Abstract: Major advances in the biological sciences are coupled to technological innovation. Here, I propose to develop IR-LAMP (IR Laser-Assisted Modulation of Proteins), a novel optigenetic technology using an infrared laser to manipulate protein function at specific sub-cellular locations with precise temporal resolution to probe fundamental questions in biology. Complex cellular behaviors, such as cell division or neuronal transmission, require a high degree of molecular regulation at a sub-cellular level. Much of the molecular machinery active during individual cell behaviors, however, is also required in other essential events, thus precluding the study of future events (e.g. during gamete production vs. morphogenesis). This limitation has been partially overcome by conditional, temperature- sensitive genetics;however, even the best temperature-controlled environment restricts ts protein inactivation spatially to a cellular or organism level. This technical limitation prevents us from determining the specific sub-cellular structure at which a given molecular pathway functions. To overcome these challenges, I will develop IR-LAMP technology using an infrared laser to target specific sub-cellular locations and inactivate fast-acting ts alleles in embryos from the nematode Caenorhabditis elegans with high temporal and spatial resolution. With intensive screening, I have obtained a bank of temperature sensitive mutations in the molecular machinery required for the highly spatially regulated process of cytokinesis. Cytokinesis is the last stage of cell division, when a mother cell is physically divided into two daughter cells. Using IR-LAMP and my collection of fast-acting temperature-sensitive mutants, I will locally inactivate the core molecular machinery required for cytokinesis in distinct sub-cellular compartments and determine the contributing functional compartments for a given essential molecular complex (e.g. at the central spindle vs. the contractile ring). I will also screen for fast-acting cold-sensitive mutations to locally activate protein function with the same spatial and temporal resolution. Once developed, IR-LAMP technology will be applicable to any complex cell behavior accessible by light microscopy--a virtually endless list of cell or developmental processes, such as studies on cell adhesion, polarity establishment, cell migration, or cell-cell signaling. Public Health Relevance: Scientific frontiers are advanced by the development of new technology;the development of IR-LAMP will open the doors for basic research aimed at probing the spatial regulation of molecular function in vivo. Further, as infrared lasers can penetrate deep into tissues, IRLAMP could also be adopted for use in humans to treat localized diseases, such as cancer, using transgenic stem cells or nanoparticles containing thermally sensitive proteins. For example, nanoparticles coupled to a thermally sensitive pro-apoptotic protein could be locally activated in cancer stem cells with IR-LAMP to induce cell death and stop tumor growth.
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