Three radioactive peaks are seen, the first from Pi, the second from ADP, and the third from ATP. For ADP and ATP the absorbance is also shown; 85% of the total absorbance is from ADP and 15% from ATP. Since F-actin contains bound-ADP and G-actin contains bound-ATP, the data indicate that 85% of the total actin in this muscle preparation was in the polymeric and 15% in the monomeric form. Determination of the Pi concentration by a colorimetric method showed that the molar ratio of Pi to ADP was close to one.

In order to quantify the extent of exchange of the actin-bound nucleotide and Pi, one has to determine their specific activity (counts /min/mol nucleotide or Pi) and compare it with those of the specific activities (s.a.) of the gamma- and beta-phosphates of the cytoplasmic ATP and that of PCr (Barany et al., 2001). With this knowledge one can calculate the percentage exchange for each of the actin components; for instance , the percentage exchange of the actin-bound- ADP is:

(s.a. of actin-ADP/s.a. of beta-P of cytoplasmic ATP) x 100

Fig. SM14 compares the exchange of the actin-bound ADP between smooth and skeletal muscles. The exchange is rapid in smooth muscle, half-time about 15 min, whereas the exchange is slow in skeletal muscle, about 15% in three hours, in agreement with the studies of Martonosi et al., 1960) in live animals.

Fig. SM13. Dowex -1 chromatography of

Extracts No. 7 and 8, shown in Fig. SM12.(From Bârâny et al., 2001). Squares correspond to Counts per ml and triangles correspond to Absorbance.

Fig. SM13. Dowex -1 chromatography of

Extracts No. 7 and 8, shown in Fig. SM12.(From Bârâny et al., 2001). Squares correspond to Counts per ml and triangles correspond to Absorbance.

Adp Artery
Fig. SM14. Time course of the exchange of the actin-bound ADP in smooth (porcine carotid artery) and skeletal (rat vastus lateralis) muscle. (From Barany et al., 2001).

Characteristics of the exchange of the actin-bound nucleotide in smooth muscle:

ATP is a prerequisite for the exchange to take place. If ATP synthesis is inhibited by azide or iodoacetamide the exchange is also inhibited. If ATP sysnthesis is reduced, by incubation of the muscles with deoxyglucose, instead of glucose, the exchange is also reduced.

Ca2+ is not required for the exchange, i.e. full exchange is observed in the muscle in the presence of EGTA.

Several smooth muscles, arteries, uteri, urinary bladder, and stomach exhibiited the exchange of the actin-bound nucleotide and phosphate, suggesting that the exchange is a property of every smooth muscle.

Upon contraction of smooth muscle, the exchange of the bound-nucleotide and phosphate decreased and upon relaxation from the contracted state it increased, suggesting that polymerization-deplolymerization of actin is a part of the contraction-relaxation cycle of smooth muscle.


Barany, M. (1996). Biochemistry of Smooth Muscle Contraction. Academic Press.

Barany, K. and Barany, M. (1996a). Myosin light chains. ]n Biochemistry of Smooth Muscle Contraction (M. Barany , Ed.), pp. 2135, Academic Press.

Barany, M. and Barany, K. (1996b). Inositol 1,4,5-trisphosphate production. ]n Biochemistry of Smooth Muscle Contraction (M. Barany, Ed.), pp. 269-282, Academic Press.

Barany, M. and Barany, K. (1996c). Protein phosphorylation during contraction and relaxation. ]n Biochemistry of Smooth Muscle Contraction (M. Barany, Ed.), pp. 321-339, Academic Press.

Barany, M., Barron, J.T., Gu, L., and Barany, K. (2001). Exchange of the actin-bound nucleotide in intact arterial smooth muscle. J. Biol. Chem., 276, 48398-48403.

Beall, A.C., Kato, K., Goldenring, J.R., Rasmussen, R., and Brophy, C.M. (1997) Cyclic nucleotide-dependent vasorelaxation is associated with the phosphorylation of a small heat shock-related protein. J. Biol. Chem. 272, 11283-11287.

Brophy, C.M., Lamb, S., and Graham, A. (1999). The small heat shock-related protein-20 is an actin-associated protein. J. Vasc. Surg. 29, 326-333.

Erdodi, F., Rokolya, A., Barany, M., and Barany, K. (1988). Phosphorylation of the 20,000 dalton myosin light chain isoforms of arterial smooth muscle by myosin light chain kinase and protein kinase C. Arch. Biochem. Biophys. 266, 583-591.

Feng, J., Ito, M., Ichikawa, K., Isaka, N., Nishikawa, M., Hartshorne, D.J., and Nakano, T. (1999). Inhibitory phosphorylation site for rho-associated kinase on smooth muscle myosin phosphatase. J. Biol. Chem. 274, 37385-37390.

Hartshorne, D.J., Ito, M., and Erdodi, F. (1998). Myosin light chain phosphatase: subunit composition, interactions and regulation. J. Muscle Res. Cell Motil. 19, 325-341.

Herrera, A.M., Kuo, K-H., and Seow, C.Y. (2002). Influence of calcium on myosin thick filament formation in intact airway smooth muscle. Am. J. Physiol. Cell Physiol., 282, C310-C316.

Hodgkinson, J.L., el-Mezgueldi, M., Craig, R., Vibert, P., Marston, S.B., and Lehman, W. (1997). 3-D image reconstruction of reconstituted smooth muscle thin filaments containing calponin : visulaization of interactions between F-actin and calponin. J. Mol. Biol., 273, 159-159.

Jones, K.A., Perkins, W.J., Lorenz, R.R., Prakash, Y.S., Sieck, G.C., Warner, D.O. (1999). F-actin stabilization increases tension cost during contraction of permeabilized airway smooth muscles in dog. J.Physiol., 519, 527-538.

Kaibuchi, K., Kuroda, S., and Amano, M. (1999). Regulation of the cytoskeleton and cell adhesion by the rho family GTPases in mammalian cells. Annu. Rev. Biochem. 68, 459-486.

Kao, C.Y. and Carsten, M. E. (1997). Cellular Aspects of Smooth Muscle Function. Cambridge University Press.

Kitazawa, T., Eto, M., Woodsome, T.P., and Brautigan, D.L. (2000). Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J. Biol. Chem., 275, 9897-9900.

Lauzon, A-M., Fagnant, P.M., Warshaw, D.M., and Trybus, K.M. (2001) Coiled-coil unwinding at the smooth muscle myosin head-rod junction is required for optimal mechanical performance. Biophys. J. 80, 1900-1904.

Lehman, W., Vibert, P., Craig, R. (1997). Visualization of caldesmon on smooth muscle thin filaments. J. Mol. Biol., 274, 310-317.

Li, X-D., Saito, J., Ikebe, R., Mabuchi, K., and Ikebe, M. (2000). The interaction between the regulatory light chain domains on two heads is critical for regulation of smooth muscle myosin. Biochemistry, 39, 2254-2260.

Martonosi, A., Gouvea, M.A., and Gergely, J. (1960). Studies on actin. III. G-F transformation of actin and muscular contraction (experiments in vivo). J. Biol. Chem. 235, 1707-1710.

Mehta, D. and Gunst, S.J. (1999). Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle. J. Physiol., 519, 820-840.

Murphy, R.A. (1999). Signal transduction in smooth muscle. Reviews of Physiology Biochemistry and Pharmacology. vol.134

Nagumo, H., Sasaki, Y., Ono, Y., Okamoto, H., Seto, M., and Takuwa, Y. (2000). Rho-kinase inhibitor HA-1077 prevents rho-mediated myosin phosphatase inhibition in smooth muscle cells. Am. J. Physiol., 278, C57-C65.

Ohki, S-Y., Eto, M., Kariya, A., Hayano, T., Hayashi, Y., Yazawa, M., Brautigan, D., and Kainosho, M. (2001). Solution NMR structure of the myosin phosphatase inhibitor protein CPI-17 shows phosphorylation-induced conformational changes responsible for activation. J. Mol. Biol. 314, 839-849.

Quevillon-Cheruel, S., Foucault, G., Desmadril, M., Lompre, A-M., and Bechet, J-J. (1999). Role of the C-terminal extremities of the smooth muscle myosin heavy chains: implication for assembly properties. FEBS Letters 454, 303-306.

Rembold, C.M., Foster, D.B., Strauss, J.D., Wingard, C.J., Van Eyk, J.E. (2000). cGMP-mediated phosphorylation of heat shock protein 20 may cause smooth muscle relaxation without myosin light chain dephosphorylation in swine carotid artery. J. Physiol., 524, 865-878.

Rovner, A.S., Fagnant, P.M., Lowey, S, and Trybus, K.M. (2002). The carboxyl-terminal isoforms of smooth muscle myosin heavy chain determine thick filament assembly properties. J. Cell. Biol. 156, 113-124.

Rowner, A.S. (1998). A long, weakly charged actin-binding loop is required for phosphorylation dependent regulation of smooth muscle myosin. J. Biol. Chem. 273, 27939-27944.

Solaro, R.J. (2000). Myosin light chain phosphatase a cinderella of cellular signaling. Circ. Res. 87, 173-175.

Somlyo, A.P. and Somlyo, A.V. (1994). Signal transduction and regulation in smooth muscle. Nature, 372, 231-236.

Somlyo, A.P. and Somlyo, A.V. (2000). Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J. Physiol., 522, 177-185.

Stull, J.T., Krueger, J.K., Kamm, K.E., Gao, Z-H., Zhi, G., and Padre, R. (1996). Myosin light chain kinase. ]n Biochemistry of Smooth Muscle Contraction (M. Barany, Ed.), pp. 119-130. Academic Press.

Sward, K., Dreja, K., Susnjar, M., Hellstrand, P., Hartshorne, D.J., and Walsh, M.P. (2000). Inhibition of rho-associated kinase blocks agonist induced Ca +sensitization of myosin phosphorylation and force in guinea pig ileum. J. Physiol. 522, 33-49.

Sweeney, H.L., Chen, L-Q., and Trybus, K.M. (2000). Regulation of asymmetric smooth muscle myosin II molecules. J.B iol. Chem. 275, 41273-41277.

Trybus, K. (2000). Biochemical studies of myosin. METHODS, 22, 327-335.


Historical Development of Cell Motility

In the first half of the 20th century, it was generally accepted that myosin and actin are proteins specific for muscle, and muscle contractility is a specific interaction of actin, myosin and ATP. It came as a surprise when in 1954 Hoffmann-Berling reported that water-glycerol extracted amnion fibroblasts contract upon addition of ATP. Figures CM1a and 1b show that the elongated fibroblast cells shrink to a small globule after addition of ATP.

Fig. CM1a, shows the fibroblast without ATP (Reprinted from Biochim. Biophys. Acta, vol. 14, Hoffmann-Berling, H., Adenosintriphosphat als Betriebsstoff von Zellbewegungen, pp. 182-194, Copyright 1954, with permission from Elsevier Science).

Fig. CM1b, shows the fibroblast after addition of ATP .(Reprinted from Biochim. Biophys. Acta, vol. 14, Hoffmann-Berling, H., Adenosintriphosphat als Betriebsstoff von Zellbewegungen, pp. 182-194, Copyright 1954, with permission from Elsevier Science).

Subsequently Hoffmann-Berling showed that glycerinated epithelial cells, hen embryos, or Jensen tumor tissues could be contracted with ATP, similarly to the contraction of glycerinated muscle fibers. The idea that actin and myosin are present in mammalian cells, other than muscle, became the working hypothesis of several laboratories:

Isolated actin-like proteins were identified by SDS gel electrophoresis, polymerization-depolymerization properties, nucleotide content, binding to myosin, activation of myosin ATPase, and tryptic peptide mapping. Actin was identified in cells by electron microscopy of fibers decorated with heavy meromyosin or subfragment-1 of myosin and immunofluorescence.

Isolated myosin-like proteins were identified by ATPase activities, SDS gel electrophoresis, subunit composition, binding to actin and by electron microscopy. Labeled antibodies against myosin provided further proof for the existence of myosin in non-muscle tissues. Thus, by the middle of 1970s actin and myosin were acknowledged as regular components of non-muscle cells.

Contractile proteins were prepared, for example, from brain, oviduct, kidney, blood platelets, liver cells, sympathetic neurons, cultures of fibroblast, plasmodial slime mold, or Dyctyostelium amoebae. From all these data it appeared that the mechanism of motion follows the same principle in biology.

Actin-Binding Proteins

In situ the polymerization-depolymerization of actin is controlled by the actin-binding proteins, which combine with actin monomers, cap the ends of the actin filaments, cross-link actin filaments, or attach actin to membranes. Fundamental cellular processes such as, cytokinesis, lamellipodial and growth cone extension, Chemotaxis, endocytosis, or exocytosis are regulated by actin-binding proteins:


The classical actin-binding protein, profilin, was discovered in the middle of 1970s. It is a small (12-15 kDa), soluble protein that is present in a high concentration (20-80 pM) throughout the cytoplasm and has a high affinity to cytoplasmic actin (Kd =10-6 M). Profilin inhibits polymerization of actin by sequestering the monomeric actin (Fig. CM2)

Fig. CM2. Schematic overview of actin polymerization and its regulation by profilin and related proteins affecting the competence of monomers to assemble into filaments (From Stossel, 1989).

The X-ray structure of profilin (Fig. CM3) reveals that the protein is bisected by an antiparallel beta-pleated sheet. Both termini are alpha-helical and pack against the same side of the central sheet, connecting to it by short loops.The X-ray structure of profilin-beta-actin is presented in Fig. CM4. Profilin forms two major contacts with actin in the crystal. The primary contact comprises a region of profilin defined by helix 3, the amino-terminal portion of helix 4, and strands 4, 5, and 6. This region makes contact with a site on actin spanning the large and the small domains of the molecule at the bases of subdomains 1 and 3.

Fig. CM3. Polypeptide fold of profilin. The four helices (H) and the seven strands (S) are shown. (From Schutt et al., reproduced with permission from Nature 365,810-816,1993, http://www.nature.com).
Fig. CM4. The profilin-actin ribbon. Profilin red, actin black. For explanation see the text. (From Schutt et al., reproduced with permission from Nature 365,810-816,1993, http://www.nature.com).

Thymosin beta4 is another actin monomer-binding protein. It is a small peptide, 43 residues, and it competes with profilin for binding to actin (Pollard et al., 2000).


Of the many proteins isolated from cell extracts, gelsolin is the most potent to solubilize gelatinous (fibrous) actin (this ability is reflected in the name of the protein). Severing of the actin-gel occurs through the weakening of sufficient bonds between actin molecules within a filament to break the filament. Severing includes binding of gelsolin to actin, structural rearrangement within gelsolin, and changing the conformation of actin. After severing, gelsolin remains attached to the barbed end of the actin filament that cannot reanneal or elongate and, thus, the actin network is disassembled. Ca2+ is required to the severing process (Sun et al.,1999).

Structure and function: Gelsolin has two tandem homologous halves, each of which contains a 3fold segmental repeat, segments S1-S3 and S4-S6, respectively (Fig. CM5).

Fig. CM5. Gelsolin structure-function domain. Amino acid residues are numbered and the 6 segments are indicated. Actin, PIP2, and Ca2+-binding segments, and the caspase-3 protease site are shown. (From Sun et al.,1999).

The crystal structure of gelsolin shows that in the absence of Ca2+ gelsolin has a compact quaternary structure. Its two halves are held together by a C-terminal S6 tail which latches onto S2 (Fig. CM6). It was predicted from this structure that "Ca2+ must induce major conformational changes in each half and in the relation between the halves to accommodate actin binding". Indeed new X-ray diffraction studies revealed domain movement in gelsolin (Robinson et al., 1999). Upon Ca2+-binding the S6 domain moved by about 40 angstroms resulting in a major structural reorganization in gelsolin, i.e. the actin-binding site on S4 became exposed enabling the severing and capping of actin filaments to proceed. The various steps of gelsolin action are illustrated in Fig. CM7.

Fig. CM6. Structural model of gelsolin in the absence of Ca . (From Sun et al.,1999).

Fig. CM7. Schematic model for gelsolin binding, severing, and capping of an actin filament (blue). The (+) barbed end and the (-) pointed end of the filaments are indicated. Left, gelsolin in the process of Ca2+ activation. This requires tail unlatching, domain separation within each half, and separation of the halves. Middlle,

S2-S3 binds to the side of an actin filament via S2. S1 wedges between two actins in the longitudinal axis. S4-S6 reaches across the filament to bind actin at the other strand. Severing occurs when sufficient actin-actin bonds are broken. Right, the terminal actin in each strand is capped by S1 and S4. PIP2 induces gelsolin to dissociate from the ends of the actin filaments; this process is called uncapping. (From Sun et al.,1999).

Phosphoinositides, particularly PIP2 ,dissociate gelsolin from actin (Fig. CM7), thus reverse the capping of the filaments. Severing of the long actin filaments by gelsolin increases the number of the filaments. Uncapping of gelsolin from these filaments generates many polymerization-capable ends from which actin can grow to rebuild the cytoskeleton to a new specification.

The barbed ends of the actin filaments are also interacting with the capping protein, called CapZ in muscle. The protein exists at micromolar concentration in the cytoplasm, and it has a high affinity to the barbed ends, Kd = 0.1 nM (Pollard et al., 2000).


The actin-depolymerizing factor (ADF), also called cofilin, has been recognized early as a widespread, small (15 -18 kDa) actin binding protein that plays important role in cytokinesis, endocytosis, and in the development of all embryonic tissues (Carlier et al., 1999). ADF/cofilins from different organisms present a high degree of sequence homology and the general mechanism of action of the different ADF/cofilins is conserved. The three-dimensional structure determined by X-ray crystallography is shown on Fig. CM8A. Five central beta-sheets are flanked by three to four alpha-helices. Image reconstruction (Fig. CM8B) shows that ADF interacts with two actin subunits along the long pitch helix, bridging subdomain 1 of the actins with subdomain 2 of the second subunit. As a result of the ADF binding to F-actin, there is a twist of 5o per subunit and, therefore, the long pitch helices crossover every 27 nm on average instead of 36 nm for standard F-actin filaments.

Camp Actin

Fig. CM8. Structure of ADF/cofilin. A, ribbon structure of cofilin showing the G- and F-actin binding domains. B, image reconstruction of a standard actin filament (a), and of a cofilin-decorated actin filament (b). (From Carlier et al., 1999)

Importantly, the stimulus-responsive function of ADF/cofilin is regulated by phosphorylation of a single serine residue. In response to stimuli, ADF is dephosphorylated. The stimuli, such as growth factors, chemotactic peptides, or agents increasing the levels of [Ca2+]i and cAMP, promote the reorganization of the actin cytoskeleton. In quiescent cells, ADF/cofilin appears diffusely distributed in the cytoplasm, the activated (dephosphorylated) protein translocates to regions of the cells where actin filaments are highly dynamic like the leading edge of ruffled membranes, the cleavage furrow of dividing cells, or the neuronal growth clone. Dephosphorylation correlates with increased motility and extension of cellular processes (Carlier et al., 1999). ADF/cofilin increases the turnover of actin filaments which powers actin motility (Fig. CM9).

Cofilin Wasp

Arp2/3 complex and WASp/Scar proteins

Fig. CM9. Enhancement of actin turnover by ADF. The F-actin filaments, containing ADF, depolymerize 30-fold faster from their pointed ends than the bare filaments, resulting in accumulation of ADF-ADP-actin pool. After dissociation of ADF, the ADP-actin assembles on the barbed end of the bare filaments. The ADF molecules are red colored. (From Carlier et al.,

Arp2/3 complex and WASp/Scar proteins

Actin related proteins (Arps) participate in a diverse array of cellular processes (Schafer and Schroer, 1999). Wiskott-Aldrich syndrome proteins (WASp/Scar proteins) stimulate the formation of new actin filaments by Arp2/3 complex (Higgs and Pollard, 1999). Fig. CM10 shows that the Arp2/3 complex contains seven protein subunits, Arp2 and Arp3 are actin related proteins, the other five subunits are novel. The Arp2/3 complex cross-links filaments in an end to side manner, with the slow growing end of one filament attached to the side of another at an angle of 70 degrees. Fig CM11 shows the binding of WASp/Scar to an actin filament and to Arp2/3, creating new filaments and cross-linking them into a branching meshwork.

Fig. CM10. Arp2/3 complex structure. Based on nearest neighbor relationship of the subunits from chemical cross linking. Molecular masses in kDa are indicated. (From Higgs and Pollard, 1999).

Fig. CM10. Arp2/3 complex structure. Based on nearest neighbor relationship of the subunits from chemical cross linking. Molecular masses in kDa are indicated. (From Higgs and Pollard, 1999).

Fig. CM11. Dendritic nucleation model. Arp2/3 complex (green) binds to the side of a preexisting actin filament (yellow), and WASp/Scar (red) bound to an actin monomer binds to Arp2/3, forming a nucleus for barbed end growth from the side of the filament (From Higgs and Pollard, 1999).

Major advances have been made in research of Arp2/3 complex recently. The crystal structure of the Arp2/3 complex has been determined at 2.0 angstrom resolution (Robinson et al., 2001). Arp2 and Arp3 are folded like actin with distinctive surface features. The structure of the five subunits has also been elucidated. Subsequently, the structure of the Arp2/3 complex in its activated state and in actin filament branch junctions has been described (Volkmann et al, 2001). Internal reflection fluorescence microscopy was used for direct real-time observation of actin filament branching mediated by Arp2/3 complex (Aman and Pollard 2001). Furthermore, it was shown that the binding of ATP to Arp2 is required for filament branching (Le Clainche et al., 2001; Dayel et al., 2001).

External stimuli drive the assembly of the actin filament network, acting through receptors and multiple signal transduction pathways, several of which converge on WSPp/Scar proteins and Arp2/3 complex. Diverse signals including those carried by the Rho family GTPases, Rac, and Cdc42 are involved (Fig. CM11A).

Fig. CM11A. Signaling pathways through WASp/Scar to Arp2/3 complex. (From Pollard et al.,with permission from the Annual Review of Biphysics and Biomolecular Sructure. vol. 29, 2000, by Annual Reviews, http://www.AnnualReviews.org).

Fig. CM11A. Signaling pathways through WASp/Scar to Arp2/3 complex. (From Pollard et al.,with permission from the Annual Review of Biphysics and Biomolecular Sructure. vol. 29, 2000, by Annual Reviews, http://www.AnnualReviews.org).

Was this article helpful?

0 0

Post a comment