Le. Tchakarov et al., LIGHT AND ELECTRON-MICROSCOPIC STUDY OF CHANGES IN THE ORGANIZATION OF THE CORTICAL ACTIN CYTOSKELETON DURING CHROMAFFIN CELL SECRETION, The Journal of histochemistry and cytochemistry, 46(2), 1998, pp. 193-203
Chromaffin cells cultured for 2 days were incubated in the absence or
presence of 10 mu M nicotine for 40 sec. Resting and stimulated cells
were fixed and either prepared for fluorescence microscopy or treated
with Triton X-100 to obtain cytoskeletons for ultrastructural studies.
Electron microscopy of cytoskeletons revealed the presence of polygon
al areas devoid of actin filaments only in nicotinic receptor-stimulat
ed cells. Staining of these cytoskeleton preparations with rhodamine-p
halloidin, a probe for filamentous actin, produced fluorescent pattern
s and three-dimensional images similar to those obtained from resting
or stimulated intact cells prepared directly for fluorescence microsco
py. Moreover, the percentage of stimulated cells showing disrupted cyt
oskeleton at the electron microscopic level was similar to the percent
age of stimulated cells showing patched rhodamine fluorescence at the
fluorescence microscopic level. In addition, cells stimulated with nic
otine for 40 sec showed a fivefold increase in amine output and a sign
ificant decrease in F-actin levels. These results provide the first ul
trastructural evidence for nicotinic receptor-evoked chromaffin cell F
-actin disassembly and show that the rhodamine-phalloidin-unstained ar
eas observed in fluorescence microscopy represent the areas devoid of
filamentous actin observed at the electron microscopic level.