This subproject is one of many research subprojects utilizing the resources provided by a Shared Instrumentation Grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the grant, which is not necessarily the institution for the investigator. DESCRIPTION (provided by applicant): This is a request for the upright epifluorescence light microscope Leica workstation DM 6000B with incubation chamber. The microscope set-up is needed for live cell studies of epithelial cell monolayers with dual-sided perfusion. The microscope is requested for a group of investigators concerned with investigating the dynamics and polarity of cell signaling, gene therapy, transport, and cellular organization of differentiated epithelial cells. It is well recognized by now that hormonal or growth factor regulation can occur not only from the blood, but also the luminal side in polarized cells. Similarly, flow on the luminal side affects cellular functions. Investigations of the dynamics and spatial organization of these regulatory processes is crucial for progress in understanding normal and pathological behavior of epithelial cells. Advances in cell culture methods have allowed us to establish in-vitro models for many different types of epithelia. To obtain highly differentiated cells and mimic the in-vivo situation, cells are grown on permeable support to form polarized, confluent, differentiated epithelial cell layers in which the dynamics of different cellular processes can be studied by fluorescence microscopy. Such epithelia are perfused with different solutions and/or rates on apical and basal side to mimic physiological conditions, i.e., nutrients, oxygen and certain regulatory agents on the basal (blood) side and other regulatory agents and flow conditions on the apical (luminal) side. In particular, we will study in live epithelia: flow dependence of receptor trafficking and signaling (renal proximal tubule cells); the intracellular fate of condensed polylysine/DNA complexes used for gene therapy (pulmonary epithelia); spatial regulation of EOF receptor signaling from wild type and polycystic kidney diseased mice (renal collecting duct cells), interactions of SI00 proteins during differentiation (epidermis), regulation of luminal pH (epithelia of the female reproductive tract), and the force required for cilia deflection (renal epithelial cells). These studies require a state-of-the-art upright epifluorescence light microscope workstation with temperature and atmospheric control for live cells and an optical chamber with dual perfusion and water immersion optics with high numerical aperture.
