Essential light chain of Drosophila nonmuscle myosin II

Summary We have cloned and sequenced a cDNA encoding the essential (alkaline) light chain of nonmuscle myosin from Drosophila melanogaster. The protein predicted from the cDNA matches partial amino acid sequence derived from essential light chain protein that copurifies with native nonmuscle myosin heavy chain. This completes the sequence of the three myosin subunits, two of which have been shown genetically to be required for morphogenesis and cytokinesis (the heavy chain encoded by zipper and the regulatory light chain encoded by spaghetti squash). The essential light chain protein is 147 amino acids in length and is 53% identical to human smooth muscle essential light chain. The sequence is consistent with the presence of four helix-loop-helix domains seen in crystallographic structures of the striated muscle myosin light chains and their close relative, calmodulin. We identified the most conserved residues among essential light chain sequences from multiple phyla and present their locations on the crystallographic structure of striated muscle essential light chain. This highlights several conserved contacts among the myosin subunits that may be important for the structure and regulation of the myosin motor. The gene encoding Drosophila nonmuscle essential light chain (Mlc-c) localizes to cytological position 5A6 and we discuss prospects for genetic analysis in this region.     

Biphasic modulation of synaptic transmission by hypertonicity at the embryonic Drosophila neuromuscular junction

Puff-application of hypertonic saline (sucrose added to external saline) causes a transient increase in the frequency of spontaneous miniature synaptic currents (mSCs) at the neuromuscular junctions of Drosophila embryos. The frequency gradually returns to pre-application levels. External Ca²⁺ is not needed for this response, but it may modify it. At 50 m m added sucrose, for example, enhanced spontaneous release was observed only in the presence of external Ca²⁺, suggesting that Ca²⁺ augments the response. In a high-K⁺ solution, in which the basal mSC frequency was elevated, higher sucrose concentrations produced an increase in mSC frequency that was followed (during and after the hypertonic exposure) by depression, with the magnitude of both effects increasing with hypertonicity between 100 and 500 m m. Evoked release by nerve stimulation showed only depression in response to hypertonicity. We do not believe that the depression of spontaneous or evoked release can be explained by the depletion of releasable quanta, however, since the frequency of quantal release did not reach levels compatible with this explanation and the enhancement and depression could be obtained independent of one another. In a mutant lacking neuronal synaptobrevin, only the depression of mSC frequency was induced by hypertonicity. Conversely, only the enhancing effect was observed in wild-type embryos when the mSC frequency was elevated with forskolin in Ca²⁺-free saline. In cultured embryonic Drosophila neurons, Ca²⁺ signals that were induced by high K⁺ and detected by Fura-2, were reduced by hypertonicity, suggesting that the depressing response is due to a direct effect of hypertonicity on Ca²⁺ influx.     

Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase

Ca2+ sensitivity of smooth muscle and nonmuscle myosin II reflects the ratio of activities of myosin light-chain kinase (MLCK) to myosin light-chain phosphatase (MLCP) and is a major, regulated determinant of numerous cellular processes. We conclude that the majority of phenotypes attributed to the monomeric G protein RhoA and mediated by its effector, Rho-kinase (ROK), reflect Ca2+ sensitization: inhibition of myosin II dephosphorylation in the presence of basal (Ca2+ dependent or independent) or increased MLCK activity. We outline the pathway from receptors through trimeric G proteins (Galphaq, Galpha12, Galpha13) to activation, by guanine nucleotide exchange factors (GEFs), from GDP. RhoA. GDI to GTP. RhoA and hence to ROK through a mechanism involving association of GEF, RhoA, and ROK in multimolecular complexes at the lipid cell membrane. Specific domains of GEFs interact with trimeric G proteins, and some GEFs are activated by Tyr kinases whose inhibition can inhibit Rho signaling. Inhibition of MLCP, directly by ROK or by phosphorylation of the phosphatase inhibitor CPI-17, increases phosphorylation of the myosin II regulatory light chain and thus the activity of smooth muscle and nonmuscle actomyosin ATPase and motility. We summarize relevant effects of p21-activated kinase, LIM-kinase, and focal adhesion kinase. Mechanisms of Ca2+ desensitization are outlined with emphasis on the antagonism between cGMP-activated kinase and the RhoA/ROK pathway. We suggest that the RhoA/ROK pathway is constitutively active in a number of organs under physiological conditions; its aberrations play major roles in several disease states, particularly impacting on Ca2+ sensitization of smooth muscle in hypertension and possibly asthma and on cancer neoangiogenesis and cancer progression. It is a potentially important therapeutic target and a subject for translational research.     

Quantal unit populations at the Drosophila larval neuromuscular junction.

Focal extracellular recording at visualized boutons of the Drosophila larval neuromuscular junction was used to determine frequency and time course of the spontaneously occurring quantal events. When simultaneous intracellular recordings from the innervated muscle cell were made, more than one class of quantal event occurred at some of the individual boutons. "True" signals (arising at the bouton within the focal macropatch electrode) were often contaminated by additional signals generated outside the lumen of the focal electrode. Inclusion of these contaminating signals gave spuriously low values for relative amplitude, and spuriously high values for spontaneous quantal emission, for the synapses within the focal electrode. The contaminating signals, which appeared to be conducted along the subsynaptic reticulum surrounding the nerve terminals, generally were characterized by relatively small extracellular signals associated with normal intracellular events in the muscle fiber. From plots of simultaneous extracellular and intracellular recordings, the individual data points were classified according to the angles they subtended with the x axis (extracellular signal axis). Statistical procedures were developed to separate the true signals and contaminants with a high level of confidence. Populations of quantal events were found to be well described by Gaussian mixtures of two or three components, one of which could be characterized as the true signal population. Separation of signals from contaminants provides a basis for improving the estimates of quantal size and spontaneous frequency for the synapses sampled by the focal extracellular electrode.     

Hippocampal synaptic transmission and plasticity are preserved in myosin Va mutant mice

Recent studies have identified myosin Va as an organelle motor that may have important functions in neurons. Abundantly expressed at the hippocampal postsynaptic density, it interacts with protein complexes involved in synaptic plasticity. It is also located in presynaptic terminals and may function to recruit vesicles in the reserve pool to the active zone. Dilute-lethal mice are spontaneous myosin Va mutants and have severe neurological symptoms. We studied hippocampal physiology at CA3-CA1 excitatory synapses in dilute-lethal mutant mice to test the hypothesis that myosin Va plays a role in pre- or postsynaptic elements of synaptic transmission. In all assays performed, the mutant synapses appeared to be functioning normally, both pre- and postsynaptically. These data suggest that myosin Va is not essential for the synaptic release machinery, postsynaptic receptor composition, or plasticity at this synapse, but does not exclude significant roles for myosin Va in other cell types nor potential compensation by other myosin V isoforms.     

Synaptic vesicle recycling at the neuromuscular junction in the presence of a presynaptic membrane marker

Staining of the presynaptic axonal membrane of the neuromuscular junction with horseradish peroxidase-labeled α-bungarotoxin was utilized as a marker for observing directly the fate of this membrane during the process of synaptic vesicle release and recycling. The neuromuscular junctions of frog sartorius-sciatic nerve preparations were stained with horseradish peroxidase-α-bungarotoxin and stimulated by electrical stimulation of the nerve, high concentration of external potassium ions, and black widow spider venom. Some preparations were stimulated in the presence of exogenous horseradish peroxidase tracer after incubation in the conjugate and were found to contain horseradish peroxidase within many synaptic vesicles, indicating that the conjugate did not affect the process of synaptic vesicle recycling. Stimulation was followed by depletion of synaptic vesicles and appearance of axolemmal infoldings and membranous cisternae. With rest after electrical and potassium stimulation, synaptic vesicles were reconstituted and terminals assumed a more normal appearance. Membrane staining after stimulation occurred in the axolemmal infoldings, some of the intra-axonal cisternae, and in a few coated vesicles. However, all synaptic vesicles were unreactive, in either rested or unrested terminals. Thus, axonal membrane labeled with horseradish peroxidase-α-bungarotoxin did not become incorporated into new synaptic vesicles.These observations support a mechanism of recycling of synaptic vesicles by specific retrieval of vesicle membrane or constituents from the axolemma.     

Drosophila Neto is essential for clustering glutamate receptors at the neuromuscular junction

Neurotransmitter receptor recruitment at postsynaptic specializations is key in synaptogenesis, since this step confers functionality to the nascent synapse. The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse, similar in composition and function to mammalian central synapses. Various mechanisms regulating the extent of postsynaptic ionotropic glutamate receptor (iGluR) clustering have been described, but none are known to be essential for the initial localization and clustering of iGluRs at postsynaptic densities (PSDs). We identified and characterized the Drosophila neto (neuropilin and tolloid-like) as an essential gene required for clustering of iGluRs at the NMJ. Neto colocalizes with the iGluRs at the PSDs in puncta juxtaposing the active zones. neto loss-of-function phenotypes parallel the loss-of-function defects described for iGluRs. The defects in neto mutants are effectively rescued by muscle-specific expression of neto transgenes. Neto clustering at the Drosophila NMJ coincides with and is dependent on iGluRs. Our studies reveal that Drosophila Neto is a novel, essential component of the iGluR complexes and is required for iGluR clustering, organization of PSDs, and synapse functionality.     

L-glutamate as an excitatory transmitter at the Drosophila larval neuromuscular junction.

The possibility that L-glutamate is the excitatory transmitter at the Drosophila larval neuromuscular junction and the ionic basis of its action on the muscle membrane are examined. 2. Iontophoretically applied L-glutamate causes muscle depolarization (L-glutamate potential) if and only if the L-glutamate pipette is within a few mum of the nerve ending. D-glutamate, substance P, ACh and GABA are ineffective. 3. Bath-applied L-glutamate produces similar changes in the time course and amplitude of miniature excitatory junctional potential (m.e.j.p.), excitatory junctional potential (e.j.p.) and the L-glutamate potential. 4. Neuromuscular transmission and excitation-contraction coupling are operative in a haemolymph-like solution containing 1 mM L-glutamate. 5. The reversal potentials of the e.j.p. and the L-glutamate potential are identical to each other, changing similarly with changes in the ionic compositions of the external medium (twelve solutions). 6. The ionic dependence of the reversal potentials is predicted from an extended constant-field equation using a ratio of sodium:potassium permeabilities of PNa/PK=1-3, and a ratio of magnesium:potassium permeabilities of PMg/PK=4-7. 7. It is concluded that L-glutamate is, or is an agonist of, the excitatory transmitter at certain Drosophila larval neuromuscular junctions.     

Structural determinants of the reliability of synaptic transmission at the vertebrate neuromuscular junction

The reliability of neuromuscular transmission depends on the size and molecular organization of the neuromuscular junction. Comparative studies show that the quantal release per unit area is similar at neuromuscular junctions in a number of species in spite of wide variation in synaptic area. They also show an inverse relationship between the size of the nerve terminal and the extent of postsynaptic folding. Evidence is presented supporting the view that the folds, and the voltage-gated sodium channels present in them, effectively amplify synaptic currents. How are the size and molecular organization of the neuromuscular junction determined? Studies with botulinum toxin, including our new work on humans, reveal striking "adaptive plasticity" of the nerve terminal. However, the links between synaptic size and effective transmission remain unclear. On the postsynaptic side, we have shown that mRNA encoding sodium channels is concentrated at the adult junction. During development, mRNA accumulates just before the protein it encodes. Throughout development the sodium channels are associated with ankyrinG and both proteins are initially excluded from the junctional acetylcholine receptor cluster, possibly accounting for the formation of the boundary between the domains occupied by the two key postsynaptic ion channels. These findings have important clinical implications. Reduced transmitter release may result from small nerve terminals as much as from defective release. Abnormal folding is likely to reduce the reliability of transmission. A better understanding of how the structural features that influence the reliability of the neuromuscular transmission are controlled should be of general interest to neuroscientists and of use to clinicians.     

Enhancement of transmission at the frog neuromuscular junction by adenosine deaminase: evidence for an inhibitory role of endogenous adenosine on neuromuscular transmission

Adenosine deaminase reversibly increased the amplitude and the quantum content of the end-plate potentials (EPPs) recorded from superficial muscle fibers of frog sartorius preparations in which twitches have been prevented with high-magnesium solutions. Adenosine deaminase prevented the inhibitory effect of exogenously applied adenosine but not that of 2-chloroadenosine on the amplitude of EPPs. The effect of adenosine deaminase was abolished by erythro-9(2-hydroxy-3-nonyl)adenine (EHNA). The results suggest that endogenous adenosine exerts an inhibitory 'tone' over neuromuscular transmission.     

Safety factor at the neuromuscular junction

Reliable transmission of activity from nerve to muscle is necessary for the normal function of the body. The term 'safety factor' refers to the ability of neuromuscular transmission to remain effective under various physiological conditions and stresses. This is a result of the amount of transmitter released per nerve impulse being greater than that required to trigger an action potential in the muscle fibre. The safety factor is a measure of this excess of released transmitter. In this review we discuss the practical difficulties involved in estimating the safety factor in vitro. We then consider the factors that influence the safety factor in vivo. While presynaptic transmitter release may be modulated on a moment to moment basis, the postsynaptic features that determine the effect of released transmitter are not so readily altered to meet changing demands. Different strategies are used by different species to ensure reliable neuromuscular transmission. Some, like frogs, rely on releasing a large amount of transmitter while others, like man, rely on elaborate postsynaptic specialisations to enhance the response to transmitter. In normal adult mammals, the safety factor is generally 3-5. Both pre- and postsynaptic components change during development and may show plasticity in response to injury or disease. Thus, both acquired autoimmune and inherited congenital diseases of the neuromuscular junction (NMJ) can significantly reduce, or even transiently increase, safety factor.     

Extracellular protons reduce quantal content and prolong synaptic currents at the Drosophila larval neuromuscular junction

Fluctuations in extracellular pH occur in the nervous system in response to a number of physiological and pathological processes, such as ischemia, hypercapnea, and high-frequency activity. Using the Drosophila larval neuromuscular junction, the author has examined acute effects of low and high pH on excitability and synaptic transmission. Acidification rapidly and reversibly reduces the size of electrically evoked excitatory junctional currents (EJCs) in a concentration-dependent manner, with transmission nearly abolished at pH 5.0. Conversely, raising pH to 7.8 increases EJC amplitude significantly. Further elevation to pH 8.5 causes an initial increase in amplitude, followed by profound, long-lasting depression of the synapse. Amplitudes of spontaneous miniature EJCs (mEJCs) are modestly, but significantly reduced at pH 5.0. It is therefore the number of quanta released per action potential, rather than the size of individual quanta, that is most strongly affected. Decay times of both EJCs and mEJCs are dramatically lengthened at low pH, suggesting that glutamate remains in the synaptic cleft for much longer than normal. Presynaptic excitability is also reduced, as indicated by increased latency between nerve shock and EJC onset. The response to low pH was not altered by mutations in genes encoding Transient Receptor Potential, Mucolipin subfamily (TRPML) and Slowpoke ion channels, which had previously been implicated as possible targets of extracellular protons. The author concludes that extracellular protons have strong effects on the release of glutamate and the time course of synaptic currents. These phenotypes can be exploited to study the mechanisms of acid-mediated changes in neuronal function, and to pursue the way in which pH modulates synaptic function in normal and pathophysiological conditions.     

Effect of calcitonin gene-related peptide on synaptic transmission at the neuromuscular junction of the frog

The effects of calcitonin gene-related peptide (CGRP) on synaptic mechanisms were studied at the frog neuromuscular junction by using classical electrophysiological techniques. CGRP reduced the quantal content of evoked neurotransmitter release, as well as the sensitivity of postsynaptic nicotinic acetylcholine receptors (AChRs). No effect on the frequency of the miniature end-plate potentials or on the desensitization of the AChRs could be observed. Both the measured effects may depend on the stimulation of the cyclic AMP second messenger system.     

Properties of the larval neuromuscular junction in Drosophila melanogaster.

The anatomy and physiology of the Drosophila larval neuromuscular junction were studied. 2. The dependence of muscle resting potentials on [K+]o and [Na+]o follows the Goldman-Hodgkin-Katz equation (PNa/PK=0-23). Chloride ions distribute passively across the membrane. 3. The mean specific membrane resistance of muscle fibres is 4-3 X 10(3) omega cm2, and the mean specific membrane capacitance is 7-1 muF/cm2. The muscle fibre is virtually isopotential. 4.Transmitter release is quantal. Both the miniature excitatory junctional potential and the evoked release follow the Poisson distribution. 5. Transmitter release depends on approximately the fourth power of [Ca2+]o. If Sr2+ replaces Ca2+, it depends on approximately the fourth power of [Sr2+]o. Mg2+ reduces transmitter release without altering the fourth power dependence on [Ca2+]o.     

Synaptic transmission in the striatum: from plasticity to neurodegeneration

Striatal neurones receive myriad of synaptic inputs originating from different sources. Massive afferents from all areas of the cortex and the thalamus represent the most important source of excitatory amino acids, whereas the nigrostriatal pathway and intrinsic circuits provide the striatum with dopamine, acetylcholine, GABA, nitric oxide and adenosine. All these neurotransmitter systems interact each other and with voltage-dependent conductances to regulate the efficacy of the synaptic transmission within this nucleus. The integrative action exerted by striatal projection neurones on this converging information dictates the final output of the striatum to the other basal ganglia structures. Recent morphological, immunohistochemical and electrophysiological findings demonstrated that the striatum also contains different interneurones, whose role in physiological and pathological conditions represents an intriguing challenge in these years. The use of the in vitro brain slice preparation has allowed not only the detailed investigation of the direct pre- and postsynaptic electrophysiological actions of several neurotransmitters in striatal neurones, but also the understanding of their role in two different forms of corticostriatal synaptic plasticity, long-term depression and long-term potentiation. These long-lasting changes in the efficacy of excitatory transmission have been proposed to represent the cellular basis of some forms of motor learning and are altered in animal models of human basal ganglia disorders, such as Parkinson's disease. The striatum also expresses high sensitivity to hypoxic-aglycemic insults. During these pathological conditions, striatal synaptic transmission is altered depending on presynaptic inhibition of transmitter release and opposite membrane potential changes occur in projection neurones and in cholinergic interneurones. These ionic mechanisms might partially explain the selective neuronal vulnerability observed in the striatum during global ischemia and Huntington's disease.     

Two synaptic vesicle pools, vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction

Drosophila neuromuscular junctions ( D NMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K(+). The ECP locates close to release sites in the periphery of presynaptic boutons. The reserve pool (RP) is loaded and unloaded only during high frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca(2+). Mobilization of internal Ca(2+) stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and D NMJs are suitable for further studies.     

Regulation of the smooth muscle contractile phenotype by nonmuscle myosin

The contractile phenotype of a smooth muscle can broadly be classified as phasic or tonic. Following activation, phasic smooth muscle exhibits an initial period of rapid force activation, following which force falls to a lower steady state level. In contrast, force generated by tonic smooth muscle rises slowly to a sustained steady state. The differences in contractile patterns cannot be explained by the time course of either the Ca(2+) transient or phosphorylation of the 20-kDa regulatory myosin light chain (MLC(20)). Therefore, a molecular marker that defines tonic and phasic smooth muscle contractile properties remains elusive. Further, smooth muscle can maintain force at low levels of MLC(20) phosphorylation; often referred to as the latch state. The mechanism for the latch state is unknown and has been hypothesized to be due to a number of mechanisms including the formation of slowly cycling dephosphorylated or latch cross-bridges (Hai and Murphy, Am J Physiol 253:H1365-H1371, 1988). This review will focus evidence suggesting that nonmuscle myosin IIB (NMIIB) are the latch cross-bridges in smooth muscle and NMIIB content could define the tonic contractile phenotype.     

Actions of polypeptides at the neuromuscular junction

The effects of several polypeptides, e.g. angiotensin II, substance P, oxytocin and vasopressin, on the isolated frog gastrocnemius, chick biventer cervicis and rat hemodiaphragm preparations were studied using electrophysiological and neurochemical techniques. The effects of angiotensin II, substance P, oxytocin and vasopressin on neuromuscular transmission and muscle contraction were investigated by studying the following parameters:

1. the directly and indirectly-elicited twitch and tetanic contractions,
2. nerve compound action potential,
3. uptake of³H-methylcholine into nerve-muscle preparations,
4. the contractures produced by depolarizing drugs, e.g. ACh or TEA.

The results showed that angiotensin II (10^?10–10^?6 M) and substance P (10^?7–10^?6 M) enhanced neuromuscular transmission and muscle contraction by increasing the amplitudes of the indirectly-elicited twitch and tetanic contractions. Oxytocin and vasopressin (1–100 mU/ml^?1) both depressed neuromuscular transmission by reducing the contractile and electrical response in the frog, chick and rat skeletal muscle. It was concluded that, like their effects on ganglionic transmission, the peptides can modify neuromuscular transmission. The mechanism by which these peptides produce their effects may be dependent on external calcium concentration. These peptides may affect both pre- and postjunctional mechanisms; prejunctionally by increasing/decreasing the release of ACh, and postjunctionally by affecting the sensitivity of the postjunctional membrane to depolarizing drugs and/or producing a contracture in the skeletal muscle.