The ontogeny of Limulus polyphemus (Xiphosura s. str., Euchelicerata) revised: looking "under the skin".

In recent years, methods for investigating the exo-morphology of zoological specimens have seen large improvements. Among new approaches, auto-fluorescence imaging offers possibilities to document specimens under high resolution without introducing additional artifacts as, for example, seen in scanning electron microscopy (SEM) imaging. Additionally, while SEM imaging is restricted to the outer morphology of the current instar, auto-fluorescence imaging can be used to document changes of the outer morphology of the next instar underneath the cuticle of the current instar. Thus, reinvestigating seemingly well known species with these methods may lead to interesting new insights. Here we reinvestigate the late embryonic development of the xiphosuran ("sword tail") Limulus polyphemus, which is often treated as a proxy for early eucheliceratan evolution. In addition to entire specimens, the appendages of the embryos were dissected off and documented separately with composite-autofluorescence microscopy. Based on these data, we can distinguish six developmental stages. These stages do not match exactly the formerly described stages, as these were largely based on SEM investigation. Our stages appear to represent earlier or later phases within what has in other studies been identified as one stage. This finer subdivision is visible as we can see the developing cuticle under the outer cuticle. In comparison to data from fossil xiphosurans, our results and those of other studies on the ontogeny of L. polyphemus point to a derived mode of development in this species, which argues against the idea of L. polyphemus as a "living fossil."

The gnathobasic spine microstructure of recent and Silurian chelicerates and the Cambrian artiopodan Sidneyia: Functional and evolutionary implications.

Gnathobasic spines are located on the protopodal segments of the appendages of various euarthropod taxa, notably chelicerates. Although they are used to crush shells and masticate soft food items, the microstructure of these spines are relatively poorly known in both extant and extinct forms. Here we compare the gnathobasic spine microstructures of the Silurian eurypterid Eurypterus tetragonophthalmus from Estonia and the Cambrian artiopodan Sidneyiainexpectans from Canada with those of the Recent xiphosuran chelicerate Limulus polyphemus to infer potential variations in functional morphology through time. The thickened fibrous exocuticle in L. polyphemus spine tips enables effective prey mastication and shell crushing, while also reducing pressure on nerve endings that fill the spine cavities. The spine cuticle of E. tetragonophthalmus has a laminate structure and lacks the fibrous layers seen in L. polyphemus spines, suggesting that E. tetragonophthalmus may not have been capable of crushing thick shells, but a durophagous habit cannot be precluded. Conversely, the cuticle of S. inexpectans spines has a similar fibrous microstructure to L. polyphemus, suggesting that S. inexpectans was a competent shell crusher. This conclusion is consistent with specimens showing preserved gut contents containing various shelly fragments. The shape and arrangement of the gnathobasic spines is similar for both L. polyphemus and S. inexpectans, with stouter spines in the posterior cephalothoracic or trunk appendages, respectively. This differentiation indicates that crushing occurs posteriorly, while the gnathobases on anterior appendages continue mastication and push food towards and into the mouth. The results of recent phylogenetic analyses that considered both modern and fossil euarthropod clades show that xiphosurans and eurypterids are united as crown-group euchelicerates, with S. inexpectans placed within more basal artiopodan clades. These relationships suggest that gnathobases with thickened fibrous exocuticle, if not homoplasious, may be plesiomorphic for chelicerates and deeper relatives within Arachnomorpha. This study shows that the gnathobasic spine microstructure best adapted for durophagy has remained remarkably constant since the Cambrian.

Zoothamnium duplicatum infestation of cultured horseshoe crabs (Limulus polyphemus).

An outbreak of the sessile peritrich Zoothamnium duplicatum in a pilot, commercial-scale Limulus polyphemus hatchery resulted in the loss of ∼96% (40,000) second/third instar larvae over a 61day period. peritrich growth was heavy, leading to mechanical obstruction of the gills and physical damage. The peritrichs were controlled without resultant loss of juvenile crabs by administering 10ppm chlorine in freshwater for 1h and the addition of aquarium grade sand; a medium into which the crabs could burrow and facilitate cleaning of the carapace. Peritrich identity was confirmed from a partial SSU rDNA contiguous sequence of 1343bp (99.7% similarity to Z. duplicatum).

Crystallographic conformers of actin in a biologically active bundle of filaments.

Actin carries out many of its cellular functions through its filamentous form; thus, understanding the detailed structure of actin filaments is an essential step in achieving a mechanistic understanding of actin function. The acrosomal bundle in the Limulus sperm has been shown to be a quasi-crystalline array with an asymmetric unit composed of a filament with 14 actin-scruin pairs. The bundle in its true discharge state penetrates the jelly coat of the egg. Our previous electron crystallographic reconstruction demonstrated that the actin filament cross-linked by scruin in this acrosomal bundle state deviates significantly from a perfect F-actin helix. In that study, the tertiary structure of each of the 14 actin protomers in the asymmetric unit of the bundle filament was assumed to be constant. In the current study, an actin filament atomic model in the acrosomal bundle has been refined by combining rigid-body docking with multiple actin crystal structures from the Protein Data Bank and constrained energy minimization. Our observation demonstrates that actin protomers adopt different tertiary conformations when they form an actin filament in the bundle. The scruin and bundle packing forces appear to influence the tertiary and quaternary conformations of actin in the filament of this biologically active bundle.

Multiple mechanisms of rhabdom shedding in the lateral eye of Limulus polyphemus.

Rhabdom shedding in horseshoe crab lateral eye photoreceptors was studied with anti-opsin and anti-arrestin immunocytochemistry. Two, possibly three, distinct shedding mechanisms were revealed in animals maintained in natural lighting. Transient rhabdom shedding, triggered by dawn, is a brief, synchronous event that removes up to 10% of the rhabdom membrane. Whorls of rhabdomeral membrane break into vesicles and form compact multivesicular bodies. These debris particles are immunoreactive for opsin and are of a relatively uniform size, averaging approximately 2 microm(2) in area. Transient shedding requires that input from circadian efferent fibers to the retina precedes the light trigger, and cutting the optic nerve blocks efferent input and transient shedding. Light-driven rhabdom shedding is a progressive process. Rhabdomeral membrane is removed by coated vesicles that accumulate into loosely packed multivesicular bodies. These debris particles label with antibodies directed against opsin, arrestin, and adaptin, and they have a large distribution of sizes, averaging almost 6 microm(2) in area and ranging up to 25 microm(2) or more. The amount of rhabdomeral membrane removed by light-driven shedding has seasonal variation and depends on latitude. Light-driven shedding does not require circadian efferent input. A possible third shedding mechanism, light-independent shedding, is observed when transient shedding is blocked either by 48 hours of darkness or by cutting the optic nerve. Small particles, averaging 1.8 microm(2) in area, exhibiting opsin but not arrestin immunoreactivity can then be found in the cytoplasm surrounding the rhabdom. The nature of light-independent shedding is not yet clear.

Protein kinase C-induced disorganization and endocytosis of photosensitive membrane in Limulus ventral photoreceptors.

Protein kinase C (PKC) desensitizes the light response in photoreceptors from the ventral optic nerve of the horseshoe crab Limulus. Photoisomerization of Limulus rhodopsin leads to phosphoinositide hydrolysis, resulting in the production of inositol trisphosphate and diacylglycerol (DAG). Inositol trisphosphate mobilizes intracellular stores of Ca(2+), resulting in photoreceptor excitation in Limulus, while DAG may activate PKC. We investigated whether PKC-mediated desensitization of the photoresponse is accompanied by ultrastructural changes in the rhodopsin-bearing photosensitive membrane (rhabdom) in Limulus ventral photoreceptors. PKC activation by (-)-indolactam V in darkness induces disorganization and swelling of the rhodopsin-containing microvilli and endocytosis of rhabdomeral membrane. The effects of (-)-indolactam V on dark-adapted photoreceptor ultrastructure are reversible, are stereospecific, are blocked by coapplication of PKC inhibitors, and closely match those induced by continuous, bright light. Rhabdom disorganization and endocytosis via PKC activation may, therefore, contribute to desensitization of the light-adapted photoreceptor.

Distal-less expression in embryos of Limulus polyphemus (Chelicerata, Xiphosura) and Lepisma saccharina (Insecta, Zygentoma) suggests a role in the development of mechanoreceptors, chemoreceptors, and the CNS.

The homeobox gene Distal-less (Dll) is well known for its participation in the development of arthropod limbs and their derivatives. Dll activity has been described for all groups of arthropods, but also for molluscs, echinoderms and vertebrates. Generally, Dll participates in the establishment of the proximo-distal-axis and differentiation along this axis. During our investigation of the expression pattern in the silverfish Lepisma saccharina and the horseshoe crab Limulus polyphemus, we found several expressions in late stages which cannot be explained with the "normal" limb-specific function. The antenna, cerci and terminal filament of the silverfish show a striped expression; single cells on the labrum, mandibles, maxillary palps and anal valves are also strongly stained by the Dll antibody. In addition to cell groups in the developing ganglia of the CNS, in the coxal endites and several nerve cells in femur and the trochanter of the prosomal limbs, the whole prosomal shield of Limulus polyphemus is surrounded by Dll-positive cell clusters. Furthermore, the lateral processes of the opisthosoma and the edges of the opisthosomal appendages are Dll positive. To get an indication of the cell fate of these regions, we examined hatched larvae and juvenile stages of both species with the SEM. We found a striking correlation of these Dll-positive areas and different sense organs, especially mechanoreceptors. Since many sense organs in arthropods are situated on the limbs, interpretation of the Dll expression in limbs is problematical. This has critical implications for comparative analysis of Dll expression patterns between arthropods and for the claim of homology between limb-like structures. Furthermore, we discuss the possibility of convergent appendage evolution in various bilaterian groups based on the improvement of spatial sensory resolution.

Localization of a putative inositol 1,4,5-triphosphate receptor in the Limulus granulocyte.

The horseshoe crab (Limulus polyphemus) granulocyte (GR) degranulates upon contact with bacteria and release factors that mediate an immune response. Stimulated cells produce IP3, which binds to receptors (IP3R, M.W.240-300 kD) that function to release stored Ca2+ into the cytoplasm that mediates degranulation. This mechanism is believed to mediate exocytosis in the Limulus GR but IP3R in the GR has not been shown. The present study utilized monoclonal antibody 4C11 and a commercially available anti-IP3R antibody, both of which label amino acids of the N-terminal of all known isoforms. Electron microscopy, immunohistochemistry, SDS-PAGE, and Western blot analysis, which employed the use of the two antibodies, demonstrates that a putative IP3R exists in the: plasma membrane, smooth surfaced vesicles, nucleus and nuclear membrane. We hypothesize that this putative IP3R is involved in mediating the immune response of the Limulus GR.

Three-dimensional structure of the acrosomal filament of Limulus sperm by 400 kV electron cryomicroscopy.

The acrosomal bundle of Limulus sperm was imaged by electron cryomicroscopy, and the three-dimensional structure of a filament computationally isolated from the bundle was determined by helical image reconstruction. The actin model of Holmes was fit into the map, and its interactions with scruin, the actin-binding and cross-linking protein, were studied. Scruin binds to two consecutive actins along the helix via subdomains 1 and 3. These interactions involve helix-loop-beta motifs that are present in both actin subdomains (in different monomers) in positions available for binding by the same scruin molecule as it wraps around the actin. Taking first the structural motif homology and then looking for sequence pattern similarities, a stretch of 12 out of 20 matches in hydrophobicity is found. Scruin itself has been found to have an internal tandem homology.

Role of hemocyte-derived granular components in invertebrate defense.

Figure 2 illustrates an outline of the cellular and humoral defense systems in limulus. On the basis of the knowledge described above, it is suggested that granular components present in L and S granules in the hemocytes play a decisive role in the biological defense for this animal. The isolated L granules contain at least three clotting factors plus coagulogen as the major component. The known anti-LPS factor and a number of additional unknown protein components are also present in the L granules. On the other hand, the isolated S granules contain antimicrobial tachyplesins as the major component, in addition to six unidentified proteins. We speculate that the L-granule-derived protein components, which probably contain all the factors essential for the Limulus clotting system participate, in immobilizing invading microbes, and that the S-granule-derived tachyplesins contribute to a self-defense system against invaders. Although we have not mentioned hemolymph plasma components, there are many humoral factors, such as proteinase inhibitors, alpha 2-macroglobulin, various lectins, C-reactive protein, and polyphemin, all of which are important for antimicrobial defense. Furthermore, Liu and colleagues have reported several endotoxin-binding proteins and a cell-adhesion protein found in the Limulus hemocytes. Although the exact functions of these substances are unknown, they may act in concert with other components to provide biological defense for the animal. Nevertheless, compared to our knowledge of mammalian blood cells, much less remains to be learned of biological/physiological events in horseshoe crab hemocytes.

The visual fields of American horseshoe crabs: two different eye shapes in Limulus polyphemus.

The optical alignment of individual cuticular cones in the dioptric array of the lateral eye of Limulus polyphemus was determined with a precision two-circle goniometer constructed and mounted to the stage of a compound microscope and using a new formaldehyde-induced fluorescence procedure. All measurements were made from the corneal surface of the excised eye mounted in seawater through an air/water interface perpendicular to the optic axis of the microscope. Our results revealed two variants of visual field and eye curvature which can actually be discriminated in casual examination of adult animals. We call animals possessing these two variants "morlocks" and "eloi." Adult male and female morlocks about 25 cm across the carapace have eyes which are relatively elongated, often darker in pigmentation, smaller, and relatively flatter in curvature. Morlocks have a monocular field of view of about 3.13 steradians or 50% of a hemisphere. The coverage averages 115 deg along the vertical axis and 168 deg along the horizontal axis of the eye, with maximum resolution in the anteroventral quadrant. Adult male and female eloi of comparable size have eyes which are relatively more round, often lighter in pigmentation, larger with more ommatidia, and relatively more bulged. Eloi have a monocular field of view of approximately 3.83 steradians or 61% of a hemisphere that covers 145 deg vertically and 185 deg horizontally. Eloi have more uniform resolution than morlocks with best resolution in the posteroventral quadrant. All horseshoe crabs examined, whether morlocks or eloi, have an identical orientation of the margin of the eye relative to the animals' coordinates.

Three-dimensional organization of endoplasmic reticulum in the ventral photoreceptors of Limulus.

Living Limulus ventral photoreceptor cells were injected with long chain lipophilic carbocyanine fluorescent dyes to label the endoplasmic reticulum (ER). The purpose of this study was to examine the continuity, dynamic changes, and structure of the ER in the living cell, using laser scanning confocal microscopy and three-dimensional image reconstruction. In this highly polarized neuron, three lines of evidence indicate that the ER is a continuous network extending throughout both lobes of the cell. First, injection of DiO or DiI results in the labeling of ER throughout both lobes of the cell. Second, three-dimensional image reconstruction of the optical sections reveals a dispersed membrane meshwork which may be the structure that serves to interconnect the ER in the two lobes. Third, in cells fixed before dye injection, the pattern of labeling was similar to that in living cells, indicating that vesicle transport was not responsible for the spread of dye throughout the cell. The overall organization of the ER in the photoreceptor cell is relatively stable; however, the fine structure changes over time. This dynamic process appears to represent continual reorganization of the intracellular membranes in the cell. Three morphological types of ER were observed. The ER of the light-sensitive lobe, identified by coinjection of rhodamine-phalloidin to label the microvillar actin, is characterized by a concentration of stratiform membranes interconnected by thin tubular cross-bridges. The perinuclear ER is characterized by a tangle of convoluted tubules sometimes terminating in bulbous structures. Finally, there is a fine tubular reticulum dispersed throughout the cell.

A 13-A map of the actin-scruin filament from the limulus acrosomal process.

We have determined the structure of the actin-scruin filament to 13-A resolution using a combination of low-dose EM and image analysis. The three-dimensional map reveals four actin-actin contacts: two within each strand and two between strands. The conformation of the actin subunit is different from that in the Holmes et al. (1990) model as refined by Lorenz et al. (1993). In particular, subdomain II is tilted in a similar way to that seen by Orlova and Egelman (1993) in F-Mg2(+)-ADP actin filaments in the absence of Ca2+. Scruin appears to consist of two domains of approximately equal volume. Each scruin subunit cross-links the two strands in the actin filament. Domain I of scruin contacts subdomain I of actin and makes a second contact at the junction of subdomains III and IV. Domain II of scruin contacts actin at subdomains I and II of a neighboring actin subunit. The two scruin domains thus bind differently to actin.

Separation of large and small granules from horseshoe crab (Tachypleus tridentatus) hemocytes and characterization of their components.

We designed a method for separating two types of granules, a smaller (S) but dense and a larger (L) but less dense granule from hemocytes of the horseshoe crab (Tachypleus tridentatus), using continuous sucrose density gradient centrifugation. The isolated L-granules contained at least three clotting factors plus a clottable protein, coagulogen, as the major component. The known anti-lipopolysaccharide factor and 7 additional unknown protein components were also present in the L-granules. Two known natural substrates, Pro-rich protein and 8.6 kDa protein, for limulus transglutaminase [Tokunaga, F., Yamada, M., Miyata, T., Ding, Y.-L., Hiranaga-Kawabata, M., Muta, T., Iwanaga, S., Ichinose, A., & Davie, E.W. (1993) J. Biol. Chem. 268, 252-261] were present in the L-granules. On the other hand, the isolated S-granules contained antimicrobial tachyplesins I and II (17 amino acids in length) as the major component, in addition to 6 unidentified proteins with molecular masses of less than 30 kDa. The structural analyses of tachyplesin analogs indicated that all these peptides of mature form are stored in the S-granules, together with a processing intermediate containing the COOH-terminal Gly-Lys sequence. We also found an Arg-rich protein of 22 kDa and a Leu-rich protein of 30 kDa in S-granules. Based on these observations, we speculate that protein components in L-granules, which probably contain all the factors essential for the limulus clotting system, participate in immobilization of invading microbes and that factors in the S-granules containing tachyplesins contribute to a self-defense system against invaders.

Crystallographic analysis of acrosomal bundle from Limulus sperm.

The acrosomal process of Limulus sperm contains a bundle of filaments composed of actin and a 102 kDa protein in a 1:1 molar ratio. The structure of the bundle in true discharge was investigated by electron cryomicroscopy, X-ray scattering and crystallographic image analysis. A bundle can be characterized as a quasi-crystal with continuously varying views along the bundle axis. Each segment of the bundle is found to obey the symmetry of space group P1, with a = b = 147 A, c = 762 A, alpha = 90 degrees, beta = 90.6 degrees, gamma = 120 degrees. A unit cell contains a helical repeat of the filament with a selection rule following that of an actin filament. A 24 A projection map based on the h0l view was reconstructed after averaging 5300 unit cells from six electron images. Filaments in this projection are well separated and clearly display a 21 screw symmetry. This screw symmetry results from the helical parameters of the bundle filament and is found to be a non-crystallographic symmetry element present in the unit cell. Our structural analysis has led to the proposal that the assembly of a stable bundle with a defined maximum diameter can be controlled by the crystallographic packing of the twisted filaments.

Limunectin. A phosphocholine-binding protein from Limulus amebocytes with adhesion-promoting properties.

A protein that binds to and precipitates with pneumococcal C-polysaccharide and a phosphocholine (PC) derivative of bovine serum albumin has been affinity purified from Limulus amebocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals that the isolated protein consists of a single polypeptide chain of approximately 50 kDa. It is an intracellular protein localized in the secretory granules of amebocytes according to immunogold staining. Although it shares the PC-binding property with C-reactive protein isolated from Limulus and other animal species, it differs from C-reactive protein in that the latter binds to PC only in the presence of Ca2+, whereas the newly isolated protein binds to PC in a Ca(2+)-independent manner. In this respect, the newly isolated PC-binding protein resembles the antibodies to PC of mouse myelomas. The gene coding for this protein has been isolated. The gene sequence predicts a protein of 54 kDa with an unusual structural feature: it consists almost entirely of 10 contiguous segments, 45 amino acids in length, with extensive homology. Some limited sequence homologies were found between the 54-kDa protein and segments of vitronectin, gelatinase, and collagenase. It binds to bacterial cells, fixed amebocytes, and a number of extracellular matrix molecules. Due to its structural and some functional similarities to other adhesion molecules, the Limulus 54-kDa protein was named "Limunectin."

Two classes of Limulus ventral photoreceptors.

Ventral photoreceptors of the horseshoe crab, Limulus polyphemus, have been important in the study of visual transduction, due to their large size and hardiness in vitro. This study shows that there are two classes of ventral photoreceptors that can be distinguished on the basis of differences in cellular and nuclear dimensions, soma and rhabdom morphology, and axon size. Large protoreceptors, which have been the subject of many physiological studies, have an extensive superficial rhabdom, a nuclear diameter of 20-24 microns, and measure 100-150 microns in length. In contrast, small photoreceptors measure 45-65 microns in length and have a nucleus 13-16 microns across. Small photoreceptors are found singly or in association with large photoreceptors. The rhabdom of isolated small photoreceptors is surrounded by a calyx originating from the soma, so that it appears to be located internally. The rhabdomeral lobe of small photoreceptors associated with large photoreceptors characteristically is divided into several segments, each of which invaginates the rhabdomeral lobe of the adjacent large photoreceptor. The entire external rhabdom of the associated small photoreceptor abuts the rhabdom of the large photoreceptor. Morphometric analysis of the ventral nerves shows that there are two size classes of photoreceptor axons, corresponding to the two classes of photoreceptors. The numbers of axons in each size class are nearly equal. Unlike the ventral eye, none of the other eyes of Limulus have been reported to have more than one morphological class of photoreceptor. Functional differences between the two classes of ventral photoreceptors are suggested by experiments, reported in the accompanying paper (Herman (1991), J. Comp. Neurol. 303:11-21), showing that the large photoreceptors exhibit light-stimulated rhabdom turnover while the small ones do not.

Light-stimulated rhabdom turnover in Limulus ventral photoreceptors maintained in vitro.

The role of light in turnover of photosensitive membranes was studied in isolated photoreceptors maintained in vitro. Ventral photoreceptors of the horseshoe crab, Limulus polyphemus, were used since they have been the subjects of many in vitro physiological studies. This study shows that the two classes of ventral photoreceptors, the large and small photoreceptors (Herman: companion paper), differ in their morphological response to light. The rhabdom of small photoreceptors is remarkable for its regularity, independent of lighting condition. The photosensitive microvilli of the rhabdom of small photoreceptors are narrow and almost always tightly packed in a hexagonal arrangement. In contrast, the morphology of the rhabdom of the large ventral photoreceptors is different in the dark and in the light, and the rhabdom undergoes turnover during lighting transitions. When fully dark-adapted, the photosensitive microvilli of large photoreceptors are narrow and well organized, sometimes in a crystalline array. However, in the light-adapted state, the microvilli are much thicker and very irregular. The transitions between the dark and light-adapted states, examined at midday, are rapid. After 5 minutes light exposure, the microvilli are dilated at their bases and shed membranes are present in the cytoplasm. By 30 minutes after light onset, the appearance of the rhabdom of large photoreceptors is indistinguishable from fully light-adapted cells. The transition to the dark-adapted state is equally rapid. Even at 5 or 12 minutes after light offset, most microvilli are narrow and quite regular, and by 30 minutes, the rhabdom usually appears to be fully dark-adapted. These experiments show that both the synthetic and degradative phases of rhabdom renewal take place in isolated photoreceptors. No efferent neural activity is required to initiate turnover; rather, changes in illumination alone are sufficient to generate rhabdom turnover in large ventral photoreceptors in vitro.

Arrangement of myosin heads on Limulus thick filaments.

The two myosin heads with a single surface subunit on thick filaments from chelicerate arthropod muscle may originate from the same, or from axially sequential molecules, as suggested by three-dimensional reconstructions. The resolution attained in the reconstructions, however, does not permit one to distinguish unequivocally between these two possible arrangements. We examined the effect of 0.6 M KCl on relaxed thick filaments separated from Limulus muscle and filaments in which nearest myosin heads were cross-linked by the bifunctional agent, 3,3'-dithio-bis[3'(2')-O-[6-propionylamino)hexanoyl]adenosine 5'-triphosphate (bis22ATP), in the presence of vanadate (Vi). In high salt, surface myosin dissolved from both native, relaxed filaments and those exposed to 1-2 mM dithiothreitol after cross-linking, but was retained on filaments with cross-linked heads. Since bis22ATP must form intermolecular bonds between myosin heads within each subunit to prevent myosin solubilization in high salt, we conclude that each of these heads originates from a different myosin molecule, as was previously predicted by the reconstructions.

The intramembrane structure of septate junctions based on direct freezing.

Smooth septate junctions from the midgut of the cricket, Acheta, and the horseshoe crab, Limulus, as well as Hydra-type septate junctions from the epidermis of Hydra have been studied by freeze-fracture after direct freezing using the liquid helium-cooled copper block/slam freezing method. The exoplasmic fracture face at both types of septate junction exhibits rows of closely packed but irregularly shaped intramembrane particles. Complementary to these particle rows, on the protoplasmic fracture face, are sharply defined grooves with a periodic variation in depth and width that was conspicuous in Hydra but less well defined in arthropods. The closely packed, irregular particles on the exoplasmic faces could represent plastically deformed portions of transmembrane proteins pulled through the bilayer during freeze-fracture. On the basis of this interpretation, the grooves on the protoplasmic faces represent a confluence of the bilayer disruptions occurring during fracturing. The structures observed here are different from those reported in replicas of glutaraldehyde-fixed and glycerol-cryoprotected tissue, in which the intramembrane junctional components partition with the protoplasmic face and often assume the appearance of continuous cylinders. This comparison illustrates some of the artifacts associated with freeze-fracturing and shadowing. On the basis of a comparison of freeze-fracture replicas and sections of lanthanum-infiltrated tissues, the relationship between intramembrane junctional components and intercellular septal elements is analysed.