The goal of the proposed research is to relate solute dynamics in intracellular aqueous compartments to biological function. A central component of this research is the continued development of optical techniques to measure solute dynamics in living cells. The biological experiments address fundamental questions about solute dynamics in cell cytoplasm, the nucleus, and mitochondria.
Aim 1 : To continue the development of novel optical techniques to examine solute dynamics in intracellular aqueous compartments. Effort will be focused on the continued development and biological application of existing methods: a. Steady-state anisotropy imaging, b. Time- resolved microfluorimetry, c. Fluorescence recovery after photobleaching, and d. Total internal reflection microfluorimetry; and the development of two new methods: e. Polarization-dependent photobleaching recovery, and f. Continuous 3-dimensional tracking of single labeled particles. These experimental methods are essential to address the biological questions below.
Aim 2 : To characterize and model the translational and rotational dynamics of solute molecules in cell cytoplasm. The goal is to construct an accurate theory/model of the dynamics of solute molecules of arbitrary size in cell cytoplasm. The """"""""sieving"""""""" properties of cytoplasm will be evaluated from the translational and rotational dynamics of a series of fluorescently labeled solutes of different sizes. For large solutes, """"""""net"""""""" translation measured by photobleaching recovery will be modeled in terms of the dynamics of individual particles measured by a novel single particle tracking method. The hypothesis that sub-plasma membrane cytoplasmic rheology differs from that in bulk cytoplasm will be tested using total internal reflection methods. Experiments will be carried out on Swiss 3T3 fibroblasts, and a newly developed explant culture of vasopressin-responsive kidney epithelial cells. The hypothesis that the vasopressin-response involves a marked change in sub-apical membrane rheology will be tested.
Aim 3 : To correlate solute dynamics with organelle function in the aqueous compartments within the cell nucleus and mitochondria. Dynamics and organization in the nucleus will be studied using microinjected, fluorescently labeled solutes of various sizes. The hypotheses that the nucleus contains a rigid """"""""scaffolding structure"""""""" and subnuclear domains with differential permeabilities will be tested. Changes in nuclear organization with cell cycle will be used to test whether a modification of nuclear sieving properties is associated with altered DNA mobility during cell division. In mitochondria, the hypothesis will be tested that high concentrations of mitochondrial proteins dramatically hinder the motion of metabolite solutes, leading to enzyme organization and metabolite channeling.
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