How actin filaments and microtubules steer growth cones to their targets

Guidance molecules steer growth cones to their targets by attracting or repelling them. Turning in a new direction requires remodeling of the growth cone and bending of the axon. This depends upon reorganization of actin filaments and microtubules, which are the primary cytoskeletal components of growth cones. This article discusses how these cytoskeletal components induce turning. The importance of each component as well as how interactions between them result in axon guidance is discussed. Current evidence shows that microtubules are influenced by both the organization and dynamics of actin filaments in the peripheral domain of growth cones. Cytoskeletal models for repulsive and attractive turning are presented. Molecular candidates that may link actin filaments with microtubules are suggested and potential signal transduction pathways that allow these cytoskeletal components to affect each other are discussed.     

Fine structural study of interstitial cells associated with the deep muscular plexus of the rat small intestine,with special reference to the intestinal pacemaker cells

Two types of interstitial cells have been demonstrated in close association in the deep muscular plexus of rat small intestine, by electron microscopy. Cells of the first type are characterized by a fibroblastic ultrastructure, i.e. a well-developed granular endoplasmic reticulum, Golgi apparatus and absence of the basal lamina. They form a few small gap junctions with the circular muscle cells and show close contact with axon terminals containing many synaptic vesicles. They may play a role in conducting electrical signals in the muscle tissue. Cells of the second type are characterized by many large gap junctions that interconnect with each other and with the circular muscle cells. Their cytoplasm is rich in cell organells, including mitochondria, granular endoplasmic reticulum and Golgi apparatus. They show some resemblance to the smooth muscle cells and have an incomplete basal lamina, caveolae and subsurface cisterns. However, they do not contain an organized contractile apparatus, although many intermediate filaments are present in their processes. They also show close contacts with axon terminals containing synaptic vesicles. These gap-junction-rich cells may be regular components of the intestinal tract and may be involved in the pacemaking activity of intestinal movement.     

Interaction of C-protein with myosin

The effect of C-protein on the assembly reaction of myosin was studied by flow birefringence, electron microscopy, and ultracentrifugation. Myosin filaments were formed by dilution to a lower ionic strength. Thinner filaments of 70-110 A in diameter were formed in the presence of C-protein. When dilution was effected by moderately slow dilution (dilution time of 0.5-2 min) or by stepwise dilution, C-protein favored the formation of longer filaments. When dilution was effected by even slower dilution (dilution time above 2 min), C-protein favored the formation of shorter filaments. Longer filaments formed by slow dilution incorporated more C-protein than shorter ones formed by faster dilution. Addition of C-protein to a solution of myosin filaments caused association of the filaments into longer filaments. The elongation effect was slower and stronger for longer filaments.     

Cross-linker system between neurofilaments, microtubules and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method

The elaborate cross-connections among membranous organelles (MO), microtubules (MT), and neurofilaments (NF) were demonstrated in unifixed axons by the quick-freeze, deep-etch, and rotary-shadowing method. They were categorized into three groups: NF-associated cross-linker, MT-associated cross-bridges, and long cross-links in the subaxolemmal space. Other methods were also employed to make sure that the observed cross-connections in the unfixed axons were not a result of artifactual condensation or precipitation of soluble components or salt during deep-etching. Axolemma were permeablized either chemically (0.1% saponin) or physically (gentle homogenization), to allow egress of their soluble components from the axon; or else the axons were washed with distilled water after fixation. After physical rupture of the axolemma or saponin treatment, most of the MO remained intact. MT were stabilized by adding taxol in the incubation medium. Axons prepared by these methods contained many longitudinally oriented NF connected to each other by numerous fine cross-linkers (4-6 nm in diameter, 20-50 nm in length). Two specialized regions were apparent within the axons: one composed of fascicles of MT linked with each other by fine cross-bridges; the other was in the subaxolemmal space and consisted of actinlike filaments and a network of long cross-links (50-150 nm) which connected axolemma and actinlike filaments with NF and MT. F-actin was localized to the subaxolemmal space by the nitrobenzooxadiazol phallacidin method. MO were located mainly in these two specialized regions and were intimately associated with MT via fine short (10-20 nm in length) cross-bridges. Cross-links from NF to MO and MT were also common. All these cross-connections were observed after chemical extraction or physical rupture of the axon; however, these procedures removed granular materials which were attached to the filaments in the fresh unextracted axons. The cross-connections were also found in the axons washed with distilled water after fixation. I conclude that the cross- connections are real structures while the granular material is composed of soluble material, probably protein in nature.     

Thin (actin) and thick (myosinlike) filaments in cone contraction in the teleost retina

The long slender retinal cones of fishes shorten in the light and elongate in the dark. Light-induced cone shortening provides a useful model for stuying nonmuscle contraction because it is linear, slow, and repetitive. Cone cells contain both thin (actin) and thick (myosinlike) filaments oriented parallel to the axis of contraction. This study examines the polarities of the cone's thin filaments and the changes in filament distribution which accompany light-induced contraction, in an attempt to elucidate the structural basis for the cone's contractile process. The proximal half of the cone is fixed to its cellular neighbors in the outer nuclear layer while the distal half is free. Thus, all shortening takes place in a necklike region (the myoid) in the distal half of the cone which extends into the space between the neural retina and the pigmented retinal epithelium. Thin filaments are found throughout the length of the cone, whereas thick filaments occur predominantly in the proximal (axon) regions of both light- and dark-adapted cones. Thus, thick filaments are primarily localized outside the region where shortening takes place. Observations from myosin subfragment-1 binding studies suggest that the cone's thin filaments are organized into two opposing sets. In the distal half of the cone (including the myoid), virtually all filaments have proximally directed arrowheads. In the more proximal regions of the axon, many thin filaments have opposite polarity, their arrowheads being distally directed. Near the synaptic proximal end of the light-adapted (contracted) cone, filaments of opposite polarities occur in approximately equal numbers. Thus, in the cone axon there appear to be two overlapping sets of actin filaments whose opposite polarities correspond to the two actin halves of a muscle sarcomere. In elongated, dark-adapted cones, thick filaments are localized throughout the axon region of the cone. In light, thick filaments accumulate towards the proximal end of the cone. These observations are consistent with a "sliding hypothesis" for cone contraction, in which thick myosinlike filaments produce sliding interdigitation of the two sets of oppositely directed actin filaments in the proximal axon region. Thus, the myoid thin filaments would be essentially reeled into the axon region to produce shortening. The mechanism of re-elongation depends on microtubules, as discussed in the companion paper.     

Structure and function of chicken gizzard myosin.

In our previous study (Onishi, H., Susuki, H., Nakamura, k., and Watanabe, S. J. Biochem. 83, 835-847, 1978), we found it to be characteristic of chicken gizzard myosin that thick filaments of gizzard myosin are readily disassembled by a stoichiometric amount of ATP (3 mol of ATP per mol of myosin), and that the ATPase activity of gizzard myosin in the ATP-disassembled state is much lower than that of gizzard myosin disassembled by a high concentration of KCl. We now report the following findings: (1) Thick filaments of (unphosphorylated) gizzard myosin can be in a bipolar structure or in a non-polar structure, depending on the method of preparing the thick filaments. (2) Thick filaments of (unphosphorylated) gizzard myosin in either the bioplar or the non-polar structure are readily disassembled by ATP. (3) Addition of rabbit skeletal C-protein does not confer ATP resistance on thick filaments of (unphosphorylated) gizzard myosin. (4) Unphosphorylated) gizzard myosin in the ATP-disassembled state is in a dimeric form as determined by ultracentrifugation. Moreover, 0.2 M KCl-dissociated gizzard myosin in monomeric form is converted to a dimeric form by ATP. (5) The Mg-ATPase activity of (unphosphorylated) gizzard myosin is much lower in its dimeric form (less than one-tenth) than in its monomeric form. The activity depression observed around 0.15 M KCl is therefore due to the formation of myosin dimers. (6) Skeletal L-meromyosin can increase the very low activity of (unphosphorylated) gizzard myosin ATPase at low ionic strength (0.13 M KCl) by forming ATP-resistant hybrid filaments with (unphosphorylated) gizzard myosin, preventing the formation of myosin dimers. (7) Gizzard myosin in which one of the light-chain components is phosphorylated by myosin light-chain kinase can form thick filaments which are resistant to the disassembling action of ATP. (8) Even in the presence of ATP, thick filaments of phosphorylated gizzard myosin do not disassembled into myosin dimers. Accordingly, the ATPase activity of phosphorylated gizzard myosin does not show activity depression at low ionic strength.     

Isolation, ultrastructure, and partial characterization of collagen from the perineurium of the Florida lobster, Panulirus argus.

Highly concentrated extracellular filaments in the perineurium of the Florida spiny lobster, Panulirus argus, were isolated using ultracentrifugation and linear sucrose gradients. The pellet obtained was highly enriched for the filaments as observed by transmission electron microscopy. Fibril diameter and axial periodicity measurements were obtained from filaments positively and negatively stained with uranyl acetate. A period between 14.0 and 25.0 nm and an average fibril diameter of 15.0 nm were observed. The filaments proved resistant to solubilization by most conventional agents and by several collagenases. NaOH (0.1 M at 100 degrees C) safely dissolved the filaments for measurements of protein content by the Lowry method and carbohydrate content with anthrone reagent. These tests revealed a protein content of approximately 84% and a high carbohydrate content of approximately 15%. Polyacrylamide electrophoresis of an acid-pepsin filament extract revealed a highly concentrated band (approximately 100,000) corresponding to the alpha-1 and alpha-2 bands of vertebrate type I collagen. Wide angle X-ray diffraction yielded meridional reflections that confirmed the filaments as collagen when compared with mammalian collagen X-ray diffraction. The amino acid composition was determined with a computer-assisted Beckman amino acid analyzer, which showed a glycine content of 279 residues/1000. Hydroxylysine and hydroxyproline were present in lower concentrations than expected.     

beta-Spectrin functions independently of Ankyrin to regulate the establishment and maintenance of axon connections in the Drosophila embryonic CNS

alpha- and beta-Spectrin are major components of a submembrane cytoskeletal network connecting actin filaments to integral plasma membrane proteins. Besides its structural role in red blood cells, the Spectrin network is thought to function in non-erythroid cells during protein targeting and membrane domain formation. Here, we demonstrate that beta-Spectrin is required in neurons for proper midline axon guidance in the Drosophila embryonic CNS. In beta-spectrin mutants many axons inappropriately cross the CNS midline, suggesting a role for beta-Spectrin in midline repulsion. Surprisingly, neither the Ankyrin-binding nor the pleckstrin homology (PH) domains of beta-Spectrin are required for accurate guidance decisions. alpha-Spectrin is dependent upon beta-Spectrin for its normal subcellular localization and/or maintenance, whereas alpha-spectrin mutants exhibit a redistribution of beta-Spectrin to the axon scaffold. beta-spectrin mutants show specific dose-dependent genetic interactions with the midline repellent slit and its neuronal receptor roundabout (robo), but not with other guidance molecules. The results suggest that beta-Spectrin contributes to midline repulsion through the regulation of Slit-Robo pathway components. We propose that the Spectrin network is playing a role independently of Ankyrin in the establishment and/or maintenance of specialized membrane domains containing guidance molecules that ensure the fidelity of axon repulsion at the midline.     

Cytoplasmic actin in neuronal processes as a possible mediator of synaptic plasticity

We have demonstrated that, after permeation with saponin and decoration with S-1 myosin subfragment, the cytoplasmic actin is organized in filaments in dendritic spines, dendrites, and axon terminals of the dentate molecular layer. The filaments are associated with the plasma membrane and the postsynaptic density with their barbed ends and also in parallel with periodical cross bridges. In the spine stalks and dendrites, the actin filaments are organized in long strands. Given the contractile properties of actin, these results suggest that the cytoplasmic actin may be involved in various forms of experimentally induced synaptic plasticity by changing the shape or volume of the pre- and postsynaptic side and by retracting and sprouting synapses.     

Tomographic reconstruction of treponemal cytoplasmic filaments reveals novel bridging and anchoring components

An understanding of the involvement of bacterial cytoplasmic filaments in cell division requires the elucidation of the structural organization of those filamentous structures. Treponemal cytoplasmic filaments are composed of one protein, CfpA, and have been demonstrated to be involved in cell division. In this study, we used electron tomography to show that the filaments are part of a complex with a novel molecular organization that includes at least two distinct features decorating the filaments. One set of components appears to anchor the filaments to the cytoplasmic membrane. The other set of components appears to bridge the cytoplasmic filaments on the cytoplasmic side, and to be involved in the interfilament spacing within the cell. The filaments occupy between 3 and 18% of the inner surface of the cytoplasmic membrane. These results reveal a novel filamentous molecular organization of independent filaments linked by bridges and continuously anchored to the membrane.     

Nerve growth factor promotes reorganization of the axonal microtubule array at sites of axon collateral branching

The localized debundling of the axonal microtubule array and the entry of microtubules into axonal filopodia are two defining features of collateral branching. We report that nerve growth factor (NGF), a branch‐inducing signal, increases the frequency of microtubule debundling along the axon shaft of chicken embryonic sensory neurons. Sites of debundling correlate strongly with the localized targeting of microtubules into filopodia. Platinum replica electron microscopy suggests physical interactions between debundled microtubules and axonal actin filaments. However, as evidenced by depolymerization of actin filaments and inhibition of myosin II, actomyosin force generation does not promote debundling. In contrast, loss of actin filaments or inhibition of myosin II activity promotes debundling, indicating that axonal actomyosin forces suppress debundling. MAP1B is a microtubule associated protein that represses axon branching. Following treatment with NGF, microtubules penetrating filopodia during the early stages of branching exhibited lower levels of associated MAP1B. NGF increased and decreased the levels of MAP1B phosphorylated at a GSK‐3β site (pMAP1B) along the axon shaft and within axonal filopodia, respectively. The levels of MAP1B and pMAP1B were not altered at sites of debundling, relative to the rest of the axon. Unlike the previously determined effects of NGF on the axonal actin cytoskeleton, the effects of NGF on microtubule debundling were not affected by inhibition of protein synthesis. Collectively, these data indicate that NGF promotes localized axonal microtubule debundling, that actomyosin forces antagonize microtubule debundling, and that NGF regulates pMAP1B in axonal filopodia during the early stages of collateral branch formation. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1441–1461, 2015     

Synaptic morphology of the carotid body of the domestic fowl

Efferent and reciprocal synapses have been demonstrated in the carotid body of the domestic fowl (Gallus gallus domesticus). Synapses were also found with purely afferent morphology, but were probably components of reciprocal synapses. The general morphology of the endings suggested the presence of two types of axon, afferent axons making reciprocal and perhaps afferent synapses with Type I cells, and efferent axons making efferent synapses with Type I cells. A few axo-dendritic synapses were also found. The dense-cored vesicles associated with the afferent components of reciprocal synapses and with the possible true afferent synapses varied in diameter and core but could belong to one population of pre-synaptic vesicles. These observations are consistent wtih a new theory for the carotid body receptor mechanism. This proposes a spontaneously discharging afferent axon inhibited by an inhibitory transmitter substance released by the Type I cell via the "afferent" component of its reciprocal synapse, the "efferent" component inhibiting this release. Besides this chemoreceptor modulation of its afferent axon, the Type I cell may also have a general secretory function.     

Axial and radial filamentous components of the neurofilamentous network

Summary The three-dimensional structure of the neurofilamentous network of the giant axon of the squid has been investigated by stereo electron microscopy and optical image analysis. The neurofilamentous network with its structural associations intact was partially purified by means of extraction of extruded axoplasm in a physiological buffer. The authors employed a double-grid mounting technique for critical point drying of axoplasm which provides high contrast preparations having great depth suitable for the analysis of spatial relations. There is a striking improvement in the perception of the continuity and three-dimensionality of the network, particularly in thin areas produced by teasing apart the specimen prior to mounting. Sidearms and cross-bridges are readily identifiable.In 1-m thick preparations of the extracted axoplasm the neurofilamentous network is composed of two structural entities: (i) long 10-nm wide filaments approximately parallel with the long axis of the extracted axoplasmic cylinder (axial), (ii) and short, finer filaments projecting from them as sidearms or crossbridges (radial). Optical analysis of micrographs of extracted axoplasm indicates that the radial filamentous components of the neurofilamentous network are distributed predominantly in the range of 32° to 67° with respect to the long axis of the axial filaments. We tentatively assign the 220,000 mol wt peptide observed by SDS-PAGE in this preparation to the radial filaments and the 68,000 mol wt peptide to the axial filaments.     

Solubility properties and specific assembly pathways of the B-type lamin from Caenorhabditis elegans

Lamins are nucleus-specific intermediate filament (IF) proteins that together with a complex set of membrane proteins form a filamentous meshwork tightly adhering to the inner nuclear membrane and being associated with the nuclear pore complexes. This so-called nuclear lamina provides mechanical stability and, in addition, has been implicated in the spatial organization of the heterochromatin. While increasing knowledge on the biological function of lamins has been obtained in recent years, the assembly mechanism of lamin filaments at the molecular level has remained largely elusive. Therefore, we have now more systematically investigated lamin assembly in vitro. Using Caenorhabditis elegans lamin, which has been reported to assemble into 10-nm filaments under low ionic strength conditions, we investigated the assembly kinetics of this protein into filaments in more detail using both His-tagged and un-tagged recombinant proteins. In particular, we have characterized distinct intermediates in the filament assembly process by analytical ultracentrifugation, electron and atomic force microscopy. In contrast to the general view that lamins assemble only slowly into filaments, we show that in vitro association reactions are extremely fast, and depending on the ionic conditions employed, significant filamentous assemblies form within seconds.     

Anomalous sedimentation behaviour of the e-coli ribosomal system: 50S-30S ⇄ 50S + 30S

An experimental and theoretical study has been made into the effect of association-dissociation reactions on the sedimentation of the E. coli ribosomal system 50S-30S ⇄ 50S + 30S. It has been found that(a) the sedimentation pattern is strongly dependent on the rotor speed;(b) the ratio of components as measured using high-speed ultracentrifugation (30000–40000 r.p.m.) is independent of rotor speed; and(c) the speed of ultracentrifugation has a strong effect on the sedimentation coefficient of the ribosomal system as determined by the mean square second moment.The results of this paper demonstrate that ribosome sedimentation at low-speed ultracentrifugation is affected by some artefactual processes. A theoretical analysis of the experimental findings has shown that the observed effects cannot be attributed to the effect of the association-dissociation reaction not to the pressure dependence of the equilibrium constant of that reaction.On the other hand, at high-speed ultracentrifugation the ribosomal system sediments as a heterogeneous mixture of non-interacting components. Consequently, the shape of the boundary in this case will reflect the equilibrium composition of the ribosomal system.     

In situ cryo-electron tomography reveals local cellular machineries for axon branch development

Neurons are highly polarized cells forming an intricate network of dendrites and axons. They are shaped by the dynamic reorganization of cytoskeleton components and cellular organelles. Axon branching allows the formation of new paths and increases circuit complexity. However, our understanding of branch formation is sparse due to the lack of direct in-depth observations. Using in situ cellular cryo-electron tomography on primary mouse neurons, we directly visualized the remodeling of organelles and cytoskeleton structures at axon branches. Strikingly, branched areas functioned as hotspots concentrating organelles to support dynamic activities. Unaligned actin filaments assembled at the base of premature branches accompanied by filopodia-like protrusions. Microtubules and ER comigrated into preformed branches to support outgrowth together with accumulating compact, ∼500-nm mitochondria and locally clustered ribosomes. We obtained a roadmap of events supporting the hypothesis of local protein synthesis selectively taking place at axon branches, allowing them to serve as unique control hubs for axon development and downstream neural network formation.     

Two distinct functions of the carboxyl-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments

Neurofilaments are the major cytoskeletal elements in the axon that take highly ordered structures composed of parallel arrays of 10-nm filaments linked to each other with frequent cross-bridges, and they are believed to maintain a highly polarized neuronal cell shape. Here we report the function of rat NF-M in this characteristic neurofilament assembly. Transfection experiments were done in an insect Sf9 cell line lacking endogenous intermediate filaments. NF-L and NF-M coassemble to form bundles of 10-nm filaments packed in a parallel manner with frequent cross-bridges resembling the neurofilament domains in the axon when expressed together in Sf9 cells. Considering the fact that the expression of either NF-L or NF-M alone in these cells results in neither formation of any ordered network of 10-nm filaments nor cross- bridge structures, NF-M plays a crucial role in this parallel filament assembly. In the case of NF-H the carboxyl-tail domain has been shown to constitute the cross-bridge structures. The similarity in molecular architecture between NF-M and NF-H suggests that the carboxyl-terminal tail domain of NF-M also constitutes cross-bridges. To examine this and to further investigate the function of the carboxyl-terminal tail domain of NF-M, we made various deletion mutants that lacked part of their tail domains, and we expressed these with NF-L. From this deletion mutant analysis, we conclude that the carboxyl-terminal tail domain of NF-M has two distinct functions. First, it is the structural component of cross-bridges, and these cross-bridges serve to control the spacing between core filaments. Second, the portion of the carboxyl- terminal tail domain of NF-M that is directly involved in cross-bridge formation affects the core filament assembly by helping them to elongate longitudinally so that they become straight.     

The actin-binding protein UNC-115 is an effector of Rac signaling during axon pathfinding in C. elegans

Rac GTPases control cell shape by regulating downstream effectors that influence the actin cytoskeleton. UNC-115, a putative actin-binding protein similar to human abLIM/limatin, has previously been implicated in axon pathfinding. We have discovered the role of UNC-115 as a downstream cytoskeletal effector of Rac signaling in axon pathfinding. We show that unc-115 double mutants with ced-10 Rac, mig-2 Rac or unc-73 GEF but not with rac-2/3 Rac displayed synthetic axon pathfinding defects, and that loss of unc-115 function suppressed the formation of ectopic plasma membrane extensions induced by constitutively-active rac-2 in neurons. Furthermore, we show that UNC-115 can bind to actin filaments. Thus, UNC-115 is an actin-binding protein that acts downstream of Rac signaling in axon pathfinding.     

Navigating the cell

Changes in cell shape are associated with a variety of processes including cell migration, axon outgrowth, cell division, and vesicle trafficking. C. elegans UNC-53 and its vertebrate homologs, the Navigators, are required for the migration of cells and the outgrowth of neuronal processes. The identification of novel molecular interactions and live imaging studies have revealed that UNC-53/NAVs are signal transducers associated with actin filaments, microtubules, and intermediate filaments. In addition to modulating cytoskeletal dynamics at the leading edge of migrating or outgrowing cells, both UNC-53 and the navigators are expressed in adult cells, conspicuously those with specialized roles in endocytosis or secretion. Collectively, these results suggest that UNC-53/NAVs may be a central regulator of cytoskeletal dynamics, responsible for integrating signaling cues to multiple components of the cytoskeleton to coordinate rearrangement during cell outgrowth or trafficking.