This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.ABSTRACTBackground -The Microsporidia are 'emerging' human and well-documented veterinary pathogens that cause disease in both immunocompromised and immunocompetent hosts. These spore-forming intracellular protistan parasites, enter their host by the extrusion of a hollow polar tube. The polar tube serves as a unique vehicle for transmission of infection by discharging from the spore, piercing a host cell, and inoculating its infective sporoplasm directly into that cell's cytoplasm.In the non-activated spore (approx. 2 X 4 m), this hollow polar 'tube' is a solid filament composed of a series of concentric 'circles' of electron lucent and dense material when viewed in cross section. This single polar filament (approx. 100- 150 nm diameter) is coiled around the inner periphery of the spore and the infective sporoplasm. The PF becomes straight in the anterior portion of the spore where it terminates in a 'mushroom' shaped anchoring disc. The straight or manubroid portion of the PF is surrounded by a series of elaborate membranes and 'tubules' termed the lamellar and tubular polaroplast, respectively. Upon activation, this 'solid' PF everts through the anterior portion of the spore, becomes hollow, and is thought to turn 'inside out' , thus becoming the polar tube, and serving as the sporoplasm transfer mechanism. Longitudinal sections of the spore reveal cross sections of the PF, and extensive membrane systems that surround the filament. The mechanism of eversion, the process of sporoplasm transfer, and the role these complex membrane systems play, are poorly understood. In addition, the structure of the sporoplasm, both inside the spore and after discharge also need extensive investigation.Goals -While the polar filament and its unique function were described almost 100 years ago, the structure and mechanism of its formation, position in the spore, attachment to the sporoplasm, and method of discharge, remain to be definitively determined. Several aspects of our knowledge of this complex series of events can be greatly advanced by the generation of 3D models of: 1) the non-activated spore structure and its contents, 2) the translocation of the spore contents during activation, 3) the structure of the extruded polar tube and sporoplasm. Project Description -Collection of data for 3-D reconstruction with the high-voltage or intermediate voltage electron microscope will greatly increase our knowledge of the microsporidian spore and its contents. Results from our recent studies have revealed several previously unknown aspects of these organisms, but we have not been able to obtain sufficient images to produce structural models. It is hoped, that images of material in 'thick' sections, will enable the production of three dimensional models by computer graphics. Based on your publications list and my discussions with your staff, I believe your facility's technologies to image cells at high resolution, using computer-facilitated image processing to reconstruct and visualize organelles from a wide range of organisms, should work with the microsporidian spores. The structural details of spore and sporoplasm organelles, obtained by electron microscope tomography, will enable us to finally have a true representation of this important entity. Suggested Methodology I will provide your facility with Epon-Araldite embedded non-activated and activated spore blocks. These will be the same or similar to the material used in our recent publication (Brachiola algerae - Cali et al. 2002). The material in the blocks has been fixed in 2.5 % cacodylate buffered gluteraldehyde (2-4 hours), post fixed in 1% buffered OsO4 , en block stained with UA for 1 hr in 70% ethanol, dehydrated in graded ethanols, transitioned through several rinses of propylene oxide, varying ratios of PO /epon, several transfers in pure resin, and then embedded. If this initial material is inadequate for your needs, I have growing cultures of infected cells in our laboratories and can provide an ample supply of material if necessary. After some experimentation, it was found that when these specimens are sectioned at 250 or 500 nm thickness, post-staining with lead citrate alone gave the best results. In the previous reporting period, two double-tilt and one single-tilt tomographic reconstructions were made. Dr Takvorian visited the RVBC three times, to use the HVEM to evaluate staining protocols, to choose specimens for tomography, and to learn about and discuss visualization methods. In the previous reporting period, Dr. Takvorian visited the resource twice. He gave a seminar on the first visit, and worked with computer visualization on both visits. Sterecon was implemented in the Takvorian lab, so that segmentation of tomograms could be done locally. This helped with the interpretation of the structure observed. The two double-tilt reconstructions from the previous reporting period were re-computed using improved image processing, and surface models of portions of the reconstructions were made, one showing a membranous area in the sporoplasm, another showing extruded membranes, another focusing on polar tubes. Animated movies were made of these models. Images of tomographic slices and surface-rendered models were prepared for presentation.
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