Centrin- and α-actinin-like immunoreactivity in the ciliary rootlets of insect sensilla

Summary Long ciliary rootlets are a characteristic feature of the dendritic inner segments of the sensory cells in insect sensilla. These rootlets are composed of highly ordered filaments and are regularly cross-striated. Collagenase digestion and immunohistochemistry reveal that the rootlets are probably not composed of collagen fibers. However, double-labeling experiments with phalloidin and anti--actinins show that antibodies to -actinin react with the ciliary rootlets of the sensilla, but do not stain the scolopale, which is composed of actin filaments as visualized by phalloidin. Antibodies to centrin, a contractile protein isolated from flagellar rootlets of green algae, also stain the ciliary rootlets. Within the ciliary rootlets of insect sensilla, -actinin may be associated with filaments other than actin filaments. The immunohistochemical localization of a centrin-like protein suggests that contractions probably occur within the rootlets. The centrin-like protein may play a role during the mechanical transduction or adaptation of the sensilla.     

Actin filaments: the main components of the scolopale in insect sensilla

Summary Two types of insect sensilla, mechanosensitive scolopidia and thermo-/hygrosensitive poreless sensilla contain a scolopale, which consists of numerous microtubules embedded in bundles of filaments (7–10 nm in diameter). The bundles are readily seen in the electron microscope in cryofixed (high-pressure freezing and rapid injection) and substituted samples. The filaments can be identified as actin filaments by using fluorescent phalloidins. Both electron microscopy and Triton-extraction exeriments reveal mechanical linkage between the main components in both types of sensilla. Since myosin appears to be absent in the scolopale, the actin filaments are unlikely to be involved in any contraction mechanism; these filaments more probably provide mechanical stability. The functional properties of the scolopale are discussed.     

Structure of the auditory system of the weta Hemideina crassidens (Blanchard, 1851) (Orthoptera,Ensifera, Gryllacridoidea,Stenopelmatidae)

Summary This study of the ultrastructure of the auditory sensilla of the New Zealand weta, Hemideina crassidens, is the first such study on a member of the orthopteran Superfamily Gryllacridoidea. Ultrastructure of the auditory sensilla is similar in all of the tibial mechanosensory organs, here called subgenual organ, intermediate organ and crista acoustica by analogy with comparable structures in Tettigoniidae.Distal to each sensory soma is a dendrite containing multiple ciliary rootlets that fuse into a single ciliary root. This splits into nine root processes that pass around the outside of the proximal basal body and then rejoin at the level of the distal basal body, distal to which the dendrite has a modified ciliary structure with a circlet of nine peripheral paired tubes and rods as it passes through the proximal extracellular space. It is then enclosed by a zone of scolopale cell cytoplasm before expanding into a dilatation within the distal extracellular space. In some sensilla this space is partially occluded by electron dense material which is part of the scolopale cell. Distal to the dilatation the cilium shrinks and ends surrounded by the scolopale cap.Accessory cells consist of glia enwrapping the sensory neuron in the region of its soma, the scolopale cell surrounding the ciliary portion of the dendrite, and the attachment cell surrounding the scolopale cell and scolopale cap and connected to them by desmosomes. The attachment cells are filled with microtubules in differing densities and orientations. Lamellae are present in the acellular matrix surrounding the attachment cells. Banded fibres, presumably of collagen, are also present in the matrix.     

Femoral chordotonal organ in the legs of an insect,Chrysoperla carnea(Neuroptera)

The femoral chordotonal organ (FCO) inChrysoperla carneais situated in the distal part of the femur and consists of two scoloparia, which are fused at their distal end. The distal scoloparium contains 17-20 scolopidia, and the proximal one six scolopidia. Each scolopidium consists of two sensory cells and three types of enveloping cells (glial, scolopale and attachment cell). The sensory cells of different scolopidia do not lie at the same level in the FCO. Therefore the attachment cells of different scolopidia have different lengths. In the FCO, three types of ciliary roots are found in different sensory cells. The dendrite of the sensory cell terminates in a distal process, which has the structure of a modified cilium (9x2+0). The very distal part of the cilium is surrounded by an extracellular electron dense material, the cap, and ends in a terminal dilation. The scolopale cell contains the electron dense scolopale rods, consisting of plentiful microtubules. In their middle third the scolopale rods are fused and form the scolopale. In the FCO septate junctions, desmosomes and hemidesmosomes are found.     

Tropomyosin is co-localized with the actin filaments of the scolopale in insect sensilla

Summary Immuno-electron microscopy confirms that the scolopale, a characteristically prominent cytoskeletal element of insect scolopidia, is composed mainly of actin filaments. Immunohistochemistry reveals that these filaments are co-localized with tropomyosin. Myosin S1-decoration shows that their polarity is unidirectional. Antibodies to -actinin do not bind within the scolopale. The association of these actin filaments with tropomyosin in the absence of myosin, together with their uniform polarity, strongly suggests that, in the scolopale, they have a stabilizing rather than contractile function. Filament elasticity would appear to be important for stimulation. The degree of elasticity may well be governed by the extent of tropomyosin binding.     

Structure of dactyl sensilla in the kelp crab,Pugettia producta

In the kelp crab, Pugettia producta, flat plate setae cover all but the ventral surfaces of the walking leg dactyls. Dendrites enter the setal shaft located inside the plate superstructure, and extend to a region of the setal tip that contains a system of minute pores resembling the pore systems found in chemosensory sensilla of insects. Presumably, much of the chemosensitivity of the dactyls in the kelp crab is mediated by the plate setae. In the interior of the dactyl, supporting cells and the neurons innervating plate setae, other types of setae, and other presumptive sensilla form scolopidia. Large scolopidia, containing as many as 12 dendrites, appear to innervate some of the plate setae and also large ventral rodlike setae that might be chemosensory. Two of the dendrites of large scolopidia usually have more densely packed microtubules, longer ciliary axonemes, slightly larger rootlets, and dark A fibers with arms, characteristics indicative of mechanosensory function. Some dactyl setae, therefore, could be both mechanosensory and chemosensory. Small scolopidia containing two or three dendrites that exhibit mechanosensory characteristics appear to innervate small, rodlike setae, which presumably are strictly mechanosensory. The two types of structures located on the epicuticular cap, elliptical structures resembling campaniform sensilla and small cones in pits resembling CAP organs, appear to be dually innervated and presumably are mechanosensory, although other functions are possible. The internal positions of the scolopidia, together with the support afforded by an extracellular dendritic sheath, by the scolopale, and by desmosomelike and septate junctions, may serve to protect internal portions of setal dendrites, some of which appear to remain functional in nonmolting adults that have abraded setae.     

The ciliary rootlet maintains long-term stability of sensory cilia

The striated ciliary rootlet is a prominent cytoskeleton originating from basal bodies of ciliated cells. Although a familiar structure in cell biology, its function has remained unresolved. In this study, we carried out targeted disruption in mice of the gene for rootletin, a component of the rootlet. In the mutant, ciliated cells are devoid of rootlets. Phototransduction and ciliary beating in sensory and motile cilia initially exhibit no apparent functional deficits. However, photoreceptors degenerate over time, and mutant lungs appear prone to pathological changes consistent with insufficient mucociliary clearance. Further analyses revealed a striking fragility at the ciliary base in photoreceptors lacking rootlets. In vitro assays suggest that the rootlet is among the least dynamic of all cytoskeletons and interacts with actin filaments. Thus, a primary function of the rootlet is to provide structural support for the cilium. Inasmuch as photoreceptors elaborate an exceptionally enlarged sensory cilium, they are especially dependent on the rootlet for structural integrity and long-term survival.     

Three-dimensional visualization of basal body structures and some cytoskeletal components in the apical zone of tracheal ciliated cells

The ciliated cells of tracheal epithelium were mechanically fragmented to remove the cytoplasmic soluble contents, and the apical zone was examined to clarify the three-dimensional structures of basal body and cytoskeletal filaments using freeze-fracture-etch approaches. The basal body was connected to the apical plasma membrane by definite laminae, formerly called alar sheets. The distal one-half of the basal foot was composed of several smooth-surfaced 12-nm fibrils. Intermediate filament networks extended to the lower half plane of the basal body, and enmeshed the basal body tightly by tiny 5- to 8-nm fibrils. Actin core bundles of microvilli also had tiny crosslinking fibrils. Some actin filaments were seen to run horizontally at the upper half plane of the basal body. Tracheal cilated cells also had circular actin filament bundles just inside the zonula adherens as many other epithelial cells. These cytoskeletal networks which enmeshed both basal bodies and core filaments of microvilli may function as a coordinator of ciliary beating.     

Ultrastructure of a possible new type of crustacean cuticular strain receptor in Carcinus maenas (crustacea,decapoda)

A hitherto unknown sensillum type, the “intracuticular sensillum” was identified on the dactyls of the walking legs of the shore crab, Carcinus maenas. Each sensillum is innervated by two sensory cells with dendrites of “scolopidial” (type I) organization. The ciliary segment of the dendrite is 5–6 μm long and contains A-tubules with an electron-dense core and dynein arm-like protuberances; the terminal segment is characterized by densely packed microtubules. The outer dendritic segments pass through the endo- and exocuticle enclosed in a dendritic sheath and a cuticulax tube (canal), which is suspended inside a slit-shaped cavity by cuticular lamellae. The dendrites and the cavity terminate in a cupola-shaped invagination of the epicuticle. External cuticular structures are lacking. Three inner and four to six outer enveloping cells are associated with each intracuticular sensillum. The innermost enveloping cell contains a large scolopale that is connected to the ciliary rootlets inside the inner dendritic segments by desmosomes. Scolopale rods are present in enveloping cell 2. Since type I dendrites and a scolopale are regarded as modality-specific structures of mechanoreceptors, and since no supracuticular endorgan is present, the intracuticular sensilla likely are sensitive to cuticular strains. The intracuticular sensilla should be regarded as analogous to insect campaniform sensilla and arachnid slit sense organs.     

Peritubular myoid cells from rat seminiferous tubules contain actin and myosin filaments distributed in two independent layers

In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.     

Cytoskeletal architecture and immunocytochemical localization of fodrin in the terminal web of the ciliated epithelial cell

In order to understand the cytoskeletal architecture at the terminal web of the ciliated cell, we examined chicken tracheal epithelium by quick-freeze deep-etch (QFDE) electron microscopy combined with immunocytochemistry of fodrin. At the terminal web, the cilia ended into the basal bodies and then to the rootlets. The rootlets were composed of several filaments and globular structures attached regularly to them. Decoration with myosin subfragment 1 (S1) revealed that some actin filaments ran parallel to the apical plasma membrane between the basal bodies, and other population traveled perpendicularly or obliquely, i.e., along the rootlets. Some actin filaments were connected to the surface of the basal bodies and the basal feet. Among the basal bodies and the rootlets there existed three kinds of fine crossbridges, which were not decorated with S1. In the deeper part of the terminal web, intermediate filaments were observed between the rootlets and were sometimes crosslinked with the rootlets. Immunocytochemistry combined with the QFDE method revealed that fodrin was a component of fine crossbridges associated with the basal bodies. We concluded that an extensive crosslinker system among the basal bodies and the rootlets along with networks of actin and intermediate filaments formed a structural basis for the effective beating of cilia.     

Action of cytochalasin D on cytoskeletal networks

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end- to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.     

Functional morphology of the subgenual organ of the carpenter ant

Using light microscopy, confocal microscopy, electron microscopy and histochemistry, the subgenual organ (SGO) of an ant, Camponutas ligniperda, is investigated. Sensory units and attachment cells together enclose a large extracellular cavity, which is filled by acid mucopolysaccharides, as revealed by staining with ruthenium red. Due to this cavity, the whole SGO has the shape of a deformed sphere and the scolopidia exhibit a distribution of angles between 0 degrees and 60 degrees with the tibial long axis (as is shown by phalloidin-rhodamin staining of the actin filaments of the scolopale, viewed in situ by laser scanning confocal microscopy). The subgenual organ is innervated by a branch of the tibial nerve, which splits within or shortly distal to the femur-tibia joint. The other features of the SGO of Camponotus ligniperda are similar as in other insects: the SGO of Camponotus ligniperda contains about 35 scolopidial sensilla; it is fixed to the subgenual nerve on its proximal end, by its attachment cells to the opposite part of the cuticle; the fixation by the attachment cells is accomplished by a vast quantity of cytoplasmic microtubules; the construction of the sensory units is the same as in other mononematic scolopidial organs. The role of the extracellular lumen inside the organ and the special shape of the SGO of Camponotus ligniperda in mechanical transmission is discussed.     

Organization of actin, myosin, and intermediate filaments in the brush border of intestinal epithelial cells

Terminal webs prepared from mouse intestinal epithelial cells were examined by the quick-freeze, deep-etch, and rotary-replication method. The microvilli of these cells contain actin filaments that extend into the terminal web in compact bundles. Within the terminal web these bundles remain compact; few filaments are separated from the bundles and fewer still bend towards the lateral margins of the cell. Decoration with subfragment 1 (S1) of myosin confirmed that relatively few actin filaments travel horizontally in the web. Instead, between actin bundles there are complicated networks of the fibrils. Here we present two lines of evidence which suggest that myosin is one of the major cross-linkers in the terminal web. First, when brush borders are exposed to 1 mM ATP in 0.3 M KCl, they lose their normal ability to bind antimyosin antibodies as judged by immunofluorescence, and they lose the thin fibrils normally found in deep-etch replicas. Correspondingly, myosin is released into the supernatant as judged by SDS gel electrophoresis. Second, electron microscope immunocytochemistry with antimyosin antibodies followed by ferritin- conjugated second antibodies leads to ferritin deposition mainly on the fibrils at the basal part of rootlets. Deep-etching also reveals that the actin filament bundles are connected to intermediate filaments by another population of cross-linkers that are not extracted by ATP in 0.3 M KCl. From these results we conclude that myosin in the intestinal cell may not only be involved in a short range sliding-filament type of motility, but may also play a purely structural role as a long range cross-linker between microvillar rootlets.     

Contraction mechanisms in composite active actin networks

Simplified in vitro systems are ideally suited for studying the principle mechanisms of the contraction of cytoskeletal actin systems. To shed light on the dependence of the contraction mechanism on the nature of the crosslinking proteins, we study reconstituted in vitro active actin networks on different length scales ranging from the molecular organization to the macroscopic contraction. Distinct contraction mechanisms are observed in polar and apolar crosslinked active gels whereas composite active gels crosslinked in a polar and apolar fashion at the same time exhibit both mechanisms simultaneously. In polar active actin/fascin networks initially bundles are formed which are then rearranged. In contrast, apolar cortexillin-I crosslinked active gels are bundled only after reorganization of actin filaments by myosin-II motor filaments.     

A novel actin-bundling kinesin-related protein from Dictyostelium discoideum

Actin filaments and microtubules are two major cytoskeletal systems involved in wide cellular processes, and the organizations of their filamentous networks are regulated by a large number of associated proteins. Recently, evidence has accumulated for the functional cooperation between the two filament systems via associated proteins. However, little is known about the interactions of the kinesin superfamily proteins, a class of microtubule-based motor proteins, with actin filaments. Here, we describe the identification and characterization of a novel kinesin-related protein named DdKin5 from Dictyostelium. DdKin5 consists of an N-terminal conserved motor domain, a central stalk region, and a C-terminal tail domain. The motor domain showed binding to microtubules in an ATP-dependent manner that is characteristic of kinesin-related proteins. We found that the C-terminal tail domain directly interacts with actin filaments and bundles them in vitro. Immunofluorescence studies showed that DdKin5 is specifically enriched at the actin-rich surface protrusions in cells. Overexpression of the DdKin5 protein affected the organization of actin filaments in cells. We propose that a kinesin-related protein, DdKin5, is a novel actin-bundling protein and a potential cross-linker of actin filaments and microtubules associated with specific actin-based structures in Dictyostelium.     

Fine structure of the terminal organ of the house fly larva,Musca domestica L.

Summary The terminal organs of the cephalic lobes of the house fly larva, Musca domestica L., were studied by scanning and transmission electron microscopy. Six different types of sensilla were found: (1) papilla sensillum, (2) pit sensillum, (3) spot sensillum, (4) modified papilla sensillum, (5) knob sensillum, and (6) scolopidium. The papilla, pit, spot, and modified papilla sensilla have the essential structure of contact chemoreceptors, i.e., the unbranched dendritic tips are exposed externally through a single opening. However, a tubular body, which is a characteristic structure of tactile setae, is also present in some of the dendritic tips. We assume these sensilla serve a dual function—contact chemo- and mechanoreception. The role of the knob sensilla is obscure. The scolopidia present in the dorsal and the terminal organ are probably stress detectors. Two basal bodies occur in the dendritic ciliary region of all sensilla. Both of the basal bodies (except in the scolopidia) give rise to the distal ciliary microtubules as well as the proximal rootlets.This research was supported in part by the Office of Naval Research, PHS Research Grant EC-246 and NIH Training Grant ES-00069. Paper No. 3608 of the North Carolina State University Agricultural Experiment Station journal series. The advise of R. A. Steinbrecht is gratefully acknowledged.     

Structure of the subgenual organ in the green lacewing, Chrysoperla carnea

REM and TEM studies of the subgenual organ in Chrysoperla carnea (Neuroptera: Chrysopidae) show that it is composed of three scolopidia, each with one sensory, one scolopale and one cap cell. The distal part of the dendrite shows a cilium with a '9 + 0' structure. The cross-handing pattern of the ciliary root has a periodicity of bands of about 61 nm. The scolopale material in a certain part of the scolopale cell is organized into five rods. The cell bodies of all three cap cells form a lens-like structure. the velum, which is fixed to the leg wall and the trachea with an extracellular material. The importance of the velum is discussed. Four types of intercellular junction are found; spot desmosomes. belt desmosomes, septate junctions and gap junctions.     

Spine-scale reorientation inApedinella radians (Pedinellales,Chrysophyceae): the microarchitecture and immunocytochemistry of the associated cytoskeleton

Summary Motile unicells ofApedinella radians have the extraordinary ability to instantaneously reorient six elongate spine-scales located on the cell surface. Extracellular striated fibrous connectors (termed microligaments) attach spine-scales to discrete regions of the plasma membrane underlain by intricate cytoplasmic plaques. A complex cytoskeleton is associated with the plaques and appears responsible for spine-scale movement. Three cytoskeletal proteins have thus far been identified by immunofluorescence using anti-tubulin, anti-actin, and anti-centrin. The three-dimensional configuration of the cytoskeleton has been established and consists of filamentous bundles of actin and centrin which form stellate systems interconnecting the plaques. Additionally, there is a network of microtubular triads which originate on the surface of the nuclear envelope and subtend the plasma membrane and also support several tentacular protrusions. It is proposed that contraction of the actin and/or centrin filamentous bundles is responsible for the reorientation of the spine-scales.     

Cytoskeletal elements in migrating and tentacle-integrated nematocytes of a marine hydrozoan

Summary— Actively migrating nematocytes of the marine polyp Stauridiosarsia producta are converted into completely immotile cells as soon as they become integrated in the ectodermal tissue of the tentacles. Immunocytochemical and electron microscopical methods revealed that cytoskeletal elements composed of actin, tubulin, centrin and a still unidentified protein are interwoven within the complete cell. While the organization of the sensory pole of the nematocyte, containing the cnidocil complex, the pseudovillar system and the distal half of a microtubular basket surrounding the nematocyst, is not affected by the transition from a motile to an immotile cell, the cytoskeletal elements in the basal portion of the cell are re-organized. Thus, the basolateral cytoplasm of migrating cells contains less organized microtubular arrays and bundles of about 10 nm-thick filaments. In the tentacle-integrated state, the 10-nm filaments are concentrated within a stalk-like foot which is stabilized by some rigid microtubular arrays derived from the microtubular basket. By elongation of the microtubular basket towards the cellular basis, the nematocyst becomes indirectly anchored at the mesoglea. As indicated by pharmacological treatments, the stiffness of the stalk depends on its microtubular content only.