FBS Colloquia No.319Physiological Laboratory

Study of molecular diffusion in the cilia as a living nano-tube structure

　Ciliary structures are widely distributed in biological systems. They express appropriate functions in varieties of regions of animal bodies. In the olfactory system, cilia are the site for signal transduction utilizing an adenylyl cyclase-cAMP pathway. To understand the details of this signal transduction, it is necessary to examine the dynamics of substances in the cilia. However, technical limitation made it difficult to observe the dynamics of substances in living cilia using light-microscope since the diameter of cilia is approximately 100 nm. In our laboratory, we monitor the activities of ciliary channels by using electrophysiological techniques. In addition, we combined laser scanning microscopy (LSM) to generate cytoplasmic cAMP by a photolysis of caged compounds. We concluded that the transduction channels are distributed evenly spanning entire cilia, and interestingly, indicated that molecular diffusions are strictly limited in the cilia.
　In the present work, we further used Ca- indicator Fluo4 to visualize cytoplasmic Ca in real-time in living cilia. We handled weak fluorescence images emitted from submicron compartments in the nanotube structure of the cilia, by surveying the actual condition of raster scanning by LSM, the adjustment of the number of stimuli and speed for excitation and imaging, the stimulus time course associated with image enhancement by sum parameters, the data processing associated with these stimuli. We would like to introduce the data processing involved and the diffusion coefficients of molecules in the cilia obtained as a result of the analysis. Furthermore, our laboratory has recently shown in electrophysiological and fluorescence photometric experiments that the diffusion of intracellular factors involved in signal transduction, such as cAMP and Ca, is extremely slow in the olfactory cilia, and together with degradation and efflux, these molecules do not move beyond a few micrometers in the long axis direction. Physiologically, this limitation of material diffusion is related to the spatial localization of signal transduction and signal amplification and plays a very important role in considering biological information conversion in the structure of the cilia. However, there is no molecular barrier that separates the long-axis direction in the cilia, and the mechanism of the restricted diffusion phenomenon is completely unknown. One hypothesis is that a large number of binding sites for these substances on the inside of the plasma membrane may prevent diffusion. This is because the cilia show a diameter of 100 nm, which is even thinner than a typical biological microtubule, and would be expected to have a higher probability of contacting with the cytoplasmic surface of the plasma membrane during internal molecular trafficking. We, therefore, created a simple digital random-walk model and investigated the mode of diffusion by allowing the diameter of the tube structure to vary while setting the strength of the membrane junctions freely. The results show that when the fiber diameter becomes narrower, the effect of membrane binding sites has a very large effect on mass diffusion, which explains well the phenomenon in the cilia.