Tropomodulin assembles early in myofibrillogenesis in chick skeletal muscle: evidence that thin filaments rearrange to form striated myofibrils

1999 ◽  
Vol 112 (8) ◽  
pp. 1111-1123 ◽  
Author(s):  
A. Almenar-Queralt ◽  
C.C. Gregorio ◽  
V.M. Fowler
Keyword(s):  
Skeletal Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Primary Cultures ◽  
General Mechanism ◽  
Thin Filaments ◽  
Phalloidin Staining ◽  
Capping Protein ◽  
Filament Bundles ◽  
End Capping

Actin filament lengths in muscle and nonmuscle cells are believed to depend on the regulated activity of capping proteins at both the fast growing (barbed) and slow growing (pointed) filament ends. In striated muscle, the pointed end capping protein, tropomodulin, has been shown to maintain the lengths of thin filaments in mature myofibrils. To determine whether tropomodulin might also be involved in thin filament assembly, we investigated the assembly of tropomodulin into myofibrils during differentiation of primary cultures of chick skeletal muscle cells. Our results show that tropomodulin is expressed early in differentiation and is associated with the earliest premyofibrils which contain overlapping and misaligned actin filaments. In addition, tropomodulin can be found in actin filament bundles at the distal tips of growing myotubes, where sarcomeric alpha-actinin is not always detected, suggesting that tropomodulin caps actin filament pointed ends even before the filaments are cross-linked into Z bodies by alpha-actinin. Tropomodulin staining exhibits an irregular punctate pattern along the length of premyofibrils that demonstrate a smooth phalloidin staining pattern for F-actin. Strikingly, the tropomodulin dots often appear to be located between the closely spaced, dot-like Z bodies that are stained for (α)-actinin. Thus, in the earliest premyofibrils, the pointed ends of the thin filaments are clustered and partially aligned with respect to the Z bodies (the location of the barbed filament ends). At later stages of differentiation, the tropomodulin dots become aligned into regular periodic striations concurrently with the appearance of striated phalloidin staining for F-actin and alignment of Z bodies into Z lines. Tropomodulin, together with the barbed end capping protein, CapZ, may function from the earliest stages of myofibrillogenesis to restrict the lengths of newly assembled thin filaments by capping their ends; thus, transitions from nonstriated to striated myofibrils in skeletal muscle are likely due principally to filament rearrangements rather than to filament polymerization or depolymerization. Rearrangements of actin filaments capped at their pointed and barbed ends may be a general mechanism by which cells restructure their actin cytoskeletal networks during cell growth and differentiation.

scholarly journals Tropomodulin caps the pointed ends of actin filaments.

1994 ◽  
Vol 127 (6) ◽  
pp. 1627-1635 ◽  
Author(s):  
A Weber ◽  
C R Pennise ◽  
G G Babcock ◽  
V M Fowler
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Critical Concentration ◽  
Thin Filaments ◽  
Capping Protein ◽  
End Capping ◽  
Pointed End ◽  
A Site

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.

scholarly journals F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments.

1996 ◽  
Vol 135 (5) ◽  
pp. 1291-1308 ◽  
Author(s):  
L G Tilney ◽  
P Connelly ◽  
S Smith ◽  
G M Guild
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Modular Design ◽  
Thin Sections ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Phalloidin Staining ◽  
Filament Bundles ◽  
End To End ◽  
As If

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.

The capactins, a class of proteins that cap the ends of actin filaments

1982 ◽  
Vol 299 (1095) ◽  
pp. 263-273 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Preliminary Evidence ◽  
The Other ◽  
Cellular Regulation ◽  
Filament Assembly ◽  
Filament Bundles

A number of proteins that bind specifically to the barbed ends of actin filaments in a cytochalasin-like manner have been purified to various degrees from a variety of muscle and non-muscle cells and tissues. Preliminary evidence also indicates that proteins that interact with the pointed ends of filaments are present in skeletal muscle. Because of their ability to cap one or the other end of an actin filament, we have designated this class of proteins as the ‘capactins’. On the basis of their effect on actin filament assembly and interaction in vitro , we propose that the capactins play important roles in cellular regulation of actin-based cytoskeletal and contractile functions. Our finding that the disappearance of actin filament bundles in virally transformed fibroblasts can be correlated with an increase in capactin activity in the extracts of these cells is consistent with this hypothesis.

scholarly journals Non Diaphanous Formin Delphilin Acts as a Barbed End Capping Protein

2016 ◽  
Author(s):  
Priyanka Dutta ◽  
Sankar Maiti
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Pdz Domains ◽  
Capping Protein ◽  
C Terminus ◽  
End Capping

ABSTRACTFormins are important for actin polymerization. Delphilin is a unique formin having PDZ domains and FH1, FH2 domains at its N and C terminus respectively. In this study we observed that Delphilin binds to actin filaments, and have negligible actin filament polymerizing activity. Delphilin inhibits actin filament elongation like barbed end capping protein CapZ. In vitro, Delphilin stabilized actin filaments by inhibiting actin filament depolymerisation. Therefore, our study demonstrates Delphilin as an actin-filament capping protein.

scholarly journals Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics

2002 ◽  
Vol 156 (6) ◽  
pp. 1065-1076 ◽  
Author(s):  
Shoichiro Ono ◽  
Kanako Ono
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Biological Properties ◽  
Actin Dynamics ◽  
Background Suppression ◽  
Thin Filaments ◽  
Actin Organization ◽  
C Elegans ◽  
Filament Dynamics

Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B–induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.

scholarly journals αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

2013 ◽  
Vol 24 (23) ◽  
pp. 3710-3720 ◽  
Author(s):  
Scott D. Hansen ◽  
Adam V. Kwiatkowski ◽  
Chung-Yueh Ouyang ◽  
HongJun Liu ◽  
Sabine Pokutta ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Binding Domain ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Filament Structure ◽  
Actin Binding Domain ◽  
Filament Bundles ◽  
Underlying Mechanisms ◽  
Cell Cell

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.

scholarly journals Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein.

1996 ◽  
Vol 134 (2) ◽  
pp. 389-399 ◽  
Author(s):  
K Barkalow ◽  
W Witke ◽  
D J Kwiatkowski ◽  
J H Hartwig
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Actin Polymerization ◽  
Residual Activity ◽  
Human Platelets ◽  
Actin Assembly ◽  
Capping Protein ◽  
Capping Proteins ◽  
End Capping ◽  
Human And Mouse

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F-actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.

Characterization of the actin filament capping state in human erythrocyte ghost and cytoskeletal preparations

2000 ◽  
Vol 349 (1) ◽  
pp. 105-111 ◽  
Author(s):  
Philip A. KUHLMAN
Keyword(s):  
Actin Filament ◽  
Human Erythrocyte ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Length Distribution ◽  
Shearing Stress ◽  
Bivalent Cation ◽  
Thin Filaments ◽  
Cation Concentration ◽  
Bivalent Cations

The narrow Gaussian-length-distribution of actin filaments forming the cytoskeleton of the human erythrocyte indicates the existence of strict mechanisms for length determination and maintenance. A similar regulation is achieved in striated muscle by the capping of both the ends of the thin filaments, which consequently prevents monomer exchange. However, the ability of erythroid cytoskeletal preparations to nucleate actin polymerization has led to the proliferation of the idea that at least the barbed ends of the actin filaments are uncapped. The mechanism by which the length of the filaments is thus maintained has been left open to debate. In an effort to resolve any doubt regarding length-maintenance in human erythrocytes we have characterized the capping state of the actin filaments in a number of different ghost and cytoskeletal preparations. Under conditions of sufficiently high bivalent-cation concentration the actin filaments retain functional caps at both the barbed and pointed ends. Hence filament capping at both ends prevents redistribution of the actin monomer in a similar manner to that proposed for the thin filaments of striated muscle. Actin filament uncapping is apparently caused by the centrifugal shearing stress imposed during ghost preparation. The uncapping is more pronounced when the bivalent-cation concentration is reduced or when the membrane is removed by detergents. The effects of bivalent cations seem to be mediated through the erythroid protein spectrin, consistent with the hypothesis of Wallis et al. [Wallis, Babitch and Wenegieme (1993) Biochemistry 32, 5045-5050] that the ability of spectrin to resist shearing stress is dependent on the degree of bound bivalent cations.

Genomic organization of mouse and human erythrocyte tropomodulin genes encoding the pointed end capping protein for the actin filaments

Gene ◽  
2000 ◽  
Vol 256 (1-2) ◽  
pp. 271-281 ◽  
Author(s):  
Xin Chu ◽  
Douglas Thompson ◽  
Leland J. Yee ◽  
Lanping Amy Sung
Keyword(s):  
Human Erythrocyte ◽  
Actin Filaments ◽  
Genomic Organization ◽  
Capping Protein ◽  
Genes Encoding ◽  
End Capping ◽  
Pointed End

scholarly journals Regulation of the acto·myosin subfragment 1 interaction by troponin/tropomyosin. Evidence for control of a specific isomerization between two acto·myosin subfragment 1 states

1991 ◽  
Vol 279 (3) ◽  
pp. 711-718 ◽  
Author(s):  
D F A McKillop ◽  
M A Geeves
Keyword(s):  
Skeletal Muscle ◽  
Active Site ◽  
Actin Filaments ◽  
Thin Filaments ◽  
Closed State ◽  
Myosin Subfragment ◽  
Actomyosin Interaction ◽  
Binding Isotherms ◽  
Myosin Subfragment 1 ◽  
Site Binding

The co-operative binding of myosin subfragment 1 (S1) to reconstituted skeletal-muscle thin filaments has been examined by monitoring the fluorescence of a pyrene probe on Cys-374 of actin. The degree of co-operativity differs when phosphate, sulphate or ADP are bound to the S1 active site. Binding isotherms have been analysed according to the Geeves & Halsall [(1987) Biophys. J. 52, 215-220] model, which proposed that troponin and tropomyosin effected regulation of the actomyosin interaction by controlling an isomerization of the actomyosin complex. The data support the proposal that seven actin monomers associated with a single tropomyosin molecule act as a co-operative unit that can be in one of two states. In the ‘closed’ state myosin can bind to actin, but the subsequent isomerization is prevented. The isomerization is only allowed after the seven-actin unit is in the ‘open’ form. Ca2+ controls the proportion of actin filaments in the ‘closed’ and ‘open’ forms in the absence of myosin heads. The ratio of ‘closed’ to ‘open’ forms is approx. 50:1 in the absence of Ca2+ and 5:1 in its presence.

Sign in / Sign up

Export Citation Format

Share Document