeleton is in close proximity to an overlying cuticular structure. This localization suggests a transmission route of the mechanical stimulus to the cell. Furthermore, the commonality of NOMPC locations in the two structurally different receptor types suggests a conserved transduction apparatus involving both the intracellular cytoskeleton and the extracellular matrix. © 2010 Wiley-Liss, Inc

Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference

BACKGROUND: Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms. RESULTS: We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain. CONCLUSION: A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis