Crosslinking of actin filaments is caused by caldesmon aggregates, but not by its dimers

A recent report by Bretscher [(1984) J. Biol. Chem. 259, 12873-12880] showed that caldesmon prepared by his method crosslinks actin filaments to form thick bundles. This is in contrast to the results of previous work that caldesmon binds to F-actin but does not cause any gelation [(1981) Proc. Natl. Acad. Sci. USA 78, 5652-5655]. The present work clearly showed that caldesmon purified according to Bretscher does not cause any gelation of F-actin. However, caldesmon aggregates formed by concentration or by freeze-thawing gelated F-actin to form bundles.     

Cytochalasin inhibits the rate of elongation of actin filament fragments

Submicromolar concentrations of cytochalasin inhibit the rate of assembly of highly purified dictyostelium discoideum actin, using a cytochalasin concentration range in which the final extent of assembly is minimally affected. Cytochalasin D is a more effective inhibitor than cytochalasin B, which is in keeping with the effects that have been reported on cell motility and with binding to a class of high-affinity binding sites from human erythrocyte membranes (Lin and Lin. 1978. J. Biol. CHem. 253:1415; Lin and Lin. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:2345); 5x10(-7) M cytochalasin B lowers it to 70 percent of the control value, whereas 10(-7) M cytochalasin B lowers the rate to 25 percent. Fragments of F-actin were used to increase the rate of assembly fivefold by providing more filament ends on to which monomers could add. Under these conditions, cytochalasin has an even more dramatic effect on the assembly rate; the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 2x10(-7) M and 10(-8) M, respectively. The assembly rate is most sensitive to cytochalasin when actin assembly is carried out in the absence of ATP (with 3 mM ADP present to stabilize the actin). In this case, the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 4x10(-8) M and 1x10(-9) M, respectively. A scatchard plot has been obtained using [(3)H]cytochalasin B binding to F-actin in the absence of ATP. The K(d) from this plot (approximately 4x10(-8) M) agrees well with the concentration of cytochalasin B required for half-maximal inhibition of the rate of assembly under these conditions. The number of cytochalasin binding sites is roughly one per F-actin filament, suggesting that cytochalasin has a specific action on actin filament ends.     

A natural oocyte component required for the reprogramming of somatic cell nuclei

Comment on: Jullien J, et al. Proc Natl Acad Sci USA 2010; 107:5483-8.     

c[D-pro-Pro-D-pro-N-methyl-Ala] adopts a rigid conformation that serves as a scaffold to mimic reverse-turns

Naturally occurring cyclic tetrapeptides (CTPs) such as tentoxin (Halloin et al., Plant Physiol 1970, 45, 310-314; Saad, Phytopathology 1970, 60, 415-418), ampicidin (Darkin-Rattray, Proc Natl Acad Sci USA 1996, 93, 13143-13147), HC-toxin (Walton, Proc Natl Acad Sci USA 1987, 84, 8444-8447), and trapoxin (Yoshida and Sugita, Jpn J Cancer Res 1992, 83, 324-328; Itazaki et al., J Antibiot (Tokyo) 1990, 43, 1524-1532) have a wide range of biological activity and potential use ranging from herbicides (Walton, Proc Natl Acad Sci USA 1987, 84, 8444-8447; Judson, J Agric Food Chem 1987, 35, 451-456) to therapeutics (Loiseau, Biopolymers 2003, 69, 363-385) for malaria (Darkin-Rattray, Proc Natl Acad Sci USA 1996, 93, 13143-13147) and cancer (Yoshida and Sugita, Jpn J Cancer Res 1992, 83, 324-328). To elucidate scaffolds that have few low-energy conformations and could serve as semirigid reverse-turn mimetics, the flexibility of CTPs was determined computationally. Four analogs of cyclic tetraproline c[Pro-pro-Pro-pro] with alternating L- and D-prolines, namely c[pro-Pro-pro-NMe-Ala], c[pip-Pro-pip-Pro], c[pro-Pip-pro-Pro], and c[Ala-Pro-pip-Pro] were synthesized and characterized by NOESY NMR. Both molecular mechanics and Density Functional Theory quantum calculations found these head-to-tail CTPs to be constrained to one or two relatively stable conformations. NMR structures, while not always yielding the same lowest energy conformation as expected by in silico predictions, confirmed only one or two highly populated solution conformations for all four peptides examined. c[pro-Pro-pro-NMe-Ala] was shown to have a single all trans-amide bond conformation from both in silico predictions and NMR characterization, and to be a reverse-turn mimetic by overlapping four Calpha-Cbeta bonds with those for approximately 6.5% (Tran, J Comput Aided Mol Des 2005, 19, 551-566) of reverse-turns in the Protein Data Bank PDB with a RMSD of 0.57 A.     

Lys-373 of actin is involved in binding to caldesmon.

Limited proteolysis of actin with trypsin removes its two or three C-terminal amino acid residues [Proc. Natl. Acad. Sci. USA 81 (1984) 3680-3684]. Carboxypeptidase B-treatment of G- and F-actin previously digested with trypsin revealed that in the first case preferential release of three and in the second two C-terminal amino acid residues takes place. Tryptic removal of three but not two C-terminal amino acid residues of actin causes weakening of its interaction with caldesmon and lowering of the caldesmon-induced inhibitory effect on actomyosin ATPase activity. Therefore, it is concluded that the third amino acid residue from the C terminus of actin, Lys-373, is important for the interaction with caldesmon.     

Kinetics of thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle: implications for regulation of muscle contraction

Making use of troponin with fluorescently labeled troponin I subunit (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-troponin I, IANBD-TnI) that had previously been described in solution studies as a probe for thin filament activation (. Proc. Natl. Acad. Sci. 77:7209-7213), we present a new approach that allows the kinetics of thin filament activation to be studied in skinned muscle fibers. After the exchange of native troponin for fluorescently labeled troponin, the fluorescence intensity is sensitive to both changes in calcium concentration and actin attachment of cross-bridges in their strong binding states (. Biophys. J. 77:000-000). Imposing rapid changes in the fraction of strongly attached cross-bridges, e.g., by switching from isometric contraction to high-speed shortening, causes changes in thin filament activation at fixed Ca(2+) concentrations that can be followed by recording fluorescence intensity. Upon changing to high-speed shortening we observed small (<20%) changes in fluorescence that became faster at higher Ca(2+) concentrations. At all Ca(2+) concentrations, these changes are more than 10-fold faster than force redevelopment subsequent to the period of unloaded shortening. We interpret this as an indication that equilibration among different states of the thin filament is rapid and becomes faster as Ca(2+) is raised. Fast equilibration suggests that the rate constant of force redevelopment is not limited by changes in the activation level of thin filaments induced by the isotonic contraction before force redevelopment. Instead, our modeling shows that, in agreement with our previous proposal for the regulation of muscle contraction, a rapid and Ca(2+)-dependent equilibration among different states of the thin filament can fully account for the Ca(2+) dependence of force redevelopment and the fluorescence changes described in this study.     

Developmentally expressed myosin heavy-chain kinase possesses a diacylglycerol kinase domain.

In Dictyostelium, an ordered actin and myosin assembly-disassembly process is necessary for proper development, differentiation, and motility (Yumura S, Fukui F, 1985, Nature 314(6007): 194-196; Ravid S, Spudich JA, 1989, J Biol Chem 264(25): 15144-15150), and phosphorylation of myosin heavy chains has been implicated in the myosin assembly-disassembly process (Egelhoff TT, Lee RJ, Spudich JA, 1993, Cell 75(2):363-371). The developmentally expressed 84-kDa myosin heavy-chain kinase (MHCK) from Dictyostelium (Ravid S, Spudich JA, 1992, Proc Natl Acad Sci USA 89(13):5877-5881) is known to be a member of the protein kinase C (PKC) family. We have observed a rather striking homology between the large central domain of MHCK and the catalytic domain of diacylglycerol kinase (DGK), indicating that MHCK is in fact a gene fusion between a DGK and a PKC, possessing two separate kinase domains. The combined diacylglycerol kinase/myosin heavy-chain kinase (DGK/MHCK) may therefore have dual functionality, possessing the ability to phosphorylate both protein and lipid. We present a hypothesis that DGK/MHCK can antagonize both actin and myosin assembly, as well as other cellular processes, by coordinated down regulation of signaling via myosin heavy-chain kinase activity and diacylglycerol kinase activity.     

Rotational dynamics of spin-labeled F-actin during activation of myosin S1 ATPase using caged ATP.

The most probable source of force generation in muscle fibers in the rotation of the myosin head when bound to actin. This laboratory has demonstrated that ATP induces microsecond rotational motions of spin-labeled myosin heads bound to actin (Berger, C. L. E. C. Svensson, and D. D. Thomas. 1989. Proc. Natl. Acad. Sci. USA. 86:8753-8757). Our goal is to determine whether the observed ATP-induced rotational motions of actin-bound heads are accompanied by changes in actin rotational motions. We have used saturation transfer electron paramagnetic resonance (ST-EPR) and laser-induced photolysis of caged ATP to monitor changes in the microsecond rotational dynamics of spin-labeled F-actin in the presence of myosin subfragment-1 (S1). A maleimide spin label was attached selectively to cys-374 on actin. In the absence of ATP (with or without caged ATP), the ST-EPR spectrum (corresponding to an effective rotational time of approximately 150 microseconds) was essentially the same as observed for the same spin label bound to cys-707 (SH1) on S1, indicating that S1 is rigidly bound to actin in rigor.(ABSTRACT TRUNCATED AT 250 WORDS)     

The chimeric VirA-tar receptor protein is locked into a highly responsive state.

The wild-type VirA protein is known to be responsive not only to phenolic compounds but also to sugars via the ChvE protein (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA 87:6708-6712, 1990, and N. Shimoda, A. Toyoda-Yamamoto, J. Nagamine, S. Usami, M. Katayama, Y. Sakagami, and Y. Machida, Proc. Natl. Acad. Sci. USA 87:6684-6688, 1990). It is shown here that the mutant VirA(Ser-44, Arg-45) protein and the chimeric VirA-Tar protein are no longer responsive to sugars and the ChvE protein. However, whereas the chimeric VirA-Tar protein was found to be locked in a highly responsive state, the VirA(Ser-44, Arg-45) mutant protein appeared to be locked in a low responsive state. This difference turned out to be important for tumorigenicity of the host strains in virulence assays on Kalanchoë daigremontiana.     

Chromosomal locations of members of a family of novel endogenous human retroviral genomes.

Human cellular DNA contains two distinguishable families of retroviral related sequences. One family shares extensive nucleotide sequence homology with infectious mammalian type C retroviral genomes (T. I. Bonner, C. O'Connell, and M. Cohen, Proc. Natl. Acad. Sci. USA 79:4709-4713, 1982; M. A. Martin, T. Bryan, S. Rasheed, and A. S. Khan, Proc. Natl. Acad. Sci. USA 78:4892-4896, 1981). The other family contains major regions of homology with the pol genes of infectious type A and B and avian type C and D retroviral genomes (R. Callahan, W. Drohan, S. Tronick, and J. Schlom, Proc. Natl. Acad. Sci. USA 79:5503-5507, 1982; I. M. Chiu, R. Callahan, S. R. Tronick, J. Schlom, and S. A. Aaronson, Science 223:364-370, 1984). Analysis of the human recombinant clone HLM-2 has shown that the pol gene in the latter family is located within an endogenous proviral genome (R. Callahan, I. M. Chiu, J. F. H. Wong, S. R. Tronick, B. A. Roe, S. A. Aaronson, and J. Schlom, Science 228:1208-1211, 1985). We show that the proviral genome in HLM-2 and the related recombinant clone HLM-25 are located, respectively, on human chromosomes 1 and 5. Other related proviral genomes are located on chromosomes 7, 8, 11, 14, and 17.     

Consistencies of individual DNA base-amino acid interactions in structures and sequences.

Amino acid-amino acid interaction energies have been derived from crystal structure data for a number of years. Here is reported the first derivation of normalized relative interaction from binding data for each of the four bases interacting with a specific amino acid, utilizing data from combinatorial multiplex DNA binding of zinc finger domains [Desjarlais, J. R. and Berg, J. M. (1994) Proc. Natl. Acad. Sci. USA, 91, 11099-11103]. The five strongest interactions are observed for lysine-guanine, lysine-thymine, arginine-guanine, aspartic acid-cytosine and asparagine-adenine. These rankings for interactions with the four bases appear to be related to base-amino acid partial charges. Also, similar normalized relative interaction energies are derived by using DNA binding data for Cro and lambda repressors and the R2R3 c-Myb protein domain [Takeda, Y., Sarai, A. and Rivera, V. M. (1989) Proc. Natl. Acad. Sci. USA, 86, 439-443; Sarai, A. and Takeda, Y. (1989) Proc. Natl. Acad. Sci. USA, 86, 6513-6517; Ogata, K. et al. (1995) submitted]. These energies correlate well with the combinatorial multiplex energies, and the strongest cases are similar between the two sets. They also correlate well with similar relative interaction energies derived directly from frequencies of bases in the bacteriophage lambda operator sequences. These results suggest that such potentials are general and that extensive combinatorial binding studies can be used to derive potential energies for DNA-protein interactions.     

Diffusion of molecules on biological membranes of nonplanar form. II. Diffusion anisotropy.

Molecules diffusing on nonplanar membranes, which have different amounts of corrugation in different directions, may experience dissimilar diffusion coefficients in each direction. Smith et al. (1979, Proc. Natl. Acad. Sci. USA, 76:5641-5644) measured diffusion anisotropy on fibroblast cell membranes in which the ratio of the diffusion coefficients, in different directions, was 0.27. In the present work we calculate the effect of anisotropic corrugation on the rate of diffusion of molecules on membranes. We find that part of the anisotropy reported by Smith et al. (1979, Proc. Natl. Acad. Sci. USA, 76:5641-5644) can be explained by the membrane nonplanarity, and we present the way of calculating this geometric factor.     

MicroRNA regulation masters basal transcription: Clash of the Titans

Comment on: Portal MM. Proc Natl Acad Sci USA 2011; 108:8686-91.     

Actin dynamics during the cell cycle in Chlamydomonas reinhardtii.

We have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349-362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335-2339, 1981) identify Chlamydomonas actin as a approximately 43,000-M(r) protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415-430, 1985) first identified Chlamydomonas actin using NBD-phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F-actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the anterior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrangement forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F-actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis.     

A novel role for Activation-induced cytidinedeaminase: Central B-cell tolerance

Comment on: Kuraoka M, et al. Proc Natl Acad Sci USA 2011; 108:11560-5     

Sensing senescence in preterm birth

Comment on: Hirota Y, et al. Proc Natl Acad Sci USA 2011; 108:18073-8     

The role of eIF5A in protein synthesis

Comment on: Henderson A, et al. Proc Natl Acad Sci USA 2011; 108:6415-9.     

You’d better catch that Wnt: Primitive hematopoiesis requires canonical Wnt signaling

Comment on: Tran HT, et al. Proc Natl Acad Sci USA 2010; 107:16160-5.     

A self-propelled biological process

Comment on : Park JE, et al. Proc Natl Acad Sci USA 2011; 108:8200-05.