Arrest of pigment granule motion in erythrophores by quick-freezing

We report the use of quick-freezing, as an alternative to conventional chemical fixation, to arrest the movement of pigment granules at various stages of the dispersion-aggregation cycle in Holocentrus erythrophores. During pigment aggregation, the granules in these cells move at up to 20 μm/sec, hence the structural changes underlying the movement are likely to be too fleeting to be captured faithfully by conventional aldehyde fixation. On the other hand, quick-frozen cells, when examined by freeze-etch electron microscopy, provide novel views of certain cytoplasmic components which appear to be involved in pigment granule movement, namely, fine (2- to 6-nm diameter) fibrils which link the granules to each other and to the radial array of microtubules. These fine crosslinking fibrils can be distinguished from thicker (8- to 15-nm diameter) strands of coherent granular material which pervade the cytoplasm of pigment-dispersed as well as pigment-aggregated cells. This granular matrix is removed by detergent permeabilization, after which it becomes apparent that the fine fibrils are insoluble and are distributed both within and distal to the aggregated pigment mass. The diameter of the specific fibrils does not change during pigment motion, which indicates that they are not contractile.