Understanding the molecular basis of developmental mechanisms ultimately requires precise knowledge of events in space and time. While the clearest view of subcellular organization has come from electron microscopy, its utility in understanding mechanism is burdened by the difficulty of mapping molecule to structure. Recent advances now offer us nanometer-scale resolution through technologies like STED, PALM/STORM and SIM that bring the resolution of light microscopy below the Abbe limit. This proposal requests support for the purchase of a Zeiss LSM710 PAL-M/SIM microscope. This instrument offers three imaging modalities;laser scanning confocal microscopy, Photo Activated Localization Microscopy (PAL-M), and Structured Illumination Microscopy (SIM). With this combination, one can obtain in a single specimen a broad 3D perspective by confocal microscopy and, in selected regions, sub-diffraction limit resolution with SIM and PAL-M. In combination with a wide array of multi-color fluorescent molecular probes, these are powerful technologies that open vast new areas of inquiry. In this proposal, the Principal Investigator Sam Kunes and five group leaders describe developmental biology projects that implement this technology. Developmental processes are a particularly suitable subject for super-resolution microscopy since they are governed and fine-tuned by spatial cues, many of which act in the sub-diffraction limited range. Three groups, Kunes, Lichtman and Dulac, propose to study the development of the ultrastructure of synaptic connections. Kunes will investigate how a developmental pathway is re-deployed to control long-term memory control axonal and dendritic branching, synapse number, and synapse morphology in the mature fruitfly brain. Lichtman plans to decipher the development of complex patterns of axonal circuitry and synaptic connectivity at the neuromuscular junction, and between neurons in the parasympathetic ganglia and cerebral cortex, using Brainbow mice. The Dulac laboratory will investigate the role of the Major Histocompatability M10 family of proteins in the developmental plasticity of vomeronasal organ sensory neuron axon terminals. Two groups, McMahon and Schier, propose to visualize morphogen movement at high resolution;McMahon will focus on Sonic Hedgehog, as it patterns the ventral neural tube during CNS development, while Schier proposes to image the morphogen Squint and its co-localization with other components of the Nodal signaling pathway during Zebrafish development. The Mango laboratory seeks to understand how the packaging of chromatin is modulated during development, in order to understand the transition from developmental plasticity to cell fate commitment. The instrument will be incorporated into a well-established imaging facility, where it will receive full-time technical support and oversight, support for maintenance and managed usage, thus providing long term availability to the co-PI group and the broader community of microscopists.
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