NORMAL GENOMIC

• Stokasimov E, Rubenstein PA
• Actin isoform-specific conformational differences observed withhydrogen/deuterium exchange and mass spectrometry.
• J Biol Chem. 2009; 284: 25421-30
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• Actin can exist in multiple conformations necessary for normal function.Actin isoforms, although highly conserved in sequence, exhibit differentbiochemical properties and cellular roles. We used amide protonhydrogen/deuterium (HD) exchange detected by mass spectrometry to analyzeconformational differences between Saccharomyces cerevisiae and muscleactins in the G and F forms to gain insight into these differences. Wealso utilized HD exchange to study interdomain and allostericcommunication in yeast-muscle hybrid actins to better understand theconformational dynamics of actin. Areas showing differences in HD exchangebetween G- and F-actins are areas of intermonomer contacts, consistentwith the current filament models. Our results showed greater exchange foryeast G-actin compared with muscle actin in the barbed end pivot regionand areas in subdomains 1 and 2 and for F-actin in monomer-monomer contactareas. These results suggest greater flexibility of the yeast actinmonomer and filament compared with muscle actin. For hybrid G-actins, themuscle-like and yeastlike parts of the molecule generally showed exchangecharacteristics resembling their parent actins. A few exceptions were apeptide on top of subdomain 2 and the pivot region between subdomains 1and 3 with muscle actin-like exchange characteristics although the areaswere yeastlike. These results demonstrate that there is cross-talk betweensubdomains 1 and 2 and the large and small domains. Hybrid F-actin datashowing greater exchange compared with both yeast and muscle actins areconsistent with mismatched yeast-muscle interfaces resulting in decreasedstability of the hybrid filament contacts.

• Sun N, Critchley DR, Paulin D, Li Z, Robson RM
• Identification of a repeated domain within mammalian alpha-synemin thatinteracts directly with talin.
• Exp Cell Res. 2008; 314: 1839-49
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• The type VI intermediate filament (IF) protein synemin is a unique memberof the IF protein superfamily. Synemin associates with the major type IIIIF protein desmin forming heteropolymeric intermediate filaments (IFs)within developed mammalian striated muscle cells. These IFs encircle andlink all adjacent myofibrils together at their Z-lines, as well as linkthe Z-lines of the peripheral layer of cellular myofibrils to thecostameres located periodically along and subjacent to the sarcolemma.Costameres are multi-protein assemblies enriched in the cytoskeletalproteins vinculin, alpha-actinin, and talin. We report herein a directinteraction of human alpha-synemin with the cytoskeletal protein talin byprotein-protein interaction assays. The 312 amino acid insert (SNTIII)present only within alpha-synemin binds to the rod domain of talin invitro and co-localizes with talin at focal adhesion sites within mammalianmuscle cells. Confocal microscopy studies showed that synemin co-localizeswith talin within the costameres of human skeletal muscle cells. Analysisof the primary sequences of human alpha- and beta-synemins revealed thatSNTIII is composed of seven tandem repeats, each containing a specificSer/Thr-X-Arg-His/Gln (S/T-X-R-H/Q) motif. Our results suggest humanalpha-synemin plays an essential role in linking the heteropolymeric IFsto adherens-type junctions, such as the costameres within mammalianstriated muscle cells, via its interaction with talin, thereby helpingprovide mechanical integration for the muscle cell cytoskeleton.

• Balasubramanian R, Karve A, Moore BD
• Actin-based cellular framework for glucose signaling by Arabidopsishexokinase1.
• Plant Signal Behav. 2008; 3: 322-4
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• Glucose functions in plants both as a metabolic resource as well as ahormone that regulates expression of many genes. Arabidopsis hexokinase1(HXK1) is the best understood plant glucose sensor/transducer, yet we areonly now appreciating the cellular complexity of its signaling functions.We have recently shown that one of the earliest detectable responses toplant glucose treatments are extensive alterations of cellular F-actin.Interestingly, AtHXK1 is predominantly located on mitochondria, yet alsocan interact with actin. A normal functioning actin cytoskeleton isrequired for HXK1 to act as an effector in glucose signaling assays. Wehave suggested that HXK1 might alter F-actin dynamics and therebyinfluence the formation and/or stabilization of cytoskeleton-boundpolysomes. In this Addendum, we have extended our initial observations onthe subcellular targeting of HXK1 and its interaction with F-actin. Wethen further consider the cellular context in which HXK1 might regulategene expression.

• Wegener KL et al.
• Structural basis of integrin activation by talin.
• Cell. 2007; 128: 171-82
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• Regulation of integrin affinity (activation) is essential for metazoandevelopment and for many pathological processes. Binding of the talinphosphotyrosine-binding (PTB) domain to integrin beta subunit cytoplasmicdomains (tails) causes activation, whereas numerous otherPTB-domain-containing proteins bind integrins without activating them.Here we define the structure of a complex between talin and themembrane-proximal integrin beta3 cytoplasmic domain and identify specificcontacts between talin and the integrin tail required for activation. Weused structure-based mutagenesis to engineer talin and beta3 variants thatinteract with comparable affinity to the wild-type proteins but inhibitintegrin activation by competing with endogenous talin. These resultsreveal the structural basis of talin's unique ability to activateintegrins, identify an interaction that could aid in the design oftherapeutics to block integrin activation, and enable engineering of cellswith defects in the activation of multiple classes of integrins.

• Cohen DM, Chen H, Johnson RP, Choudhury B, Craig SW
• Two distinct head-tail interfaces cooperate to suppress activation ofvinculin by talin.
• J Biol Chem. 2005; 280: 17109-17
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• Vinculin is autoinhibited by an intramolecular interaction that masksbinding sites for talin and F-actin. Although a recent structural modelexplains autoinhibition solely in terms of the interaction betweenvinculin tail (Vt) and residues 1-258 (D1), we find an absoluterequirement for an interface involving the D4 domain of head (Vh residues710-836) and Vt. Charge-to-alanine mutations in Vt revealed a class ofmutants, T12 and T19, distal to the V-(1-258) binding site, which showedincreases in their Kd values for head binding of 100- and 42-fold,respectively. Reciprocal mutation of residues in the D4 domain thatcontact Vt yielded a head-tail interaction mutant of comparable magnitudeto T19. These findings account for the approximately 120-fold differencein Kd values between Vt binding to V-(1-258), as opposed to full-lengthVh-(1-851). The significance of a bipartite autoinhibitory site isevidenced by its effects on talin binding to Vh. Whereas Vt fails tocompete with the talin rod domain for binding to V-(1-258), competitionoccurs readily with full-length Vh, and this requires the D4 interface.Moreover in intact vinculin, mutations in the D4-Vt interface stabilizeassociation of vinculin and talin rod. In cells, these head-tailinteraction mutants induce hypertrophy and elongation of focal adhesions.Definition of a second autoinhibitory site, the D4-Vt interface, supportsthe competing model of vinculin activation that invokes cooperative actionof ligands at two sites. Together the D1-Vt and D4-Vt interfaces providethe high affinity (approximately 10(-9)) autoinhibition observed infull-length vinculin.

• Kranewitter WJ, Ylanne J, Gimona M
• UNC-87 is an actin-bundling protein.
• J Biol Chem. 2001; 276: 6306-12
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• The Caenorhabditis elegans unc-87 gene product is essential for the maintenance of the nematode body wall muscle where it is found colocalized with actin in the I band. The molecular domain structure of the protein reveals similarity to the C-terminal repeat region of the smooth muscle actin-binding protein calponin. In this study we investigated the in vitro function of UNC-87 using both the full-length recombinant molecule and several truncated mutants. According to analytical ultracentrifugation UNC-87 occurs as a monomer in solution. UNC-87 cosedimented with both smooth and skeletal muscle F-actin, but not with monomeric G-actin, and exhibited potent actin filament bundling activity. Actin binding was independent of the presence of tropomyosin and the actin cross-linking proteins filamin and alpha-actinin. Consistent with its actin bundling activity in vitro, UNC-87 tagged with green fluorescent protein associated with and promoted the formation of actin stress fiber bundles in living cells. These data identify UNC-87 as an actin-bundling protein and highlight the calponin-like repeats as a novel actin-binding module.

• Melms AS, Gausmann U, Swoboda RK, Dominguez A, Kurischko C
• Sequence analysis of SLA2 of the dimorphic yeasts Candida albicans andYarrowia lipolytica.
• Yeast. 1999; 15: 1519-28
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• We report the complete nucleotide sequence of SLA2 of the dimorphic yeastsCandida albicans and Yarrowia lipolytica. In Saccharomyces cerevisiae,SLA2 codes for an actin binding protein. The deduced amino acid (aa)sequences of C. albicans CaSla2p and Y. lipolytica YlSla2p consist of 1063and 1054 aa, respectively. The alignment of the deduced proteins ofSaccharomyces cerevisiae, Y. lipolytica and C. albicans shows regions ofidentity in the N-terminal part of the proteins, which are essential forgrowth at 37 degrees C, endocytosis and actin organization in S.cerevisiae. The Sla2p proteins have also several conserved regions in theC-terminal moiety, the I/LWEQ boxes, displaying homology to the talinprotein of mouse, Dictyostelium discoideum, Caenorhabditis elegans and tohuman huntingtin interacting protein (Hip 1p). The sequence data of C.albicans SLA2 are registered in the EMBL database (AJ009556), and for theY. lipolytica gene in GenBank (U65409).

• Tempel M, Goldmann WH, Isenberg G, Sackmann E
• Interaction of the 47-kDa talin fragment and the 32-kDa vinculin fragmentwith acidic phospholipids: a computer analysis.
• Biophys J. 1995; 69: 228-41
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• In recent in vitro experiments, it has been demonstrated that the 47-kDafragment of the talin molecule and the 32-kDa fragment of the vinculinmolecule interact with acidic phospholipids. By using a computer analysismethod, we determined the hydrophobic and amphipathic stretches of thesefragments and, by applying a purpose-written matrix method, we ascertainedthe molecular amphipathic structure of alpha-helices. Calculations for the47-kDa mouse talin fragment (residues 1-433; NH2-terminal region) suggestspecific interactions of residues 21-39, 287-342, and 385-406 with acidicphospholipids and a general lipid-binding domain for mouse talin (primaryamino acid sequence 385-401) and for Dictyostelium talin (primary aminoacid sequence 348-364). Calculations for the 32-kDa chicken embryovinculin fragment (residues 858-1066; COOH-terminal region) and fromnematode vinculin alignment indicate for chicken embryo vinculin residues935-978 and 1020-1040 interactions with acidic phospholipids. Experimentalconfirmation has been given for vinculin (residues 916-970), and futuredetailed experimental analyses are now needed to support the remainingcomputational data.

• Hagmann J, Grob M, Burger MM
• The cytoskeletal protein talin is O-glycosylated.
• J Biol Chem. 1992; 267: 14424-8
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• Talin is a 215-kDa cytoskeletal protein implicated in linking actinfilaments to the plasma membrane. We show here that chicken gizzard talinis galactosylated by incubation with UDP-[3H]galactose andgalactosyl-transferase. The labeled carbohydrate moiety is removed bybeta-elimination and comigrates with Gal beta 1-4GlcNAcitol, indicatingthat talin belongs to a recently discovered class of cytosolic proteinscarrying N-acetylglucosamine (GlcNAc) O-linked to serine or threonine(Holt, G. D., and Hart, G. W. (1986) J. Biol. Chem. 261, 8049-8057). Twoglycosylated sequences were identified in the tail domain of talin:ANQAIQMAXQNLVDPAXTQ and GILANQLTNDYGQLAQQ, corresponding to amino acids1470-1488 and 1883-1899, respectively, of the mouse talin amino acidsequence (Rees, D. J. G., Ades, S. E., Singer, S. J., and Hynes, R. O.(1990) Nature 347, 685-689). The putative glycosylation sites are PAXTQand QLTND. At most 6% of chicken gizzard talin and 3% of porcine stomachtalin are galactosylated by galactosyltransferase. Furthermore, humanplatelet talin is not labeled at all by the procedure, indicating that itmay not be glycosylated.

• Matsudaira P
• Modular organization of actin crosslinking proteins.
• Trends Biochem Sci. 1991; 16: 87-92
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• A family of actin-crosslinking proteins share a conserved 125 residue sequence that lies within a 250 residue actin-binding domain. This domain is combined with spacer segments consisting of a variable number of repeated alpha-helical or beta-sheet motifs and other functional domains, which generate proteins that differ in their ability to form actin bundles or networks and to associate with the plasma membrane. These functional domains are not in other actin-crosslinking proteins, one of which is elongation factor 1a (EF-1a) suggesting there are several pathways for the evolution of actin-crosslinking function.

• Muguruma M, Matsumura S, Fukazawa T
• Direct interactions between talin and actin.
• Biochem Biophys Res Commun. 1990; 171: 1217-23
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• Talin was purified from chicken gizzard by a modification of the method of L. Molony et al. [J. Biol. Chem.(1987) 262, 7790-7795]. Unlike the talin purified by the previous method, the talin purified by the new method was found to bind to both F- and G-actin: Talin cosedimented with F-actin. On gel filtration of a mixture of talin and G-actin, a complex of talin and action was obtained. Talin stimulated the polymerization rate of G-actin. A major proteolytic fragment of talin that retained the binding ability to F-actin was also identified. These results indicate that talin can bind directly to actin and suggest that talin plays a key role in the organization of actin filaments at the actin-membrane attachment sites in vivo also.