Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins

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.     

Studies of the alpha-actinin/actin interaction in the Z-disk by using calpain

Both mu- and m-calpain (the micro- and millimolar Ca(2+)-requiring Ca(2+)-dependent proteinases) can completely remove Z-disks from skeletal muscle myofibrils and leave a space devoid of filaments in the Z-disk area. alpha-Actinin, a principal protein component of Z-disks, is removed from myofibrils by the calpains, and a 100-kDa polypeptide that comigrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the alpha-actinin subunit is released into the supernatant. Purified calpain does not degrade purified actin or purified alpha-actinin as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N- and C-terminal amino acid analysis of calpain-treated and untreated alpha-actinin and actin. The 100-kDa polypeptide released from myofibrils by calpain elutes identically with native alpha-actinin off DEAE-cellulose and hydroxyapatite columns and, after purification, binds to pure F-actin in the same manner that untreated, native alpha-actinin binds. Calpain-released alpha-actinin also accelerates the rate of superprecipitation of reconstituted actomyosin, a sensitive property characteristic of native alpha-actinin. Consequently, the calpains release alpha-actinin from the Z-disk of myofibrils without degrading it or without altering its ability to bind to actin. These results indicate that alpha-actinin does not simply cross-link thin filaments across the Z-disk but that at least one additional protein (or perhaps an altered actin or alpha-actinin) is involved in the alpha-actinin/actin interaction in Z-disks.     

Simultaneous interaction of actin with alpha-actinin and calponin

Interaction of calponin and alpha-actinin with actin was analyzed by means of cosedimentation and electron microscopy. G-actin was polymerized in the presence of calponin, alpha-actinin, or both of these actin-binding proteins (ABPs). The single and bundled actin filaments were separated, and the stoichiometry of ABPs and actin in both types of filaments was determined. Binding of calponin to the single or bundled actin filaments was not dependent on the presence of alpha-actinin and did not displace alpha-actinin from actin. In the presence of calponin, however, less alpha-actinin was bound to the bundled actin filaments, and the binding of alpha-actinin was accompanied by a partial decrease in the calponin/actin stoichiometry in the bundles of actin filaments. Calponin had no influence on the binding of alpha-actinin to the single actin filaments. The structure of actin bundles formed in the presence of the two ABPs differed from that formed in the presence of either one singly. We conclude that calponin and alpha-actinin can coexist on actin and that nearly each actin monomer can bind one of these ABPs.     

Three-dimensional structure of a vertebrate muscle Z-band: implications for titin and alpha-actinin binding

The Z-band in vertebrate striated muscles, mainly comprising actin filaments, alpha-actinin, and titin, serves to organise the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation. Different Z-band thicknesses, formed from different numbers of zigzag crosslinking layers and found in different fibre types, are thought to be associated with the number of repetitive N-terminal sequence domains of titin. In order to understand myofibril formation it is necessary to correlate the ultrastructures and sequences of the actin filaments, titin, and alpha-actinin in characteristic Z-bands. Here electron micrographs of the intermediate width, basketweave Z-band of plaice fin muscle have been subject to a novel 3D reconstruction process. The reconstruction shows that antiparallel actin filaments overlap in the Z-band by about 22-25 nm. There are three levels of Z-links (probably alpha-actinin) in which at each level two nearly diametrically opposed links join an actin filament to two of its antiparallel neighbours. One set of links is centrally located in the Z-band and there are flanking levels orthogonal to this. A 3D model of the observed structure shows how Z-bands of different widths may be formed and it provides insights into the structural arrangements of titin and alpha-actinin in the Z-band. The model shows that the two observed symmetries in different Z-bands, c2 and p12(1), may be attributed respectively to whether the number of Z-link levels is odd or even.     

Phosphoinositide binding inhibits alpha-actinin bundling activity

alpha-Actinin is an abundant actin-bundling and adhesion protein that directly links actin filaments to integrin receptors. Previously, in platelet-derived growth factor-treated fibroblasts, we demonstrated that phosphoinositides bind to alpha-actinin, regulating its localization (Greenwood, J. A., Theibert, A. B., Prestwich, G. D., and Murphy-Ullrich, J. E. (2000) J. Cell Biol. 150, 627- 642). In this study, phosphoinositide binding and regulation of alpha-actinin function is further characterized. Phosphoinositide binding specificity, determined using a protein-lipid overlay procedure, suggests that alpha-actinin interacts with phosphates on the 4th and 5th position of the inositol head group. Binding assays and mutational analyses demonstrate that phosphoinositides bind to the calponin homology domain 2 of alpha-actinin. Phosphoinositide binding inhibited the bundling activity of alpha-actinin by blocking the interaction of the actin-binding domain with actin filaments. Consistent with these results, excessive bundling of actin filaments was observed in fibroblasts expressing an alpha-actinin mutant with decreased phosphoinositide affinity. We conclude that the interaction of alpha-actinin with phosphoinositides regulates actin stress fibers in the cell by controlling the extent to which microfilaments are bundled.     

Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins

When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the host's cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning regions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody staining of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit a number of focal adhesion proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli.     

Novel structures for alpha-actinin:F-actin interactions and their implications for actin-membrane attachment and tension sensing in the cytoskeleton

We have applied correspondence analysis to electron micrographs of 2-D rafts of F-actin cross-linked with alpha-actinin on a lipid monolayer to investigate alpha-actinin:F-actin binding and cross-linking. More than 8000 actin crossover repeats, each with one to five alpha-actinin molecules bound, were selected, aligned, and grouped to produce class averages of alpha-actinin cross-links with approximately 9-fold improvement in the stochastic signal-to-noise ratio. Measurements and comparative molecular models show variation in the distance separating actin-binding domains and the angle of the alpha-actinin cross-links. Rafts of F-actin and alpha-actinin formed predominantly polar 2-D arrays of actin filaments, with occasional insertion of filaments of opposite polarity. Unique to this study are the numbers of alpha-actinin molecules bound to successive crossovers on the same actin filament. These "monofilament"-bound alpha-actinin molecules may reflect a new mode of interaction for alpha-actinin, particularly in protein-dense actin-membrane attachments in focal adhesions. These results suggest that alpha-actinin is not simply a rigid spacer between actin filaments, but rather a flexible cross-linking, scaffolding, and anchoring protein. We suggest these properties of alpha-actinin may contribute to tension sensing in actin bundles.     

The Z-band: 85,000-dalton amorphin and alpha-actinin and their relation to structure

The conclusions arrived at as a result of this work can be summarized as follows: (a) We have found that there is an 85,000-dalton protein, which we have called 85K amorphin, associated with the Z-band of chicken pectoralis muscle myofibrils. We have isolated and purified this protein. It is not a structural component of the Z-filaments since it can be extracted completely without extraction of the Z-filaments. Extraction of 85K amorphin results in loss of specific staining of the Z-band with fluorescence specific anti-85K amorphin. (b) We have found that alpha-actinin is the structural component of the Z-filaments, since extraction of alpha-actinin is accompanied by loss of the Z- filament structure.     

An interaction between alpha-actinin and the beta 1 integrin subunit in vitro

A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1-integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha-actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha-actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.     

Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments.

Phosphatidylinositol 4,5-bisphosphate (PIP2) reorganizes actin filaments by modulating the functions of a variety of actin-regulatory proteins. Until now, it was thought that bound PIP2 is hydrolyzed only by tyrosine-phosphorylated phospholipase Cgamma (PLCgamma) after the activation of tyrosine kinases. Here, we show a new mechanism for the hydrolysis of bound PIP2 and the regulation of actin filaments by PIP2 phosphatase (synaptojanin). We isolated a 150-kDa protein (p150) from brains that binds the SH3 domains of Ash/Grb2. The sequence of this protein was found to be homologous to that of synaptojanin. The expression of p150 in COS 7 cells produces a decrease in the number of actin stress fibers in the center of the cells and causes the cells to become multinuclear. On the other hand, the expression of a PIP2 phosphatase-negative mutant does not disrupt actin stress fibers or produce the multinuclear phenotype. We have also shown that p150 forms the complexes with Ash/Grb2 and epidermal growth factor (EGF) receptors only when the cells are treated with EGF and that it reorganizes actin filaments in an EGF-dependent manner. Moreover, the PIP2 phosphatase activity of native p150 purified from bovine brains is not inhibited by profilin, cofilin, or alpha-actinin, although PLCdelta1 activity is markedly inhibited by these proteins. Furthermore, p150 suppresses actin gelation, which is induced by smooth muscle alpha-actinin. All these data suggest that p150 (synaptojanin) hydrolyzes PIP2 bound to actin regulatory proteins, resulting in the rearrangement of actin filaments downstream of tyrosine kinase and Ash/Grb2.     

In vitro cleavage of eIF4GI but not eIF4GII by HIV-1 protease and its effects on translation in the rabbit reticulocyte lysate system

The vertebrate muscle Z-band organizes and tethers antiparallel actin filaments in adjacent sarcomeres and hence propagates the tension generated by the actomyosin interaction during muscular contraction. The axial width of the Z-band varies with fibre and muscle type: fast twitch muscles have narrow (approximately 30-50 nm) Z-bands, while slow-twitch and cardiac muscles have wide (approximately 100-140 nm) Z-bands. In electron micrographs of longitudinal sections of fast fibres like those found in fish body white muscle, the Z-band appears as a characteristic zigzag layer of density connecting the mutually offset actin filament arrays in adjacent sarcomeres. Wide Z-bands in slow fibres such as the one studied here (bovine neck muscle) show a stack of three or four zigzag layers. The variable Z-band width incorporating variable numbers of zigzag layers presumably relates to the different mechanical properties of the respective muscles. Three-dimensional reconstructions of Z-bands reveal that individual zigzag layers are often composed of more than one set of protein bridges, called Z-links, probably alpha-actinin, between oppositely oriented actin filaments. Fast muscle Z-bands comprise two or three layers of Z-links. Here we have applied Fourier reconstruction methods to obtain clear three-dimensional density maps of the Z-bands in beef muscle. The bovine slow muscle investigated here reveals a Z-band comprising six sets of Z-links, which, due to their shape and the way their projected densities overlap, appear in longitudinal sections as either three or four zigzag layers, depending on the lattice view. There has been great interest recently in the suggestion that Z-band variability with fibre type may be due to differences in the repetitive region (tandem Z-repeats) in the Z-band part of titin (also called connectin). We discuss this in the context of our results and present a systematic classification of Z-band types according to the numbers of Z-links and titin Z-repeats.     

Bundling of actin filaments by alpha-actinin depends on its molecular length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.     

Muscle Z-band ultrastructure: titin Z-repeats and Z-band periodicities do not match

Vertebrate muscle Z-bands show zig-zag densities due to different sets of alpha-actinin cross-links between anti-parallel actin molecules. Their axial extent varies with muscle and fibre type: approximately 50 nm in fast and approximately 100 nm in cardiac and slow muscles, corresponding to the number of alpha-actinin cross-links present. Fish white (fast) muscle Z-bands have two sets of alpha-actinin links, mammalian slow muscle Z-bands have six. The modular structure of the approximately 3 MDa protein titin that spans from M-band to Z-band correlates with the axial structure of the sarcomere; it may form the template for myofibril assembly. The Z-band-located amino-terminal 80 kDa of titin includes 45 residue repeating modules (Z-repeats) that are expressed differentially; heart, slow and fast muscles have seven, four to six and two to four Z-repeats, respectively. Gautel et al. proposed a Z-band model in which each Z-repeat links to one level of alpha-actinin cross-links, requiring that the axial extent of a Z-repeat is the same as the axial separation of alpha-actinin layers, of which there are two in every actin crossover repeat. The span of a Z-repeat in vitro is estimated by Atkinson et al. to be 12 nm or less; much less than half the normal vertebrate muscle actin crossover length of 36 nm. Different actin-binding proteins can change this length; it is reduced markedly by cofilin binding, or can increase to 38.5 nm in the abnormally large nemaline myopathy Z-band. Here, we tested whether in normal vertebrate Z-bands there is a marked reduction in crossover repeat so that it matches twice the apparent Z-repeat length of 12 nm. We found that the measured periodicities in wide Z-bands in slow and cardiac muscles are all very similar, about 39 nm, just like the nemaline myopathy Z-bands. Hence, the 39 nm periodicity is an important conserved feature of Z-bands and either cannot be explained by titin Z-repeats as previously suggested or may correlate with two Z-repeats.     

Palladin is an actin cross-linking protein that uses immunoglobulin-like domains to bind filamentous actin

Palladin is a recently described phosphoprotein that plays an important role in cell adhesion and motility. Previous studies have shown that palladin overexpression results in profound changes in actin organization in cultured cells. Palladin binds to the actin-associated proteins alpha-actinin, vasodilator-stimulated phosphoprotein, profilin, Eps8, and ezrin, suggesting that it may affect actin organization indirectly. To determine its molecular function in generating actin arrays, we purified palladin and asked if it is also capable of binding to F-actin directly. In co-sedimentation and differential sedimentation assays, palladin was found to both bind and cross-link actin filaments. This bundling activity was confirmed by fluorescence and electron microscopy. Palladin fragments were then purified and used to determine the sequences necessary to bind and bundle F-actin. The Ig3 domain of palladin bound to F-actin, and a palladin fragment containing Ig3, Ig4, and the region linking these domains was identified as a fragment that was able to bundle F-actin. Because palladin has multiple Ig domains, and only one of them binds to F-actin, this suggests that different Ig domains may be specialized for distinct biological functions. In addition, our results suggest a potential role for palladin in generating specialized, actin-based cell morphologies via both direct actin cross-linking activity and indirect scaffolding activity.     

Rat heart glycogen phosphorylase II is genetically distinct from phosphorylase I

Previous studies in our laboratory have shown that rat heart glycogen phosphorylase (1,4-alpha-D-glucan: orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) separates into two forms upon ion-exchange chromatography. Both forms could be shown to have the same subunit Mr and to incorporate one molecule of phosphate per subunit. The studies reported here were done to check whether both forms are native isoenzymes and, further, which form might represent the heart-specific phosphorylase. Firstly, the iso-electric points of the purified enzymes are compared with those associated with phosphorylase activity in crude extracts from rat heart. Two out of four major bands coincided with the bands of purified phosphorylase Ib and IIb (isoelectric points: 5.5 and 6.25), indicating apparent identity. Secondly, antibodies to rat skeletal muscle phosphorylase reacted with rat heart phosphorylase I, whereas phosphorylase II was neither inhibited nor precipitated by the antibody. Thirdly, peptide maps obtained after proteolytic digestion of SDS-denatured phosphorylase I and II showed different patterns. In addition to the kinetic differences between these two forms reported earlier, phosphorylase IIa was inhibited by glucose 6-phosphate, whereas phosphorylase Ia was not. These results suggest that phosphorylase II is a heart-specific isoenzyme which is presumably encoded by a different gene.     

Interaction of alpha-actinin, filamin and tropomyosin with F-actin

The abilities of alpha-actinin, filamin and tropomyosin to bind F-actin were examined by cosedimentation experiments. Results indicated that smooth muscle alpha-actinin and filamin can bind to actin filaments simultaneously with little evidence of competition. In contrast, tropomyosin exhibits marked competition with either filamin or alpha-actinin for sites on actin filaments.     

Binding of Protein Kinase C Isoforms to Actin in Aplysia

Abstract: Recently, two of the 10 vertebrate protein kinase C (PKC) isoforms, PKCβII and PKCε, have been shown to bind specifically to actin filaments, suggesting that these kinases may regulate cytoskeletal dynamics. Here, we present evidence that two PKCs from the marine mollusk Aplysia californica , PKC Apl I, a Ca²⁺-activated PKC, and PKC Apl II, a Ca²⁺-independent PKC most similar to PKCε and η, also bind F-actin. First, they both cosedimented with purified actin filaments in a phorbol ester-dependent manner. Second, they both translocated to the Triton-insoluble fraction of the nervous system after phorbol ester treatment. PKC Apl II could also partially translocate to actin filaments and associate with the Triton-insoluble fraction in the absence of phorbol esters. Translocation to purified actin filaments was increased in the presence of a PKC inhibitor, suggesting that PKC phosphorylation reduces PKC bound to actin. Although both kinases bound F-actin, actin was not sufficient to activate the kinases. In support of a physiological role for actin-PKC interactions, immunochemical localization of PKC Apl II in neuronal growth cones revealed a striking colocalization with F-actin. Our results are consistent with a role for actin-PKC interactions in regulating cytoskeletal dynamics in Aplysia .     

Affinity of alpha-actinin for actin determines the structure and mechanical properties of actin filament gels.

Proteins that cross-link actin filaments can either form bundles of parallel filaments or isotropic networks of individual filaments. We have found that mixtures of actin filaments with alpha-actinin purified from either Acanthamoeba castellanii or chicken smooth muscle can form bundles or isotropic networks depending on their concentration. Low concentrations of alpha-actinin and actin filaments form networks indistinguishable in electron micrographs from gels of actin alone. Higher concentrations of alpha-actinin and actin filaments form bundles. The threshold for bundling depends on the affinity of the alpha-actinin for actin. The complex of Acanthamoeba alpha-actinin with actin filaments has a Kd of 4.7 microM and a bundling threshold of 0.1 microM; chicken smooth muscle has a Kd of 0.6 microM and a bundling threshold of 1 microM. The physical properties of isotropic networks of cross-linked actin filaments are very different from a gel of bundles: the network behaves like a solid because each actin filament is part of a single structure that encompasses all the filaments. Bundles of filaments behave more like a very viscous fluid because each bundle, while very long and stiff, can slip past other bundles. We have developed a computer model that predicts the bundling threshold based on four variables: the length of the actin filaments, the affinity of the alpha-actinin for actin, and the concentrations of actin and alpha-actinin.     

Dynamics of Z-band based proteins in developing skeletal muscle cells

During myofibril formation, Z-bodies, small complexes of alpha-actinin and associated proteins, grow in size, fuse and align to produce Z-bands. To determine if there were changes in protein dynamics during the assembly process, Fluorescence Recovery after Photobleaching was used to measure the exchange of Z-body and Z-band proteins with cytoplasmic pools in cultures of quail myotubes. Myotubes were transfected with plasmids encoding Yellow, Green, or Cyan Fluorescent Protein linked to the Z-band proteins: actin, alpha-actinin, cypher, FATZ, myotilin, and telethonin. Each Z-band protein showed a characteristic recovery rate and mobility. All except telethonin were localized in both Z-bodies and Z-bands. Proteins that were present both early in development in Z-bodies and later in Z-bands had faster exchange rates in Z-bodies. These results suggest that during myofibrillogenesis, molecular interactions develop between the Z-band proteins that decrease their mobility and increase the stability of the Z-bands. A truncated construct of alpha-actinin, which localized in Z-bands in myotubes and exhibited a very low rate of exchange, led to disruption of myofibrils, suggesting the importance of dynamic, intact alpha-actinin molecules for the formation and maintenance of Z-bands. Our experiments reveal the Z-band to be a much more dynamic structure than its appearance in electron micrographs of cross-striated muscle cells might suggest.