Comparative scanning, transmission and atomic force microscopy of the microtubular cytoskeleton in fenestrated liver endothelial cells

Endothelial fenestrae control the exchange of fluids, solutes and particles between the sinusoidal lumen and the microvillous surface of the parenchym al cells. Fenestrae have a critical dimension in the order of 150-200 nm, m aking it necessary to use microscopes with a resolution better than the lig ht microscope. Comparative whole-mount preparations of isolated, purified a nd cultured rat liver sinusoidal endothelial cells (LEC) were studied by sc anning electron microscopy (SEM), transmission electron microscopy (TEM) an d atomic force microscopy (AFM). Examination of detergent-extracted LEC by SEM and TEM shows an integral cytoskeleton: sieve plates are delineated by a sieve plate-associated cytoskeleton ring and fenestrae by a fenestrae-ass ociated cytoskeleton ring. By using microtubule altering agents we could de monstrate: (1) the architectural role of microtubules in arranging fenestra e, (2) the existence of a population of microtubules resistant against low temperature and colchicine, (3) the ability of LEC to shift the microtubule assembly-disassembly steady state under various conditions, (4) and the ne cessity of an intact microtubular cytoskeleton to support the increase in t he number of fenestrae after cytochalasin B. Topographical examinations of AFM images revealed that sieve plates are delineated by elevated borders, p robably projections of the underlying tubular cytoskeleton.