Visualization of Melanosome Dynamics within Wild-Type and Dilute Melanocytes Suggests a Paradigm for Myosin V Function In Vivo

Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va⁻) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid (∼1.5 μm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va–dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This “capture” model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an ∼120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, ∼0.14 μm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do not drive obvious spreading of melanosomes over 90 min. We conclude that long-range, bidirectional, microtubule-dependent melanosome movements, coupled with actomyosin Va–dependent capture of melanosomes in the periphery, is the predominant mechanism responsible for the centrifugal transport and peripheral accumulation of melanosomes in mouse melanocytes. This mechanism represents an alternative to straightforward transport models when interpreting other myosin V mutant phenotypes.     

Transglutaminase inhibitors,calmodulin antagonists,and calcium channel blockers: Influence on Arbacia sperm motility

Sea urchin spermatozoa react to transglutaminase inhibitors, which may antagonize the function of calmodulin, in dose-dependent fashion. The optimum concentration of diallyl-amino propionyl benzothiophenc (DAPBT), a potent, noncompetitive inhibitor of transglutaminase, 0.01 mM, causes a 3 1/2-fold increase in the forward swimming speed of Arbacia sperm. This effect apparently involves calcium-dependent enzymes since manipulation of both extracellular and intracellular calcium by means of chelating agents, calcium-channel blockers, and calmodulin antagonists depresses the stimulatory effect of DAPBT. These results suggest that in the spermatozoa the interaction of flagellar sliding filaments may be mediated by reversible cross-linking of the contractile proteins catalyzed by a calcium- dependent transglutaminase.     

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.     

Myosin V exhibits a high duty cycle and large unitary displacement.

Myosin V is a double-headed unconventional myosin that has been implicated in organelle transport. To perform this role, myosin V may have a high duty cycle. To test this hypothesis and understand the properties of this molecule at the molecular level, we used the laser trap and in vitro motility assay to characterize the mechanics of heavy meromyosin-like fragments of myosin V (M5(HMM)) expressed in the Baculovirus system. The relationship between actin filament velocity and the number of interacting M5(HMM) molecules indicates a duty cycle of > or =50%. This high duty cycle would allow actin filament translocation and thus organelle transport by a few M5(HMM) molecules. Single molecule displacement data showed predominantly single step events of 20 nm and an occasional second step to 37 nm. The 20-nm unitary step represents the myosin V working stroke and is independent of the mode of M5(HMM) attachment to the motility surface or light chain content. The large M5(HMM) working stroke is consistent with the myosin V neck acting as a mechanical lever. The second step is characterized by an increased displacement variance, suggesting a model for how the two heads of myosin V function in processive motion.     

Myosin V and the endoplasmic reticulum: the connection grows

In this issue, Estrada et al. (2003) provide new and important insights into how the endoplasmic reticulum (ER) of budding yeast cells is inherited. Together with other studies in plant and animal cells, the results of Estrada et al. (2003) support the idea that myosin V acts as a universal motor for the transport of ER membranes.     

Thermodynamic analyses of calcium binding to troponin C, calmodulin and parvalbumins by using microcalorimetry

Results of microcalorimetric titrations of calcium-binding proteins with calcium or magnesium have been reviewed and evaluated. Results were analyzed mostly in terms of heat capacity changes, which is most closely related to the structural changes of the molecule on metal binding. Two high-affinity sites of rabbit skeletal troponin C are distinguishable in terms of their affinity to calcium and associated enthalpy changes. Heat capacity changes on calcium binding to one of the two high-affinity sites is negative and is in the range ascribed to the ligand binding. In contrast, that to the other of the high-affinity sites is large and positive, indicating that a substantial area of hydrophobic groups become exposed to the solvent. In frog skeletal troponin C, the anomalous positive heat capacity changes occur in one of the low-affinity calcium-specific sites, so that this may be involved in the regulation of contraction. Unlike skeletal troponin C, both of the two high-affinity sites of cardiac troponin C show negative heat capacity changes. In calmodulin, heat capacity changes are positive but small, indicating that calcium binding may induce clustering of the hydrophobic residues on the surface of the molecule. In parvalbumins, heat capacity changes are negative, characteristic of most ligand binding.     

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter

Summary In rabbit ileum, Ca²⁺/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca²⁺/CaM stimulate linked Na⁺ and Cl^– absorption. The role of Ca²⁺/CaM-dependent phosphorylation in regulation of the brush-border Na⁺/H⁺ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na⁺/H⁺ antiporter and Ca²⁺/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca²⁺, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca²⁺/CaM and ATP inhibited Na⁺/H⁺ exchange by 45±13%. This effect was specific since Ca²⁺/CaM and ATP did not alter diffusive Na⁺ uptake, Na⁺-dependent glucose entry, or Na⁺ or glucose equilibrium volumes. The inhibition of the Na⁺/H⁺ exchanger by Ca²⁺/CaM/ATP was due to an effect on theV _max and not on theK _m for Na⁺. In the presence of CaM and ATP, Ca²⁺ caused a concentration-dependent inhibition of Na⁺ uptake, with an effect 50% of maximum occurring at 120nm. This Ca²⁺ concentration dependence was similar to the Ca²⁺ concentration dependence of Ca²⁺/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca²⁺/CaM/ATP-inhibition of Na⁺/H⁺ exchange was reversed by W₁₃, a Ca²⁺/CaM antagonist, but not by a hydrophobic control, W₁₂, or by H-7, a protein kinase C antagonist. we conclude that Ca²⁺, acting through CaM, regulates ileal brush-border Na⁺/H⁺ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.     

Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.     

Structure and Regulation of the Movement of Human Myosin VIIA

Human myosin VIIA (HM7A) is responsible for human Usher syndrome type 1B, which causes hearing and visual loss in humans. Here we studied the regulation of HM7A. The actin-activated ATPase activity of full-length HM7A (HM7AFull) was lower than that of tail-truncated HM7A (HM7AΔTail). Deletion of the C-terminal 40 amino acids and mutation of the basic residues in this region (R2176A or K2179A) abolished the inhibition. Electron microscopy revealed that HM7AFull is a monomer in which the tail domain bends back toward the head-neck domain to form a compact structure. This compact structure is extended at high ionic strength or in the presence of Ca²⁺. Although myosin VIIA has five isoleucine-glutamine (IQ) motifs, the neck length seems to be shorter than the expected length of five bound calmodulins. Supporting this observation, the IQ domain bound only three calmodulins in Ca²⁺, and the first IQ motif failed to bind calmodulin in EGTA. These results suggest that the unique IQ domain of HM7A is important for the tail-neck interaction and, therefore, regulation. Cellular studies revealed that dimer formation of HM7A is critical for its translocation to filopodial tips and that the tail domain (HM7ATail) markedly reduced the filopodial tip localization of the HM7AΔTail dimer, suggesting that the tail-inhibition mechanism is operating in vivo. The translocation of the HM7AFull dimer was significantly less than that of the HM7AΔTail dimer, and R2176A/R2179A mutation rescued the filopodial tip translocation. These results suggest that HM7A can transport its cargo molecules, such as USH1 proteins, upon release of the tail-dependent inhibition.     

Oxidation of calmodulin alters activation and regulation of CaMKII

Increases in reactive oxygen species and mis-regulation of calcium homeostasis are associated with various physiological conditions and disease states including aging, ischemia, exposure to drugs of abuse, and neurodegenerative diseases. In aged animals, this is accompanied by a reduction in oxidative repair mechanisms resulting in increased methionine oxidation of the calcium signaling protein calmodulin in the brain. Here, we show that oxidation of calmodulin results in an inability to: (1) activate CaMKII; (2) support Thr(286) autophosphorylation of CaMKII; (3) prevent Thr(305/6) autophosphorylation of CaMKII; (4) support binding of CaMKII to the NR2B subunit of the NMDA receptor; and (5) compete with alpha-actinin for binding to CaMKII. Moreover, oxidized calmodulin does not efficiently bind calcium/calmodulin-dependent protein kinase II (CaMKII) in rat brain lysates or in vitro. These observations contrast from past experiments performed with oxidized calmodulin and the plasma membrane calcium ATPase, where oxidized calmodulin binds to, and partially activates the PMCA. When taken together, these data suggest that oxidative stress may perturb neuronal and cardiac function via a decreased ability of oxidized calmodulin to bind, activate, and regulate the interactions of CaMKII.     

Characterization of cocaine-elicited cell vacuolation: the involvement of calcium/calmodulin in organelle deregulation

The sizes of organelles are tightly regulated in the cells. However, little is known on how cells maintain the homeostasis of these intracellular compartments. Using cocaine as a model compound, we have characterized the mechanism of deregulated vacuolation in cultured rat liver epithelial Clone 9 cells. The vacuoles were observed as early as 10 min following cocaine treatment. Removal of cocaine led to vacuole degeneration, indicating vacuolation is a reversible process. The vacuoles could devour intracellular materials and the vacuoles originated from late endosome/lysosome as indicated by immunofluorescence studies. Instant calcium influx and calmodulin were required for the initiation of vacuole formation. The unique properties of these late endosome/lysosome-derived vacuoles were further discussed. In summary, cocaine elicited a new type of deregulated vacuole and the involvement of calcium/calmodulin in vacuolation could shed light on prevention or treatment of cocaine-induced cytotoxicity.     

Binding of chara Myosin globular tail domain to phospholipid vesicles

Binding of Chara myosin globular tail domain to phospholipid vesicles was investigated quantitatively. It was found that the globular tail domain binds to vesicles made from acidic phospholipids but not to those made from neutral phospholipids. This binding was weakened at high KCl concentration, suggesting that the binding is electrostatic by nature. The dissociation constant for the binding of the globular tail domain to 20% phosphatidylserine vesicles (similar to endoplasmic reticulum in acidic phospholipid contents) at 150 mM KCl was 273 nM. The free energy change due to this binding calculated from the dissociation constant was -37.3 kJ mol(-1). Thus the bond between the globular tail domain and membrane phospholipids would not be broken when the motor domain of Chara myosin moves along the actin filament using the energy of ATP hydrolysis (DeltaG degrees ' = -30.5 kJ mol(-1)). Our results suggested that direct binding of Chara myosin to the endoplasmic reticulum membrane through the globular tail domain could work satisfactorily in Chara cytoplasmic streaming. We also suggest a possible regulatory mechanism of cytoplasmic streaming including phosphorylation-dependent dissociation of the globular tail domain from the endoplasmic reticulum membrane.     

Reciprocal regulation of calcium dependent and calcium independent cyclic AMP hydrolysis by protein phosphorylation

The hydrolysis of cyclic nucleotide second messengers takes place through multiple cyclic nucleotide phosphodiesterases (PDEs). The significance of this diversification is not fully understood. Here we report the differential regulation of low K(m) Ca2+-activated (PDE1C) and Ca2+-independent, rolipram-sensitive (PDE4) PDEs by protein phosphorylation in the neuroendocrine cell line AtT20. Incubation of cells with 8-(4-chlorophenylthio)-cyclic AMP (CPT-cAMP) enhanced PDE4 and reduced PDE1C activity. These effects were blocked by H89 indicating mediation by cAMP-dependent protein kinase (PKA), furthermore in broken cell preparations PKA produced the same reciprocal changes of PDE activities. Calyculin A, an inhibitor of protein phosphatases 1 and 2 A, stimulated PDE4 and enhanced the inhibitory effect of CPT-cAMP on PDE1C. The reduction of PDE1C activity was characterized by a marked attenuation of the activation by Ca2+/calmodulin. Stimulation of PDE4 activity by CPT-cAMP or calyculin A was attributable to PDE4D3 and these effects could also be reproduced in human embryonic kidney cells expressing epitope-tagged PDE4D3. Together, these data show reciprocal regulation of PDE1C and PDE4D by PKA, which represents a novel scheme for plasticity in intracellular signalling.     

A role for calcium/calmodulin kinase(s) in the regulation of GABA exocytosis

A possible role for protein kinases in the regulation of GABA exocytosis in nerve endings was investigated. The effect on the release of the radioactive neurotransmitter ([³H]GABA) from mouse brain synaptosomes of several protein kinase inhibitors was estimated after treatment with 37 mM K⁺ in the absence of external Na⁺, a condition under which [³H]GABA release is completely Ca²⁺ dependent. Among the inhibitors one group inhibit the kinases by binding to the catalytic site (i.e. staurosporine and H7) and others (TFP, sphingosine and W7) act on the regulatory site of protein kinases. The compounds of the second group, which are reported to inhibit calmodulin dependent events and the increase in cytosolic Ca²⁺ (Ca _i ) induced by high K⁺ depolarization, were the most efficient inhibitors of [³H]GABA release. The selective inhibitor of CaMPK II, KN-62, also markedly diminished [³H]GABA release as well as the increase in Ca _i induced by high K⁺. The kinase inhibitors from the first group that are unable to diminish the increase in Ca _i induced by high K⁺ were also less efficient inhibitors of [³H]GABA release even at high concentrations. The present results indicate that at the doses tested all the drugs inhibit to some extent the release of the Ca²⁺ dependent fraction of [³H]GABA perhaps by inhibiting a CaMPK II mediated phosphorylation step triggered by depolarization and facilitated by the elevation of Ca _i . In addition, the second group of antagonists and KN-62 inhibit the elevation of Ca _i to high K⁺ thus exhibiting a higher efficiency on [³H]GABA release than the first group of antagonists.     

Effect of calcium and calmodulin modulators on the development of Erysiphe pisi on pea leaves

The effect of calcium and calmodulin modulators, viz., ethylene glycol bis (beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), a calcium chelator; verapamil, a plasma membrane Ca2+ channel blocker; ruthenium red, an organelle Ca2+ channel blocker; and chlorpromazine, a calmodulin antagonist; on the development of Erysiphe pisi was studied by floating the inoculated leaves on the respective solutions of chemicals. All the modulators affected the development of E. pisi by inhibiting the colony diameter, number of secondary branches, number of hyphal cells per colony and number of haustoria. The calmodulin antagonist was more effective in reducing E. pisi development. The results suggest the possible involvement of calcium and calmodulin in the development of E. pisi on pea leaves.     

Lymphocyte calmodulin and its participation in the stimulation of T lymphocytes by mitogenic lectins.

Calmodulin was purified from human tonsillar lymphocytes utilizing calcium-dependent binding of calmodulin to fluphenazine-Sepharose. The molecular weight and phosphodiesterase activation of the lymphocyte calmodulin were very similar to those of purified bovine brain calmodulin. Trifluoperazine (TFP), a calmodulin inhibitor, suppressed lymphocyte stimulation as assessed by 3H-thymidine incorporation into DNA of lectin-stimulated lymphocytes. TFP had no effect on the early 45Ca2+ uptake induced by mitogenic lectins, although this latter was inhibited by verapamil which also suppressed the 3H-thymidine incorporation. The results are in keeping with the interpretation that the inhibition of T cell stimulation by TFP was not due to suppression of Ca2+ uptake, but due to inactivation of Ca(2+)-calmodulin complex which might be formed subsequent to Ca2+ entry into the cell.     

Possible involvement of calmodulin in the regulation of ATPase activity in guard cells

Calcium ions play an important role in the regulation of stomatal movement and the mechanism underlying this action is yet to be determined. It is suggested that guard cell plasma membrane ATPase is a target for calcium action and that this effect is mediated by calmodulin. In this study, the effects of calcium and two calmodulin antagonists on ATPase activity in a crude homogenate of Commelina communis L. guard cell protoplasts were examined. The homogenate contained Mg²⁺-dependent, K⁺-simulated ATPase activity, which was inhibited by CaCl² while stimulated by the calmodulin antagonists, compound 48/80 and chlorpromazine. The calmodulin antagonists partially reversed the inhibitory effect of calcium ions. The results support the possibility of calmodulin involvement in the regulation of guard cell ATPase activity by calcium ions.     

Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short‐term synaptic plasticity

Ca²⁺ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca²⁺–CaM binds a conserved region in the priming proteins Munc13‐1 and ubMunc13‐2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca²⁺ signals. We solved the structure of Ca²⁺₄–CaM in complex with the CaM‐binding domain of Munc13‐1, which features a novel 1‐5‐8‐26 CaM‐binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13‐2 isoform. The N‐module can be dissociated with EGTA to form the half‐loaded Munc13/Ca²⁺₂–CaM complex. The Ca²⁺ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca²⁺–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca²⁺]_i, whereas the N‐module acts as a sensor at micromolar [Ca²⁺]_i. This Ca²⁺/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca²⁺‐dependent modulation of short‐term synaptic plasticity.     

Myosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretion

The presence of myosin II and V in chromaffin cells and their subcellular distribution is described. Myosin II and V distribution in sucrose density gradients showed only a strong correlation between the distribution of myosin V and secretory vesicle markers. Confocal microscopy images demonstrated colocalization of myosin V with dopamine beta-hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca2+ and time-dependent manner, the dissociation of myosin V from chromaffin vesicles suggesting that this association was not permanent but determined by secretory cycle requirements. Myosin II was also found in the crude granule fraction, however, its distribution was not affected by cell depolarization. Myosin V head antibodies were able to inhibit secretion whereas myosin II antibodies had no inhibitory effect. The pattern of inhibition indicated that these treatments interfered with the transport of vesicles from the reserve to the release-ready compartment, suggesting the involvement of myosin V and not myosin II in this transport process. The results described here suggest that myosin V is a molecular motor involved in chromaffin vesicle secretion. However, these results do not discard an indirect role for myosin II in secretion through its interaction with F-actin networks.