Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Deep-etch visualization of proteins involved in clathrin assembly

Assembly proteins were extracted from bovine brain clathrin-coated vesicles with 0.5 M Tris and purified by clathrin-Sepharose affinity chromatography, then adsorbed to mica and examined by freeze-etch electron microscopy. The fraction possessing maximal ability to promote clathrin polymerization, termed AP-2, was found to be a tripartite structure composed of a relatively large central mass flanked by two smaller mirror-symmetric appendages. Elastase treatment quantitatively removed the appendages and clipped 35 kD from the molecule's major approximately 105-kD polypeptides, indicating that the appendages are made from portions of these polypeptides. The remaining central masses no longer promote clathrin polymerization, suggesting that the appendages are somehow involved in the clathrin assembly reaction. The central masses are themselves relatively compact and brick-shaped, and are sufficiently large to contain two copies of the molecule's other major polypeptides (16- and 50-kD), as well as two copies of the approximately 70-kD protease-resistant portions of the major approximately 105-kD polypeptides. Thus the native molecule seems to be a dimeric, bilaterally symmetrical entity. Direct visualization of AP-2 binding to clathrin was accomplished by preparing mixtures of the two molecules in buffers that marginally inhibit AP-2 aggregation and cage assembly. This revealed numerous examples of AP-2 molecules binding to the so-called terminal domains of clathrin triskelions, consistent with earlier electron microscopic evidence that in fully assembled cages, the AP's attach centrally to inwardly-directed terminal domains of the clathrin molecule. This would place AP-2s between the clathrin coat and the enclosed membrane in whole coated vesicles. AP-2s linked to the membrane were also visualized by enzymatically removing the clathrin from brain coated vesicles, using purified 70 kD, uncoating ATPase plus ATP. This revealed several brick-shaped molecules attached to the vesicle membrane by short stalks. The exact stoichiometry of APs to clathrin in such vesicles, before and after uncoating, remains to be determined.     

Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation

Two seemingly unrelated experimental treatments inhibit receptor mediated endocytosis: (a) depletion of intracellular K+ (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson. 1983. Cell. 33:273-285); and (b) treatment with hypertonic media (Daukas, G., and S. H. Zigmond. 1985. J. Cell Biol. 101:1673-1679). Since the former inhibits the formation of clathrin-coated pits (Larkin, J. M., W. D. Donzell, and R. G. W. Anderson, 1986. J. Cell Biol. 103:2619-2627), we were interested in determining whether hypertonic treatment has the same effect, and if so, why. Fibroblasts (human or chicken) were incubated in normal saline made hypertonic with 0.45 M sucrose, then broken open by sonication and freeze-etched to generate replicas of their inner membrane surfaces. Whereas untreated cells display typical geodesic lattices of clathrin under each coated pit, hypertonic cells display in addition a number of empty clathrin "microcages". At first, these appear around the edges of normal coated pit lattices. With further time in hypertonic medium, however, normal lattices largely disappear and are replaced by accumulations of microcages. Concomitantly, low density lipoprotein (LDL) receptors lose their normal clustered distribution and become dispersed all over the cell surface, as seen by fluorescence microscopy and freeze-etch electron microscopy of LDL attached to the cell surface. Upon return to normal medium at 37 degrees C, these changes promptly reverse. Within 2 min, small clusters of LDL reappear on the surfaces of cells and normal clathrin lattices begin to reappear inside; the size and number of these receptor/clathrin complexes returns to normal over the next 10 min. Thus, in spite of their seeming unrelatedness, both K+ depletion and hypertonic treatment cause coated pits to disappear, and both induce abnormal clathrin polymerization into empty microcages. This suggests that in both cases, an abnormal formation of microcages inhibits endocytosis by rendering clathrin unavailable for assembly into normal coated pits.     

3,4-Dehydroproline inhibits cell wall assembly and cell division in tobacco protoplasts.

We investigated the function of cell wall hydroxyproline-rich glycoproteins by observing the effects of a selective inhibitor of prolyl hydroxylase, 3,4-dehydro-L-proline (Dhp), on wall regeneration by Nicotiana tabacum mesophyll cell protoplasts. Protoplasts treated with micromolar concentrations of Dhp do not develop osmotic stability and do not initiate mitosis. The architecture of regenerated cell walls was examined using deep-etch, freeze-fracture electron microscopy of rapidly frozen tobacco cells. Untreated protoplasts assemble a dense fibrillar cell wall consisting of laterally associating subelementary fibrils. In contrast, treatment of protoplasts with Dhp alters the structure of the regenerated wall fibrils in several ways: first, the microfibrils are coated with globular knobs; second, some larger fiber bundles have an open ribbon-like appearance; and third, the smallest subelementary fibrils were not visible. Tobacco cells develop an abnormal morphology as a consequence of this abnormal cell wall structure. Thus, inhibition of prolyl hydroxylase results in the regeneration of a cell wall with abnormal structural and functional properties. These data provide experimental evidence that hydroxyproline-rich glycoproteins are important for the structural integrity of primary cell walls and for the correct assembly of other wall polymers, and that wall structure is an important regulator of cell division and cell morphology.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

The inside and outside of gap-junction membranes visualized by deep etching

We have viewed the membrane specializations that occur at gap junctions from the inside and outside of cells in replicas of quick-frozen and deep-etched samples. Gap junctions were split to expose the normally apposed outside surfaces of their membranes, which displayed uniform 8-9 nm protrusions with central pores. Such pores were also observed in the protoplasmic-face particles of freeze-fractured gap junctions, even after the junctions were induced to crystallize by treatment with metabolic inhibitors or by homogenization. Crystallized junctions have been shown to be in the closed, high-resistance state; hence the channel-closing mechanism must not be located in the regions viewed so far. In washed-out broken cells, the inner surfaces of gap junctions possess smooth surfaces with no visible pores. These surfaces are devoid of special undercoatings of cytoskeletal elements, suggesting that crystallization observed during uncoupling is an intramembrane phenomenon. Hypertonicity, in itself, may produce the same sort of hexagonal crystallization of gap-junction components that is usually observed after uncoupling.     

Membrane fusion during secretion: cortical granule exocytosis in sex urchin eggs as studied by quick-freezing and freeze-fracture

Exocytosis of cortical granules was observed in sea urchin eggs, either quick-frozen or chemically fixed after exposure to sperm. Fertilization produced a wave of exocytosis that began within 20 s and swept across the egg surface in the following 30 s. The front of this wave was marked by fusion of single granules at well-separated sites. Toward the rear of the wave, granule fusion became so abundant that the egg surface left with confluent patches of granule membrane. The resulting redundancy of the egg surface was accommodated by elaboration of characteristic branching microvilli, and by an intense burst of coated vesicle formation at approximately 2 min after insemination. Freeze-fracture replicas of eggs fixed with glutaraldehyde and soaked in glycerol before freezing displayed forms of granule membrane interaction with the plasma membrane which looked like what other investigators have considered to be intermediates in exocytosis. These were small disks of membrane contact or membrane fusion, which often occurred in multiple sites on one granule and also between adjacent granules. However, such membrane interactions were never found in eggs that were quick-frozen fixation, or in eggs fixed and frozen without exposure to glycerol. Glycerination of fixed material appeared to be the important variable; more concentrated glycerol produced a greater abundance of such "intermediates." Thus, these structures may be artifacts produced by dehydrating chemically fixed membranes, and may not be directly relevant to the mechanism by which membranes naturally fuse.     

Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry

The structure of rat liver vault ribonucleoprotein particles was examined using several different staining techniques in conjunction with EM and digestion with hydrolytic enzymes. Quantitative scanning transmission EM demonstrates that each vault particle has a total mass of 12.9 +/- 1 MD and contains two centers of mass, suggesting that each vault particle is a dimer. Freeze-etch reveals that each vault opens into delicate flower-like structures, in which eight rectangular petals are joined to a central ring, each by a thin hook. Vaults examined by negative stain and conventional transmission EM (CTEM) also reveal the flower-like structure. Trypsin treatment of vaults resulted exclusively in cleavage of the major vault protein (p104) and concurrently alters their structure as revealed by negative stain/CTEM, consistent with a localization of p104 to the flower petals. We propose a structural model that predicts the stoichiometry of vault proteins and RNA, defines vault dimer-monomer interactions, and describes two possible modes for unfolding of vaults into flowers. These highly dynamic structural variations are likely to play a role in vault function.