Monoclonal antibodies against surface components of the mechano-and chemosensitive cnidocil complex of Hydra vulgaris

Summary Two monoclonal antibodies (Gc3.2 and Bd 2.2) against surface components of the cnidocil complex of Hydra vulgaris have been produced. In indirect immunofluorescence and in immunogold-labelling, the Gc 3.2-antibody stains the complete surface of all nematocytes, whereas other cellular surfaces are not labelled. The Bd 2.2-antibody, in contrast, produces only a small band of fluorescence on isolated cnidocils. This pattern of fluorescence and the corresponding immunogold-labelling indicate that the Bd 2.2-antibody exclusively binds to those intermembrane connectors that link the cnidocil and stereovillar cone in situ. In isolated and decnidociliated nematocytes, the tips of the stereovilli are also labelled by the Bd 2.2-antibody. Physiological experiments suggest that the Bd 2.2-antibody disturbs the reconstitution of intermembrane connectors during cnidocil regeneration. These data confirm the hypothesis that the intermembrane connectors are formed by two identical subunits located at the cnidociliar and stereovillar surfaces.     

Surface charges of the membrane and cell adhesion substances determine the structural integrity of hair bundles from the inner ear of fish

Summary The hairs (stereocilia = stereovilli) of sensory cells from the inner ear of vertebrates are interconnected by several types of connectors, whose role is unknown. They appear to stabilize the hair bundle mechanically, and may be directly involved in mechano-electric transduction. Our transmission electron-microscopical investigation of sensory epithelia from two species of fish (Rutilus rutilus, Scardinius erythrophthalmus, both Leuciscidae) has shown that not only the connectors but also the surface charges of the membrane are important factors for determining the shape of the hair bundle and the spatial interrelation of the stereovilli. A reduction of the ionic strength in the medium leads to an increase in distance between the stereovilli. This may be the result of an extension of the spread of the surface potential of the membrane at low ionic strength. The connectors are not broken by the increase in distance between the stereovilli. They are EDTA (ethylene-diamine-tetra-acetic-acid) resistant as are some cell adhesion molecules such as N-CAM (nerve-cell adhesion molecule) and protein A from Dictyostelium discoideum. The connectors do not prevent polycation-induced fusion of adjacent stereovillar membranes.     

Ultrastructural evidence for the occurrence of three types of mechanosensitive cells in the tentacles of the cubozoan polypCarybdea marsupialis

Summary The ectodermal cell layer in the tentacles of the cubozoan polypCarybdea marsupialis contains four types of cells (types 1–4) bearing specialized cilia. Epitheliomuscular cells (type 1) are characterized by motile cilia with dynein-decorated axonemes. 200 nm long extramembranous filaments of unknown function are restricted to a belt-like region distal to the transition zone. Up to 40 rn long rigid cilia formed by a slender epithelial cell type (type 2) are surrounded by rings of short microvilli. The axonemes of these cilia are composed of incomplete microtubules and lack dynein. Microvilli and cilia are linked by intermembrane connectors. Microtubuledoublets and ciliary membrane are interconnected by microtubule-associated cross-bridges only within this contact region. At the tip of each tentacle a single nematocyte (type 3) is surrounded by 7–10 accessory cells (type 4). These both cell types are equipped with similar cilium-stereovilli-complexes consisting of a cone-like arrangement of stereovilli and a modified cilium. The axonemal modifications of the cilium, its interconnections with the surrounding stereovilli and the linkages between ciliary axoneme and ciliary membrane are similar to those known from the cnidocil-complexes of hydrozoons and other epithelial mechanosensitive cells of the collar-receptor type. Our data indicate that besides the nematocyte two other types of mechanosensory cells (types 2 and 4) are integrated in the ectodermal cell layer ofCarybdea which possibly affect the triggering mechanism of nematocyst discharge.     

Cytoskeletal modifications of the sensorimotor interneurons ofHydra vulgaris (Cnidaria,Hydrozoa), indicating a sensory function similar to chordotonal receptors of insects

Summary The battery mother cell complexes in the tentacles ofHydra vulgaris contain a neuronal cell known as sensorimotor interneuron that is characterized by a modified cilium lying parallel to the mesoglea. The cilium is surrounded by up to three rings of microvilli. Microvilli and cilium arise in an unusual antiparallel orientation from the opposite poles of a central cellular cavity. The lumen of this cavity communicates with the extracellular environment by way of a straight channel-like opening that is encircled by the microvillar rings. The modified cilium extends into the channel and terminates outside in the intercellular space. The wall of the cavity and the channel are stabilized by bundles of microtubules. A prominent glycocalyx interconnects all microvilli and links the innermost microvillar ring to the cilium. Within this contact region approximately 0.7 m in length the ciliary axoneme is specifically modified: all nine microtubule doublets and up to six additional microtubules are embedded in electron-dense material. The microtubule doublets are connected to the ciliary membrane by ledges of Y-shaped cross-bridging elements. These axonemal modifications resemble those known from the hydrozoan cnidocil complex or from the outer segments of insect mechanoreceptor cells. Distribution and orientation of the sensorimotor interneuron within the tentacles indicate a mechanosensory function of the cell similar to that of chordotonal receptors of insects.     

Interconnections between the stereovilli of the fish inner ear

Summary The maculae utriculi and sacculi of the inner ear from the European roach (Rutilus rutilus) were investigated by transmission electron microscopy. The stereovilli of peripherally and centrally located sensory cells differ in several features that suggest a developmental gradient. The stereovilli of the peripheral sensory cells, shown to be differentiating cells by other research groups, are short and less steeply graded in height than in central hair cells. All stereovilli in both kinds of hair bundles are interconnected. In the central bundles of stereovilli basal, tip, and vertical connectors are separated by unconnected regions. In contrast, filaments and sometimes other additional structures connect the stereovilli of peripheral bundles over their entire length, but vertical connectors are usually absent. Osmiophilic material occurring inside peripheral stereovilli is interpreted to be monomeric actin. Central and peripheral hair bundles also differ in their reaction to ruthenium red and cationized ferritin. Only the stereovilli of the central cells can be fused by these polycations. Ruthenium red also discriminates between supporting and sensory cells indicating differences in amount or distribution of extracellular material. Hair bundles, intermediate in properties and position between central and peripheral sensory cells, were also found, so that it became possible to propose a scheme of developmental steps leading from microvilli or microvillus-like stereovilli to the fully differentiated hair bundle.     

The ciliated sensory cell of Stauridiosarsia producta (Cnidaria,Hydrozoa) — a nematocyst-free nematocyte?

Summary A mechanosensitive ciliated cell type of the polyp Stauridiosarsia producta (Hydrozoa) was investigated by means of electron microscopy. These cells bear at their apical cell surface a modified cilium, a set of seven stereovilli, a so-called pseudovillar system and a large vacuole. Cilium and stereovilli are interconnected like the cnidocil apparatus of hydrozoan nematocytes which is responsible for mechanoelectric transduction. The vacuole is enclosed by and linked to the pseudovillar system by a microtubular basket. Considering its structural organization and physiological activities the ciliated sensory cell closely resembles a nematocyte that has lost its ability ot produce a nematocyst.     

Structures transmitting stimulatory force to the sensory hairs of vestibular ampullae of fishes and frog

Summary Serial sections of the vestibular ampullae of two species of fish and one species of frog were investigated by electron microscopy. The kinocilium is the only connection between the sensory cells and the auxiliary structure (cupula). The cupula possesses canals that traverse its entire height. Each canal contains a single kinocilium in its proximal part; distally, it is filled with material that stains with colloidal silver. The matrix of the cupula consists of filaments running perpendicular to the canals. These filaments do not stain with colloidal silver. The kinocilium is connected to the wall of the canal via structures that differ in the studied species of fish and frog. The filamentous links between the kinocilium and the longest stereovilli of the sensory hair bundle are similar in all the investigated species. The stereovilli are interconnected by basal and shaft links, and by horizontal and oblique tip connectors, similar to those described by other authors for macula organs and the organ of Corti, although differences in structural details, especially of the horizontal tip and the shaft connectors, are present. Some of these are species specific and some are related to the position of the sensory cell in the epithelium and/or specific to the organ (ampulla or macula organ). Some attachment sites of the links are associated with osmiophilic submembranous material. These differences in the structure, distribution and attachment sites of the links are possibly of functional importance.     

Arrangement of accessory cells and nematocytes bearing mastigophores in the tentacles of the cubozoan Chironex fleckeri

Nematocytes containing microbasic mastigophores are intimately associated with accessory cells in the epidermis of Chironex fleckeri. Large microbasic mastigophores may be surrounded by seven to nine such cells. Each accessory cell possesses an apical portion containing secretory droplets and a basal portion which carries a radially oriented fibre linking the cell to the underlying mesogloea. The fibre is capable of projecting and retracting the accessory cell. Junctional complexes occur between accessory cells and the apical regions of neighbouring mastigophores. Each nematocyte bearing a mastigophore contains a triggering apparatus consisting of a cnidocil surrounded by microvilli. This apparatus protrudes from an invagination in the apical region of the nematocyte and is exposed when the mastigophore is in the fire-ready position. A basket of filaments which make contact with microvilli surrounds the apical end of the nematocyst like a collar. The basket is linked via fibrous bundles which envelop the mastigophore to radially oriented fibres basally. These fibres are capable of projecting and retracting the mastigophore and its associated triggering apparatus. Up to nine such fibres were observed to be associated with a single large microbasic mastigophore. Microtubules averaging 25 nm in diameter and linked via cross bridges to electrondense material were detected in the radial fibres of both nematocytes and accessory cells. Retraction of the accessory cells and projection of nematocytes result in mastigophores being brought to the firing line and in the exposure of the cnidocil apparatus.     

Interconnections between the stereovilli of the fish inner ear

Summary We investigated at the electron-microscopic level the hair bundles of the macula organs in the inner ear of two species of teleost fish (Rutilus rutilus, Scardinius erythrophthalmus). All hairs (stereovilli) are interconnected by extracellular material that, similar to the cell coat, is well contrasted by application of tannic acid/osmium.The kinocilium is connected to the longest stereovilli via filaments. Filaments also connect the stereovilli at their bases. More distally all stereovilli are linked by thin filaments or very short solid connectors.The interconnections of the stereovilli in fish show greater variations, and are different in structural detail and distribution, compared to the interconnections described for amphibians and mammals. The interconnections are discussed in the context of their structural and functional significance and variations in terms of organ- and species specificity. Certain forms of interconnections are tentatively interpreted to be characteristic for developing hair bundles.     

Ultrastructure of paired coniform 9 + 0 sensory cilia: a new type in the organ of Bellonci of the marine amphipod Gammarus setosus

Summary The structure of modified 9 + 0 cilia in the organ of Bellonci was studied in Gammarus setosus from late embryonic development to adult after routine fixation, fixation with lanthanum treatment, and prefixation with ethylene diamine tetraacetic acid and sodium dodecyl sulphate. The cilia are distinct from known sensory cilia in that they occur in pairs and lack centrioles. The basal bodies are at right angles to each other. The basal body cylinders consist of dense microtubule doublets and have 3 regions: the basal cartwheel, the middle pinwheel and the distal transitional. The pinwheel, which has 9 fins of dense material attached to the doublets, is differentiated into a spiral attachment of the ciliary roots whose periodicity is 70 nm. The scanning electron microscope shows the roots as beaded, tapering ribbons. The coniform outer segments give rise to tubules, each with 1 or 2 single or double microtubules in its core. The tubules are in contact with extracellular chains of calcium granules inside the organ. A bend in the axoneme brings the paired outer segments together. Lamellar bodies develop from the ciliary tubules in embryos and juveniles, but not in adults, except after exposure to lanthanum.     

Apical surface of hydrozoan nematocytes: Structural adaptations to mechanosensory and exocytotic functions

At the apical cell pole of the nematocytes of a hydrozoan (Hydra vulgaris), cytoskeletal elements of different molecular families (microfilaments, microtubules, cross-striated rootlets) are combined in a complex framework. As determined by transmission and scanning electron microscopy, the stimulus-transducing cnidocil apparatus of these mechanosensitive cells is forced into a lateral position, facilitating the close apposition of the nematocyst to the apical cell membrane. The nematocyst, a single, extraordinarily large, exocytotic organelle, is held in position by a microtubular basket. The cnidocil apparatus and microtubular basket are linked to an ellipsoid arrangement of pseudovilli, i.e., small surface protrusions containing cross-striated rods. These nematocyte-specific, cytoskeletal elements mediate the anchorage of the entire cytoskeletal framework to the apical cell membrane. The apical membranes of the nematocyte and nematocyst are separated by a distance of only ～ 50 nm. Structural modifications on the external side of the cyst membrane resemble those of synaptic membranes. © 1994 Wiley-Liss, Inc.     

Freeze-fracture observations on the plasma membrane,the cell wall and the cuticle of growing protonemata of Adiantum capillus-veneris L.

Using freeze-fracture electron microscopy we have examined the morphology of the plasma membrane and the cell wall of single-celled protonemal filaments of the fern Adiantum capillus-veneris grown under continuous red light. The surface of the protonemal cell wall is completely covered by a multilayered, lipid-like coat, probably consisting of cuticular waxes. The rhizoid seems to lack this type of coat. The cell walls of the protonemata contain 8-nm thick, randomly oriented fibrils. In rapidly growing protonemata the P-face of the plasma membrane contains both randomly distributed particles and distinct particle rosettes. The rosettes consist of six 8–9-nm-wide particles in a ring-like configaration and have an outer diameter of 24 nm. They closely resemble the particle rosettes seen on the P-face of the plasma membrane of green algae and of higher plants, which recently have been implicated in the synthesis of cellulose fibrils. Within 20 m from the tip of the protonemata, and coinciding with the region of maximal cell-wall growth and expansion and thus cellulose-fibril synthesis, the greatest density of rosettes (20/m²) is observed. Beyond 20 m from the tip this number drops rapidly to near zero at 50 m. The rosettes have a tendency to form small, irregular clusters, but only very rarely are three or more rosettes found in a row or in a geometrical pattern. Our measurements of the size and the density of the randomly distributed plasma membrane particles indicate that the tip region must be specialized with respect to other plasma-membrane activities as well. Thus the tip region contains not only the highest density of randomly destributed intramembrane particles, but also particles of different sizes than those found elsewhere in the plasma membrane.     

Biogenesis of mitochondrialc-type cytochromes

Cytochromesc andc ₁ are essential components of the mitochondrial respiratory chain. In both cytochromes the heme group is covalently linked to the polypeptide chain via thioether bridges. The location of the two cytochromes is in the intermembrane space; cytochromec is loosely attached to the surface of the inner mitochondrial membrane, whereas cytochromec ₁ is firmly anchored to the inner membrane. Both cytochromec andc ₁ are encoded by nuclear genes, translated on cytoplasmic ribosomes, and are transported into the mitochondria where they become covalently modified and assembled. Despite the many similarities, the import pathways of cytochromec andc ₁ are drastically different. Cytochromec ₁ is made as a precursor with a complex bipartite presequence. In a first step the precursor is directed across outer and inner membranes to the matrix compartment of the mitochondria where cleavage of the first part of the presequence takes place. In a following step the intermediate-size form is redirected across the inner membrane; heme addition then occurs on the surface of the inner membrane followed by the second processing reaction. The import pathway of cytochromec is exceptional in practically all aspects, in comparison with the general import pathway into mitochondria. Cytochromec is synthesized as apocytochromec without any additional sequence. It is translocated selectively across the outer membrane. Addition of the heme group, catalyzed by cytochromec heme lyase, is a requirement for transport. In summary, cytochromec ₁ import appears to follow a conservative pathway reflecting features of cytochromec ₁ sorting in prokaryotic cells. In contrast, cytochromec has invented a rather unique pathway which is essentially non-conservative.     

Organization of cytoskeleton during the tentacle contraction and cytostome movement in the dinoflagellate Noctiluca scintillans McCartney

Summary Electron microscopy of Noctiluca scintillans reveals that the cytoskeleton of the tentacle involved in the motor action of the prey capture consists of three characteristic elements: a deformable peripheral fibrillo-granular ectosarc, abundant underlying microtubules organized in several rows on the convex side, and helicoid filaments about 8 nm in diameter organized into striated myonemes. Microtubules of the external row are crossed-linked with each other by fibrous elements 5 nm in diameter and 10–15 nm long, their links with the second row result in a Y-shaped binding. Bonds of the other rows are linked to each other irregularly between those of the same row. Striated myonemes are regularly inserted between the rows of microtubules on the ectosarc and between its pleats, joining together in a knot of disarrayed filaments with multidirectional orientation in the central axis of the tentacle. Striation of myonemes is based on an alternation of thick striae (TS) 40 nm wide with a periodicity of about 200 nm, and of some intermediary fine striae (FS) 10 nm wide. The events during tentacle contraction are: (1) Rotation of the tentacle, bringing the convex side to the inner side of it. Here, large numbers of microtubules have been visualized by optical immunocytochemistry after labelling with Paramecium antitubulin antiserum. (2) Increase of pleat amplitude (200–300 nm to 600 nm) in concomitance with a decrease of its period (500–700 nm to 250 nm). (3) Apparent modification of the microtubule orientation. (4) Transformation of some TS in several FS without modification of the striation periodicity.Near the cytostome, the cytoskeleton consists of a number quantity of microtubules underlying a non-pleated ectosarc and long tracts of contractile myonemes formed by 6-nm helicoid filaments linking the internal side of the cytostome of the supporting rod. Semirelaxed myonemes show an alternation of fine striae (FS) 35 nm wide between two clear areas (CA) with a periodicity of about 300 nm, plus an incipient dark area (DA) lying between them; together they are transformed into a thick stria (TS) during maximal contraction; the striation periodicity thus decreases by one half. These two systems are compared with one another and with other motile systems.     

Structural interactions of actin filaments and endoplasmic reticulum in honeybee photoreceptor cells

Summary Fluorescent phallotoxins and heavy meromyosin were used to reveal the organization of the actin cytoskeleton in honeybee photoreceptor cells, and the relationship of actin filaments to the submicrovillar, palisade-like cisternae of the endoplasmic reticulum (ER). Bundles of unipolar actin filaments (pointed end towards the cell center) protrude from the microvillar bases and extend through cytoplasmic bridges that traverse the submicrovillar ER. Within the cytoplasmic bridges, the filaments are regularly spaced and tightly apposed to the ER membrane. In addition, actin filaments are deployed close to the microvillar bases to form a loose web. Actin filaments are scarce in cell areas remote from the rhabdom; these areas contain microtubule-associated ER domains. The results suggest that the actin system of the submicrovillar cytoplasm shapes the submicrovillar ER cisternae, and that the distinct ER domains interact with different cytoskeletal elements.     

The formation and development of cellulose-synthesizing linear terminal complexes (TCs) in the plasma membrane of the marine red algaErythrocladia subintegra Rosenv.

Summary The formation and development of linear terminal complexes (TCs), the putative cellulose synthesizing units of the red algaErythrocladia subintegra Rosenv., were investigated by a freeze etching technique using both rotary and unidirectional shadowing. The ribbon-like cellulose fibrils ofE. subintegra are 27.6 ± 0.8 nm wide and only 1–1.5 nm thick. They are synthesized by TCs which are composed of repeating transverse rows formed of four particles, the TC subunits. About 50.4 ± 1.7 subunits constitute a TC. They are apparently more strongly interconnected in transverse than in longitudinal directions. Some TC subunits can be resolved as doublets by Fourier analysis. Large globular particles (globules) seem to function as precursor units in the assembly and maturation of the TCs. They are composed of a central hole (the core) with small subunits forming a peripheral ridge and seem to represent zymogenic precursors. TC assembly is initiated after two or three gobules come into close contact with each other, swell and unfold to a nucleation unit resembling the first 2–3 transverse rows of a TC. Longitudinal elongation of the TC occurs by the unfolding of globules attached to both ends of the TC nucleation unit until the TC is completed. The typical intramembranous particles observed inErythrocladia (unidirectional shadowing) are 9.15 ± 0.13 nm in diameter, whereas those of a TC have an average diameter of 8.77 ± 0.11 nm. During cell wall synthesis membranes of vesicles originating from the Golgi apparatus and which seem to fuse with the plasma membrane contain large globules, 15–22 nm in diameter, as well as tetrads with a particle diameter of about 8 nm. The latter are assumed to be involved in the synthesis of the amorphous extracellular matrix cell wall polysaccharides. The following working model for cellulose fibril assembly inE. subintegra is suggested: (1) the ribbon-like cellulose fibril is synthesized by a single linear TC; (2) the number of glucan chains per microfibril correlates with the number of TC subunits; (3) a single subunit synthesizes 3 glucan chains which appear to stack along the 0.6 nm lattice plane; (4) lateral aggregation of the 3-mer stacks leads to the crystalline microfibril.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Mechanoreception and synaptic transmission of hydrozoan nematocytes

Mechanoelectric transduction and its ultrastuctural basis were studied in the cnidocil apparatus of stenotele nematocytes of marine and freshwater Hydrozoa (Capitata and Hydra) as a paradigm for invertebrate hair cells with concentric hair bundles. The nematocytes respond to selective deflection of their cnidocil with phasic-tonic receptor currents and potentials, similar to vertebrate hair cells but without directional dependence of sensitivity. Ultrastructural studies and the use of monoclonal antibodies allowed correlating the mechanoelectric transduction with structural components of the hair bundle. Two other types of depolarising current and voltage changes in nematocytes are postsynaptic, as concluded from their ionic and pharmacological characteristics. One of these types is induced by mechanical stimulation of distant nematocytes and sensory hair cells. It is graded in amplitude and duration, but different from the presynaptic receptor potential. Adequate chemical stimulation of the stenoteles strongly increases the probability of discharge of their cnidocyst, if the chemical stimulus precedes the mechanical one. Simultaneously, the probability of synaptic signalling induced by mechanical stimulation is increased, reaching nearly 100%. The chemoreception of the phospholipids used could be localized in the shaft of the cnidocil, because of the water-insolubility of the stimulant. This chemical stimulation itself does not cause a receptor potential; its action is classified as a modulatory process. Electron microscopy of serial sections of the tentacular spheres of Coryne revealed synapses that are efferent to nematocytes and hair cells besides neurite–neurite synapses, each containing 3–10 clear and/or dense-core vesicles of 70–150 nm diameter. The only candidates to explain the graded afferent signal transmission of nematocytes and hair cells are regularly occurring cell contacts associated with 1(–4) clear vesicles of 160–1100 nm diameter. Transient fusion and partial depletion of stationary vesicles are discussed as mechanisms to reconcile functional and structural data of many cnidarian synapses. Review contributed to the Symposium on Neuro-Anatomy and -Physiology of Coelenterates; 7th International Conference on Coelenterate Biology, Lawrence, Kansas, USA; July 6–11, 2003.     

Spermiogenesis and chromatin condensation in the common tree shrew, <Emphasis Type="Italic">Tupaia glis</Emphasis>

We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80–100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15–16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12–16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells. This work was supported by the Thailand Research Fund (Senior Research Fellowship to Prof. Prasert Sobhon).     

Aspects of the structure and development of monotreme spermatozoa and their relevance to the evolution of mammalian sperm morphology

Summary The elongated spermatid nuclei of monotremes exhibit a circumferentially arranged spiral pattern of chromatin condensation, and ultimately form helical filiform sperm heads up to 50 m long and either circular or slightly oval in transverse section. The acrosome is formed by the collapse of the proacrosomal vacuole onto the rostral surface of the elongating nucleus. However, genesis of acrosomal material occurs in the absence of a prominent proacrosomal granule. The flagellum becomes inserted into the distal extremity of the nucleus, the most proximal mitochondria of the midpiece directly abutting the nuclear membrane, so that a prominent neck region is absent. The axoneme is simple and, in the midpiece, small dense peripheral fibres are closely applied to the outer surface of each of the nine microtubule doublets. The cortical fibrous sheath of the principal piece is an anastomosing spiral that lacks lateral columnar elements.The spermatozoal cytoplasmic droplet undergoes migration and is lost during epididymal passage.Monotreme spermatozoa exhibit a montage of features, some of these being also found in marsupials and some in sauropsidan vertebrates, as well as a number of their own distinctive features. It is concluded that monotreme spermatozoa also have a close affinity with the unspecialised spermatozoa of some eutherian mammals.We wish to thank Dr. Mervyn E. Griffiths for providing the Zaglossus material. Permits to collect these native mammals were provided by the National Parks and Wildlife Services, and State Fisheries Services of New South Wales and Queensland. Portion of this work was supported by grants from the Australian Research Grants Committee. The provision of skilled technical assistance and access to electron microscope facilities were kindly provided by Dr. C.D. Shorey, formerly Acting Director of the University of Sydney Electron Microscope Unit, and by Mr. John Hardy, Senior Lecturer-in-Charge of the University of Queensland Electron Microscope Centre. Members of the technical staff of the Departments of Anatomy and Veterinary Anatomy are also acknowledged for their assistance     

Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear

Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed.