Organelle transport in melanophores analyzed by white light image correlation spectroscopy.

Intracellular transport of organelles, vesicles and proteins is crucial in all eukaryotic cells, and is accomplished by motor proteins that move along cytoskeletal filaments. A widely used model of intracellular transport is Xenopus laevis melanophores. These cells help the frog to change color by redistributing melanin-containing organelles in the cytoplasm. The high contrast of the pigment organelles permits changes in distribution to be observed by ordinary light microscopy; other intracellular transport systems often require fluorescence labeling. Here we have developed white light Image Correlation Spectroscopy (ICS) to monitor aggregation and dispersion of pigment. Hitherto in ICS, images of fluorescent particles from Confocal Laser Scanning Microscopy (CLSM) have been used to calculate autocorrelation functions from which the density can be obtained. In the present study we show that ICS can be modified to enable analysis of light-microscopy images; it can be used to monitor pigment aggregation and dispersion, and distinguish between different stimuli. This new approach makes ICS applicable not only to fluorescent but also to black-and-white images from light or electron microscopy, and is thus very versatile in different studies of movement of particles on the membrane or in the cytoplasm of cells without potentially harmful fluorescence labeling and activation.

Height changes associated with pigment aggregation in Xenopus laevis melanophores.

Melanophores are pigment cells found in the skin of lower vertebrates. The brownish-black pigment melanin is stored in organelles called melanosomes. In response to different stimuli, the cells can redistribute the melanosomes, and thereby change colour. During melanosome aggregation, a height increase has been observed in fish and frog melanophores across the cell centre. The mechanism by which the cell increases its height is unknown. Changes in cell shape can alter the electrical properties of the cell, and thereby be detected in impedance measurements. We have in earlier studies of Xenopus laevis melanophores shown that pigment aggregation can be revealed as impedance changes, and therefore we were interested in investigating the height changes associated with pigment aggregation further. Accordingly, we quantified the changes in cell height by performing vertical sectioning with confocal microscopy. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that the elevation of plasma membrane is caused by local swelling due to influx of water through HgCl2-sensitive aquaporins. We also measured the height of the microtubule structures to assess whether they are involved in the height increase. Our results show that pigment aggregation in X. laevis melanophores resulted in a significant height increase, which was substantially larger when aggregation was induced by latrunculin than with melatonin. Moreover, the elevation of the plasma membrane did not correlate with influx of water through aquaporins or formation of new microtubules, Rather, the accumulation of granules seemed to drive the change in cell height.