Myosin-linked calcium regulation in squid mantle muscle. Light-chain components of squid myosin.

As reported by Kendrick-Jones et al. (1976), myosin from squid mantle muscle contains two types of light-chain components, different in size but similar in net charge. We were able to separate the two types of light chains by a five-step procedure, yielding LC-1 (17,000 daltons) and LC-2 (15,000 daltons). It was found that squid mantle LC-1 and LC-2 function exactly like SH-light chains and EDTA-light chains of scallop adductor myosin, respectively. In functional tests, we used "desensitized" myosin of scallop adductor muscle, simply because "EDTA washing" removed neither LC-1 nor LC-2 from squid mantle myosin. The removal and recombination of light chains were examined by gel electrophoresis, and Ca or Sr sensitivity was determined by measuring the Mg-ATPase activity of skeletal acto-scallop or squid myosin. It was found that EDTA washing readily released the EDTA-light chains of scallop myosin completely, and that the EDTA-washed scallop myosin was capable of regaining its full content of EDTA-LC as well as its full sensitivity to calcium. We also found that as regards combining with, and conferring calcium sensitivity on the EDTA-washed myosin of scallop adductor, squid mantle LC-2 could effectively replace scallop adductor EDTA-LC. In addition, calcium or strontium ions were found to induce changes in the UV absorption spectrum of scallop adductor EDTA-LC, although the apparent binding constants estimated from the difference spectrum were too low to account for the Ca or Sr sensitivity of scallop actomyosin-ATPase. The divalent cations also induced changes in the UV absorption spectrum of squid LC-2, and the apparent binding constants estimated from the difference spectrum were sufficiently high (1.5 X 10(5) M-1 for Ca binding, and 1.6 X 10(3) M-1 for Sr binding) to account for the Ca and Sr sensitivities of squid mantle myosin B-ATPase. The findings with scallop adductor myosin are in conflict with those reported by Kendrick-Jones et al., and must be accounted for in formulating the molecular mechanism of myosin-linked calcium regulation in molluscan muscles.     

Comparative expression and tissue distribution analyses of astacin-like squid metalloprotease in squid and cuttlefish

Astacin-like squid metalloprotease (ALSM) is a member of the astacin family of metalloproteases. In the present study, we investigated the expression and tissue distribution of ALSM in bigfin reef squid (Sepioteuthis lessoniana) and golden cuttlefish (Sepia esculenta). Myosin heavy chain hydrolysis tests showed ALSM-I-like activity in both species. We isolated partial cDNA clones showing high sequence similarity to ALSM-I and -III, suggesting that ALSM is common to squid and cuttlefish. Phylogenetic analysis showed that ALSMs are classified into two clades: ALSM-I forms one clade, and ALSM-II and -III form the other. ALSM was expressed in several tissues in bigfin reef squid, though expression was confined to the liver in cuttlefish. ALSMs are distributed in digestive organs but not in mantle muscle of squid and cuttlefish. Immunofluorescence analysis further showed that cellular localization of ALSM is evident not only in hepatic cells but also in pancreatic cells of bigfin reef squid. Thus, ALSM is commonly expressed in squid and cuttlefish, but its expression levels and distribution are distinct.     

The journey of squid sperm

Sperm storage is common in internally fertilizing animals, but is also present in several external fertilizers, such as many cephalopods. Cephalopod males attach sperm packets (spermatangia) to female conspecifics during mating. Females of eight externally fertilizing families comprising 25% of cephalopod biodiversity have sperm-storage organs (seminal receptacles) in their buccal area, which are not in direct physical contact with the deposited spermatangia. The mechanism of sperm transmission between the implantation site and the storage organ has remained a major mystery in cephalopod reproductive biology. Here, jumbo squid females covering almost the entire life cycle, from immature to a laboratory spawned female, were used to describe the internal structure of the seminal receptacles and the process of sperm storage. Seminal fluid was present between the spermatangia and seminal receptacles, but absent in regions devoid of seminal receptacles. The sperm cellular component was formed by spermatozoa and round cells. Although spermatozoa were tracked over the buccal membrane of the females to the inner chambers of the seminal receptacles, round cells were not found inside the seminal receptacles, suggesting that spermatozoa are not sucked up by the muscular action of the seminal receptacles. This finding supports the hypothesis that spermatozoa are able to actively migrate over the female skin. Although further experimental support is needed to fully confirm this hypothesis, our findings shed light on the elusive process of sperm storage in many cephalopods, a process that is fundamental for understanding sexual selection in the sea.     

Mastigoteuthis–the whip-lash squid

Features of the brain of this oceanic squid have been investigated and related, as far as possible, to its habits and mode of life. The body and arms are much vacuolated for buoyancy and the animal probably lives with the head upwards. The very long whip-like tentacles are not vacuolated and perhaps hang downwards. They are covered by numerous minute pedunculated suckers, perhaps providing a sticky surface. A special nerve running outside the brain carries signals from the arms and tentacles to the magnocellular lobe, which is very large and of complex structure. However, there are no giant cells and the mantle is weak. Propulsion is mainly by the large fins, which are controlled from the magnocellular lobe, presumably using the information from the arms and tentacles.     

Automated Resonance Assignment of Proteins: 6 DAPSY-NMR

The 6-dimensional (6D) APSY-seq-HNCOCANH NMR experiment correlates two sequentially neighboring amide moieties in proteins via the C′ and C^α nuclei, with efficient suppression of the back transfer from C^α to the originating amide moiety. The automatic analysis of two-dimensional (2D) projections of this 6D experiment with the use of GAPRO (Hiller et al., 2005) provides a high-precision 6D peak list, which permits automated sequential assignments of proteins with the assignment software GARANT (Bartels et al., 1997). The procedure was applied to two proteins, the 63-residue 434-repressor(1–63) and the 115-residue TM1290. For both proteins, complete sequential assignments for all NMR-observable backbone resonances were obtained, and the polypeptide segments thus identified could be unambiguously located in the amino acid sequence. These results demonstrate that APSY-NMR spectroscopy in combination with a suitable assignment algorithm can provide fully automated sequence-specific backbone assignments of small proteins.Francesco Fiorito and Sebastian Hiller - Both authors contributed equally to this work     

The squid accessory nidamental gland: Ultrastructure and association with bacteria

The structure of the accessory nidamental gland of the female squid, Loligo pealei, has been investigated using transmission and scanning electron microscopy. The accessory gland has many of the structural features of a secretory organ. The basic structural unit is a tubule composed of a single layer of epithelial cells containing ordered arrays of rough endoplasmic reticulum and a lumenal surface covered with microvilli, cilia, and structural specializations presumed to be involved in secretion. The lumen of each tubule is filled with a dense population of bacteria. During sexual maturation of the squid, the accessory gland changes in color from white to mottled red. The accessory gland of the sexually mature squid has a mixture of red, white, and yellow tubules ; in each case, the color of the tubule is due to the bacterial population occupying the tubule. Since the red color of the gland is due to the pigmentation of the bacteria, the bacteria must be responsive to the sexual state of the host, possibly through a change in the nature of the material secreted into the tubule lumen. The bacteria can be cultured easily, but in culture they fail to synthesize the red pigment.     

Magnesium efflux in dialyzed squid axons

The efflux of Mg++ from squid axons subject to internal solute control by dialysis is a function of ionized [Mg], [Na], [ATP], and [Na]o. The efflux of Mg++ from an axon with physiological concentrations of ATP, Na, and Mg inside into seawater is of the order of 2-4 pmol/cm2s but this efflux is strongly inhibited by increases in [Na]i, by decreases in [ATP]i, or by decreases in [Na]o. The efflux of Mg++ is largely independent of [Mg]i when ATP is at physiological levels, but in the absence of ATP reaches half the value of Mg efflux in be presence of ATP when [Mg]i is about 4 mM and [Na] 40 mM. Half-maximum responses to ATP occur at about 350 micronM ATP into seawater with Na either present or absent. The Mg efflux mechanism has many similarities to the Ca efflux system in squid axons especially with respect to the effects of ATP, Nao, and Na on the flux. The concentrations of free Mg and Ca in axoplasm differ, however, by a factor of 10(5) while the observed fluxes differ by a factor of 10(2).     

The rhodopsin system of the squid

Squid rhodopsin (λ_max 493 mµ)—like vertebrate rhodopsins—contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (λ_max 500 mµ) near neutrality, yellow (λ_max 380 mµ) in alkaline solution. The two forms—acid and alkaline metarhodopsin—are interconverted according to the equation, Alkaline metarhodopsin + H⁺ acid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.     

Frequency entrainment of squid axon membrane

Summary Sinusoidally varying stimulating currents were applied to space-clamped squid giant axon membranes in a double sucrose gap apparatus. Stimulus parameters varied were peak-to-peak current amplitude, frequency, and DC offset bias. In response to these stimuli, the membranes produced action potentials in varying patterns, according to variation of input stimulus parameters. For some stimulus parameters the output patterns were stable and obviously periodic with the periods being simple multiples of the input period; for other stimulus parameters no obvious periodicity was manifest in the output. The experimental results were compared with simulations using a computer model which was modified in several ways from the Hodgkin-Huxley model to make it more representative of our preparation. The model takes into account K⁺ accumulation in the periaxonal space, features of Na⁺ inactivation which are anomalous to the Hodgkin-Huxley model, sucrose gap hyperpolarization current, and membrane current noise. Many aspects of the experiments are successfully simulated but some are not, possibly because some very slow process present in the preparation is not included in the model.     

N-Glycan structures of squid rhodopsin.

To determine the glycoforms of squid rhodopsin, N-glycans were released by glycoamidase A digestion, reductively aminated with 2-aminopyridine, and then subjected to 2D HPLC analysis [Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y. & Tomiya, N. (1995) Anal. Biochem.226, 139-146]. The major glycans of squid rhodopsin were shown to possess the alpha1-3 and alpha1-6 difucosylated innermost GlcNAc residue found in glycoproteins produced by insects and helminths. By combined use of 2D HPLC, electrospray ionization-mass spectrometry and permethylation and gas chromatography-electron ionization mass spectrometry analyses, it was revealed that most (85%) of the N-glycans exhibit the novel structure Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4(Galbeta1-4Fucalpha1-6)(Fucalpha1-3)GlcNAc.     

Locomotory function of the squid mantle

A detailed kinematic analysis of the mantle movements of swimming Lolliguncula brevis was made. Some data were also obtained on Loligo pealei. The qualitative and quantitative data provided are of use in discussing mechanisms of squid mantle function.
Several possible mechanisms of squid mantle re-expansion were proposed and investigated. The inhalant phase of jet propulsion is probably effected by contraction of the radial muscles, since thinning of the mantle wall accompanies re-expansion. The radial muscles may cause mantle re-expansion by contracting alternately with the circular muscles in response to nerve impulses, or by contracting alternately with the circular muscles in response to stretch cycles effected by mantle wall thickening in the power stroke, or by contracting continuously through both power and recovery strokes. Elasticity of the mantle tissue may contribute to mantle re-expansion. Neither pressure pumps nor a Bernoulli effect mechanism are effectors of mantle re-expansion.     

Ionized calcium concentrations in squid axons

Values for ionized [Ca] in squid axons were obtained by measuring the light emission from a 0.1-mul drop of aequorin confined to a plastic dialysis tube of 140-mum diameter located axially. Ionized Ca had a mean value of 20 x 10(-9) M as judged by the subsequent introduction of CaEGTA/EGTA buffer (ratio ca. 0.1) into the axoplasm, and light measurement on a second aequorin drop. Ionized Ca in axoplasma was also measured by introducing arsenazo dye into an axon by injection and measuring the Ca complex of such a dye by multichannel spectrophotometry. Values so obtained were ca. 50 x 10(-9) M as calibrated against CaEGTA/EGTA buffer mixtures. Wth a freshly isolated axon in 10 mM Ca seawater, the aequorin glow invariably increased with time; a seawater [Ca] of 2-3 mM allowed a steady state with respect to [Ca]. Replacement of Na+ in seawater with choline led to a large increase in light emission from aequorin. Li seawater partially reversed this change and the reintroduction of Na+ brought light levels back to their initial value. Stimulation at 60/s for 2-5 min produced an increase in aequorin glow about 0.1% of that represented by the known Ca influx, suggesting operationally the presence of substantial Ca buffering. Treatment of an axon with CN produced a very large increase in aequorin glow and in Ca arsenazo formation only if the external seawater contained Ca.     

Physiological color change in squid iridophores

Evidence is presented that changes in the optical properties of active iridophores in the dermis of the squid Lolliguncula brevis are the result of changes in the ultrastructure of these cells. At least two mechanisms may be involved when active cells change from non-iridescent to iridescent or change iridescent color. One is the reversible change of labile, detergent-resistant proteinaceous material within the iridophore platelets, from a contracted gel state (non-iridescent) to an expanded fluid or sol state when the cells become iridescent. The other is a change in the thickness of the platelets, with platelets becoming significantly thinner as the optical properties of the iridophores change from non-iridescent to iridescent red, and progressively thinner still as the observed iridescent colors become those of shorter wavelengths. Optical change from Rayleigh scattering (non-iridescent) to structural reflection (iridescent) may be due to the viscosity change in the platelet material, with the variations in observed iridescent colors due to changes in the dimensions of the iridophore platelets.     

Locomotory aspects of squid mantle structure

Morphological aspects of squid ( Loligo, Lolliguncula ) mantle relevant to locomotory function were studied. Methods used included polarized light microscopy of frozen sections of untreated tissue taken from animals immediately after death and electron microscopy.
The mantle consists of circular and radial muscles arranged in alternating rings along the whole length of the mantle. The muscle is obliquely striated. Connective tissue fibres are found in the body of the muscle and in the outer and inner tunics. The outer tunic consists of layers of large collagenous fibres. The fibres run in superimposed right- and left-handed helical courses that lie at an angle of 27° to the long axis of the animal. The tunics and the intramuscular connective fibres are thought to resist length changes in the mantle while permitting the changes in girth required for the jet power stroke. Both the intramuscular and the tunic fibre systems may provide elastic energy for the return phase of the jet cycle. Tunic fibres appear to be a geodesic tensile reinforcing system ensuring smooth shape changes in the mantle.