James D. Baker - US grants

Little is known of the molecular components that transduce mechanical stimuli in specialized sensory cells. The goal of this project is to investigate the function of the unc gene product in the mechanosensory cells of Drosophila. Disruption of this gene eliminates touch and hearing; in addition, unc mutant males have immotile sperm. The sensory defects in unc mutants occur in a class of ciliated cells that includes vertebrate photoreceptors and olfactory cells as well as many invertebrate sensory receptors. The combination of sensory and sperm defects suggests that unc is required for normal ciliary differentiation or operation. Defects in ciliary components are potentially involved in human conditions such as retinal degeneration, deafness, male sterility, or situs inversus. The unc gene has recently been cloned and while it includes domains found in cytoskeletal and axonemal motor proteins such as myosins and dyneins- a nucleotide-binding P-loop and a coiled-coil domain-it is not clearly related by sequence similarity to these or to any known proteins. It is proposed to: 1) Study the cellular and subcellular distribution of UNC during development. 2) Use electron microscopy to study the axonemal structure of unc mutant sperm. 3) Test if nucleotide binding is required for unc function, by altering conserved residues in the P-loop and coiled coil domain- it is not cellular and subcellular distribution of UNC during development. 2) Use electron microscopy to study the axonemal structure of unc mutant sperm. 3) Test if nucleotide binding is required for unc function, by altering conserved residues in the P-loop. 4) Search for proteins that interact with the UNC protein using the yeast two hybrid screen.

This project aims at the design and preparation of molecules that can be transported to target locations in organisms and emit light only after stimulation with an activating optical beam. Subsequent detection of the emitted light offers the opportunity to locate the position of the activated molecules in a sample of interest and follow their movements in real time. These light emitters can ultimately become analytical tools for biological studies, such as the tracking of moving cells during the growth of the organism, and contribute valuable insights on organismal development. These fundamental studies in chemistry may have implications in biology and medicine. Additionally, the activities teach the participating undergraduate and graduate students how to prepare molecules from commercial reagents, characterize their structures and investigate their interactions with light. Furthermore, they contribute to strengthening an existing educational collaboration between the laboratory of the principal investigator at the University of Miami and a faculty member of Miami-Dade College. This collaboration exposes high-school and undergraduate students to research. Considering that both institutions have minority enrollments in excess of 50%, these training opportunities may have a significant educational impact on members of underrepresented groups.

The goal of this research is the development of biocompatible probes with photoactivatable fluorescence in the deep-red and near-infrared regions of the electromagnetic spectrum. Specifically, this project involves the synthesis of molecules incorporating a fluorescent borondipyrromethene chromophore and a photoswitchable oxazine auxochrome, together with their photochemical/photophysical characterization and incorporation within polymer nanoparticles. Upon illumination at an appropriate activation wavelength, the photoinduced and irreversible opening of the oxazine heterocycle of these compounds is expected to extend the electronic conjugation of the adjacent borondipyrromethene chromophore. This structural transformation is designed to bathochromically shift the main absorption band of the latter component and allow its selective excitation at a suitable wavelength with concomitant fluorescence. On the basis of these operating principles, the translocation of the photochemical and emissive product, across a given sample of interest, can then be probed in real time with the sequential acquisition of fluorescence images. These light emitters can ultimately become analytical tools for biological studies, such as the tracking of moving cells during the growth of the organism, and contribute valuable insights on organismal development.