Tropomodulin 3 binds to actin monomers

Regulation of the actin cytoskeleton by filament capping proteins is critical to myriad dynamic cellular functions. The ability of these proteins to bind both filaments as well as monomers is often central to their cellular functions. The ubiquitous pointed end capping protein Tmod3 (tropomodulin 3) acts as a negative regulator of cell migration, yet mechanisms behind its cellular functions are not understood. Analysis of Tmod3 effects on kinetics of actin polymerization and steady state monomer levels revealed that Tmod3, unlike previously characterized tropomodulins, sequesters actin monomers with an affinity similar to its affinity for capping pointed ends. Furthermore, Tmod3 is found bound to actin in high speed supernatant cytosolic extracts, suggesting that Tmod3 can bind to monomers in the context of other cytosolic monomer binding proteins. The Tmod3-actin complex can be efficiently cross-linked with 1-ethyl-3-(dimethylaminopropyl)carbodiimide/N-hydroxylsulfosuccinimide in a 1:1 complex. Subsequent tryptic digestion and liquid chromatography/tandem mass spectrometry revealed two binding interfaces on actin, one distinct from other actin monomer binding proteins, and two potential binding sites in Tmod3, which are independent of the previously characterized leucine-rich repeat structure involved in pointed end capping. These data suggest that the Tmod3 isoform may regulate actin dynamics differently in cells than the previously described tropomodulin isoforms.     

Tropomodulin caps the pointed ends of actin filaments

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.     

Bovine filensin possesses primary and secondary structure similarity to intermediate filament proteins

The cDNA coding for calf filensin, a membrane-associated protein of the lens fiber cells, has been cloned and sequenced. The predicted 755- amino acid-long open reading frame shows primary and secondary structure similarity to intermediate filament (IF) proteins. Filensin can be divided into an NH2-terminal domain (head) of 38 amino acids, a middle domain (rod) of 279 amino acids, and a COOH-terminal domain (tail) of 438 amino acids. The head domain contains a di- arginine/aromatic amino acid motif which is also found in the head domains of various intermediate filament proteins and includes a potential protein kinase A phosphorylation site. By multiple alignment to all known IF protein sequences, the filensin rod, which is the shortest among IF proteins, can be subdivided into three subdomains (coils 1a, 1b, and 2). A 29 amino acid truncation in the coil 2 region accounts for the smaller size of this domain. The filensin tail contains 6 1/2 tandem repeats which match analogous motifs of mammalian neurofilament M and H proteins. We suggest that filensin is a novel IF protein which does not conform to any of the previously described classes. Purified filensin fails to form regular filaments in vitro (Merdes, A., M. Brunkener, H. Horstmann, and S. D. Georgatos. 1991. J. Cell Biol. 115:397-410), probably due to the missing segment in the coil 2 region. Participation of filensin in a filamentous network in vivo may be facilitated by an assembly partner.     

APC binds intermediate filaments and is required for their reorganization during cell migration

Intermediate filaments (IFs) are components of the cytoskeleton involved in most cellular functions, including cell migration. Primary astrocytes mainly express glial fibrillary acidic protein, vimentin, and nestin, which are essential for migration. In a wound-induced migration assay, IFs reorganized to form a polarized network that was coextensive with microtubules in cell protrusions. We found that the tumor suppressor adenomatous polyposis coli (APC) was required for microtubule interaction with IFs and for microtubule-dependent rearrangements of IFs during astrocyte migration. We also show that loss or truncation of APC correlated with the disorganization of the IF network in glioma and carcinoma cells. In migrating astrocytes, vimentin-associated APC colocalized with microtubules. APC directly bound polymerized vimentin via its armadillo repeats. This binding domain promoted vimentin polymerization in vitro and contributed to the elongation of IFs along microtubules. These results point to APC as a crucial regulator of IF organization and confirm its fundamental role in the coordinated regulation of cytoskeletons.     

Assembly of intermediate filaments

The assembly of intermediate filaments is a fundamental property of the central rod domain of the individual subunit proteins. This rod domain, with its high propensity for α-helix formation, is the common and identifying feature of this family of proteins. Assembly occurs in vitro in the absence of other proteins or exogenous sources of energy; in vivo, it appears as if other factors, as yet poorly understood, modulate the assembly of intermediate filaments. Parallel, in-register dimers form via coiled-coil interactions of the rod domain. Tetramers may form from staggered arrays of parallel or antiparallel arrangements of dimers. Higher-order polymerization, which occurs spontaneously if the ionic strength of a mixture of dimers and tetramers is raised, proceeds rapidly through poorly described intermediates to the final 10 nm filament. This process is dependent on and modulated by the non-α-helical end domains, as well as those amino acids present at the very beginning and end of the rod domain. The interactions governing tetramer formation are most probably the same ones that are responsible for the lateral and longitudinal associations within intermediate filaments.     

Structure of intermediate filaments

Recent amino acid sequence data have revealed that the microfibrils in hard α-keratin contain proteins with highly significant homologies and closely similar structural characteristics to the intermediate filament (IF) proteins known as desmin and vimentin. This result implies that microfibrils in hard α-keratin may be classified as a member of the IF and that the major features of these various filamentous structures are the same. Consequently, data obtained using X-ray diffraction, electron microscopy, amino acid sequence structural analysis and physicochemical techniques have been collated from the hitherto diverse fields of keratin and IF structure and used to formulate a more detailed model for the 7–8 nm diameter filaments than has previously been possible. Two models consisting of four-chain units arranged with the helical symmetry deduced for hard α-keratin¹ (Fraser et al. J. Mol. Biol. 1976, 108, 435–452) are in accord with the data. The structural unit comprises an oppositely directed pair of molecules each consisting of a two-stranded parallel-chain coiled-coil rope of length ～45 nm stabilized by both interchain and intermolecular ionic interactions. For a perfectly regular structure the filament may be likened either to a seven-stranded cable with a supercoil pitch length of about 345 nm (pitch angle ～2.9°), or a ten-stranded cable (Fraser, R. D. B. and MacRae, T. P. Polymer 1973, 14, 61–67) with a supercoil pitch length of about 1293 nm (pitch angle ～0.8°). The models also provide some insight into the self-assembly mechanism of the IF.     

Myosin-Va binds to and mechanochemically couples microtubules to actin filaments

Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.     

Tropomodulin binds two tropomyosins: a novel model for actin filament capping

Tropomodulin, a tropomyosin-binding protein, caps the slow-growing (pointed) end of the actin filament regulating its dynamics. Tropomodulin, therefore, is important for determining cell morphology, cell movement, and muscle contraction. For the first time we show that one tropomodulin molecule simultaneously binds two tropomyosin molecules in a cooperative manner. On the basis of the tropomodulin solution structure and predicted secondary structure, we introduced a series of point mutations in regions important for tropomyosin binding and actin capping. Capping activity of these mutants was assayed by measuring actin polymerization using pyrene fluorescence. Using direct methods (circular dichroism and native gel electrophoresis) for detecting tropomodulin/tropomyosin binding, we localized the second tropomyosin-binding site to residues 109-144. Despite previous reports that the second binding site is for erythrocyte tropomyosin only, we found that both short nonmuscle and long muscle alpha-tropomyosins bind there as well, though with different affinities. We propose a model for actin capping where one tropomodulin molecule can bind to two tropomyosin molecules at the pointed end.     

Biomechanical properties of intermediate filaments: from tissues to single filaments and back

The animal cell cytoskeleton consists of three interconnected filament systems: actin-containing microfilaments (MFs), microtubules (MTs), and the lesser known intermediate filaments (IFs). All IF proteins share a common tripartite domain structure and the ability to assemble into 8-12 nm wide filaments. Electron microscopy data suggest that IFs are built according to a completely different plan from that of MFs and MTs. IFs are known to impart mechanical stability to cells and tissues but, until recently, the biomechanical properties of single IFs were unknown. However, with the discovery of naturally occurring micrometer-wide IF bundles and the development of new methodologies to mechanically probe single filaments, it is now possible to propose a more unified view of IF biomechanics. Unlike MFs and MTs, single IFs can now be described as flexible, extensible and tough, which has important implications for our understanding of cell and tissue mechanics. Furthermore, the molecular mechanisms at play when IFs are deformed point toward a pivotal role for them in mechanotransduction.     

GCP6 binds to intermediate filaments: a novel function of keratins in the organization of microtubules in epithelial cells

In simple epithelial cells, attachment of microtubule-organizing centers (MTOCs) to intermediate filaments (IFs) enables their localization to the apical domain. It is released by cyclin-dependent kinase (Cdk)1 phosphorylation. Here, we identified a component of the gamma-tubulin ring complex, gamma-tubulin complex protein (GCP)6, as a keratin partner in yeast two-hybrid assays. This was validated by binding in vitro of both purified full-length HIS-tagged GCP6 and a GCP6(1397-1819) fragment to keratins, and pull-down with native IFs. Keratin binding was blocked by Cdk1-mediated phosphorylation of GCP6. GCP6 was apical in normal enterocytes but diffuse in K8-null cells. GCP6 knockdown with short hairpin RNAs (shRNAs) in CACO-2 cells resulted in gamma-tubulin signal scattered throughout the cytoplasm, microtubules (MTs) in the perinuclear and basal regions, and microtubule-nucleating activity localized deep in the cytoplasm. Expression of a small fragment GCP6(1397-1513) that competes binding to keratins in vitro displaced gamma-tubulin from the cytoskeleton and resulted in depolarization of gamma-tubulin and changes in the distribution of microtubules and microtubule nucleation sites. Expression of a full-length S1397D mutant in the Cdk1 phosphorylation site delocalized centrosomes. We conclude that GCP6 participates in the attachment of MTOCs to IFs in epithelial cells and is among the factors that determine the peculiar architecture of microtubules in polarized epithelia.     

Molecular architecture of intermediate filaments

Together with microtubules and actin microfilaments, approximately 11 nm wide intermediate filaments (IFs) constitute the integrated, dynamic filament network present in the cytoplasm of metazoan cells. This network is critically involved in division, motility and other cellular processes. While the structures of microtubules and microfilaments are known in atomic detail, IF architecture is presently much less understood. The elementary 'building block' of IFs is a highly elongated, rod-like dimer based on an alpha-helical coiled-coil structure. Assembly of cytoplasmic IF proteins, such as vimentin, begins with a lateral association of dimers into tetramers and gradually into the so-called unit-length filaments (ULFs). Subsequently ULFs start to anneal longitudinally, ultimately yielding mature IFs after a compaction step. For nuclear lamins, however, assembly starts with a head-to-tail association of dimers. Recently, X-ray crystallographic data were obtained for several fragments of the vimentin dimer. Based on the dimer structure, molecular models of the tetramer and the entire filament are now a possibility.     

Apoptosis and keratin intermediate filaments

Intermediate filament (IF) proteins utilize central alpha-helical domains to generate polymeric fibers intermediate in size between actin microfilaments and microtubules. The regions flanking the central structural domains have diverged greatly to permit IF proteins to adopt specialized functions. Keratins represent the largest two groups of IF proteins. Most keratins serve structural functions in hair or epidermis. Intracellular epidermal keratins also provide strength to epithelial sheets. The intracellular type I keratins and other IF proteins are cleaved by caspases during apoptosis to ensure the disposal of the relatively insoluble cellular components. However, recent studies have also revealed an unexpected protective role for keratin 8 during TNF and Fas mediated apoptosis. Evidence for possible functions of keratins both upstream and downstream of apoptotic signaling are considered.     

An alternatively spliced exon links intermediate filaments to adhesions

Anchorage of the contractile actomyosin apparatus to the plasma membrane at discrete sites in muscle and non-muscle cells enables the transmission and conversion of force into work, such as muscle contraction and membrane deformation to regulate cell and tissue shape. Assembly, stabilization and turnover of adhesion sites are complex processes that involve structural components, a variety of signalling and adapter molecules, diverse kinases and phosphatases, and phospholipids. The dynamic turnover of adhesions also requires the frequent interaction with other filament systems of the cytoskeleton, in particular with microtubules. How the delivery and activation of all the required components is co-ordinated, however, remains to be fully understood. In the current issue of Biochemical Journal, Sun et al. provide evidence that a specific exon that is exclusively present in the alpha variant of the type IV intermediate filament protein synemin interacts directly with the focal adhesion protein vinculin in its active state. Interaction of adhesion components with intermediate filaments could serve as a general mechanism to regulate cell- and tissue-specific cytoskeleton-membrane attachment.     

Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin

Human erythrocytes contain a Mr 43,000 tropomyosin-binding protein that is unrelated to actin and that has been proposed to play a role in modulating the association of tropomyosin with spectrin-actin complexes based on its stoichiometry in the membrane skeleton of one Mr 43,000 monomer per short actin filament (Fowler, V. M. 1987. J. Biol. Chem. 262:12792-12800). Here, we describe an improved procedure to purify milligram quantities to 98% homogeneity and we show that this protein inhibits tropomyosin binding to actin by a novel mechanism. We have named this protein tropomodulin. Unlike other proteins that inhibit tropomyosin-actin interactions, tropomodulin itself does not bind to F-actin. EM of rotary-shadowed tropomodulin-tropomyosin complexes reveal that tropomodulin (14.5 +/- 2.4 nm [SD] in diameter) binds to one of the ends of the rod-like tropomyosin molecules (33 nm long). In agreement with this observation, Dixon plots of inhibition curves demonstrate that tropomodulin is a non-competitive inhibitor of tropomyosin binding to F-actin (Ki = 0.7 microM). Hill plots of the binding of the tropomodulin-tropomyosin complex to actin indicate that binding does not exhibit any positive cooperativity (n = 0.9), in contrast to tropomyosin (n = 1.9), and that the apparent affinity of the complex for actin is reduced 20-fold with respect to that of tropomyosin. These results suggest that binding of tropomodulin to tropomyosin may block the ability of tropomyosin to self-associate in a head-to-tail fashion along the actin filament, thereby weakening its binding to actin. Antibodies to tropomodulin cross-react strongly with striated muscle troponin I (but not with troponin T) as well as with a nontroponin Mr 43,000 polypeptide in muscle and in other nonerythroid cells and tissues, including brain, lens, neutrophils, and endothelial cells. Thus, erythrocyte tropomodulin may be one member of a family of tropomyosin-binding proteins that function to regulate tropomyosin-actin interactions in non-muscle cells and tissues.     

Nucleus-associated intermediate filaments from chicken erythrocytes

Chicken erythrocyte nuclei prepared by isolation in isotonic KCl and Nonidet P-40 detergent were found to contain numerous attached filaments with a mean diameter of 11.0 nm. In polypeptide content and solubility properties, they resembled the vimentin type of intermediate filament found in cells of mesenchymal origin. Examination of their association with the nucleus suggests that more than a simple membrane attachment is involved.