Joseph M. Corless

The direct measurement of temperature changes within freeze-fracture specimens during rapid quenching in liquid coolants

We have evaluated the cooling rates of specimens mounted in a variety of freeze‐fracture holders when plunged into a series of liquid coolants. These rates were measured using miniature thermocouples placed within the mounted specimens. The most rapid cooling rates were obtained using propane at 83 K as the coolant. When mounted on a newly devised ‘copper sandwich’ holder, specimen cooling rates in excess of 4500 K/s have been recorded.

On the asymmetry of frog retinal rod outer segment disk membranes

Abstract Freeze-fracture studies on intact frog rod outer segments have been carried out using a device designed to minimize surface contamination and inadvertent specimen etching. When fractured and shadowed at −170°C, each double-membrane disk gives rise to two membrane fracture faces. In replicas with particularly favorable directions of heavy metal deposition, it is possible to infer the axial spacings between the membrane fracture faces by analyzing the widths of the oblique fracture jumps. The distance of each membrane fracture plane from the center of the disk can then be determined from these axial spacings and the structural centrosymmetry of the disk. Our analysis indicates that each membrane fracture plane is located at a distance of ∼47 A from the coordinate origin at the center of the disk. This coordinate corresponds in position to the electron density minimum of the membrane. Our results therefore support the original hypothesis by Clark and Branton (1968) that disk membranes fracture along some interior hydrophobic plane, presumably the region of terminal methyl groups at the center of the lipid bilayer. Fracture face A is characterized by a rough, particulate appearance, and represents the hydrophobic face of that half of the disk membrane adjacent to the cytoplasmic matrix. Face B is smooth, and represents the hydrophobic face of that half of the membrane adjacent to the intradisk compartment. When these membrane fracture faces are compared with the characteristic textures observed in pure lipid bilayers and in rhodopsin-lipid recombinant membranes, it appears that in frozen disk membranes rhodopsin is restricted to the cytoplasmic half of the membrane. The smooth B face has the appearance of a lipid monolayer. Although the A face particles have a rather low surface relief, it is still possible to estimate their apparent size and shape. These particles are roughly circular; the range of diameters is about 120–140 A, when measured in the projected direction of heavy metal shadowing. Optical diffraction patterns of the A face show broad intensity bands centered at ± ( ∼ 1 130 A ). When examining the published equatorial X-ray diffraction data from frog outer segments, it is apparent that there is no broad band of intensity within the interval ( 1 150 A ) to ( 1 70 A ), corresponding to the optical diffraction data. Accordingly, it must be postulated that at physiological temperatures, the distribution of matter within the plane of the disk membrane does not correspond to the distribution evident in the A fracture face. Hence, the A face particles appear to represent artifactual, derivative phenomena. An explanation in terms of lateral phase separations of disk membrane components is developed. The consequences of such an hypothesis for the etching behavior of the B face are derived in the context of those parameters which are known to affect the passage of water molecules across lipid monolayers.

Paracrystalline inclusions associated with the disk membranes of frog retinal rod outer segments

Abstract Freeze-fracture replicas were prepared from freshly isolated, ultra-rapidly frozen frog retinal rod outer segments (ROSs), which were neither fixed nor exposed to antifreeze agents prior to freezing. Embedded within the lamellar array of ROS disk membranes, a series of rare paracrystalline inclusions were observed which displayed both irregular and cylindrical geometries. These inclusions appear to be formed from elongated lipid cylinders similar to those present in hexagonal (HII) lipid phases. In thin sections through the ROS, corresponding images have not been detected. However, lacunae with similar shapes and dimensions have been found. We believe these paracrystalline inclusions provide the first case in which contiguous bilayer and non-bilayer organizations of lipid have been visualized within a naturally occurring membrane system. The inclusions illustrate a unique and unpredicted mode of structural interaction between several adjacent ROS disks. The absence of freeze-fracture particles within such inclusions further suggests that only the lipid phases of these disks may be structurally continuous. Although the functional significance of these inclusions is unknown, they may have relevance to the enhanced rates of lipid redistribution and turnover in ROSs, compared to those of disk membrane proteins.

Structural interpretation of the birefringence gradient in retinal rod outer segments

The birefringence of frog retinal rod outer segments is analyzed in terms of a three-dielectric layer model. The possibility that the birefringence gradient found in such cells is due to changes in the disk membrane-pair spacing is investigated using previously published glycerol imbibition data (Kaplan et al., 1978. Biophys. J. 23: 59-70). The higher net birefringence of the basal end compared to the midpoint of rod outer segments can be accounted for by a smaller negative form birefringence term due to either a smaller or larger intradiskal space, depending upon the assumed relative solids contents of the intradiskal and cytoplasmic spaces.

Orientation of acidic polysaccharides and rhodopsin-oligosaccharides in frog retinal rod outer segments

SummaryUsing topo-optical staining reactions, the presence and molecular order of three structural components of outer segments of frog retina were studied. These components included (1) an acidic polysaccharide texture, (2) free aldehyde groups which arise during formalin fixation and (3) the oligosaccharide chains of rhodopsin. Quantitative measurements of the dye binding and birefringence effects arising from the individual structural components in rod outer segments were made. Results indicated that all three structural components had a rather well-defined orientation within the ROS.The spherulites phagocytized from the apical ends of ROSs by the pigment epithelium also demonstrate preferred orientation of the three structural components investigated.

Three-dimensional membrane crystals in amphibian cone outer segments: 2. Crystal type associated with the saddle point regions of cone disks.

In light-adapted, perfusion fixed retinas of the Congo eel salamander, Amphiuma, we have observed distinctive 3-D crystalline domains within the axial array of cone outer segment disks. These crystalline domains, each involving 2-12 disks, have been observed in the distal half of cone outer segment, and are associated with saddle point regions and immediately adjacent segments of the cone disk perimeter. In longitudinal sections, the crystals typically display an axially oriented array of cytoplasmic filaments with lateral spacings in the range of 12-13 nm. The width of the intradiskal compartment is expanded to 8-10 nm within the crystal, and approximates the width of the cytoplasmic compartment. In some sections, the cytoplasmic filaments are axially aligned with intradiskal filaments of similar length and width. In transverse sections, the projected lattice appears to be approximately rectangular, with unit cell dimensions of approximately 12 nm x 12.5 nm. In shape, orientation, dimension, location, projection symmetry and associated membrane spacing relationships, the cone outer segment crystal filaments share a strong resemblance with lattice filaments located along the perimeters and preincisures of rod disks. These similarities suggest that the cone crystal filaments may be related to the rim protein of rod disks. Lastly, the preferential association of these crystals with saddle point regions indirectly supports the hypothesis that reductions in COS disk area with apical displacement are accomplished by resorption of disk membrane components through the saddle points.

Cone Outer Segments: A Biophysical Model of Membrane Dynamics, Shape Retention, and Lamella Formation

An hypothesis is developed to explain how the unique, right circular conical geometry of cone outer segments (COSs) in Xenopus laevis and other lower vertebrates is maintained during the cycle of axial shortening by apical phagocytosis and axial elongation via the addition of new basal lamellae. Extension of a new basal evagination (BE) applies radial (lateral) traction to membrane and cytoplasmic domains, achieving two coupled effects. 1), The bilayer domain is locally stretched/dilated, creating an entropic driving force that draws membrane components into the BE from the COSs distributed bilayer phase, i.e., plasmalemma and older lamellae (membrane recycling). Membrane proteins, e.g., opsins, are carried passively in this advective, bilayer-driven process. 2), With BE stretching, hydrostatic pressure within the BE cytoplasm is reduced slightly with respect to that of the axonemal cytoplasmic reservoir, allowing cytoplasmic flow into the BE. Attendant lowering of the reservoirs hydrostatic pressure facilitates the subsequent transfer of cytoplasm from lamellar domains to the reservoir (cytoplasmic recycling). The geometry of the BE reflects the membrane/cytoplasm ratio needed for its construction, and essentially specifies the ratio of components recycled from older lamellae. Length and taper angle of the COS reflect the ratio of recycled/new components constructing a new BE. The model also integrates the trajectories and dynamics of lamella open margin lattice components. Although not fully evaluated, the initial model has been assessed against the relevant literature, and three experimental predictions are derived.

Scanning interferometry of sunfish cones. I. Longitudinal variation in single-cone refractive index

Interferometric measurements of cone inner segment–cone outer segments (CIS–COS’s) isolated from the green sunfish (Lepomis cyanellus) were made with a He–Ne laser focused to a 0.3-μm spot. Multiple measurements were obtained by moving the spot across the CIS–COS’s in a raster pattern; adjacent measurements were spaced approximately 0.2 μm apart. The CIS–COS’s were oriented with their longitudinal (z) axes parallel to one of the raster directions. By assuming that the CIS–COS’s have circular cross sections in the orthogonal (x−y) planes, we calculated from the interferometric measurements average refractive indices as a function of z. In the cone inner segments these average refractive indices rise monotonically with z in the direction along which light travels through the living photoreceptor. The average refractive index was typically between 1.36 and 1.39 at the base of the photoreceptor and between 1.42 and 1.44 at its junction with the outer segment. These findings confirm previous qualitative assessments of density variations evident in electron micrographs [ Rowe et al., J. Opt. Soc. Am. A11, 55 ( 1994)].

[80] Isolation, rapid freezing, and freeze-fracture methods for frog retinal photoreceptors

Publisher Summary This chapter discusses the isolation, rapid freezing, and freeze-fracture methods for frog retinal photoreceptors. The freeze-fracture technique involves four essential steps: (1) rapid freezing of the specimen; (2) specimen fracture; (3) heavy metal replication of the exposed fracture surfaces; followed by deposition of a reinforcing film such as carbon; and (4) recovery of the replica, which is then examined in a transmission or scanning electron microscope. Freeze-fracture methods are employed in morphological studies when one wishes to preserve a structure in a state that most closely approximates its native configuration. Fixation of the structure is achieved by specimen freezing. Specimen preparation is primarily dictated by experimental objective. Specimen freezing rates are determined by the mass, geometry, and material properties of the sample and supporting planchette and by the freezing method employed.

Simple and inexpensive fabrication of small-volume density gradients.

Abstract Conventional large-volume gradient makers can be used in conjunction with peristaltic pumps to produce density gradients with total volumes as small as desired. The described approach involves continuous sampling of the large-volume gradient at a specific rate.