Stanley Bullivant

Endothelial Cell Membranes: Polarity of Particles as Seen by Freeze-Fracturing

Freeze-fracturing shows particles within membranes. In plasma membranes of most cells the particles are more strongly bound to the inner half. In unfixed endothelial cells, this polarity is reversed. Glutaraldehyde fixation results in conventional polarity. The reverse polarity may be related to a mechanism for preferential fusion of pinocytotic vesicles with the plasma membrane.

Lens gap junctions and orthogonal arrays are unrelated

Gap junctions are particularly abundant in calf eye lens tibre cells [l-4], and characteristically for isolated lens gap junctions, the connexons are randomly dispersed in the plane of the junctions: thus they differ from isolated mouse liver gap junctions, where the connexons are ordered on a hexagonal lattice [5-91. Exposure of isolated lens junctions to 5 X 10m7 M Ca*+ induces the randomly dispersed connexons to arrange themselves on a hexagonal lattice [lo]. Similar concentrations of intracellular Ca2+ in Chironomus salivary gland cells (applied by micro-injection) cause electrical uncoupling of adjacent cells [ 11 ,I 21. Therefore it has been proposed that the Ca2’-induced hexagonal connexon arrangement of the isolated lens junction represents the high resistance state, when the permeable channels are closed, whilst the initial unordered connexon arrangement characterizes the low resistance (= permeable) configuration [ lo,1 31. Conductivity measurements also have revealed that lowering the intracellular pH increases the electrical resistance between neighbouring Xenopus embryonic cells [ 141. When isolated lens plasma membranes were incubated at pH 6-6.5, orthogonal arrays were observed in freeze-fractured preparations, and by analogy it has been proposed that these orthogonal arrays represent the lens gap junctions in another uncoupled configuration induced by low pH treatment [15]. We have investigated the nature of the orthogonal arrays which copurify with gap junctions (unordered connexon arrangement) using the basic isolation protocol for eye lens fibre junctions designed in [2]. These orthogonal arrays appear to be the same as reported to be formed from gap junctions upon incubating crude lens membrane preparations at pH 6-6.5 [15], since they have essentially the same lattice

Viral RNA Polymerases: Electron Microscopy of Reovirus Reaction Cores

The Kleinschmidt technique has been used to observe reovirus cores that have synthesized messenger RNA. Some individual viral cores probably synthesize all ten messenger RNA molecules in vitro. Each messenger RNA molecule appears to be attached to a different site on the core surface, implying that there are probably a number of different enzymic sites in each core.

Structural and molecular biology of the eye lens membranes

Lens transparency is associated with a unique design in tissue development and architecture. The fiber plasma membrane has domains which link with the cytoskeleton, thus maintaining cell shape. Other membrane regions form processes which interlock adjacent lens fibers, and intercellular junctions contain transmembrane pores which allow passage of metabolites between cells. Much interest has recently focused on the study of lens membrane structure and function, mainly because membrane dysfunction may be associated with cataract formation. This article reviews what is known about the structure of membrane domains, about the identification of domain-specific proteins, and describes current attempts to relate these results to function. Much of the presently available data is controversial, and an attempt will be made to reconcile them in revised models and testable hypotheses.

Freeze-fracturing of biological materials

Abstract The freeze- discussed particularly with respect to the faces exposed by fractures along the plane of membranes. Two distinct faces are seen. One is covered with many small particles and the other is relatively free of particles but may have some small depressions. Plasma membranes seen from the outer aspect exhibit the particle covered face and from the inner aspect the smoother face. The reverse of this holds for membrane bound organelles within the cytoplasm, as would be expected. It appears most likely that these two distinct faces are the complementary ones produced by the fracture. The problem of the path of the fracture plane with respect to membranes is discussed.

An anastomosing septate junction in endothelial cells of the phylum echinodermata

As part of an extensive study of the structure and interrelationships of the various forms of septate junction, several species of the Echinodermata have been investigated using conventional thin-section, lanthanum tracer, and freeze-fracture techniques. In the endothelial tissues of this phylum, a new type of junction has been identified: an anastomosing septate junction. It has the usual 15- to 18-nm spacing between membranes characteristic of a septate junction, and occurs in the same relative position around the apical circumference of cells lining an outside or luminal edge. However, the septa form an anastomosing pleated network, which in tangential view is not unlike that seen in the freeze fracture of tight junctions in vertebrates, though generally it is more extensive. The finding of this structural intermediate between septate and tight junctions adds weight to the idea that both of these junctional types may have the same occluding function.

The connexon order in isolated lens gap junctions.

We have purified plasma membranes from calf eye lens fiber cells and have examined the arrangement of the connexons in the gap junctions by the freeze-fracture method. Lens gap junctions with almost entirely random connexon arrangement were obtained when 10 mM EDTA was present throughout the isolation protocol. However we failed to isolate lens gap junctions in which the connexon order was entirely crystalline: a maximum yield of approximately 50% crystalline gap junctions was achieved by solubilization of lens cytoplasmic matrix proteins in 7 M urea/1 mM Ca2+. Treatment of crude lens plasma membranes with proteinase to remove the matrix material yielded only low levels of crystalline gap junctions upon freeze-fracture. We discuss our results on the connexon order of isolated lens gap junctions with regard to the possible use of these junctions as a model system for the regulation of gap junctions in general.

Molecular portrait of lens gap junction protein MP70

A 70-kDa membrane protein (MP70) is a component of the lens fiber gap junctions. Its membrane topology and its N-terminal sequence are similar to those of the connexin family of proteins. Some features of MP70 containing fiber gap junctions are, however, distinct from gap junctions in other mammalian tissues: (i) Lens connexons form crystalline arrays only after cleavage of junctional proteins in vitro. These hexagonal arrays have a periodicity of 13.6 nm which is significantly larger than the 8- 9-nm spacing of liver and heart gap junctions. (ii) Lens fiber gap junctions dissociate in low concentrations of nonionic detergent and this provides an avenue to purify MP70 directly from a membrane mixture. Isolated MP70 in the form of 17 S structures has an appearance consistent with connexon pairs. (iii) The C-terminal half of MP70 is cleaved in situ by a lens endogenous calcium-dependent protease. The processed from MP38 remains in the membrane and is abundant in the central region of the lens. A testable hypothesis for MP70 function is presented.