Effects of channel blocking on information transmission and energy efficiency in squid giant axons.

Action potentials are the information carriers of neural systems. The generation of action potentials involves the cooperative opening and closing of sodium and potassium channels. This process is metabolically expensive because the ions flowing through open channels need to be restored to maintain concentration gradients of these ions. Toxins like tetraethylammonium can block working ion channels, thus affecting the function and energy cost of neurons. In this paper, by computer simulation of the Hodgkin-Huxley neuron model, we studied the effects of channel blocking with toxins on the information transmission and energy efficiency in squid giant axons. We found that gradually blocking sodium channels will sequentially maximize the information transmission and energy efficiency of the axons, whereas moderate blocking of potassium channels will have little impact on the information transmission and will decrease the energy efficiency. Heavy blocking of potassium channels will cause self-sustained oscillation of membrane potentials. Simultaneously blocking sodium and potassium channels with the same ratio increases both information transmission and energy efficiency. Our results are in line with previous studies suggesting that information processing capacity and energy efficiency can be maximized by regulating the number of active ion channels, and this indicates a viable avenue for future experimentation.

Self-coacervation of modular squid beak proteins - a comparative study.

The beak of the Humboldt squid is a biocomposite material made solely of organic components - chitin and proteins - which exhibits 200-fold stiffness and hardness gradients from the soft base to the exceptionally hard tip (rostrum). The outstanding mechanical properties of the squid beak are achieved via controlled hydration and impregnation of the chitin-based scaffold by protein coacervates. Molecular-based understanding of these proteins is essential to mimic the natural beak material. Here, we present detailed studies of two histidine-rich beak proteins (HBP-1 and -2) that play central roles during beak bio-fabrication. We show that both proteins have the ability to self-coacervate, which is governed intrinsically by the sequence modularity of their C-terminus and extrinsically by pH and ionic strength. We demonstrate that HBPs possess dynamic structures in solution and achieve maximum folding in the coacervate state, and propose that their self-coacervation is driven by hydrophobic interactions following charge neutralization through salt-screening. Finally, we show that subtle differences in the modular repeats of HBPs result in significant changes in the rheological response of the coacervates. This knowledge may be exploited to design self-coacervating polypeptides for a wide range of engineering and biomedical applications, for example bio-inspired composite materials, smart hydrogels and adhesives, and biomedical implants.

Characterization of the cell polarity gene crumbs during the early development and maintenance of the squid-vibrio light organ symbiosis.

The protein Crumbs is a determinant of apical-basal cell polarity and plays a role in apoptosis of epithelial cells and their protection against photodamage. Using the squid-vibrio system, a model for development of symbiotic partnerships, we examined the modulation of the crumbs gene in host epithelial tissues during initiation and maintenance of the association. The extracellular luminous symbiont Vibrio fischeri colonizes the apical surfaces of polarized epithelia in deep crypts of the Euprymna scolopes light organ. During initial colonization each generation, symbiont harvesting is potentiated by the biochemical and biophysical activity of superficial ciliated epithelia, which are several cell layers from the crypt epithelia where the symbionts reside. Within hours of crypt colonization, the symbionts induce the cell death mediated regression of the remote superficial ciliated fields. However, the crypt cells directly interacting with the symbiont are protected from death. In the squid host, we characterized the gene and encoded protein during light organ morphogenesis and in response to symbiosis. Features of the protein sequence and structure, phylogenetic relationships, and localization patterns in the eye supported assignment of the squid protein to the Crumbs family. In situ hybridization revealed that the crumbs transcript shows opposite expression at the onset of symbiosis in the two different regions of the light organ: elevated levels in the superficial epithelia were attenuated whereas low levels in the crypt epithelia were turned up. Although a rhythmic association in which the host controls the symbiont population over the day-night cycle begins in the juvenile upon colonization, cycling of crumbs was evident only in the adult organ with peak expression coincident with maximum symbiont population and luminescence. Our results provide evidence that crumbs responds to symbiont cues that induce developmental apoptosis and to symbiont population dynamics correlating with luminescence-based stress throughout the duration of the host-microbe association.

The ultrastructure and contractile properties of a fast-acting, obliquely striated, myosin-regulated muscle: the funnel retractor of squids.

We investigated the ultrastructure, contractile properties, and in vivo length changes of the fast-acting funnel retractor muscle of the long-finned squid Doryteuthis pealeii. This muscle is composed of obliquely striated, spindle-shaped fibers ~3 mum across that have an abundant sarcoplasmic reticulum, consisting primarily of membranous sacs that form 'dyads' along the surface of each cell. The contractile apparatus consists of 'myofibrils' approximately 0.25-0.5 microm wide in cross section arrayed around the periphery of each cell, surrounding a central core that contains the nucleus and large mitochondria. Thick myofilaments are approximately 25 nm in diameter and approximately 2.8 microm long. 'Dense bodies' are narrow, resembling Z lines, but are discontinuous and are not associated with the cytoskeletal fibrillar elements that are so prominent in slower obliquely striated muscles. The cells approximate each other closely with minimal intervening intercellular connective tissue. Our physiological experiments, conducted at 17 degrees C, showed that the longitudinal muscle fibers of the funnel retractor were activated rapidly (8 ms latent period following stimulation) and generated force rapidly (peak twitch force occurred within 50 ms). The longitudinal fibers had low V(max) (2.15 +/-0.26 L(0) s(-1), where L(0) was the length that generated peak isometric force) but generated relatively high isometric stress (270+/-20 mN mm(-2) physiological cross section). The fibers exhibited a moderate maximum power output (49.9 W kg(-1)), compared with vertebrate and arthropod cross striated fibers, at a V/V(max) of 0.33+/-0.044. During ventilation of the mantle cavity and locomotion, the funnel retractor muscle operated in vivo over a limited range of strains (+0.075 to -0.15 relative to resting length, L(R)) and at low strain rates (from 0.16 to 0.91 L(R) s(-1) ), corresponding to a range of V/V(max) from 0.073 to 0.42. During the exhalant phase of the jet the range of strains was even narrower: maximum range less than +/-0.04, with the muscle operating nearly isometrically during ventilation and slow, arms-first swimming. The limited length operating range of the funnel retractor muscles, especially during ventilation and slow jetting, suggests that they may act as muscular struts.

The argonaut shell: gas-mediated buoyancy control in a pelagic octopus.

Argonauts (Cephalopoda: Argonautidae) are a group of rarely encountered open-ocean pelagic octopuses with benthic ancestry. Female argonauts inhabit a brittle 'paper nautilus' shell, the role of which has puzzled naturalists for millennia. The primary role attributed to the shell has been as a receptacle for egg deposition and brooding. Our observations of wild argonauts have revealed that the thin calcareous shell also functions as a hydrostatic structure, employed by the female argonaut to precisely control buoyancy at varying depths. Female argonauts use the shell to 'gulp' a measured volume of air at the sea surface, seal off the captured gas using flanged arms and forcefully dive to a depth where the compressed gas buoyancy counteracts body weight. This process allows the female argonaut to attain neutral buoyancy at depth and potentially adjust buoyancy to counter the increased (and significant) weight of eggs during reproductive periods. Evolution of this air-capture strategy enables this negatively buoyant octopus to survive free of the sea floor. This major shift in life mode from benthic to pelagic shows strong evolutionary parallels with the origins of all cephalopods, which attained gas-mediated buoyancy via the closed-chambered shells of the true nautiluses and their relatives.

Cartilage differentiation in cephalopod molluscs.

Amongst the various metazoan lineages that possess cartilage, tissues most closely resembling vertebrate hyaline cartilage in histological section are those of cephalopod molluscs. Although elements of the adult skeleton have been described, the development of these cartilages has not. Using serial histology of sequential developmental stages of the European cuttlefish, Sepia officinalis, we investigate these skeletal elements and offer the first description of the formation of any cellular invertebrate cartilage. Our data reveal that cuttlefish cartilage most often differentiates from uncondensed mesenchymal cells near the end of embryonic development, but that the earliest-forming cartilages differentiate from a cellular condensation which goes through a protocartilage stage in a manner typical of vertebrate primary cartilage formation. We further investigate the distribution and degree of differentiation of cartilages at the time of hatching in an additional four cephalopod species. We find that the timing of cartilage development varies between elements within a single species, as well as between species. We identify a tendency towards cartilage differentiation from uncondensed connective tissue in elements that form at the end of embryogenesis or after hatching. These data suggest a form of metaplasia from connective tissue is the ancestral mode of cartilage formation in this lineage.

Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters.

Cadmium (Cd) and mercury (Hg) levels were measured in the tissue samples of two loliginid (Alloteuthis sp. and Loligo forbesi) and two ommastrephid (Todarodes sagittatus and Todaropsis eblanae) squid species collected from research cruise and fishery (market) samples in UK waters during 2004-05. Concentrations of Cd were generally higher in the ommastrephids, in all tissues except muscle. Hg concentrations were higher in T. sagittatus than in the loliginids. In L. forbesi, metal concentrations differed between tissues and also varied in relation to body size, geographic origin, and season. Cd levels decreased with increasing body size. This may be related to a shift in the diet with growth, since small L. forbesi feed on benthic invertebrates that have relatively high Cd concentrations, whereas larger individuals prey mainly on fish that have low Cd concentrations. Hg levels increased with body size, indicating its retention, and they were highest at the end of the spawning season and in squid from the English Channel and the Scottish West Coast. It is likely that the ambient concentration of Hg in seawater plays an important part in its accumulation in squid tissues. As it is a short-lived species, L. forbesi may therefore function as a bioindicator species for Hg contamination of the marine environment. Our results indicate that there is no significant danger to humans from consuming squid from UK waters.

Characterization and role of p53 family members in the symbiont-induced morphogenesis of the Euprymna scolopes light organ.

Within hours of hatching, the squid Euprymna scolopes forms a specific light organ symbiosis with the marine luminous bacterium Vibrio fischeri. Interactions with the symbiont result in the loss of a complex ciliated epithelium dedicated to promoting colonization of host tissue, and some or all of this loss is due to widespread, symbiont-induced apoptosis. Members of the p53 family, including p53, p63, and p73, are conserved across broad phyletic lines and p63 is thought to be the ancestral gene. These proteins have been shown to induce apoptosis and developmental morphogenesis. In this study, we characterized p63-like transcripts from mRNA isolated from the symbiotic tissues of E. scolopes and described their role in symbiont-induced morphogenesis. Using degenerate RT-PCR and RACE PCR, we identified two p63-like transcripts encoding proteins of 431 and 567 amino acids. These transcripts shared identical nucleotides where they overlapped, suggesting that they are splice variants of the same gene. Immunocytochemistry and Western blots using an antibody specific for E. scolopes suggested that the p53 family members are activated in cells of the symbiont-harvesting structures of the symbiotic light organ. We propose that once the symbiosis is initiated, a symbiont-induced signal activates p53 family members, inducing apoptosis and developmental morphogenesis of the light organ.

Squid (Loligo pealei) giant fiber system: a model for studying neurodegeneration and dementia?

In many neurodegenerative disorders that lead to memory loss and dementia, the brain pathology responsible for neuronal loss is marked by accumulations of proteins in the form of extracellular plaques and intracellular filamentous tangles, containing hyperphosphorylated cytoskeletal proteins. These are assumed to arise as a consequence of deregulation of a normal pattern of topographic phosphorylation-that is, an abnormal shift of cytoskeletal protein phosphorylation from the normal axonal compartment to cell bodies. Although decades of studies have been directed to this problem, biochemical approaches in mammalian systems are limited: neurons are too small to permit separation of cell body and axon compartments. Since the pioneering studies of Hodgkin and Huxley on the giant fiber system of the squid, however, the stellate ganglion and its giant axons have been the focus of a large literature on the physiology and biochemistry of neuron function. This review concentrates on a host of studies in our laboratory and others on the factors regulating compartment-specific patterns of cytoskeletal protein phosphorylation (primarily neurofilaments) in an effort to establish a normal baseline of information for further studies on neurodegeneration. On the basis of these data, a model of topographic regulation is proposed that offers several possibilities for further studies on potential sites of deregulation that may lead to pathologies resembling those seen in mammalian and human brains showing neurodegeneration, dementia, and neuronal cell death.

Confocal microscopy of the light organ crypts in juvenile Euprymna scolopes reveals their morphological complexity and dynamic function in symbiosis.

In the hours to days following hatching, the Hawaiian bobtail squid, Euprymna scolopes, obtains its light-emitting symbiont, Vibrio fischeri, from the surrounding environment and propagates the bacteria in the epithelial crypts of a specialized light organ. Three-dimensional analyses using confocal microscopy revealed that each of the three crypts on either side of the juvenile light organ is composed of four morphological regions. Progressing from the lateral pore to the medial blind end of each crypt, the regions consist of 1) a duct, 2) an antechamber, 3) a bottleneck, and 4) a deep region. Only the deep region houses a persistent bacterial population, whereas the duct, antechamber, and bottleneck serve as conduits through which the bacteria enter during initial colonization and exit during diel venting, a behavior in which approximately 90% of the symbionts are expelled each dawn. Our data suggest that, like the duct, the antechamber and bottleneck may function to promote and maintain the specificity of the symbiosis. Pronounced structural and functional differences among the deep regions of the three crypts, along with previously reported characterizations of embryogenesis, suggest a continued developmental progression in the first few days after hatching. Taken together, the results of this study reveal a high degree of complexity in the morphology of the crypts, as well as in the extent to which the three crypts and their constituent regions differ in function during the early stages of the symbiosis.

Specific accumulation of polychlorinated biphenyls and organochlorine pesticides in Japanese common squid as a bioindicator.

Organochlorines (OCs) representing Persistent Organic Pollutants (POPs) such as PCBs (polychlorinated biphenyls), DDTs (DDT and its metabolites), CHLs (chlordane compounds), HCHs (hexachlorocyclohexane isomers) and HCB (hexachlorobenzene) were determined in the liver of Japanese common squid (Todarodes pacificus) collected from the waters around Japan (Japan Sea and western North Pacific Ocean). Among OCs concentrations, PCBs (upto 5600 ng/g lipid wt.) were the highest, and those of other OCs were in the order of DDT> CHLs > HCHs > HCB. Studies on growth trend and seasonal variation of OCs in this species suggest a rapid reflection of the pollution levels in seawater where and when they were collected, regardless of body-length and time of collection. These results indicate that Japanese common squid is a suitable bioindicator for monitoring OCs pollution in waters around Japan. With regard to the geographical distribution of OCs in this species collected from waters around Japan, OCs concentrations in specimens from Japan Sea were higher than those from the Pacific Ocean. This result might reflect some existing of local pollution sources of OCs around Japan Sea, and slower water exchange between Japan Sea and open ocean.

Vibrio fischeri lipopolysaccharide induces developmental apoptosis, but not complete morphogenesis, of the Euprymna scolopes symbiotic light organ.

During initiation of the association between the squid host Euprymna scolopes and its bacterial partner Vibrio fischeri, the bacteria induce dramatic morphogenesis of the host symbiotic organ, a portion of which involves the signaling of widespread apoptosis of the cells in a superficial ciliated epithelium on the colonized organ. In this study, we investigated the role in this process of lipopolysaccharide (LPS), a bacterial cell-surface molecule implicated in the induction of animal cell apoptosis in other systems. Purified V. fischeri LPS, as well as the LPS of V. cholerae, Haemophilus influenzae, Escherichia coli, and Shigella flexneri, added in the concentration range of pg/ml to ng/ml, induced apoptosis in epithelial cells 10- to 100-fold above background levels. The absence of species specificity suggested that the conserved lipid A portion of the LPS was the responsible component of the LPS molecule. Lipid A from V. fischeri, E. coli, or S. flexneri induced apoptosis. In addition, strains of H. influenzae carrying a mutation in the htrB gene, which is involved in the synthesis of virulent lipid A, showed a diminished ability to induce apoptosis of host cells. Confocal microscopy using fluorescently labeled LPS indicated that the LPS behaves similar to intact bacterial symbionts, interacting with host cells in the internal crypt spaces and not directly with the superficial epithelium. Although LPS was able to induce apoptosis, it did not induce the full morphogenesis of the ciliated surface, suggesting that multiple signals are necessary to mediate the development of this animal-bacterial mutualism.

Vesicle transport: the role of actin filaments and myosin motors.

The transport of vesicles and the retention of organelles at specific locations are fundamental processes in cells. Actin filaments and myosin motors have been shown to be required for both of these tasks. Most of the organelles in cells associate with actin filaments and some of the myosin motors required for movement on actin filaments have been identified. Myosin V has been shown to transport endoplasmic reticulum (ER) vesicles in neurons, pigment granules in melanocytes, and the vacuole in yeast. Myosin I has been shown to be involved in the transport of Golgi-derived vesicles in epithelial cells. Myosin VI has been shown to be associated with Golgi-derived vesicles, and cytoplasmic vesicles in living Drosophila embryos. Myosin II may be a vesicle motor but its role in vesicle transport has not been resolved. Secretory vesicles, endosomes and mitochondria appear to be transported on actin filaments but the myosin motors on these organelles have not been identified. Mitochondria in yeast may be transported by the dynamic assembly of an actin "tail." The model that has unified all of these findings is the concept that long-range movement of vesicles occurs on microtubules and short-range movement on actin filaments. The details of how the microtubule-dependent and the actin-dependent motors are coordinated are important questions in the field. There is now strong evidence that two molecular motors, kinesin and myosin V, interact with each other and perhaps function as a complex on vesicles. An understanding of the interrelationship of microtubules and actin filaments and the motors that move cargo on them will ultimately establish how vesicles and organelles are transported to their specific locations in cells.

Microscopia electrónica de fibras nerviosas gigantes / Electron microscopy of giant nerve fibers

El presente trabajo es una revisión breve sobre los detalles ultraestructurales de las fibras nerviosas gigantes y su posible correlación con hallazgos fisiológicos hechos en el axón gigante del calamar. Tanto en secciones finas, como en réplicas obtenidas por crío-fractura, el aspecto más llamativo ha sido la gran cantidad de membranas apareadas que llenan la capa de Schwann. Estas membranas limitan hendiduras intercelulares permeables que conectan la superficie axónica con el espacio extracelular del endoneuro, dejando, así, al axolema como la única barrera continua interpuesta entre el axoplasma y el exterior de la neurona. Se ha observado, tanto en las secciones como en las réplicas, zonas de adosamiento íntimo del axón y de su célula de Schwann a nivel d elos llamados complejos estructurales, en los cuales aparecen involucradas las membranas plasmáticas de ambas células adyacentes. Estas zonas podrían constituir la expresión morfológica del acoplamiento funcional dado a conocer en la misma preparación. Además, la célula de Shwann parece ser muy activa a juzgar por la cantidad de perfiles de exo-endocitosis observados en todas sus superficies de fractura. Finalmente, las células del endoneuro aparecen diferentes en los varios tipos de fibras gigantes estudiados: en el calamar, ellas se muestran como células esponjosas, mientras que en la langosta, ellas exhiben una cantidad extraordinaria de imágenes de exo-endocitosis entremezcladas con algunas uniones epiteliales de baja resistencia y con uniones estrechas incompletas. Por último, en el camarón de río, la célula endoneurales muestran el mismo aspecto que el de la glía adaxónica

Neurofilament protein is phosphorylated in the squid giant axon.

We have observed the phosphorylation of neurofilament protein from squid axoplasm. Phosphorylation is demonstrated by 32P labeling of protein during incubation of axoplasm with [gamma-32P]ATP. When the labeled proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two bands, at 2.0 x 10(5) daltons and greater than 4 x 10(5) daltons, contain the bulk of the 32P. The 2.0 x 10(5)-dalton phosphorylated polypeptide comigrates on SDS-PAGE with one of the subunits of squid neurofilament protein. Both major phosphorylated polypeptides co-fractionate with neurofilaments in discontinuous sucrose gradient centrifugation and on gel filtration chromatography on Sepharose 4B. The protein-phosphate bond behaves like a phospho-ester, and labeled phospho-serine is identified in an acid hydrolysate of the protein. The generality of this phenomenon in various species and its possible physiological significance are discussed.

The cells that secrete the beaks in octopods and squids (Mollusca, Cephalopoda).

A single layer of cell secrets the hard cephalopod beaks. The beccublasts are tall columnar cells that separate the beak from the surrounding buccal muscles, and must serve to attach these muscles to the beak. Within the cell layer there are three types of cells. The first, and most frequently found contain cell-long fibrils. These fibrils may have contractile and tensile properties. Complex trabeculae extend from the beccublasts into the matrix of the beak. The fibrils are attached to these trabeculae and at the other end of the cells they are anchored near to the beccublast-muscle cell interface, closely associated with the muscles that move the beak. The second group of cells contain masses of endoplasmic reticulum the cysternae of which are arranged along the long axis of the cell. These cells also contain dense granules and are probably the major source of beak hard tissue. It is probable that each cell secretes its own column of beak hard tissue. The third group of cells cells contains a mixture of fibrils and secretory tissue. In the beccublast layer there are changes in the proportion of the three types of cells depending upon the region sampled. In the region where growth is most active there are mostly secretory cells, whereas near the biting and wearing tip there are mainly anchoring type cells.

A light and electron microscope study of intra-epithelial putative mechanoreceptors in squid suckers.

Putative mechanoreceptor neurons in the cuticularized epithelium of the suckers of the squid Lolliguncula brevis, were investigated using light and electron microscope techniques. These neurons were found to have a multipolar shape, thick unbranched axon, glial cell ensheathment, and accessory nerve fiber innervation. The need for electrophysiological and/or behavioral studies on these putative mechanoreceptors is emphasized.