Effect of various hepatic membrane fractions on microtubule assembly-with special emphasis on the role of membrane phospholipids.

Abstract

This report describes an interaction between rat brain microtubule protein and various hepatic fractions in vitro . Purified preparations of Golgi membranes, plasma membranes, rough and smooth endoplasmic reticulum, nuclear membranes, and mitochondria were obtained from the livers of 200-g rats . Several concentrations of fresh or sonicated frozen membranes were incubated with twice-cycled rat brain microtubule protein in a microtubule assembly buffer for 60 min at 30°C. Changes in microtubule assembly were assessed either by quantitative electron microscopy on negatively stained samples or by spectrophotometric methods. The results show that all the tested membranes "bound" microtubule protein, preventing assembly : Golgi and plasma membranes, as well as mitochondria, were especially potent in this regard . To identify the membrane-associated components responsible for microtubule protein binding, the membranes were extracted with methanol-chloroform, and liposomes were prepared from the resulting lipids . Microtubule protein incubated with these liposomes showed a differential ability to assemble that was similar to the effect obtained with intact membranes . Membrane-extracted phospholipids were identified as the lipid component responsible for these changes, with the negatively charged phospholipids (cardiolipin and phosphatidylserine) being uniquely active . These findings indicate that hepatic membranes differentially interact with brain microtubule protein ; this interaction may be dependent on membrane phospholipids .In a previous in vivo study (32), we noted a unique association between microtubules and Golgi complexes in rat hepatocytes following colchicine treatment. This led to the suggestion that microtubules may normally aid in maintaining the structural integrity of the cisternal membranes in Golgi complexes and, in so doing, play a crucial, if permissive, role in hepatic lipoprotein secretion. In the present study, we wish to see whether isolated Golgi complexes show any special interactions with microtubule protein in vitro. Accordingly, Golgi complexes and various other hepatic membranes were isolated and incubated with twice-cycled brain microtubule protein . The results show that all liver membranes "bind" microtubule protein, but that some membranes, such as those derived from the Golgi complex and cell surface, are particularly effective in this regard . Subsequent incubation with the lipids of the various membrane preparations show an effect similar to that obtained with the intact membranes themselves and suggests that mem-300 brane phospholipids may play an important role in microtubule protein-membrane interactions . MATERIALS AND METHODS Incubation of Microtubule Protein with Liver MembranesVarious concentrations of fresh, intact, or frozen-sonicated hepatic membranes were preincubated with twice-cycled rat brain microtubule protein (120 pg per incubation vial or 460 kg/ml) for 30 min at 30°C, pH 6.4, in the following buffer : 100 mM 2-[N-morphohno]ethane sulfonic acid [MES]; ...

Phosphatidylinositol 4,5-Bisphosphate Modifies Tubulin Participation in Phospholipase Cβ1Signaling

“…Membrane-or phospholipid-associated tubulin has been reported (Bhattacharyya and Wolff, 1976;Klausner et al, 1981;Kumar et al, 1981;Reaven and Azhar, 1981;Regula et al, 1986;Caron and Berlin, 1987). It appeared that this "membrane" tubulin was similar to the soluble form (Bhattacharyya and Wolff, 1976;Stephens, 1977).…”

“…Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) as well as the second messenger IP 3 can bind to certain PIP 2 -binding protein domains (Ferguson et al, 1995;Kavran et al, 1998), and the negative charge of these molecules was presumed responsible for such interaction. The anionic phospholipid constituents of hepatic membranes have also been reported to account for membrane binding of brain microtubule protein and inhibition of assembly (Reaven and Azhar, 1981). A hydrophobic interaction of tubulin (Andreu, 1986) with the uncharged phospholipid phosphatidylcholine at the lipid phase transition temperature Kumar et al, 1981) has been found as well.…”

“…Although it is not clear how tubulin associates with membranes (a subject of some controversy), it is possible that PIP 2 is involved in the process (Reaven and Azhar, 1981). This was investigated using both membranes prepared from Sf9 cells expressing recombinant m1 muscarinic receptors, G␣q, and PLC␤ 1 (Popova et al, 1997) and membranes from SK-N-SH neuroblastoma cells, which normally Where indicated, PIP 3 , PIP 2 , PIP, PI, PC, PE, and the inositol phosphate IP 3 (final molar concentration of 75 M) were preincubated with tubulin (Tub) as described in Materials and Methods.…”

Morphological evidence that high density lipoproteins are not internalized by steroid-producing cells during in situ organ perfusion.

“…These fragments are sometimes empty but usually have a loose fibrillar interior similar to the microvillus cavities seen in the intact tissue: more certain identification came from the fact that many of these structures still retained the double membranes with fibrillar connections (see arrows) that are seen so clearly in Figs. [8][9][10]. By taking advantage of the characteristic appearance of these membrane fragments, we were able to quantitatively assess their content in the special mitochondrial fraction and their degree of labeling with radioactive HDL (Table III).…”

“…Different sets of ARGs from each sample were developed at weekly intervals until one set proved to have sufficient grains for quantification. At this point, 10 nucleated cells (chosen at random) of each of three blocks per sample were photographed at a low magnification (X 4,000) so as to include an image of the entire cell: such micrographs were printed on 11 X 14 sheets of paper. Each exposed grain that was determined to be associated with the LC in question was delineated with a tightfitting circle: the number of grains varied from -15-30/cell.…”