Ultrastructural studies of hepatocyte cytoskeletons of phalloidin-treated rats by quick-freezing and deep-etching method

Abstract

We observed hepatocyte cytoskeletons in phalloidin-treated rats by the quick-freezing and deep-etching method in three dimensions and compared them with the ultrastructural findings on conventional ultrathin sections. The numbers of microvilli in dilated bile canaliculi were decreased in the rats treated with phalloidin for 1 wk. In hepatocytes the cytoplasm around bile canaliculi could be divided into three layers, increased microfilament layer, cell organelle layer of secretory system and increased smooth surface endoplasmic reticulum layer. In the rats treated with phalloidin for 4 wk, microfilaments were extended into the cytoplasm near the nucleus in addition to the increased number of large lysosomes and microtubules. In both groups, three-dimensional structures of microfilaments could be visualized around bile canaliculi and along cell borders by the quick-freezing and deep-etching method. The branching microfilaments with the diameters of 7 to 10 nm were directly attached to other filaments, cell organelles or cytoplasmic sides of cell membranes. Moreover, bundled intermediate filaments were increased around peribiliary microfilaments associated with long-term cholestasis. It is suggested that excessive accumulation of peribiliary microfilaments disturb the secretion of bile components into bile canaliculi. The cytoskeletal reorganizations of intermediate filaments seem to alter the arrangements of various cell organelles.

Different organization of intermediate filaments in columnar cells of rat large intestinal mucosa as revealed by confocal laser scanning microscopy and quick-freezing and deep-etching method

“…They were then quickly frozen by the metal contact method using a quickfreezing machine (JEOL JFD-RFA, Japan), in which the copper metal is cooled in liquid nitrogen (-196°C). They were fractured in the liquid nitrogen with a scalpel to expose well-preserved surface areas [27,28] and transferred into an EIKO FD-3AS etching machine (Eiko Company, Ibaragi, Japan). They were deeply etched under vacuum conditions (1-4x10 7 Torr) at -95°C for 10-20 min and rotary shadowed with platinum at the angle of 30°and with carbon at the angle of 90 °.…”

“…CLSM also offers improved elimination of out-of-focus noise and greater resolution than conventional fluorescence microscopy [33]. The quick-freezing and deepetching (QFDE) method is a morphological technique by which the ultrastructure of cytoskeletons and cell organelles in cells and tissues can be observed three-dimensionally at high resolution [1,15,20,21,27,28]. It is well known that the cytoskeleton cannot be clearly observed using transmission electron microscopy with conventional Epon-embedded specimens, because the plastic embedding material interferes with its visualization.…”

Changes in the localization of the rat canalicular conjugate export pump mrp2 in phalloidin-induced cholestasis

“…Failure of recycling vesicles or transport vesicles carrying newly formed proteins to reach the canalicular membrane after phalloidin poisoning has been suggested as a mechanism in chronic phalloidin-induced cholestasis. 46,47 However, this possibility cannot explain the described intracellular accumulation of canalicular proteins in acute phalloidin poisoning for the following reasons: (1) vacuoles do not accumulate with time around the canaliculi, as would be expected, but deformations begin at the canalicular domain, and vacuoles then spread over the hepatocyte; (2) the time interval (30 minutes after phalloidin treatment) is too short to allow de novo synthesis of most proteins accumulating intracellularly; (3) the canalicular proteins investigated colocalized to the same vacuoles, although recent evidence suggests that not only secretory proteins and membrane proteins 48 but also different apical membrane proteins 49 and (4) studies on acute phalloidin toxicity in isolated rat hepatocyte couplets reported vacuole formation from the bile canalicular region within minutes after addition of phalloidin. 39,40 Thus, in addition to the early impairment of canalicular contractility, a loss of ATP-dependent export pumps from the canalicular domain is involved in the pathogenesis of acute phalloidin-induced cholestasis.…”

Histochemical analyses of living mouse liver under different hemodynamic conditions by “in vivo cryotechnique”

“…However, chemicalWxation often produces artifacts of morphology, because of the relatively long Wxation time (Fraser et al 1980;Kellenberger et al 1992;Hippe-Sanwald 1993;Chan and Inoue 1994;Shiurba 2001). To minimize such artifacts, quick-freezing (QF) methods have often been used, especially for analyses at the ultrastructural level (Naramoto et al 1991;Furuta et al 1992a, b;Fujimoto 1995). In the QF methods, liver tissues of living animals are perfused or resected before QF, and so there is inevitably morphological modiWcation due to perfusion or loss of blood supply (van Harreveld and Biber 1962;von Zglinicki et al 1986).…”