scholarly journals Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.

1984 ◽  
Vol 98 (3) ◽  
pp. 825-833 ◽  
Author(s):  
J W Sanger ◽  
B Mittal ◽  
J M Sanger
Keyword(s):  
Actin Filaments ◽  
Striated Muscle ◽  
Contractile Proteins ◽  
Actin Binding ◽  
Thin Filaments ◽  
In Vitro System ◽  
Self Association ◽  
Cross Bridges

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A-band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha-actinin and, if actin is added subsequently, the exogenous alpha-actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.

scholarly journals Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

2008 ◽  
Vol 19 (5) ◽  
pp. 1837-1847 ◽  
Author(s):  
Christopher T. Pappas ◽  
Nandini Bhattacharya ◽  
John A. Cooper ◽  
Carol C. Gregorio
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Actin Polymerization ◽  
Striated Muscle ◽  
Solid Phase ◽  
Molecular Model ◽  
Direct Interaction ◽  
Tryptophan Fluorescence ◽  
Actin Binding

The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant “molecular ruler” nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160–164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160–164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.

scholarly journals Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

2011 ◽  
Vol 22 (8) ◽  
pp. 1290-1299 ◽  
Author(s):  
Simren Mehta ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Actin Filaments ◽  
Gliding Motility ◽  
Conditional Knockout ◽  
Host Cells ◽  
Actin Binding ◽  
Actin Depolymerizing Factor

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.

scholarly journals The toxofilin–actin–PP2C complex of Toxoplasma: identification of interacting domains

2007 ◽  
Vol 401 (3) ◽  
pp. 711-719 ◽  
Author(s):  
Gaelle Jan ◽  
Violaine Delorme ◽  
Violaine David ◽  
Celine Revenu ◽  
Angelita Rebollo ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Actin Filaments ◽  
Function Analysis ◽  
Protozoan Parasite ◽  
Actin Binding ◽  
Protein Protein Interactions ◽  
The Third ◽  
Parasite Motility

Toxofilin is a 27 kDa protein isolated from the human protozoan parasite Toxoplasma gondii, which causes toxoplasmosis. Toxofilin binds to G-actin, and in vitro studies have shown that it controls elongation of actin filaments by sequestering actin monomers. Toxofilin affinity for G-actin is controlled by the phosphorylation status of its Ser53, which depends on the activities of a casein kinase II and a type 2C serine/threonine phosphatase (PP2C). To get insights into the functional properties of toxofilin, we undertook a structure–function analysis of the protein using a combination of biochemical techniques. We identified a domain that was sufficient to sequester G-actin and that contains three peptide sequences selectively binding to G-actin. Two of these sequences are similar to sequences present in several G- and F-actin-binding proteins, while the third appears to be specific to toxofilin. Additionally, we identified two toxofilin domains that interact with PP2C, one of which contains the Ser53 substrate. In addition to characterizing the interacting domains of toxofilin with its partners, the present study also provides information on an in vivo-based approach to selectively and competitively disrupt the protein–protein interactions that are important to parasite motility.

scholarly journals Overexpression of human fibroblast caldesmon fragment containing actin-, Ca++/calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments.

1994 ◽  
Vol 125 (2) ◽  
pp. 359-368 ◽  
Author(s):  
K S Warren ◽  
J L Lin ◽  
D D Wamboldt ◽  
J J Lin
Keyword(s):  
Cho Cells ◽  
Actin Filaments ◽  
Actin Binding ◽  
Expression Vectors ◽  
Binding Domains ◽  
Microfilament Bundles ◽  
The Stability ◽  
Triton X

Fibroblast caldesmon is a protein postulated to participate in the modulation of the actin cytoskeleton and the regulation of actin-based motility. The cDNAs encoding the NH2-terminal (aa.1-243, CaD40) and COOH-terminal (aa.244-538, CaD39) fragments of human caldesmon were subcloned into expression vectors and we previously reported that bacterially produced CaD39 protein retains its actin-binding properties as well as its ability to enhance low M(r) tropomyosin (TM) binding to actin and to inhibit TM-actin-activated HMM ATPase activity in vitro (Novy, R. E., J. R. Sellers, L.-F. Liu, and J. J.-C. Lin. 1993. Cell Motil. Cytoskeleton. 26:248-261). Bacterially produced CaD40 does not bind actin. To study the in vivo effects of CaD39 expression on the stability of actin filaments in CHO cells, we isolated and characterized stable CHO transfectants which express varying amounts of CaD39. We found that expression of CaD39 in CHO cells stabilized microfilament bundles as well as endogenous TM. CaD39-expressing clones displayed an increased resistance to cytochalasin B and Triton X-100 treatments and yielded increased amounts of TM-containing actin filaments in microfilament isolation procedures. In addition, analysis of these clones with immunoblotting and indirect immunofluorescence microscopy with anti-TM antibody revealed that stabilized endogenous TM and enhanced TM-containing microfilament bundles parallel increased amounts of CaD39 expression. The increased TM observed corresponded to a decrease in TM turnover rate and did not appear to be due to increased synthesis of endogenous TM. Additionally, the phenomenon of stabilized TM did not occur in stable CHO clones expressing CaD40. Therefore, it is likely that CaD39 can enhance TM's binding to F-actin in vivo, thus reducing TM's rate of turnover and stabilizing actin microfilament bundles.

scholarly journals Reassociation of microvillar core proteins: making a microvillar core in vitro.

1989 ◽  
Vol 108 (2) ◽  
pp. 495-502 ◽  
Author(s):  
L M Coluccio ◽  
A Bretscher
Keyword(s):  
Actin Filaments ◽  
Direct Proof ◽  
Intestinal Epithelia ◽  
The Core ◽  
Cross Bridge ◽  
Core Proteins ◽  
The Cross ◽  
Cross Bridges

Intestinal epithelia have a brush border membrane of numerous microvilli each comprised of a cross-linked core bundle of 15-20 actin filaments attached to the surrounding membrane by lateral cross-bridges; the cross-bridges are tilted with respect to the core bundle. Isolated microvillar cores contain actin (42 kD) and three other major proteins: fimbrin (68 kD), villin (95 kD), and the 110K-calmodulin complex. The addition of ATP to detergent-treated isolated microvillar cores has previously been shown to result in loss of the lateral cross-bridges and a corresponding decrease in the amount of the 110-kD polypeptide and calmodulin associated with the core bundle. This provided the first evidence to suggest that these lateral cross-bridges to the membrane are comprised at least in part by a 110-kD polypeptide complexed with calmodulin. We now demonstrate that purified 110K-calmodulin complex can be readded to ATP-treated, stripped microvillar cores. The resulting bundles display the same helical and periodic arrangement of lateral bridges as is found in vivo. In reconstitution experiments, actin filaments incubated in EGTA with purified fimbrin and villin form smooth-sided bundles containing an apparently random number of filaments. Upon addition of 110K-calmodulin complex, the bundles, as viewed by electron microscopy of negatively stained images, display along their entire length helically arranged projections with the same 33-nm repeat of the lateral cross-bridges found on microvilli in vivo; these bridges likewise tilt relative to the bundle. Thus, reconstitution of actin filaments with fimbrin, villin, and the 110K-calmodulin complex results in structures remarkably similar to native microvillar cores. These data provide direct proof that the 110K-calmodulin is the cross-bridge protein and indicate that actin filaments bundled by fimbrin and villin are of uniform polarity and lie in register. The arrangement of the cross-bridge arms on the bundle is determined by the structure of the core filaments as fixed by fimbrin and villin; a contribution from the membrane is not required.

scholarly journals A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

2006 ◽  
Vol 17 (4) ◽  
pp. 1971-1984 ◽  
Author(s):  
Michael G. Clark ◽  
Joseph Teply ◽  
Brian K. Haarer ◽  
Susan C. Viggiano ◽  
David Sept ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Actin Filaments ◽  
Random Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Actin Binding ◽  
Interacting Protein ◽  
Actin Binding Site

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal β-propeller and a secondary actin binding site lies in a comparable location on its C-terminal β-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding- and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N- and C-terminal propeller domains.

scholarly journals Forced expression of chimeric human fibroblast tropomyosin mutants affects cytokinesis.

1995 ◽  
Vol 129 (3) ◽  
pp. 697-708 ◽  
Author(s):  
K S Warren ◽  
J L Lin ◽  
J P McDermott ◽  
J J Lin
Keyword(s):  
Cho Cells ◽  
Actin Filaments ◽  
Human Fibroblasts ◽  
Fine Tuning ◽  
Actin Binding ◽  
Functional Consequences ◽  
Alternatively Spliced ◽  
Carboxy Terminal

Human fibroblasts generate at least eight tropomyosin (TM) isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsm alpha) from four distinct genes, and we have previously demonstrated that bacterially produced chimera hTM5/3 exhibits an unusually high affinity for actin filaments and a loss of the salt dependence typical for TM-actin binding (Novy, R.E., J. R. Sellers, L.-F. Liu, and J.J.-C. Lin, 1993. Cell Motil. & Cytoskeleton. 26: 248-261). To examine the functional consequences of expressing this mutant TM isoform in vivo, we have transfected CHO cells with the full-length cDNA for hTM5/3 and compared them to cells transfected with hTM3 and hTM5. Immunofluorescence microscopy reveals that stably transfected CHO cells incorporate force-expressed hTM3 and hTM5 into stress fibers with no significant effect on general cell morphology, microfilament organization or cytokinesis. In stable lines expressing hTM5/3, however, cell division is slow and sometimes incomplete. The doubling time and the incidence of multinucleate cells in the stable hTM5/3 lines roughly parallel expression levels. A closely related chimeric isoform hTM5/2, which differs only in the internal, alternatively spliced exon also produces defects in cytokinesis, suggesting that normal TM function may involve coordination between the amino and carboxy terminal regions. This coordination may be prevented in the chimeric mutants. As bacterially produced hTM5/3 and hTM5/2 can displace hTM3 and hTM5 from actin filaments in vitro, it is likely that CHO-expressed hTM5/3 and hTM5/2 can displace endogenous TMs to act dominantly in vivo. These results support a role for nonmuscle TM isoforms in the fine tuning of microfilament organization during cytokinesis. Additionally, we find that overexpression of TM does not stabilize endogenous microfilaments, rather, the hTM-expressing cells are actually more sensitive to cytochalasin B. This suggests that regulation of microfilament integrity in vivo requires stabilizing factors other than, or in addition to, TM.

scholarly journals The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex

2013 ◽  
Vol 202 (2) ◽  
pp. 251-260 ◽  
Author(s):  
Sara Solinet ◽  
Kazi Mahmud ◽  
Shannon F. Stewman ◽  
Khaled Ben El Kadhi ◽  
Barbara Decelle ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Metastatic Potential ◽  
Actin Binding ◽  
Cell Cortex ◽  
Shape Transformation ◽  
Erm Proteins ◽  
Cellular Processes ◽  
Anaphase Onset

Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM’s role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. In this paper, we show that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. We identified two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs that mediated the association with microtubules. This ERM–microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs.

scholarly journals Specific Interaction of the Actin-binding Domain of Dystrophin with Intermediate Filaments Containing Keratin 19

2005 ◽  
Vol 16 (9) ◽  
pp. 4280-4293 ◽  
Author(s):  
Michele R. Stone ◽  
Andrea O'Neill ◽  
Dawn Catino ◽  
Robert J. Bloch
Keyword(s):  
Striated Muscle ◽  
Specific Interaction ◽  
Binding Domain ◽  
Actin Binding ◽  
Glycoprotein Complex ◽  
Actin Binding Domain ◽  
Dystrophin Glycoprotein Complex ◽  
Related Proteins

Cytokeratins 8 and 19 concentrate at costameres of striated muscle and copurify with the dystrophin-glycoprotein complex, perhaps through the interaction of the cytokeratins with the actin-binding domain of dystrophin. We overexpressed dystrophin's actin-binding domain (Dys-ABD), K8 and K19, as well as closely related proteins, in COS-7 cells to assess the basis and specificity of their interaction. Dys-ABD alone associated with actin microfilaments. Expressed with K8 and K19, which form filaments, Dys-ABD associated preferentially with the cytokeratins. This interaction was specific, as the homologous ABD of βI-spectrin failed to interact with K8/K19 filaments, and Dys-ABD did not associate with desmin or K8/K18 filaments. Studies in COS-7 cells and in vitro showed that Dys-ABD binds directly and specifically to K19. Expressed in muscle fibers in vivo, K19 accumulated in the myoplasm in structures that contained dystrophin and spectrin and disrupted the organization of the sarcolemma. K8 incorporated into sarcomeres, with no effect on the sarcolemma. Our results show that dystrophin interacts through its ABD with K19 specifically and are consistent with the idea that cytokeratins associate with dystrophin at the sarcolemma of striated muscle.

Abstract 1464: Cardiac Myosin Binding Protein-C (cMyBP-C) Directly Modulates Actomyosin Binding and Kinetics Independent of LMM and S2 Binding.

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Walid Saber ◽  
Kelly J Begin ◽  
David M Warshaw ◽  
Peter VanBuren
Keyword(s):  
Actin Filament ◽  
Thin Filament ◽  
Motility Assay ◽  
Actin Binding ◽  
Maximal Velocity ◽  
In Vitro Motility Assay ◽  
Thin Filaments ◽  
Myosin Binding ◽  
Cross Bridges

BACKGROUND: While mutations in cMyBP-C constitute a common cause of FHC, its role in sarcomere contraction remains unclear. cMyBP-C binds to actin, titin and the S2 and LMM proteolytic domains of myosin. Through its numerous binding interactions cMyBP-C may act as a tether, restricting myosin and/or actomyosin function. We directly tested this hypothesis in the in vitro motility assay using either whole myosin or the myosin subfragments, HMM and S1 (which lack LMM and LMM-S2, respectively). METHODS AND RESULTS: The motility assay is an in vitro model of muscle contraction in which thin filaments are propelled across a myosin coated surface. The addition of cMyBP-C to the motility assay resulted in a concentration dependent reduction in actin filament velocity when using either whole myosin, HMM or S1, demonstrating that cMyBP-C inhibits thin filament velocity independent of LMM or S2 binding. Using whole myosin and thin filaments reconstituted with troponin/tropomyosin, the addition of cMyBP-C resulted in a 29% reduction in maximal velocity (P=0.002) with no effect on maximal force. At sub-maximal calcium, the pCa50 for velocity was increased (6.64 ± 0.06 vs. control, 6.44 ± 0.03, P=0.003) whereas the pCa50 for force was decreased (6.25 ± 0.09 vs. control, 6.55 ± 0.02, P=0.008). Thin filament activation by myosin strong-binding demonstrated an increased amount of myosin required to half maximally activate the thin filament in the presence of cMyBP-C, indicating that myosin binding to the thin filament is reduced with cMyBP-C. These findings were supported by co-sedimentation experiments which demonstrate that cMyBP-C competes with S1 for actin binding in the presence of ATP, with no effect on S1/actin binding in the absence of ATP. Finally, while the number of cross-bridges interacting with the thin filament is rate limiting for velocity at shorter filament lengths, this was not observed at longer filament lengths indicating that cMyBP- C directly modulates the kinetics of actomyosin. CONCLUSIONS: The effects of cMyBP-C on velocity and force demonstrate that cMyBP-C does not simply act as a tether but likely affects both the kinetics and the recruitment of myosin cross-bridges through its direct interaction with the myosin head and/or the actin filament.

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