Fast axonal transport of foreign synaptic vesicles in squid axoplasm

Translocation of intracellular organelles requires interaction with the cellular cytoskeleton, but the membrane and cytoskeletal proteins involved in movement are unknown. Here we show that highly purified synaptic vesicles from electric fish added to extruded squid axoplasm can show ATP-dependent movement. The movement is indistinguishable from that of endogenous vesicles and has a slight preference for the orthograde direction. In the presence of a nonhydrolyzable ATP analog, the synaptic vesicles bind to axoplasmic fibers but do not move. Elastase treatment of vesicles inhibits both binding and movement. We conclude that a protein component on the surface of cholinergic synaptic vesicles from electric fish is conserved during evolution and so can be recognized by the organelle-translocating machinery of the squid axon, resulting in ATP-dependent movement. Synaptic vesicles apparently retain the capacity for fast axonal transport, even after they reach their intracellular destination.     

Characterization of the phosphorylation sites of the squid (Loligo pealei) high-molecular-weight neurofilament protein from giant axon axoplasm

Axonal caliber in vertebrates is attributed, in part, to the extensive phosphorylation of NFM and NFH C-terminal tail domain KSP repeats by proline-directed kinases. The squid giant axon, primarily involved in rapid impulse conduction during jet propulsion motility, is enriched in squid-specific neurofilaments, particularly the highly phosphorylated NF-220. Of the 228 serine-threonine candidate phosphate acceptor sites in the NF-220 tail domain (residues 401-1220), 82 are found in numerous repeats of three different motifs SAR/K, SEK/R, K/RSP, with 62 of these tightly clustered in the C-terminal repeat segment (residues 840-1160). Characterization of the in vivo NF-220 phosphorylated sites should provide clues as to the relevant kinases. To characterize these sites, proteolytic digests of NF-220 were analyzed by a combination of HPLC, electrospray tandem mass spectrometry and database searching. A total of 53 phosphorylation sites were characterized, with 47 clustered in the C-terminal repeat segment (residues 840-1160), representing 76% (47/62) of the total acceptor sites in the region. As in mammalian NFH, approximately 64% of the K/RSP sites (14/22) in this region were found to be phosphorylated implicating proline-directed kinases. Significantly, 78% of serines (31/40) in the KAES*EK and EKS*ARSP motifs were also phosphorylated suggesting that non proline-directed kinases such as CKI may also be involved. This is consistent with previous studies showing that CKI is the principal kinase associated with axoplasmic NF preparations. It also suggests that phosphorylation of large macromolecules with multiple phospho-sites requires sequential phosphorylation by several kinases.     

Resistivity of axoplasm. I. Resistivity of extruded squid axoplasm

Six methods have given squid axoplasm resistivities of from 1.0 to 6.9 times seawater (X SW), so another was tried. A 100-mum platinized electrode was to be inserted from each end of an axion in iso-osmotic sucrose and impedance between them measured vs. separation. But observations that the resistance of axons in sucrose increased steadily ruled this out. Axoplasm from two or three axons was transferred to a glass capillary, 0.6 mm ID, and the 1-kHz series resistance and reactance were measured at electrode separations from 16 to 2 mm. The resistance was linear vs. distance, giving the resistivity, while the reactance was nearly constant, implying constant electrode contributions. Frequency runs from 10 Hz to 30 kHz at 10 mm gave electrode impedances of the form (jomega)-alpha, allowing 1-2% effects on the axoplasm resistivities. In nine experiments, one was discarded for cause, the range and average resistivities were, respectively, 1.2-1.6 and 1.4 times those of artificial seawater (19.7 omegacm at 24.4 degrees C). No single cause for the variability was apparent. These experiments essentially confirm the means and variations of two early experiments with intact axons and recent results with a single internal electrode to give overall resistivities of 1.4 +/- 0.2 X SW.     

Cell junctions in early embryos of squid (Loligo pealei)

Summary Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7–8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12–13). During germinal layer establishment (stages 12–13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavagestage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.     

The locomotory function of the fins in the squid Loligo pealei

Kinematic data of high spatial and temporal resolution, acquired from image sequences of adult long-finned squid, Loligo pealei, during steady swimming in a flume, were used to examine the role of fins and the coordination between fin and jet propulsion in squid locomotion. Fin shape and body outlines were digitized and used to calculate fin wave speed, amplitude, frequency, angle of attack, body deformation, speed, and acceleration. L. pealei were observed to have two fin gait patterns with a transition at 1.4-1.8 mantle lengths per second (L_m s^-1) marked by alternation between the two patterns. Fin motion in L. pealei exhibited characteristics of both traveling waves and flapping wings. At low speeds, fin motion was more wave-like; at high speeds, fin motion was more flap-like and was marked by regular periods during which the fins were wrapped tightly against the mantle. Fin cycle frequencies were dependent on swimming speed and gait, and obvious coordination between the fins and jet were observed. Fin wave speed, angle of attack, and body acceleration confirmed the role of fins in thrust production and revealed a role of fins at all swimming speeds by a transition from drag-based to lift-based thrust when fin wave speed dropped below swimming speed. Estimates of peak fin thrust were as high as 0.44-0.96 times peak jet thrust in steady swimming over the range of swimming speeds observed. Fin downstrokes generally contributed more to thrust than did upstrokes, especially at high speeds.     

The locomotory function of the fins in the squid Loligo pealei

Kinematic data of high spatial and temporal resolution, acquired from image sequences of adult long-finned squid, Loligo pealei, during steady swimming in a flume, were used to examine the role of fins and the coordination between fin and jet propulsion in squid locomotion. Fin shape and body outlines were digitized and used to calculate fin wave speed, amplitude, frequency, angle of attack, body deformation, speed, and acceleration. L. pealei were observed to have two fin gait patterns with a transition at 1.4-1.8 mantle lengths per second (Lm s-1) marked by alternation between the two patterns. Fin motion in L. pealei exhibited characteristics of both traveling waves and flapping wings. At low speeds, fin motion was more wave-like; at high speeds, fin motion was more flap-like and was marked by regular periods during which the fins were wrapped tightly against the mantle. Fin cycle frequencies were dependent on swimming speed and gait, and obvious coordination between the fins and jet were observed. Fin wave speed, angle of attack, and body acceleration confirmed the role of fins in thrust production and revealed a role of fins at all swimming speeds by a transition from drag-based to lift-based thrust when fin wave speed dropped below swimming speed. Estimates of peak fin thrust were as high as 0.44-0.96 times peak jet thrust in steady swimming over the range of swimming speeds observed. Fin downstrokes generally contributed more to thrust than did upstrokes, especially at high speeds.     

An ultrastructural and cytochemical analysis of oogenesis in the squid, Loligo pealei

Oogenesis has been investigated utilizing both light and electron microscopical techniques in the squid, Loligo pealei. This complex process has been divided into five stages according to the structure of the follicle. Because of the highly coordinated differentiation of the follicle cells (and follicular syncytium) and the oocyte, their development is described in concert. Specific attention is given to the contribution of the follicular syncytium to vitellogenesis and the formation of the extracellular egg envelope or chorion. Our observations indicate heterosynthetic yolk production and the synthesis of the secondary envelope by the follicular syncytium.     

Deltamethrin induced changes in choline transport and phosphorylation activities in synaptosomes from the optic lobe of squid, Loligo pealei

1. Deltamethrin, a powerful synthetic pyrethroid causes a significant change in choline transport in freshly prepared synaptosomes from squid optic lobes. 2. At resting state (nondepolarized) such an effect manifested as a reduction of 14C-choline uptake in a short term (1 min) uptake experiment. 3. At depolarized state, or under conditions where synaptosomes are subjected to osmotic, aging and other stress conditions, deltamethrin caused stimulation of 14C-choline uptake, resulting in elevation of the levels of total radiocarbons in synaptosomes. 4. Such changes are accompanied with changes in overall phosphorylation activities in synaptosomes.     

Laser light scattering determination of size and dispersity of synaptosomes and synaptic vesicles isolated from squid (Loligo pealei) optic lobes

Quasi-elastic laser light scattering has been used to investigate the size and dispersity of synaptosomes and synaptic vesicles isolated from optic lobes of the squid Loligo pealei. Synaptosomal fractions were highly polydisperse ( ) and the mean diameter ( ) ranged from 0.5–2.0 m. Size distribution histograms yielded two major components — smaller particles ( ) and a larger group of particles ( ). The heterogeneity of the synaptosomal particles detected in solution is in agreement with published data obtained using electron microscopy. Purified synaptic vesicle fractions also yielded complex particle size distribution data. A component with a mean diameter in the range 150–250 nm was detected, though a smaller particle ( ) dominated the scattering signal. This smaller particle closely resembles in size the electron lucent vesicles seen in the majority of squid optic lobe nerve terminals when examined by electron microscopy. Osmotically-induced shirnkage and swelling of the synptosomes was detected. Depolarization by veratridine (1.0×10^–4 M) did not result in a detectable change in the size of synaptosomal particles.     

Light-induced changes in the content of inositol phosphates in squid (Loligo pealei) retina.

Illumination induced an increase in inositol polyphosphates, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, in the photoreceptors of the squid, Loligo pealei. There was a concomitant decrease in phosphatidylinositol 4,5-bisphosphate, but no light-induced change in any other phosphoinositide. None of these stimulus-induced changes were altered by treatment in vivo with pertussis toxin, which ADP-ribosylated a Mr-39000-peptide. These findings support the hypothesis that inositol 1,4,5-trisphosphate participates as either a messenger or a modulator in transduction in invertebrate photoreceptors.     

Topographic regulation of phosphorylation in giant neurons of the squid, Loligo pealei: role of phosphatases

In previous studies of phosphorylation in squid stellate ganglion neurons, we demonstrated that a specific multimeric phosphorylation complex characterized each cellular compartment. Although the endogenous protein profile of cell body extracts (giant fiber lobe, GFL), as determined by Coomassie staining, was similar to that of axoplasm from the giant axon, in this study we show that the protein phosphorylation profiles are qualitatively different. Whereas many axoplasm proteins were phosphorylated, including most cytoskeletal proteins, virtually all phosphorylation in perikarya was confined to low molecular weight compounds (<6 kDa). Because phosphorylation of exogenous substrates, histone and casein, was equally active in extracts from both compartments, failure to detect endogenous protein phosphorylation in cell bodies was attributed to the presence of more active phosphatases. To further explore the role of phosphatases in these neurons, we studied phosphorylation in the presence of serine/threonine and protein tyrosine phosphatase (PTP) inhibitors. We found that phosphorylation of axonal cytoskeletal proteins was modulated by okadaic acid-sensitive ser/thr phosphatases, whereas cell body phosphorylation was more sensitive to an inhibitor of protein tyrosine phosphatases, such as vanadate. Inhibition of PTPs by vanadate stimulated endogenous phosphorylation of GFL proteins, including cytoskeletal proteins. Protein tyrosine kinase activity was equally stimulated by vanadate in cell body and axonal whole homogenates and Triton X-100 free soluble extracts, but only the Triton X soluble fraction (membrane bound proteins) of the GFL exhibited significant activation in the presence of vanadate, suggesting higher PTP activities in this fraction than in the axon. The data are consistent with the hypothesis that neuronal protein phosphorylation in axons and cell bodies is modulated by different phosphatases associated with compartment-specific multimeric complexes.     

Characterization of phospholipase A2 and acyltransferase activities in squid (Loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions.

Phospholipase A₂ and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A₂ activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A₂ was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating.

The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K_m (apparent) and V_max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide.

Phospholipase A₂ activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A₂ activity was stimulated by Ca⁺⁺ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca⁺⁺-independent phospholipase A₂, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.     

Cation concentrations in the hemolymph of Loligo pealei.

Hemolymph and protein-free hemolymph obtained from Loligo pealei were analyzed for cation concentration by the method of atomic absorption spectroscopy, for the following ions: Na+, K+, Ca++, and Mg++. No significant differences were seen in ion concentration between hemolymph and protein-free hemolymph. Of particular neurophysiological significance is that K+ ion concentration is much closer to that of seawater than previously reported.