Kinetics Of Intracellular And Cell-To-Cell Diffusion Of Fluorescent Tracers

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Physiology and Biophysics


This study analyzes in detail the kinetics of diffusion of fluorescent tracers within and among cells of the Chironomus salivary gland, and investigates changes in the cell-to-cell diffusion kinetics in response to changes in membrane potential. Tracers of different size and fluorescence characteristics were injected simultaneously into a cell, and the resulting fluorescence changes in this and a neighboring cell were measured using dual-wavelength microfluorometry. Compartment analysis of the fluorescence data yielded relative rate constants for intracellular tracer diffusion (k(,C)), cell-to-cell tracer diffusion (k(,J)), loss of tracer (or fluorescence) from the cells (k(,L)), and apparent tracer sequestration (k(,S)). This analysis allowed comparison of membrane potential effects on the junctional permeability to tracers of different size, and to small inorganic ions (measured as the electrical conductance of a junction), in order to determine if changes in junctional permeability are accompanied by changes in selectivity.Intracellular tracer diffusion was found to last tens of seconds, several times slower than diffusion in agar, and to differ considerably for different tracers. Cell-to-cell tracer diffusion within healthy glands occurred with time constants ranging from one or two minutes for a small tracer (carboxyfluorescein, M.W. 376) to about forty minutes for a large fluorescein-labelled sugar (M.W. 2327). The time constants for loss and apparent sequestration of tracers were often similar to those for cell-to-cell tracer diffusion.Cell membrane depolarization elicited a reversible reduction in cell-to-cell tracer diffusion which paralleled a reduction in junctional conductance. Compartment analysis suggests that such changes were non-selective: changes in the diffusion kinetics of large and small tracer molecules could be simulated by the same fractional change in k(,J) for both; and when junctional conductance was changed by a factor of two, a change in accompanying cell-to-cell tracer diffusion could be simulated by the same fractional change in K(,J). The simplest explanation for these results is that the channels that mediate cell-to-cell diffusion typically open and close in an all-or-none manner.


Biology, Animal Physiology

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