Formation of two-dimensional complexes of F-actin and crosslinking proteins on lipid monolayers: demonstration of unipolar alpha-actinin-F-actin crosslinking.

A method is described for forming two-dimensional (2-D) paracrystalline complexes of F-actin and bundling/gelation proteins on positively charged lipid monolayers. These arrays facilitate detailed structural studies of protein interactions with F-actin by eliminating superposition effects present in 3-D bundles. Bundles of F-actin have been produced using the glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase, the cytoskeletal protein erythrocyte adducin as well as smooth muscle alpha-actinin from chicken gizzard. All of the 2-D bundles formed contain F-actin with a 13/6 helical structure. F-actin-aldolase bundles have an interfilament spacing of 12.6 nm and a superlattice arrangement of actin filaments that can be explained by expression of a local twofold axis in the neighborhood of the aldolase. Well ordered F-actin-alpha-actinin 2-D bundles have an interfilament spacing of 36 nm and contain crosslinks 33 nm in length angled approximately 25-35 degrees to the filament axis. Images and optical diffraction patterns of these bundles suggest that they consist of parallel, unipolar arrays of actin filaments. This observation is consistent with an actin crosslinking function at adhesion plaques where actin filaments are bound to the cell membrane with uniform polarity.     

Structure of actin paracrystals induced by nerve growth factor

When nerve growth factor is added to F-actin, well-ordered bundles of filaments are formed. These bundles are observed even at low concentrations of NGF²¹, but when N-bromosuccinimide-treated NGF, a biologically inactive form of the protein is used, a much higher concentration is required to produce aggregation. Moreover, the bundles induced by the modified NGF are not very well ordered and show amorphous aggregates attached at various points.Electron microscopy of paracrystals induced by native NGF shows that, although they resemble pure actin paracrystals induced by Mg²⁺, the interfilament spacing is larger and bridges connect the filaments. Optical diffraction patterns show, in addition to the off-meridional reflections characteristic of the actin helix, meridional reflections on the first and fourth layer-lines, at axial spacings of 37 and 9 nm. Measurements of the axial positions of the layer-lines show that the actin helical symmetry is not significantly different from that in pure actin paracrystals. The presence of the meridional reflections indicates that groups of two or three bridges with spacing 9 nm or nearly 9 nm are arranged along the bundles at a repeating interval of 37 nm.Actin filament bundles have been observed in several non-muscle cells, and specific actin-binding proteins have been identified as responsible for this aggregation. Our in vitro observations show that the biologically active form of NGF interacts with actin and organizes it into well-ordered paracrystalline arrays. The in vitro formation of NGF-actin complexes may be related to the in vivo mechanism of action of this growth factor.     

An electron microscope study of the interaction between fructose diphosphate aldolase and actin-containing filaments

The interaction of fructose diphosphate aldolase with F-actin, F-actin- tropomyosin, and F-actin-tropomyosin-troponin has been studied by using negative staining. In the absence of troponin, minor aggregates of aldolase and the F-actin filaments are formed. A well-ordered lattice structure is only formed in the case of the fully reconstituted filament when the filament-to-filament spacing is 18nm, and the cross- bridge spacing is 38.7 nm. Evidence is presented that the lattice is due to an interaction between troponin and aldolase. The minimum subunit structure of troponin, still capable of giving rise to a lattice, is the troponin-IT complex, which indicates that troponin-C is not involved in aldolase binding.     

Electron microscopy study of the interaction of F-protein (phosphofructokinase) with actin-containing filaments

By the use of electron microscopy and optical diffraction the interaction of F-protein (phosphofructokinase) with F-actin and reconstructed thin filaments has been revealed. F-protein distributes itself along the filament bundles at regular intervals of 36-38 nm.     

Preferential binding of alpha-actinin to actin bundles.

At 37 degrees C, the alpha-actin-F-actin binding isotherm is anomalous. In 6.7% polyethylene glycol 6000, concomitantly with the formation of actin bundles, the binding isotherm becomes hyperbolic (Kdiss. = 11.3 microM). alpha-Actinin increases the rigidity of the networks formed by actin bundles in polyethylene glycol and by paracrystalline actin in 16 mM MgCl2 but not by F-actin. It is proposed that in the cell alpha-actinin functions are mostly carried on by interaction with actin bundles.     

Assemblies of psoriatic keratin and their relation to normal intermediate filament structures

Protein extracts from normal human epidermis reassemble in vitro into 8-10 nm diameter filaments characteristic of intermediate filaments, whereas extracts from psoriatic epidermal scales reassemble, under identical conditions, into a variety of paracrystalline bundles. Optical diffraction and image analysis of these paracrystalline bundles reveal an axial repeat of 16.5 nm, which subdivides into three bands of 5.5 nm, and a lateral spacing of 5.1 nm. This information, together with available sequence studies of intermediate filaments and biochemical data, suggests that the subunit of psoriatic keratin is made up essentially from the coiled-coil alpha-helical rod domain of the normal keratin subunits, whereas the random coil domains are missing or greatly reduced in size.     

Microtubule-associated proteins (MAPs) and the organization of actin filaments in vitro

When purified muscle actin was mixed with microtubule-associated proteins (MAPs) prepared from brain microtubules assembled in vitro, actin filaments were organized into discrete bundles, 26 nm in diameter. MAP-2 was the principal protein necessary for the formation of the bundles. Analysis of MAP-actin bundle formation by sedimentation and electrophoresis revealed the bundles to be composed of approximately 20% MAP-2 and 80% actin by weight. Transverse striations were observed to occur at 28-nm intervals along negatively stained MAP- actin bundles, and short projections, approximately 12 nm long and spaced at 28-nm intervals, were resolved by high-resolution metal shadowing. The formation of MAP-actin bundles was inhibited by millimolar concentrations of ATP, AMP-PCP (beta, gamma-methylene- adenosine triphosphate), and pyrophosphate but not by AMP, ADP, or GTP. The addition of ATP to a solution containing MAP-actin bundles resulted in the dissociation of the bundles into individual actin filaments; discrete particles, presumably MAP-2, were periodically attached along the splayed filaments. These results demonstrate that MAPs can bind to actin filaments and can induce the reversible formation of actin filament bundles in vitro.     

Increase in actin contents and elongation of apical projections in retinal pigmented epithelial cells during development of the chicken eye

The structural and biochemical changes of cytoskeletal components of retinal pigmented epithelial cells were studied during the development of chicken eyes. When the cytoskeletal components of the pigmented epithelial cells from various stages of development were examined by SDS PAGE, actin contents in the cells markedly increased between the 15-d-old and hatching stages. Immunofluorescence microscopy showed that chicken pigmented epithelial cells have two types of actin bundles. One is the circumferential bundle associated with the zonula adherens region as previously reported (Owaribe, K., and H. Masuda, 1982, J. Cell Biol., 95:310-315). The other is the paracrystalline bundle forming the core of the apical projections. The increase in actin contents after the 15-d-old stage is accompanied by the formation and elongation of core filaments of apical projections in the cells. During this period the apical projections extend into extracellular space among outer and inner segments of photoreceptor cells. Accompanying this change is an elongation of the paracrystalline bundles of actin filaments in the core of the projection. By electron microscopy, the bundles decorated with muscle heavy meromyosin showed unidirectional polarity, and had transverse striations with approximately 12-nm intervals, as determined by optical diffraction of electron micrographs. Since the shape of these bundles was not altered in the presence or absence of Ca2+, they seemed not to have villin-like proteins. Unlike the circumferential bundles, the paracrystalline bundles did not contract when exposed to Mg-ATP. These observations indicate that the paracrystalline bundles are structurally and functionally different from the circumferential actin bundles.     

Supramolecular forms of actin from amoebae of Dictyostelium discoideum.

Actin purified from amoebae of Dictyostelium discoideum polymerizes into filaments at 24 degrees upon addition of KCl, as judged by a change in optical density at 232 nm and by electron microscopy. The rate and extent of formation of this supramolecular assembly and the optimal KCl concentrations (0.1 M) for assembly are similar to those of striated muscle actin. The apparent equilibrium constant for the monomer-polymer transition is 1.3 muM for both Dictyostelium and muscle actin. Although assembly of highly purified Dictyostelium actin monomers into individual actin filaments resembles that of muscle actin, Dictyostelium actin but not muscle actin was observed to assemble into two-dimensional nets in 10 mM CaCl2. The Dictyostelium actin also forms filament bundles which are 0.1 mum in diameter and which assemble in the presence of 5 mM MgCl2. These bundles formed from partially purified Dictyostelium actin preparations but not from highly purified preparations, suggesting that their formation may depend on the presence of another component. These actin bundles reconstituted in vitro resemble the actin-containing bundles found in situ by microscopy in many non-muscle cells.     

Molecular architecture of the neurofilament. I. Subunit arrangement of neurofilament L protein in the intermediate-sized filament

Using the smallest subunit (NF-L) of a neurofilament and a glial fibrillary acidic protein, the subunit arrangement in intermediate filaments was studied by low-angle rotary shadowing. NF-L formed a pair of 70 to 80 nm rods in a low ionic strength solution at pH 6.8. Two 70 to 80 nm rods appeared to associate in an antiparallel manner with an overlap of about 55 nm, almost the same length as the alpha-helix-rich central rod domain of intermediate filament proteins. The overlap extended for three-beaded segments, present at 22 nm intervals along the pairs of rods. The observations that (1) 70 to 80 nm rods were a predominant structure in a low ionic strength solution at pH 8.5, (2) the molecular weights of the rod and the pair were measured by sedimentation equilibrium as 190,000 and 37,000 respectively, and (3) the rods formed from the trypsin-digested NF-L had a length of about 47 nm, indicated that the 70 to 80 nm rod is the four-chain complex and the pair of rods is the eight-chain complex. Similar structures were observed with glial fibrillary acidic protein, indicating that these oligomeric structures are common to other intermediate filament proteins. NF-L assembled into short intermediate-sized filaments upon dialysis against a low-salt solution containing 1 to 2 mM-MgCl2 at 4 degrees C. The majority of these short filaments possessed four or five-beaded segments, suggesting that the pair of rods were arranged in a half-staggered fashion in neurofilaments. On the basis of these observations, we propose the following model for the intermediate filament subunit arrangement. (1) The four-chain complex is the 70 to 80 nm rod, in which two coiled-coil molecules align in parallel and in register. (2) Two four-chain complexes form the eight-chain complex by associating in an antiparallel fashion with the overlap of the entire central rod domain. (3) The eight-chain complex is the building block of the intermediate filament. The eight-chain complexes are arranged in a half-staggered fashion within the intermediate filament.     

The actin filament severing protein actophorin promotes the formation of rigid bundles of actin filaments crosslinked with alpha-actinin

The actin filament severing protein, Acanthamoeba actophorin, decreases the viscosity of actin filaments, but increases the stiffness and viscosity of mixtures of actin filaments and the crosslinking protein alpha-actinin. The explanation of this paradox is that in the presence of both the severing protein and crosslinker the actin filaments aggregate into an interlocking meshwork of bundles large enough to be visualized by light microscopy. The size of these bundles depends on the size of the containing vessel. The actin filaments in these bundles are tightly packed in some areas while in others they are more disperse. The bundles form a continuous reticulum that fills the container, since the filaments from a particular bundle may interdigitate with filaments from other bundles at points where they intersect. The same phenomena are seen when rabbit muscle aldolase rather than alpha-actinin is used as the crosslinker. We propose that actophorin promotes bundling by shortening the actin filaments enough to allow them to rotate into positions favorable for lateral interactions with each other via alpha-actinin. The network of bundles is more rigid and less thixotropic than the corresponding network of single actin filaments linked by alpha-actinin. One explanation may be that alpha-actinin (or aldolase) normally in rapid equilibria with actin filaments may become trapped between the filaments increasing the effective concentration of the crosslinker.     

In vitro reconstitution of recombinant lamin A and a lamin A mutant lacking the carboxy-terminal tail

Xenopus lamin A and a lamin A mutant lacking the complete 280 amino acid long carboxy-terminal tail were expressed in bacteria and purified from inclusion bodies. Electron microscopic analysis of lamin A dimers revealed that the carboxy-terminal 280 amino acids correspond to the globular domain seen in rotary-shadowed wild-type lamin and that the rodlike domain consists of the short non-helical amino terminus and the alpha-helical region. During reconstitution lamin A dimers first formed polar head to tail aggregates which then associated laterally resulting in paracrystals with periodic repeats of 25 nm. In the mutant, the longitudinal and lateral association of dimers had not been influenced, however, periodic repeats were absent in the filament bundles formed. Thus our data clearly demonstrate that carboxy-terminal tails are localized in light-stained regions of negatively stained paracrystals and that they are responsible for the alternating light dark staining of paracrystals. Fibrils, 2 to 3 nm thick, were a common structural element of paracrystals and filament bundles.     

Interaction of aldolase with actin-containing filaments. Structural studies.

Electron micrographs of the paracrystals formed when fructose bisphosphate aldolase (EC 4.1.2.13) is added to actin-containing filaments were analysed by computer methods so that ultrastructural changes could be correlated with the various stoicheiometries of binding determined in the preceding paper [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98]. Paracrystals formed with aldolase and either F-actin or F-actin-tropomyosin have a single light transverse band every 38 nm, which is due to aldolase molecules cross-linking the filaments. In contrast, the paracrystals formed between aldolase and F-actin-tropomyosin-troponin filaments show two transverse bands every 38 nm: a major band, interpreted as aldolase binding to troponin, and a minor band, interpreted as aldolase cross-linking the filaments. The intensity of the minor band varies with Ca2+ concentration, being greatest when the Ca2+ concentration is low. A model for the different paracrystal structures which relates the various patterns and binding stoicheiometries to structural changes in the actin-containing filaments is proposed.     

Growth conditions control the size and order of actin bundles in vitro.

The bonding rules for actin filament bundles do not lead to a particular packing symmetry, but allow for either regular or disordered filament packing. Indeed, both hexagonal and disordered types of packing are observed in vivo. To investigate factors which control bundle order, as well as size, we examined the effect of protein concentration on the growth of actin-fascin bundles in vitro. We found that bundles require 4-8 d to achieve both maximum size and order. The largest and best ordered bundles were grown at low fascin and high actin concentrations (an initial fascin/actin ratio of 1:200). In contrast, a much larger number of poorly ordered bundles were formed at ratios of 1:25 and 1:50, and most surprisingly, no bundles were formed at 1:300 or 1:400. Based on these observations we propose a two-stage mechanism for bundle growth. The first stage is dominated by nucleation, which requires relatively high concentrations of fascin and which is therefore accompanied by rapid growth. Below some concentration threshold, nucleation ceases and bundles enter the second stage of slow growth, which continues until the supply of fascin is exhausted. By analogy with crystallization, we hypothesize that slower growth produces better order. We are able to use this mechanism to explain our observations as well as previous observations of bundle growth both in vitro and in vivo.     

Structure of helical RecA-DNA complexes. II. Local conformational changes visualized in bundles of RecA-ATP gamma S filaments

Complexes of RecA-DNA filaments, formed in the presence of a non-hydrolyzable ATP analog, ATP gamma S, aggregate together into regular bundles in the presence of Mg2+. Electron micrographs of several different forms of RecA-double-stranded DNA bundles have been analyzed: bundles of six supercoiled filaments at two different concentrations of Mg2+, and bundles of three supercoiled filaments at a single concentration of Mg2+. The bundles are all characterized by a regular left-handed supercoiling of the component filaments arising from the non-integral number of RecA subunits per turn of the RecA helix in these aggregates, about 6.15 units/turn. When single-stranded DNA is used instead of double-stranded DNA, regular aggregates composed of many filaments are formed. These aggregates do not supercoil, consistent with a symmetry of the component filaments of close to 6.0 units/turn. These different structures have provided a strong confirmation of the analysis of isolated RecA filaments. Since different RecA protomers within the component filaments of these aggregates are in different environments, they have provided a direct view of different conformations that RecA subunits may adopt within the same filament as a result of nonequivalent contacts. The conformational changes we have visualized are quite large, with apparent movements of mass over distances greater than 2 nm. The RecA-mediated strand exchange reaction is a highly dynamic process, which involves both the unwinding and stretching of DNA, in addition to the physical movement of DNA strands. It is quite likely, therefore, that the different conformations of RecA subunits seen in these aggregates represent different states of RecA during its enzymatic strand exchange activity.     

The Supramolecular Organization of the C. elegans Nuclear Lamin Filament

Nuclear lamins are involved in most nuclear activities and are essential for retaining the mechano-elastic properties of the nucleus. They are nuclear intermediate filament (IF) proteins forming a distinct meshwork-like layer adhering to the inner nuclear membrane, called the nuclear lamina. Here, we present for the first time, the three-dimensional supramolecular organization of lamin 10 nm filaments and paracrystalline fibres. We show that Caenorhabditis elegans nuclear lamin forms 10 nm IF-like filaments, which are distinct from their cytoplasmic counterparts. The IF-like lamin filaments are composed of three and four tetrameric protofilaments, each of which contains two partially staggered anti-parallel head-to-tail polymers. The beaded appearance of the lamin filaments stems from paired globular tail domains, which are spaced regularly, alternating between 21 nm and 27 nm. A mutation in an evolutionarily conserved residue that causes Hutchison-Gilford progeria syndrome in humans alters the supramolecular structure of the lamin filaments. On the basis of our structural analysis, we propose an assembly pathway that yields the observed 10 nm IF-like lamin filaments and paracrystalline fibres. These results serve also as a platform for understanding the effect of laminopathic mutations on lamin supramolecular organization.     

Structural polymorphism of the RecA protein from the thermophilic bacterium Thermus aquaticus.

The Escherichia coli RecA protein has served as a model for understanding protein-catalyzed homologous recombination, both in vitro and in vivo. Although RecA proteins have now been sequenced from over 60 different bacteria, almost all of our structural knowledge about RecA has come from studies of the E. coli protein. We have used electron microscopy and image analysis to examine three different structures formed by the RecA protein from the thermophilic bacterium Thermus aquaticus. This protein has previously been shown to catalyze an in vitro strand exchange reaction at an optimal temperature of about 60 degrees C. We show that the active filament formed by the T. aquaticus RecA on DNA in the presence of a nucleotide cofactor is extremely similar to the filament formed by the E. coli protein, including the extension of DNA to a 5.1-A rise per base pair within this filament. This parameter appears highly conserved through evolution, as it has been observed for the eukaryotic RecA analogs as well. We have also characterized bundles of filaments formed by the T. aquaticus RecA in the absence of both DNA and nucleotide cofactor, as well as hexameric rings of the protein formed under all conditions examined. The bundles display a very large plasticity of mass within the RecA filament, as well as showing a polymorphism in filament-filament contacts that may be important to understanding mutations that affect surface residues on the RecA filament.     

The blood cell: an expensive disappointment: Atlas of Human Hemopoietic Development. By E. Kelemen,W. Calvo and T. M. Fliedner. Berlin,Heidelberg, New York: Springer-Verlag, 266 pp. $198.00

We have utilized a cracking procedure followed by a rotary shadow technique to examine the ultrastructure of cultured ovarian granulosa cells. We have demonstrated that the structures observed are not artifacts of fixation or cracking by generating equivalent images following freezing and deep etching as well as by fixation prior to cracking. The cytoplasm of granulosa cells exhibits a complex cytoskeletal lattice composed of many 40–55 nm filaments. This filamentous network is continuous with the plasma membrane and appears to incorporate all formed elements within the cytoplasm. Filaments are organized in three ways: first, in large bundles, second, in individual filaments that are in direct association with organelles, and third, in a complex branching and anastomosing configuration. S-1 decoration revealed that the predominant filament species is actin.     

Intermediate filament proteins in nonfilamentous structures: Transient disintegration and inclusion of subunit proteins in granular aggregates

The intermediate filament cytoskeleton of cultured bovine kidney epithelial cells and human HeLa cells changes dramatically during mitosis. The bundles of cytokeratin and vimentin filaments progressively unravel into protofilament-like threads of 2–4 nm diameter, and intermediate filament protein is included in numerous, variously sized (2–15 μm) spheroidal aggregates containing densely stained granular particles of 5–16 nm diameter. We describe these mitotic bodies in intact cells and in isolated cytoskeletons. In metaphase to anaphase of normal mitosis and after colcemid arrest of mitotic stages, many cells contain all their detectable cytokeratin and vimentin material in the form of such spheroidal aggregate bodies, whereas in other mitotic cells such bodies occur simultaneously with bundles of residual intermediate filaments. In telophase, the extended normal arrays of intermediate filament bundles are gradually reestablished. We find that vimentin and cytokeratins can be organized in structures other than intermediate filaments. Thus, at least during mitosis of some cell types, factors occur that promote unraveling of intermediate filaments into protofilament-like threads and organization of intermediate filament proteins into distinct granules that form large aggregate bodies. Some cells, at least certain epithelial and carcinoma cells, may contain factors effective in structural modulation and reorganization of intermediate filaments.     

Polymeric Structures and Dynamic Properties of the Bacterial Actin AlfA

AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 Å, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 Å. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5′-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure small angle X-ray scattering experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes.