Improved method for mapping the binding site of an actin-binding protein in the actin sequence. Use of a site-directed antibody against the N-terminal region of actin as a probe of its N-terminus

An antibody was raised against the N-terminal 18 residues of rabbit skeletal muscle actin. By the use of this antibody as the N-terminal probe of actin and the fluorescent label at Cys-374 as its C-terminal probe, binding sites of depactin (an actin-depolymerizing protein from starfish oocytes) were identified in the actin sequence according to the method of Sutoh [Sutoh, K. (1982) Biochemistry 21, 3654-3661]. Cross-linking of the one-to-one complex of actin and depactin with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC) generated two types of cross-linked products with slightly different apparent molecular weights, denoted as 60KU and 60KL. By the use of the N-terminal probe, it was unequivocally revealed that the C-terminal actin segment of residues 357-375 participated in cross-linking with depactin to form 60KL. On the other hand, by the use of the C-terminal probe it was revealed that the N-terminal actin segment of residues 1-12 participated in cross-linking with depactin to form 60KU. Since EDC cross-links Lys residue with Asp or Glu residue only when they are in direct contact, the result indicates that some of the N-terminal residues 1-12 and the C-terminal residues 357-375 of actin participate in binding depactin. The introduction of the N-terminal probe (the antibody recognizing the actin N-terminus) has increased the flexibility of the mapping method for locating binding sites of actin-binding proteins in the actin sequence.     

An actin-depolymerizing protein (depactin) from starfish oocytes: properties and interaction with actin

Physico-chemical properties and interaction with actin of an actin- depolymerizing protein from mature starfish oocytes were studied. This protein, which is called depactin, exists in a monomeric form under physiological conditions. Its molecular weight is approximately 20,000 for the native protein and approximately 17,000 for denatured protein. The Glu + Asp/Lys + Arg molar ratio of this protein is 1.55. The apparent pl of the denatured depactin is approximately 6. The extent of actin polymerization is reduced by the presence of depactin; however, the rate of polymerization seems to be accelerated as measured spectrophotometrically at 238nm. This effect is interpreted to indicate that depactin cut the newly formed filaments into small fragments, thereby increasing the number of the filament ends to which monomers are added. The apparent critical concentration of actin for polymerization, as determined by viscometry or flow birefringence measurement, is increased by the presence of depactin in a concentration-dependent manner. Raising the pH of the solution does not reverse the action of depactin. The molar ratio of actin and depactin, which interact with each other, is estimated to be 1:1 by means of a cross-linking experiment using a water-soluble carbodiimide. Depactin binds to a DNase I-Sepharose column via actin and is selectively eluted with 0.6 M KCl or 0.6 M Kl. The association constant between actin and depactin is estimated, using the column, to be 2-3 X 10(6) M-1. The content of depactin in the high-speed supernatant of the oocyte extract is determined to be 1%; this can act upon approximately 63% of the actin in the supernatant.     

Actin-fragmin interactions as revealed by chemical cross-linking

A one to one complex of actin and fragmin (a capping protein from Physarum polycephalum plasmodia) was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linking reaction generated two cross-linked products with slightly different molecular weights (88 000 and 90 000) as major species. They were cross-linked products of one actin and one fragmin. The cross-linking site of fragmin in the actin sequence was determined by peptide mappings [Sutoh, K. (1982) Biochemistry 21, 3654-3661] after partial chemical cleavages of cross-linked products with hydroxylamine. The results indicated that the N-terminal segment of actin spanning residues 1-12 participated in cross-linking with fragmin. The cross-linker used in this study covalently bridges lysine side chains and side chains of acidic residues when they are in direct contact. Therefore, it seems that acidic residues in the N-terminal segment of actin (Asp-1, Glu-2, Asp-3, Glu-4, and Asp-11), at least some of them, are in the binding site of fragmin. It has already been shown that the same acidic segment of actin is in the binding site of myosin or depactin (an actin-depolymerizing protein isolated from starfish oocytes). We suggest that the unusual amino acid sequence of the N-terminal segment of actin makes its N-terminal region a favorable anchoring site for various types of actin-binding proteins.     

End-label fingerprintings show that the N- and C-termini of actin are in the contact site with gelsolin

Gelsolin was cleaved by chymotrypsin or thermolysin into an N-terminal Mr 45,000 fragment (45N) and a C-terminal Mr 38,000 fragment (38C). The N-terminal half was further cleaved into two fragments with Mr 17,000 (17N) and Mr 28,000 (28N). These fragments were complexed with actin and cross-linked with 1-ethyl-3-[3-(dimethylamino)prophyl]carbodiimide (EDC) to introduce covalent bonds into their contact sites. The location of these bonds was mapped along the actin sequence by end-label fingerprinting with highly sensitive probes for the N- and C-termini of actin. The mapping studies revealed that two gelsolin N-terminal fragments (17N and 28N) were cross-linked with the actin C-terminal segment. The result indicates that the actin N- and C-terminal segments are in the binding site of gelsolin.     

Physarum actin is phosphorylated as the actin-fragmin complex at residues Thr203 and Thr202 by a specific 80 kDa kinase.

The Physarum EGTA-resistant actin-fragmin complex, previously named cap 42(a+b), is phosphorylated in the actin subunit by an endogenous kinase [Maruta and Isenberg (1983) J. Biol. Chem., 258, 10151-10158]. This kinase has been purified and characterized. It is an 80 kDa monomeric enzyme, unaffected by known kinase regulators. Staurosporine acts as a potent inhibitor. The actin-fragmin complex is the preferred substrate. The phosphorylation is inhibited by micromolar Ca2+ concentrations, but only in the presence of additional actin. Polymerized actin (vertebrate muscle and non-muscle isoforms) and actin complexes with various actin-binding proteins are poorly phosphorylated. The heterotrimer consisting of two actins and one fragmin, which is formed from cap 42(a+b) and actin in the presence of micromolar concentrations of Ca2+, is also a poor substrate. From the other substrates tested, only histones were significantly phosphorylated, in particular histone H1. In the same manner, casein kinase I could also phosphorylate the actin-fragmin complex. The major phosphorylation site in actin is Thr203. A second minor site is Thr202. These residues constitute one of the contact sites for DNase I [Kabsch et al. (1990) Nature, 347, 37-44] and are also part of one of the predicted actin-actin contact sites in the F-actin model [Holmes et al. (1990) Nature, 347, 44-49].     

Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons

We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X 10(4) sites/skeleton with an association rate constant of approximately 1 X 10(6) M-1 S-1 and a KD of 1.9 X 10(-9) M at 20 degrees C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The DNase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to DNase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested. Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerization of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely inhibited by 35 nM DNase I but not by 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament.     

Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin

Cofilin is an F-actin side-binding and -depolymerizing protein with an apparent molecular mass of 21 kDa. By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide, was identified as Lys112 and/or Lys114. A synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin (Trp104-Met115) inhibited the binding of cofilin to actin. Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization. Thus, we conclude that the dodecapeptide sequence constitutes the region essential for the actin-binding and -depolymerizing activity of cofilin. A sequence similar to the dodecapeptide is found in other actin-depolymerizing proteins, destrin, actin-depolymerizing factor, and depactin. Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin-binding and -depolymerizing activity in actin-depolymerizing proteins.     

Actin cross-linking and inhibition of the actomyosin motor.

Intrastrand cross-linking of actin filaments by ANP, N-(4-azido-2-nitrophenyl) putrescine, between Gln-41 in subdomain 2 and Cys-374 at the C-terminus, was shown to inhibit force generation with myosin in the in vitro motility assays [Kim et al. (1998) Biochemistry 37, 17801-17809]. To clarify the immobilization of which of these two sites inhibits the actomyosin motor, the properties of actins with partially overlapping cross-linked sites were examined. pPDM (N,N'-p-phenylenedimaleimide) and ABP [N-(4-azidobenzoyl) putrescine] were used to obtain actin filaments cross-linked ( approximately 50%) between Cys-374 and Lys-191 (interstrand) and Gln-41 and Lys-113 (intrastrand), respectively. ANP, ABP, and pPDM cross-linked filaments showed similar inhibition of their sliding speeds and force generation with myosin ( approximately 25%) in the in vitro motility assays. In analogy to ANP cross-linking of actin, pPDM and ABP cross-linkings did not change the strong S1 binding to actin and the V(max) and K(m) parameters of actomyosin ATPase. The similar effects of these three cross-linkings reveal the tight coupling between structural elements of the subdomain 2/subdomain 1 interface and show the importance of its dynamic flexibility to force generation with myosin. The possibility that actin cross-linkings inhibit rate-limiting steps in motion and force generation during myosin cross-bridge cycle was tested in stopped-flow experiments. Measurements of the rates of mantADP release from actoS1 and ATP-induced dissociation of actoS1 did not reveal any differences between un-cross-linked and ANP cross-linked actin in these complexes. These findings are discussed in terms of the uncoupling between force generation and other aspects of actomyosin interactions due to a constrained dynamic flexibility of the subdomain 2/subdomain 1 interface in cross-linked actin filaments.     

Radiolabel-transfer cross-linking demonstrates that protein 4.1 binds to the N-terminal region of beta spectrin and to actin in binary interactions

Erythrocyte protein 4.1 plays a major role in stabilizing the spectrin-actin junction of the erythrocyte membrane skeleton. The particular sites on spectrin responsible for the binding of actin and protein 4.1 have not been specifically defined, although the general region of the 'tail' end, opposite the self-association site, has been deduced by electron microscopy. Using a photoactivatable, radiolabel-transfer cross-linker, 1-[N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)- succinate, we have determined that the binding site for protein 4.1 on spectrin resides in the N-terminal region of beta spectrin within a sequence homologous to the actin-binding region of alpha actinin. Moreover, this technique provided clear evidence for a direct binding interaction between actin filaments and protein 4.1 that was confirmed by rapid-sedimentation assays. In summary, use of radiolabel-transfer cross-linking has enabled assignment of the protein-4.1-binding site on erythrocyte spectrin and has identified a previously ill-defined binary interaction between protein 4.1 and F-actin.     

Binding Properties of 3-[125I]Iodophencyclidine, a New Radioligand for N-Methyl-D-Aspartate-Gated Ionic Channels

The binding properties of the 125I-labeled phencyclidine derivative N-[1-(3-[125I]iodophenyl)cyclohexyl]piperidine (3-[125I]iodo-PCP), a new ligand of the N-methyl-D-aspartate (NMDA)-gated ionic channel, were investigated. Association and dissociation kinetic curves of 3-[125I]iodo-PCP with rat brain homogenates were well described by two components. About 32% of the binding was of fast association and fast dissociation, and the remaining binding was of slow association and slow dissociation. Saturation curves of 3-[125I]iodo-PCP also were well described using two binding sites: one of a high affinity (KDH = 15.8 +/- 2.3 nM) and the other of a low affinity (KDL = 250 +/- 40 nM). 3-Iodo-PCP inhibited the binding of 3-[125I]iodo-PCP with inhibition curves that were well fitted by a two-site model. The binding constants (KiH, BmaxH; KiL, BmaxL) so obtained were close to those obtained in saturation experiments. Ligands of NMDA-gated ionic channels also inhibited the binding of 3-[125I]iodo-PCP with two constants, KiH and KiL. There was a very good correlation (r = 0.987) between the affinities of these ligands to bind to NMDA-gated ionic channels and their potencies to inhibit the binding of 3-[125I]iodo-PCP with a high affinity. Moreover, the regional distribution of the high-affinity binding of 3-[125I]-iodo-PCP paralleled that of tritiated N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP). In contrast to that of [3H] TCP, the binding of 3-[125I]iodo-PCP to well-washed rat brain membranes was fast and insensitive to glutamate and glycine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fentanyl derivatives bearing aliphatic alkaneguanidinium moieties: a new series of hybrid molecules with significant binding affinity for mu-opioid receptors and I2-imidazoline binding sites

A new series of fentanyl derivatives [i.e., N-[1-(2-phenethyl)-4-piperidyl]-N-(guanidinoalkyl)propanamide] bearing aliphatic alkaneguanidinium moieties were prepared. Their affinities for the micro opioid receptors and for the I(2)-imidazoline binding sites (I(2)-IBS) were determined on human post-mortem prefrontal cortex membranes. All of these hybrid compounds had significant and/or very high affinity for both receptors in the nanomolar range, meaning an improvement compared to the prototype N-[1-(2-phenethyl)-4-piperidyl]-N-(guanidinopropyl)propanamide previously reported.     

Targeted Disruption of an EH‐domain Protein Endocytic Complex,Pan1‐End3

Pan1 is a multi‐domain scaffold that enables dynamic interactions with both structural and regulatory components of the endocytic pathway. Pan1 is composed of Eps15 Homology (EH) domains which interact with adaptor proteins, a central region that is responsible for its oligomerization and C‐terminal binding sites for Arp2/3, F‐actin, and type‐I myosin motors. In this study, we have characterized the binding sites between Pan1 and its constitutive binding partner End3, another EH domain containing endocytic protein. The C‐terminal End3 Repeats of End3 associate with the N‐terminal part of Pan1's central coiled‐coil region. These repeats appear to act independently of one another as tandem, redundant binding sites for Pan1. The end3‐1 allele was sequenced, and corresponds to a C‐terminal truncation lacking the End3 Repeats. Mutations of the End3 Repeats highlight that those residues which are identical between these repeats serve as contact sites for the interaction with Pan1.      

Carbodiimide-catalyzed cross-linking sites in the heads of gizzard heavy meromyosin attached to F-actin

In the rigor complex between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), the direct contact between two HMM heads was demonstrated by using a zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]maleimide (EDC) [Onishi, H., Maita, T., Matsuda, G., & Fujiwara, K. (1989) Biochemistry (preceding paper in this issue)]. Here, the 60K peptide which was a product of the EDC cross-linking between two 24K heavy chain (tryptic) fragments of HMM was further fragmented with cyanogen bromide, and the location of the cross-linking sites on the amino acid sequence of the HMM heavy chain was investigated. The result showed that one site resided within the 77-residue peptide region (residues 1-77) on one head of HMM, whereas the other site belonged to the 40-residue peptide region (residues 164-203) on the other head. This finding suggests that the two HMM heads are in contact with each other at different sites. Ultracentrifugal fractionation revealed that the head-to-head cross-linked gizzard HMM could be reversibly released from F-actin in the presence of Mg-ATP. The yield of the head-to-head cross-linking was not significantly changed with the acto-HMM complex between actin/HMM head molar ratios of 1 and 4, and it was very slightly decreased even at a molar ratio of 8, where HMM molecules were attached sparsely to actin filaments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of angiotensin converting enzyme: mechanism and substrate dependence

The interaction of angiotensin converting enzyme with six metal-coordinating [(D-3-mercapto-2-methylpropanoyl)-L-Pro (captopril), N-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Pro (MK-422), N-(phenylphosphoryl)-L-Phe-L-Phe, N alpha-(3-mercaptopropanoyl)-L-Arg, N alpha-[1(S)-carboxy-3-phenylpropyl]-Ala-L-Lys, and N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly] and three dipeptide inhibitors (Gly-L-Trp, L-Phe-L-Arg, and L-Ala-L-Pro) was examined at pH 7.5 in the presence of 300 mM NaCl. Inhibition modes, apparent Ki [Ki(app)] values, and shapes of 1/v vs. [I] plots were found to vary with the substrate employed. All inhibitors except Phe-Arg were competitive with the substrate furanacryloyl (Fa)-Phe-Gly-Gly, while five of seven tested with Fa-Phe-Phe-Arg as substrate produced mixed patterns. Ki-(app) values for N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly, N-(phenylphosphoryl)-L-Phe-L-Phe, Gly-Trp, and MK-422 were 8.3-, 5.5-, 4.7-, and 2.6-fold lower, respectively, when Fa-Phe-Gly-Gly was substrate, compared with values measured with Fa-Phe-Phe-Arg. In contrast, Ki(app) values for Phe-Arg and (3-mercaptopropanoyl)-Arg were lower (2.8- and 2.2-fold, respectively) when Fa-Phe-Phe-Arg was the substrate. Plots of 1/v vs. [I] for most of the inhibitors were nonlinear, to an extent which was also substrate dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody against the actin-binding protein depactin attenuates Ca signaling in starfish eggs

Being present in starfish oocytes, the cofilin/ADF (actin-depolymerizing factor) family protein depactin severs actin filaments. Previously, we reported that exogenous cofilin microinjected into starfish eggs significantly augmented the Ca²⁺ release in response to inositol 1,4,5-trisphosphate (InsP₃) or fertilizing sperm, raising the possibility that intracellular Ca²⁺ signaling could be modulated by the actin cytoskeleton. In this communication, we have targeted the endogenous depactin by use of the specific antibody that was raised against its actin-binding domain. The anti-depactin antibody microinjected into the starfish oocytes and eggs effectively altered the structure of the actin cytoskeleton, and significantly delayed the meiotic progression induced by 1-methyladenine. When microinjected into the mature eggs, the anti-depactin antibody markedly reduced the amplitude of the Ca²⁺ response in a dose-dependent manner, corroborating the results of our previous study with cofilin. In addition, the eggs microinjected with the anti-depactin antibody displayed reduced rate of successful elevation of the fertilization envelope and an elevated tendency of polyspermic interaction. Taken together, our data suggest that the actin cytoskeleton is implicated not only in meiotic maturation and intracellular Ca²⁺ signaling, but also in the fine regulation of gametes interaction and cortical granules exocytosis.     

The cytosolic class II chaperonin CCT recognizes delineated hydrophobic sequences in its target proteins

The nonhomologous proteins actin and alpha- and beta-tubulin need the assistance of the cytosolic chaperonin containing TCP-1 (CCT) to reach their correct native state, and their folding requires a transient binary complex formation with CCT. We show that separate or combined deletion of three delineated hydrophobic sequences in actin disturbs the interaction with CCT. These sites are situated between residues 125-179, 244-285, and 340-375. Also, alpha- and beta-tubulin contain at least one recognition region, and intriguingly, it has a similar distribution of hydrophobic residues as region 244-285 in actin. Internal deletion of the sites in actin favor a model for cooperative binding of target proteins to CCT. Peptide mimetics, representing the binding regions, inhibit target polypeptide binding to CCT, suggesting that actin and tubulin contact similar CCT subunits. In addition, we show that actin recognition by class II chaperonins is different from that by class I.     

Difference between subfragment-1 and heavy meromyosin in their interaction with F-actin

To elucidate the difference between subfragment-1 and heavy meromyosin in their interaction with F-actin, we used limited tryptic digestion and cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The binding of actin to subfragment-1 lowers the susceptibility of the 50K-20K junction of its heavy chain to tryptic digestion. At a molar ratio of one actin to one subfragment-1, all the sites were gradually cleaved by trypsin whereas the sites were completely protected in the presence of a 2-fold molar excess of actin over subfragment-1. In the case of heavy meromyosin, nearly half of the sites were protected completely by the presence of an equimolar amount of actin to its heads suggesting that the two heads of heavy meromyosin bound actin in a different manner. The rate of the cross-linking reaction between subfragment-1 heavy chain and actin with 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide also depended on the molar ratio of actin to subfragment-1. The rate was maximum at a molar ratio of about 5 actin to 1 subfragment-1. When heavy meromyosin was cross-linked to actin, the maximum rate was observed at a molar ratio of about 3 actin to 1 heavy meromyosin head, the level being about 60% that for subfragment-1 and actin. It was suggested that the presence of the subfragment-2 portion of heavy meromyosin caused these differences by restricting the motion of the two heads.     

A novel actin label: a fluorescent probe at glutamine-41 and its consequences

By peptide isolation and analysis, it has been shown that the dansyl fluorophore of dansylcadaverine [N-(5-aminopentyl)-5-(dimethylamino)naphthalene-1-sulfonamide] transfers to Gln-41 of actin from rabbit skeletal muscle when the reaction is catalyzed by guinea pig liver transglutaminase. As a function of time, the degree of labeling asymptotically approaches 1 mol of dansyl/l mol of actin. About 80-85% of the attached dansyl fluorophore was found at Gln-41. Such labeled G-actin polymerizes to the same extent as control actin, but the polymerization rate is greater and the critical concentration is less than for control actin. Complete polymerization is accompanied by a 1.5-2.0-fold increase in the emission intensity of the attached fluorophore. Labeled F-actin thus obtained activates myosin subfragment 1 (S-1) Mg2+-ATPase activity with the same Kapp, and to the same Vmax, as control actin; moreover, when such labeled F-actin is cross-linked to S-1 by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, the resulting superactivation of Mg2+-ATPase is the same as that attained with control actin. The attributes of this label thus make it an ideal reporter of events in the N-terminal 10-kilodalton region of actin, and a new topological point for proximity mapping.     

Mechanism of formation of actomyosin interface

Force generation in muscle results from binding of myosin to F-actin. ATP binding to myosin provides energy to dissociate actomyosin complex while the hydrolysis of ATP is needed for re-binding of myosin to F-actin. At the end of each cycle myosin and actin form a tight complex with a substantial interface area. We investigated the dynamics of formation of actomyosin interface in presence and absence of nucleotides by quenched flow cross-linking technique. We showed previously that myosin head (subfragment 1, S1) directly interacts with at least two monomers in the actin filament. The quenched flow cross-linking experiments revealed that the initial contact (in presence or absence of nucleotides) occurs between loop 635-647 of S1 and 1-12 N-terminal residues of one actin and, then, the second contact forms between loop 567-574 of S1 and the N terminus of the second actin. The distance between these two loops in S1 corresponds to the distance between N termini of two actins in the same strand (53 A) but is smaller than that between two actins from the different strands (102 A). The formation of the actomyosin complex proceeds in ordered sequence: S1 initially binds to one actin then binds with the second actin located in the same strand but probably closer to the barbed end of F-actin. The presence of nucleotides slows down the interaction of S1 with the second actin, which correlates with recently proposed cleft movement in a 50 kDa domain of S1. The sequential mechanism of formation of actomyosin interface starting from one end and developing towards the barbed end might be involved in force generation and directional movement in actin-myosin system.     

Expression of various scinderin domains in chromaffin cells indicates that this protein acts as a molecular switch in the control of actin filament dynamics and exocytosis

Stimulation-induced chromaffin cell cortical F-actin disassembly allows the movement of vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+-dependent protein, controls actin dynamics. Sc six domains have three actin, two PIP2 and two Ca2+-binding sites. F-actin severing activity of Sc is Ca2+-dependent, whereas Sc-evoked actin nucleation is Ca2+-independent. Sc domain role in secretion was studied by co-transfection of human growth hormone (hGH) reporter gene and green fluorescent protein (GFP)-fusion Sc constructs. Cells over-expressing actin severing Sc1-6 or Sc1-2 (first and second actin binding sites) constructs, increased F-actin disassembly and hGH release upon depolarization. Over-expression of nucleating Sc5-6, Sc5 or ScABP3 (third actin site) constructs decreased F-actin disassembly and hGH release upon stimulation. Over-expression of ScL5-6 or ScL5 (lack of third actin site) produced no changes. During secretion, actin sites 1 and 2 are involved in F-actin severing, whereas site 3 is responsible for nucleation (polymerization). Sc functions as a molecular switch in the control of actin (disassembly left arrow over right arrow assembly) and release (facilitation left arrow over right arrow inhibition). The position of the switch (severing left arrow over right arrow nucleation) may be controlled by [Ca2+]i. Thus, increase in [Ca2+]i produced by stimulation-induced Ca2+ entry would increase Sc-evoked cortical F-actin disassembly. Decrease in [Ca2+]i by either organelle sequestration or cell extrusion would favor Sc-evoked actin nucleation.