N-Ethylmaleimide-modified subfragment-1 and heavy meromyosin inhibit reactivated contraction in motile models of retinal cones

The mechanism of contraction in motile models of teleost retinal cones has been examined by using N-ethylmaleimide (NEM)-modified myosin fragments (NEM-S-1 and NEM-heavy meromyosin [HMM]) to prevent access of native myosin to actin filaments during reactivation of contraction. In the diurnal light/dark cycle, retinal cones of green sunfish (Lepomis cyanellus) and bluegill (lepomis macrochirus) exhibit length changes of more than 90 mum. The motile myoid region of the cone contracts from 100 mum in the dark to 6 mum in the light. Motile models for cone contraction have been obtained by lysis of dark-adapted retinas with the non-ionic detergent, Brij-58. These cone motile models undergo Ca(++)-and ATP-dependent reactivated contraction, with morphology and rate comparable to those observed in vivo (Burnside, B.,B. Smith, M. Nagata, and K. Porrello, 1982, J. Cell Biol., 92:198-206). The cone myoids contain longitudinally oriented actin filaments which bind myosin subfragment-1 (S-1) to form characteristic “arrowhead” complexes which dissociate in the presence of MgATP (Burnside, B., 1978, J. Cell Biol., 78:227-246). Modification of S-1 or HMM with the sulfhydryl reagent, NEM, produces new species, NEM-S-1 or NEM-HMM, which still bind actin but which fail to detach in the presence of MgATP (Meeusen, R.L., and W.Z. Cande, 1979, J. Cell Biol., 82:57-65). We have used NEM-S-1 and NEM-HMM to test whether cone contraction depends on an actomyosin force- generating system. We find that reactivated contraction of cone models is inhibited by NEM-S-1 and NEM-HMM but not by the unmodified species, S-1 and HMM. Thus, reactivated cone contraction exhibits NEM-S-1 and NEM-HMM sensitivity as well as Ca(++)- and ATP- dependence. These observations are consistent with and actimyosin-mediated mechanism for force production during cone contraction.     

Regulation of reactivated contraction in teleost retinal cone models by calcium and cyclic adenosine monophosphate

We have been using lysed cell models of teleost retinal cones to examine the mechanism of contraction in nonmuscle cells. We have previously reported that dark-adapted retinas can be lysed with the detergent Brij-58 to obtain cone motile models that undergo Ca++- and adenosine triphosphate (ATP)-dependent reactivated contraction. In this report we further dissect the roles of ATP and Ca++ in activation of contraction and force production by (a) characterizing the Ca++ and nucleotide requirements in more detail, (b) by analyzing the effects of inosine triphosphate (ITP) and the ATP analog ATP gamma S and (c) by testing effects of cyclic adenosine monophosphate (cAMP) on reactivated cone contraction. Exposing lysed cone models to differing free Ca++ concentrations produced reactivated contraction at rates proportional to the free Ca++ concentration between 3.16 X 10(-8) and 10(-6) M. A role for calmodulin (CaM) in this Ca++ regulation was suggested by the inhibition of reactivated contraction by the calmodulin inhibitors trifluoperazine and calmidazolium ( R24571 ). The results of analysis of nucleotide requirements in lysed cone models were consistent with those of smooth muscle studies suggesting a role for myosin phosphorylation in Ca++ regulation of contraction. ATP gamma S and ITP are particularly interesting in that ATP gamma S, on the one hand, can be used by kinases to phosphorylate proteins (e.g., myosin light chains) but resists cleavage by phosphatases or adenosine triphosphatases (ATPases), e.g., myosin ATPase. ITP, on the other hand, can be used by myosin ATPase but does not support Ca++/calmodulin mediated phosphorylation of myosin light chains by myosin light chain kinase. Thus, these nucleotides provide an opportunity to distinguish between the kinase and myosin ATPase requirements for ATP. When individual nucleotides were tested with cone motile models, the nucleotide requirement was highly specific for ATP; not only ITP and ATP gamma S, but also guanosine triphosphate, cytosine triphosphate, adenylyl-imidodiphosphate (AMPPNP) failed to support reactivated contraction when substituted for ATP throughout the incubation. However, if lysed cones were initially incubated with ATP gamma S and then subsequently incubated with ITP, the cones contracted to an extent that was comparable to that observed with ATP. As observed in skinned smooth muscle, adding cAMP to contraction medium strongly inhibited contraction in lysed cone models.     

Regulation of reactivated elongation in lysed cell models of teleost retinal cones by cAMP and calcium

Teleost retinal cones elongate in the dark and contract in the light. In isolated retinas of the green sunfish Lepomis cyanellus, cone myoids undergo microtubule-dependent elongation from 5 to 45 micron. We have previously shown that cone contraction can be reactivated in motile models of cones lysed with Brij-58. Reactivated contraction is both actin and ATP dependent, activated by calcium, and inhibited by cAMP. We report here that we have obtained reactivated cone elongation in lysed models prepared by the same procedures. Reactivated elongation is ATP dependent, activated by cAMP, and inhibited by calcium. The rate of reactivated elongation is proportional to the cAMP concentration between 10 microM and 0.5 mM, but is constant between 10 microM and 1.0 mM Mg-ATP. No elongation occurs if cAMP or Mg-ATP concentration is less than or equal to 5 microM. Mg-ATP is required for both cAMP-dependent and cAMP-independent processes, suggesting that Mg-ATP is required both for a regulatory process entailing cAMP-dependent phosphorylation and for a force-producing process. Free calcium concentrations greater than or equal to 10(-7) reduce the elongation rate by 78% or more, completely inhibiting elongation at 10(-5) M. This inhibition is not due to competition from calcium-activated contraction. Cytochalasin D blocks reactivated contraction, but does not abolish calcium inhibition of reactivated elongation. Thus calcium directly affects the elongation mechanism. Calcium inhibition is calmodulin dependent. The calmodulin inhibitor trifluoperazine abolishes calcium inhibition of elongation. Furthermore, calcium blocks elongation only if present during the lysis step; subsequent calcium addition has no effect. However, if calcium plus exogenous calmodulin are subsequently added, elongation is again inhibited. Thus calcium inhibition appears to require a soluble calmodulin which is lost shortly after lysis.     

Calcium-independent contraction in lysed cell models of teleost retinal cones: activation by unregulated myosin light chain kinase or high magnesium and loss of cAMP inhibition

The retinal cones of teleost fish contract at dawn and elongate at dusk. We have previously reported that we can selectively induce detergent-lysed models of cones to undergo either reactivated contraction or reactivated elongation, with rates and morphology comparable to those observed in vivo. Reactivated contraction is ATP dependent, activated by Ca2+, and inhibited by cAMP. In addition, reactivated cone contraction exhibits several properties that suggest that myosin phosphorylation plays a role in mediating Ca2+-activation (Porrello, K., and B. Burnside, 1984, J. Cell Biol., 98:2230-2238). We report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with trypsin-digested, unregulated myosin light chain kinase (MLCK) obtained from smooth muscle. This observation provides further evidence that MLCK plays a role in regulating cone contraction. We also report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with high concentrations of MgCl2 (10-20 mM). Mg2+-induced reactivated contraction is supported by inosine triphosphate (ITP) just as well as by ATP. Because ITP will not serve as a substrate for MLCK, this finding suggests that Mg2+-activation of contraction does not require myosin phosphorylation. Although Ca2+-induced contraction is completely blocked by cAMP at concentrations less than 10 microM, cAMP has no effect on cone contraction activated by unregulated MLCK or by high Mg2+ in the absence of Ca2+. Because trypsin digestion of MLCK cleaves off not only the Ca2+/calmodulin-binding site but also the site phosphorylated by cAMP-dependent protein kinase, and because Mg2+ activation of cone contraction circumvents MLCK action altogether, both these observations would be expected if cAMP inhibits reactivated cone contraction by catalyzing the phosphorylation of MLCK and thus reducing its affinity for Ca2+, as has been described for smooth muscle. Together our results suggest that in lysed cone models, myosin phosphorylation is sufficient for activating cone contraction, even in the absence of other Ca2+-mediated events, that cAMP inhibition of contraction is mediated by cAMP-dependent phosphorylation of MLCK, and that 10-20 mM Mg2+ can activate actin-myosin interaction to produce contraction in the absence of myosin phosphorylation.     

Microtubles and actin filaments in teleost visual cone elongation and contraction

Teleost retinal cones contract in light and elongate in darkness. This paper describes the disposition of microtubules and cytoplasmic filaments in cone cells of 2 species of fish (Haemulon sciurus and Lutjanus griseus). In Haemulon, the neck-like “myoid” region of the cone changes in length from 5 μ to 75 μ. Maximal observed rates of elongation and contraction are comparable to that of chromosome movement in mitosis (2–3 μ/min). Microtubules presumably participate in cone elongation, since numerous longitudinal microtubules are present in the myoid region, and colchicine blocks dark-induced elongation. Myoid shortening, on the other hand, appears to be an active contractile process. Disruption of microtubules in dark-adapted cones does not produce myoid shortening in the absence of light, and light-induced myoid shortening is blocked by cytochalasin-B. Cone cells possess longitudinally-oriented thin filaments which bind myosin subfragment-1 to form arrowhead complexes typical of muscle actin. Myoid thin filaments are clearly observed in negatively stained preparations of isolated cones which have been disrupted with detergent after attachment to grids. These myoid filaments are not, however, generally preserved by conventional fixation, though bundles of thin filaments are preserved in other regions of the cell. Thus, actin filaments are poorly retained by fixation in precisely the region of the cone cell where contraction occurs. Cone cells also possess longitudinally-oriented thick filaments 130–160Å in diameter. That these thick filaments may be myosin is suggested by the presence of side-arms with approximately 150 Å periodicity. The linear organization of the contractile apparatus of the retinal cone cell makes this cell a promising model for morphological characterization of the disposition of actin and myosin filaments during contraction in a nonmuscle cell.     

Elevation of cyclic AMP activates an actin-dependent contraction in teleost retinal rods

Agents which elevate cyclic AMP (cAMP) cause teleost retinal rods to contract. We have characterized this cAMP effect and have evaluated the role of the cytoskeleton in cyclic nucleotide-induced contraction, using actin and microtubule inhibitors. The necklike myoid region of the rod contracts in the dark and elongates in the light. If long, light-adapted rods are cultured with cAMP analogs and IBMX, rods contract to their short dark-adapted position. Cyclic nucleotide- induced rod contraction occurs in constant light, requires a phosphodiesterase inhibitor, and is specific to cAMP (db cyclic GMP, 8- bromocyclic GMP, 5'AMP, and adenosine have no effect on rod myoid length). Cyclic AMP effects on rod length are consistent with observations from several species that cAMP levels are higher in dark- adapted than in light-adapted retinas. Since rod myoids contain paraxially aligned actin filaments and microtubules, we have used the motility inhibitors cytochalasin D and cold and nocodazole to investigate the roles of these cytoskeletal elements in rod contraction. Cyclic nucleotide-induced contraction is not inhibited when myoid microtubules are disrupted with cold and nocodazole treatments, but contraction is blocked if myoid actin filaments are disrupted with cytochalasin D. Thus, we conclude that actin filaments, but not microtubules, are required for rod contraction. We propose that rod contraction in vivo is triggered by a rise of cytoplasmic cAMP at onset of darkness and that this contraction is mediated by an actin- dependent mechanism.     

Effects of extracellular Ca++, K+, and Na+ on cone and retinal pigment epithelium retinomotor movements in isolated teleost retinas

We have examined the effects of changes in extracellular ionic composition on cone and retinal pigment epithelium (RPE) retinomotor movements in cultured isolated teleost retinas. In vivo, the myoid portion of teleost cones contracts in the light and elongates in the dark; RPE pigment disperses in the light and aggregates in the dark. In vitro, cones of dark-adapted (DA) retinas cultured in constant darkness contracted spontaneously to their light-adapted (LA) positions if the culture medium contained greater than or equal to 10(-3)M Cao++. DA cones retained their long DA positions in a medium containing less than or equal to 10(-6)M Cao++. Low [Ca++]o (10(-5)-10(-7)M) also permitted darkness to induce cone elongation and RPE pigment aggregation. Light produced cone contraction even in the absence of Cao++, but the extent of contraction was reduced if [Ca++]o was less than 10(-3) M. Thus, full contraction appeared to require the presence of external Ca++. High [K+]o (greater than or equal to 27 mM) inhibited both light-induced and light-independent Ca++-induced cone contraction. However, low [Na+]o (3.5 mM) in the presence of less than or equal to 10(-6)M Cao++ did not mimic light onset by inducing cone contraction in the dark. High [K+]o also promoted dark-adaptive cone and RPE movements in LA retinas cultured in the light. All results obtained in high [K+]o were similar to those observed when DA or LA retinas were exposed to treatments that elevate cytoplasmic cyclic 3',5'-adenosine monophosphate (cAMP) content.     

Thin (actin) and thick (myosinlike) filaments in cone contraction in the teleost retina

The long slender retinal cones of fishes shorten in the light and elongate in the dark. Light-induced cone shortening provides a useful model for stuying nonmuscle contraction because it is linear, slow, and repetitive. Cone cells contain both thin (actin) and thick (myosinlike) filaments oriented parallel to the axis of contraction. This study examines the polarities of the cone's thin filaments and the changes in filament distribution which accompany light-induced contraction, in an attempt to elucidate the structural basis for the cone's contractile process. The proximal half of the cone is fixed to its cellular neighbors in the outer nuclear layer while the distal half is free. Thus, all shortening takes place in a necklike region (the myoid) in the distal half of the cone which extends into the space between the neural retina and the pigmented retinal epithelium. Thin filaments are found throughout the length of the cone, whereas thick filaments occur predominantly in the proximal (axon) regions of both light- and dark-adapted cones. Thus, thick filaments are primarily localized outside the region where shortening takes place. Observations from myosin subfragment-1 binding studies suggest that the cone's thin filaments are organized into two opposing sets. In the distal half of the cone (including the myoid), virtually all filaments have proximally directed arrowheads. In the more proximal regions of the axon, many thin filaments have opposite polarity, their arrowheads being distally directed. Near the synaptic proximal end of the light-adapted (contracted) cone, filaments of opposite polarities occur in approximately equal numbers. Thus, in the cone axon there appear to be two overlapping sets of actin filaments whose opposite polarities correspond to the two actin halves of a muscle sarcomere. In elongated, dark-adapted cones, thick filaments are localized throughout the axon region of the cone. In light, thick filaments accumulate towards the proximal end of the cone. These observations are consistent with a "sliding hypothesis" for cone contraction, in which thick myosinlike filaments produce sliding interdigitation of the two sets of oppositely directed actin filaments in the proximal axon region. Thus, the myoid thin filaments would be essentially reeled into the axon region to produce shortening. The mechanism of re-elongation depends on microtubules, as discussed in the companion paper.     

Evidence for D4 Receptor Regulation of Retinomotor Movement in Isolated Teleost Cone Inner-Outer Segments

Abstract: In the retinas of teleost fish, cone photoreceptors change shape in response to light and circadian signals. They elongate in the dark, contract in the light, and under conditions of constant darkness undergo appropriate movements at expected dusk and dawn. Dopamine induces cones to contract, thus mimicking the effect of light or expected dawn. To identify the receptor subtype responsible for mediating dopamine regulation of cone retinomotor movements, we have carried out pharmacological studies using isolated fragments of teleost cones consisting of cone inner segments-cone outer segments (CIS-COS). Isolated CIS-COS retain the ability to elongate in dark culture and contract when subsequently exposed to light or dopamine. We report that dark-induced elongation of CIS-COS was inhibited by dopamine and its agonists with an effectiveness ranking of dopamine = quinpirole > bromocriptine ⋙SKF-38393. After 60 min of elongation in dark culture, CIS-COS myoids contracted when subsequently cultured in the dark with dopamine or quinpirole. Quinpirole-induced inhibition of elongation and quinpirole-induced contraction were completely blocked by clozapine at 1 µ M or by sulpiride at 100 µ M . These effectiveness profiles for dopamine agonists and antagonists suggest that dopamine regulation of cone retinomotor movement is mediated by a D₄-like receptor.     

Reactivation of cell-free models of Physarum plasmodia after myosin reconstitution

Thin-spread glycerol-extracted Physarum plasmodia were treated with N-ethylmaleimide (NEM) to block myosin-ATPase and contractility. After supplementing the models with purified plasmodial myosin, they could be reactivated and contracted upon addition of ATP. Fluorescently labeled actomyosin fibers ruptured during contraction, resulting in beaded or rod-like contraction centers. Glycerol-extracted plasmodia lose their negative Ca++-dependence during extraction. Reconstitution of NEM-treated models with plasmodial myosin partly restored this Ca++-sensitivity. Thus, either myosin or a factor associated with it seems to be involved in the Ca++-dependent regulation of cytoplasmic actomyosin contraction in Physarum. NEM-blocked models reconstituted with skeletal muscle myosin were not reactivated by ATP. The same plasmodia subsequently incubated with plasmodial myosin were able to contract.     

Adenosine Stimulates Cone Photoreceptor Myoid Elongation via an Adenosine A2-Like Receptor

In several parts of the nervous system, adenosine has been shown to function as an extracellular neuromodulator binding to surface receptors on target cells. This study examines the possible role of adenosine in mediating light and circadian regulation of retinomotor movements in teleost cone photoreceptors. Teleost cones elongate in the dark and contract in the light. In continuous darkness, the cones continue to elongate and contract at subjective dusk and dawn in response to circadian signals. We report here that exogenous adenosine triggers elongation (the dark/night movement) in isolated cone inner segment-cone outer segment preparations (CIS-COS) in vitro. Agonist/antagonist potency profiles indicate that adenosine's effect on cone movement is mediated by an A2-like adenosine receptor, which like other A2 receptors enhances adenylate cyclase activity. Although closest to that expected for A2 receptors, the antagonist potency profile for CIS-COS does not correspond exactly to any known A2 receptor subtype, suggesting that the cone receptor may be a novel A2 subtype. Our findings are consistent with previous reports that retinal adenosine levels are higher in the dark, and further suggest that adenosine could act as a neuromodulatory "dark signal" influencing photoreceptor metabolism and function in the fish retina.     

Inhibition by ATP and activation by ADP in the regulation of flagellar movement in sea urchin sperm

ATP and ADP are known to play inhibitory and activating roles, respectively, in the regulation of dynein motile activity of flagella. To elucidate how these nucleotide functions are related to the regulation of normal flagellar beating, we examined their effects on the motility of reactivated sea urchin sperm flagella at low pH. At pH 7.0-7.2 which is lower than the physiological pH of 8, about 90% of reactivated flagella were motionless at 1 mM ATP, while about 60% were motile at 0.02 mM ATP. The motionless flagella at 1 mM ATP maintained a single large bend or an S-shaped bend, indicating formation of dynein crossbridges in the axoneme. The ATP-dependent inhibition of flagellar movement was released by ADP, and was absent in outer arm-depleted flagella. Similar inhibition was also observed at 0.02 mM ATP when demembranated flagella were reactivated in the presence of Li+ or pretreated with protein phosphatase 1 (PP1). ADP also released this type of ATP-inhibition. In PP1-pretreated axonemes the binding of a fluorescent analogue of ADP to dynein decreased. Under elastase-treatment at pH 8.0, the beating of demembranated flagella at 1 mM ATP and 0.02 mM ATP lasted for approximately 100 and 45 s, respectively. The duration of beating at 0.02 mM ATP was prolonged by Li+, and that at 1 mM ATP was shortened by removal of outer arms. These results indicate that the regulation of on/off switching of dynein motile activity of flagella involves ATP-induced inhibition and ADP-induced activation, probably through phosphorylation/dephosphorylation of outer arm-linked protein(s).     

Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos

Sensory neurons from chick embryos were cultured on substrata that support neurite growth, and were fixed and prepared for both cytochemical localization of actin and electron microscopic observation of actin filaments in whole-mounted specimens. Samples of cells were treated with the detergent Triton X-100 before, during, or after fixation with glutaraldehyde to determine the organization of actin in simpler preparations of extracted cytoskeletons. Antibodies to actin and a fluorescent derivative of phallacidin bound strongly to the leading margins of growth cones, but in neurites the binding of these markers for actin was very weak. This was true in all cases of Triton X- 100 treatment, even when cells were extracted for 4 min before fixation. In whole-mounted cytoskeletons there were bundles and networks of 6-7-nm filaments in leading edges of growth cones but very few 6-7-n filaments were present among the microtubules and neurofilaments in the cytoskeletons of neurites. These filaments, which are prominent in growth cones, were identified as actin because they were stabilized against detergent extraction by the presence of phallacidin or the heavy meromyosin and S1 fragments of myosin. In addition, heavy meromyosin and S1 decorated these filaments as expected for binding to F-actin. Microtubules extended into growth cone margins and terminated within the network of actin filaments and bundles. Interactions between microtubule ends and these actin filaments may account for the frequently observed alignment of microtubules with filopodia at the growth cone margins.     

Inhibition of the ATP-induced reactivation of demembranated hamster spermatozoa by the action of free ATP4- and MgATP2-

Hamster spermatozoa collected from the caput and cauda epididymidis were washed, diluted in a medium containing Triton X-100 to dissolve the cell membrane and reactivated with various concentrations of MgSO4 and ATP. Stepwise increase in the concentrations of free ATP4- from 0.08 to 1.1 mM at constant concentrations of MgATP2- caused a dose-dependent delay of reactivation but the maximal percentage of motile spermatozoa was inhibited only at 1.1 mM. The inhibitory effect on caput spermatozoa was greater than that on cauda spermatozoa. When concentrations of ATP4- were fixed at 0.2 mM, 2.9 mM-MgATP2- suppressed the reactivation of cauda spermatozoa. When compared to 0.9 mM-MgATP2-, reactivation of caput spermatozoa was delayed at 1.9 mM and almost completely blocked by 2.9 mM-MgATP2-. Inhibition of cauda sperm reactivation by ATP4- and MgATP2- were both prevented by the presence of trypsin (50 ng/ml). Incubation of cauda spermatozoa in the reactivation medium for 1 and 2 min before the addition of ATP progressively reduced the inhibitory effect of ATP4-; inhibition by MgATP2- was reduced to a lesser extent. Addition of 100 microM-cyclic AMP to the medium abolished the delay of reactivation by ATP4- but not that by MgATP2-. Before reactivation occurred, inhibitory concentrations of ATP4- and MgATP2- both induced large-angle coiling of sperm tails but in opposite direction to each other with reference to the asymmetric sperm head. The results suggest that free-ATP4- and superoptimal concentrations of MgATP2- inhibit flagellar movement by different mechanisms.     

Isolation and reactivation of highly-coupled newt lung cilia

The paired lungs of the newt, Taricha granulosa, are simple, unbranched sacs, 3.5-5.0 cm in length. The inner epithelium overlying the pulmonary vein is differentiated into a mucociliary tract that extends the entire length of the lung. Populations of single, demembranated ciliary axonemes, 12-13 micron in length, can be isolated by extracting whole lungs or primary cultures of the ciliated epithelium with Triton X-100. The motile capabilities of the isolated axonemes are the highest yet obtained for any ciliary model. When exposed to a suitable reactivating medium containing Mg2+ and ATP, nearly 100% of the axonemes become motile. Uniform reactivation of high quality requires short extraction times, minimization of mechanical damage, and strict adherence to optimal conditions throughout the extraction, storage, and reactivation procedures. Significant deviations from either pH 7.0 or 0.12 M salt can lead to a rapid, irreversible decrease in the beat frequency of reactivated axonemes. Both DTT and EDTA serve to stabilize their motility. The isolated axonemes beat at 29.5 Hz in the presence of 1.75 mM ATP at 21 degrees C, matching the beat frequencies measured for cultured cells at the same temperature. With 5 mM ATP, beat frequencies over 40 Hz are measured. Our results show that neither the plasma membrane, accessory structures, nor hydrodynamic coupling of cilia are required for this activity and imply that the lack of these factors is not responsible for the low motile capabilities of ciliary models isolated previously.     

Caffeine-stimulated ATP-reactivated motility in a detergent-treated bovine sperm model.

Mammalian sperm cells contain most of the components of a cyclic AMP-mediation system. To determine if the cyclic AMP-dependent protein kinase has a role in the control of bovine sperm motility, a sperm model was developed that was permeable to exogenously added ATP. Treatment of bovine epididymal spermatozoa with dithiothreitol and Brij-35 (polyoxyethylene alcohol), a nonionic surfactant, resulted in a sperm model with caffeine-stimulated, ATP-reactivatable motility. The results of the data obtained using this sperm model can be summarized as follows. (1) Brij partially solubilized the cyclic AMP-dependent protein kinase activity and released nearly half of the total acid-extractable nucleotides of the cells. (2) Brij treatment severely damaged the sperm mitochondria as judged by their lack of respiration. (3) Brij-treated spermatozoa lose their motility but were reactivated with ATP; the reactivated motility was stimulated by caffeine. (4) Despite the caffeine stimulation of motility in Brij-treated spermatozoa, increased protein phosphorylation did not accompany reactivation of motility, nor could a cyclic AMP effect be demonstrated on reactivated motility or on kinase activity in the sperm model.     

Effect of triton X-100 on ultrastructure,reactivation, and motility characteristics of ram spermatozoa

For optimal extraction and reactivation of ram sperm, glutamate, dithiothreitol, magnesium, and cyclic AMP were required in a medium of pH 7.9. On extraction with 0.01 % Triton X-100, ram sperm were only partially demembranated, and extensive areas of plasma membrane remained intact especially in the midpiece region. Treatment with 0.1% Triton X-100 removed all plasma membranes and extracted the mitochondrial membrane and matrix. In the absence of ATP, 16.6% ± 0.4 of the partially demembranated sperm were motile, but sperm extracted with 0.1% Triton X-100 were completely immotile. On adding ATP partially demembranated sperm reactivated better (81.6% ± 2.8) than sperm completely demembranated in 0.1 % Triton X-100 (39.5% ± 4.6). The release of intracellular LDH rose linearly with increasing concentrations of the detergent from 0.01 to 0.05%, at which it plateaued. There was a significant increase in beat frequency and forward velocity of partially demembranated sperm when treated with ATP. Partially demembranated sperm had intact mitochondria that presumably were still able to produce ATP, although the spermatozoan movement was stimulated by exogenous ATP.     

Filaments associated with the endoplasmic reticulum in the streaming cytoplasm of Chara corallina

Perfused Chara cells capable of resuming ATP-dependent cytoplasmic streaming in low free Ca++ solutions have been examined by electron microscopy for myosin-like filaments. Filaments 44 nm in diameter and up to 3 micron in length have been found associated with the endoplasmic reticulum that along with mitochondria, microbodies and dictyosomes from the endoplasm becomes immobilised around the sub-cortical actin bundles when ATP is depleted. Such endoplasmic filaments have not been detected in association with mitochondria or microbodies and they have not been found in the stationary cortex. These filaments are extracted from the perfused cell by ATP unless motility-inhibiting levels of cytochalasin B are present. The filaments are not detectable in cells inactivated in solutions containing high (10(-4) M) Ca++ concentrations even when the Ca++ level is subsequently lowered. Consistent with their being required for motility, cytoplasmic streaming cannot be effeiciently reactivated by ATP in such filament-depleted cells. The possibility is discussed that the filaments contain myosin and that the endoplasmic reticulum with which they are associated has a major role in generating and transmitting the motive force for streaming.