Acta histochemica. Supplementband最新文献

Two proteins from crayfish (Orconectes limosus) retinae bind Ca(2+)-dependently to phospholipid membranes and crossreact with an antibody against calelectrin (annexin IV). These proteins (p48 and p40) are prominent substrates for the protein carboxyl methyl transferase (PCMT) which we propose to be an intracellular protein crosslinking enzyme. Methylation and consequent crosslinking of p40 and p48 to the cytoskeleton or to the plasma membrane seem to be regulated by phosphorylation. In vivo inhibition of the PCMT abolished reversibly the phototransduction in Limulus ventral photoreceptors and solubilized the rhodopsin from the cortical cytoskeleton of the microvilli. We propose a model showing the annexins to regulate the organization of the microvillar cytoskeleton the integrity of which in turn is essential for an unhindered phototransduction.

The purpose of our investigations was to answer the question of whether an increased number of cytoskeletal structures (CSS) in endothelial cells (EC) of the mesenteric terminal vascular bed will be seen in the early phase of a traumatic haemorrhagic shock (THS). A THS was induced in 32 adult R-rats according to a standardized model (fracture of right heel and withdrawal of one third blood volume through carotid catheter). Eight animals each time were killed 15, 30, 60, and 120 minutes after shock induction. Tissue samples of mesenteric microvessels were taken and flatly embedded in Epon. The mesenterium of 8 animals was used as control. CSS formed in the perinuclear and/or peripheric regions of endothelial cytoplasm were visually evaluated and divided into 3 degrees on the basis of 1,500 micrographs. Results were statistically analyzed. A significantly higher degree of CSS (microfilaments, intermediate filaments and microtubules) in EC was found in all shock groups as compared with the control group. CSS are frequently clearly seen in oedematous and swollen EC as well as in EC rich in organelles. Capillaries with stasis are especially predisposed to CSS. Different factors as changed interaction between vascular content and EC, hypoxia and ischemia as well an increased metabolic activity of EC have to be considered as possible causes.

On the ultrastructural level, the role of Ca2+ and cAMP in the rat spermatogenesis was studied. Using a K-pyroantimonate method for intracellular localization of Ca2+, precipitates were found within (i) the vesicular component of the chromatoid body, (ii) vesicular elements of the trans-Golgi area including coated vesicles, (iii) smooth endoplasmic reticulum vesicles and cisterns accompanying the perinuclear (manchette) microtubules, and (iv) mid-piece mitochondria of germ and Sertoli cells. The results suggest a close relationship between the Ca(2+)-containing smooth endoplasmic reticulum and the control of microtubule assembly within microdomains. Theophylline (a cAMP-phosphodiesterase inhibitor) treatment of rats (96 mg/kg daily i.p. over 5 days) caused a significant increase of (a) the GERL-related coated vesicles and (b) the number of manchette microtubules.

In this report we describe the different forms of motile behavior of individual native microtubules from squid giant axons. The three major types of motile behavior of native microtubules are gliding, fishtailing and circling. Gliding, the type of movement observed most often, is the straight translocation of an unbent microtubule segment. Gliding velocities observed in the population ranged from 0.2 to 0.7 microns/s with an average velocity of 0.45 microns/s. The direction of gliding was random with respect to the surface suggesting that physical features of the surface did not influence the direction of gliding. Microtubules are able to glide over objects on the surface and over each other without changing velocity or direction. These observations prove that gliding can continue under conditions where direct contact of the microtubule with the glass surface is not possible along its entire length. When a frontal segment of a microtubule becomes slowed down or attached to the surface, the microtubule begins to fishtail, a process whereby bends form in the frontal part and propagate rearward. The shapes of a fishtailing microtubule resemble that of a beating flagellum. Microtubules with focal attachment near the tip do not propagate bending waves but assume a spiral or circular shape and rotate horizontally (circling). The frontal end of these microtubules stays or rotates in place as pushing forces from the rear turn the microtubule in a circular pattern. An analysis of these data shows that all forms of motion can be explained by pushing forces due to kinesin acting along the length of the microtubule. In an attempt to transport the kinesin-covered cover glass as if it were a big organelle, microtubules translocate themselves in the opposite direction. We estimated the minimum density of force generating enzymes on the surfaces of our preparations as well as that required to maintain active gliding of microtubules. We concluded that the heads of the surface-bound kinesin molecules must display extreme rotatory freedom in order to explain the observed smoothness and straightness of microtubule motion. Few, but usually at least two molecules of kinesin have to work simultaneously to generate the forms of motility observed.

The kinetics of in-vitro assembly of microtubule protein (MTP) to microtubules (MTs) was followed under various conditions (temperature, GTP, ultrasonic treatment) by dynamic light scattering (DLS) and turbidimetric measurements. The results of both methods roughly coincide, but DLS additionally allows to differentiate between MTs and aggregates and to follow their growth. The complexity of these investigations, however, causes serious restrictions in the interpretation of the DLS data.

The binding and internalization of gold-labelled mistletoe lectin I (ML I), its A (ML I-A) and B (ML I-B) subunits, as well as of an immunotoxin consisting of an monoclonal anti L 1210 antibody and the cytotoxic A chains of ML I, were studied on murine L 1210 leukemia cells by a preembedding electron microscopic technique. We found that receptor-mediated endocytosis differs remarkably between the whole lectin, its subunits, and the immunotoxin. Whereas ML I, its A chain, as well as the immunotoxin are internalized by coated pits/coated vesicles or in combination with uncoated membranes, the B chain is exclusively endocytosed via uncoated deep invaginations of the cell membrane. The endocytosis via clathrin-coated or uncoated vesicles is discussed taking into account the binding and internalization kinetics of ligand-receptor complexes related with the movement by the cytoskeleton.

In order to get insight into the topological relationship of phytochrome and the actin cytoskeleton in Mougeotia, phytochrome was localized by indirect immunofluorescence in fixed protoplasts of Mougeotia with the monoclonal antibody Z-3B1, raised against purified Zea mays phytochrome (Schneider-Poetsch et al 1988, Planta 173, 61-72). So far no detection of phytochrome in the immunoblot was possible by this antibody, in contrast to the detection of actin by the monoclonal anti-actin C4 (Lessard 1988, Cell Motil. Cytoskeleton 10, 349-362). Preliminary results are presented on attempts to enrich plant factors which interfere with the G-/F-actin equilibrium, as probed by the viscometric falling ball assay.

The cytoskeletal proteins from erythrocytes, lymphoid cells, unstimulated and stimulated platelets, HeLa cells, and Ehrlich ascites cells were prepared as Triton X 100 insoluble residues. The pellet was extracted using the Bligh-Dyer procedure. After separation of the lipids by thin-layer chromatography, phospholipids and neutral lipids were estimated and the lipid pattern was compared with the lipid composition of the total cell. The percentage of the lipids associated with the Triton X 100 insoluble pellet ranged between 10 and 50 depending on the lipid and the cell type. Despite of the heterogenous protein composition of the residue in the different cells involving microfilaments and intermediate filaments together with associated proteins and minor components, in all cells sphingomyeline (Sph) and free fatty acids (FA) could be found in outstanding contents. In HeLa cells we found beside the high proportion of Sph a different species pattern of diacyl-, alkylacyl-, and alkenylacyl classes of endogenous diacylglycerol (DG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The discussion involved the data from literature showing lipid associations with all 3 classes of cytoskeletal filaments: microtubules, intermediate filaments, and microfilaments. These results were obtained by histological observation, by in vitro binding studies between cytoskeletal proteins and purified lipids, and--as we have practised--by lipid analysis after extraction of the more or less purified cytoskeleton. Artefacts could not be excluded, but the different lipid pattern in the total cell compared with the cytoskeletal let us assume that the results can not be explained by coprecipitation of micelles or organelle remnants with the Triton X 100 insoluble residue alone. An in vivo association of lipids, mainly of Sph, with F-actin and/or associated proteins might be concluded.

Using an ultrathin-sectioning electron microscopic procedure, no efficient binding of coated vesicles to microtubules (both purified from brain tissue) could be achieved, independently of the presence of microtubule-associated proteins. Addition of the ATP analogue AMP-PNP or the inorganic tripolyphosphate, known to cause tight associations of (uncoated) vesicles to microtubules by means of specific motor proteins, could not enhance the binding efficiency. Moreover, crude preparations of clathrin, the major protein of the coat, did not affect the turbidity course of microtubule assembly. These results were confirmed by electrophoresis indicating that within the preparations of microtubule protein, obtained by temperature-dependent cycles of disassembly/reassembly, constituents of coated vesicles were not present. Beside this, coated vesicles have never been found among microtubules reconstituted in vitro. Vice versa, preparations of coated vesicles were completely free of microtubules. Our results suggest that further proteins should be involved in the binding of coated vesicles to microtubules, if there is indeed a biologically relevant interaction.

The phenotype of the osmotically dependent S. cerevisiae mutant VY1160 is caused by a single chromosomal mutation, termed srb, with pleiotropic effect. Compared with cells of the parental strain S288C, it was shown that the size and surface structure of the mutant cells are changed. The latter are sensitive to elevated cultivation temperatures as well as to hypotonic pressure and mechanical stress. In these cases, specific plasma membrane alteration were revealed by freeze-fracture electron microscopy. The total actin content is only 88% (21.4 micrograms actin/mg protein) of that of S288C cells. Remarkably, the mutant cells contain only 2.2 micrograms F-actin/mg protein, whereas the S288C cells have 10.3 micrograms F-actin/mg protein. Moreover, the level of reduced glutathione is found to be higher in the mutant cells (23.4 nmole/10(10) cells) than in the parental cells (15.2 nmole/10(10) cells). These results implicate that the srb mutation is localized in the actin gene.