Neurospecific cytoskeletal proteins

Data available in literature on neurospecific proteins of cytoskeletal structures--microtubules, microfilaments and intermediate filaments are generalized. Properties of tissue-specific cytoskeletal proteins which are typical of nerve cells are summarized. The structure, physicochemical properties, cell localization, metabolism and function of cytoskeletal proteins are characterized. The coexpression and interaction of different cytoskeletal structures are considered. An analysis of neurospecific cytoskeletal proteins is of great practical importance for neurobiology, neurooncology, neurosurgery. The proteins can be used as markers of different pathologies in the nervous system.     

The cytoskeletal proteins of erythrocytes

The erythrocyte membrane skeleton is composed of the number of proteins isolated and characterized. One of the major proteins of cytoskeleton is actin presented in erythrocytes in the form of short protofilaments. This review will focus on the manner of attachment of actin protofilaments to the red cell membrane, and on the relationships between skeleton membrane proteins. Membrane skeleton proteins in erythrocytes are not unique. Recently a lot of proteins similar to the red cell membrane skeleton proteins were found in a wide variety of non-erythroid cells. This fact gives the opportunity to suppose the existence of a unique protein system in erythroid and non-erythroid cells which provides the attachment of actin filaments to cell membranes and which might be the centre for the assembling of actin structures in the cortical cytoplasm.     

Mechanical properties of cytoskeletal polymers

The mechanical properties of cytoplasm are dominated by microfilaments, microtubules, and intermediate filaments, collectively termed the cytoskeleton. This review discusses how the physical properties of these biopolymer systems are related to their molecular structures and interactions, and how remodelling of these biopolymers in vivo affects cell shape and motility.     

Structural interaction of cytoskeletal components

Three-dimensional cytoskeletal organization of detergent-treated epithelial African green monkey kidney cells (BSC-1) and chick embryo fibroblasts was studied in whole-mount preparations visualized in a high voltage electron microscope. Stereo images are generated at both low and high magnification to reveal both overall cytoskeletal morphology and details of the structural continuity of different filament types. By the use of an improved extraction procedure in combination with heavy meromyosin subfragment 1 decoration of actin filaments, several new features of filament organization are revealed that suggest that the cytoskeleton is a highly interconnected structural unit. In addition to actin filaments, intermediate filaments, and microtubules, a new class of filaments of 2- to 3-nm diameter and 30- to 300-nm length that do not bind heavy merymyosin is demonstrated. They form end-to-side contacts with other cytoskeletal filaments, thereby acting as linkers between various fibers, both like (e.g., actin- actin) and unlike (e.g., actin-intermediate filament, intermediate filament-microtubule). Their nature is unknown. In addition to 2- to 3-nm filaments, actin filaments are demonstrated to form end-to-side contacts with other filaments. Y-shaped actin filament “branches” are observed both in the cell periphery close to ruffles and in more central cell areas also populated by abundant intermediate filaments and microtubules. Arrowhead complexes formed by subfragment 1 decoration of actin filaments point towards the contact site. Actin filaments also form end-to-side contacts with microtubules and intermediate filaments. Careful inspection of numerous actin-microtubule contacts shows that microtubules frequently change their course at sites of contact. A variety of experimentally induced modifications of the frequency of actin-microtubule contacts can be shown to influence the course of microtubules. We conclude that bends in microtubules are imposed by structural interactions with other cytoskeletal elements. A structural and biochemical comparison of whole cells and cytoskeletons demonstrates that the former show a more inticate three-dimensional network and a more complex biochemical composition than the latter. An analysis of the time course of detergent extraction strongly suggests that the cytoskeleton forms a structural backbone with which a large number of proteins of the cytoplasmic ground substance associate in an ordered fashion to form the characteristic image of the “microtrabecular network” (J.J. Wolosewick and K.R. Porter. 1979. J. Cell Biol. 82: 114-139).     

Intermediate filaments mediate cytoskeletal crosstalk

Intermediate filaments, actin-containing microfilaments and microtubules are the three main cytoskeletal systems of vertebrate and many invertebrate cells. Although these systems are composed of distinctly different proteins, they are in constant and intimate communication with one another. Understanding the molecular basis of this cytoskeletal crosstalk is essential for determining the mechanisms that underlie many cell-biological phenomena. Recent studies have revealed that intermediate filaments and their associated proteins are important components in mediating this crosstalk.     

Characterization of human erythrocyte cytoskeletal ATPase

Human erythrocyte cytoskeletal ATPase was extracted with 0.2 mM ATP (pH 8.0) from Triton X-100 treated ghosts. The ATPase fraction contained mainly spectrin, actin, and band 4.1. When the ATPase fraction was applied to a Sepharose 4B column, 90% of the ATPase activity was recovered in a spectrin, actin, and band 4.1 complex fraction and none was detected in the spectrin fraction. A specific activity of the complex ATPase was 60-120 nmol/(mg protein X h). No ATPase activity was detected in the presence of EDTA. The presence of magnesium in the incubation medium was essential for the ATPase activity. The activity was activated by KCl and was almost completely inhibited by 10(-5) M free calcium in the presence of 0.2 mM MgCl2. The Ki for Ca2+ was 7 X 10(-7) M. Phalloidin and DNase 1, which affect actin, inhibited this K,Mg-ATPase activity by 95%, but cytochalasin B did not inhibit it. N-Ethylmaleimide activated the ATPase 1.6-fold. The order of affinity for nucleotides was ATP greater than ITP greater than CTP, ADP, AMP-PNP, GTP. A specific ATPase activity of purified actin was 50 nmol/(mg X h). These results suggest that the cytoskeletal ATPase is actin ATPase and the actin ATPase is activated by spectrin and band 4.1.     

Glial shape and cytoskeletal protein synthesis

We investigated whether the shape of astroglial derived cells influences the expression of cytoskeletal proteins. In reaggregating cultures GFAP, vimentin and actin synthesis was approximately 52%, 50% and 37% the level found in monolayer cultures, respectively. Monolayer cultures consisted of polygonal shaped cells adhering to plastic, while reaggregating cultures were comprised of round cells growing in a suspension like culture. Additionally, human glioma cells induced to grow as round cells on poly-2-hydroxyethyl methacrylate (polyhema) coated plastic exhibited a level of GFAP synthesis that was approximately 20% the level displayed by polygonal shaped cells grown on uncoated plastic. Glioma cells initially grown on a polyhema surface and replated onto uncoated plastic were capable of reinitiating GFAP synthesis. Thus, aterations in the synthesis of GFAP and other cytoskeletal proteins can occur when astrocytes change their shape.     

Focal adhesions - the cytoskeletal connection

Cellular contacts with the extracellular matrix are regulated by the Rho family of GTPases through their effects on both the actin and the microtubule cytoarchitecture. Recent genetic, biochemical and structural data have highlighted the role played by a subset of actin-binding proteins in coupling integrins to cytoskeletal actin and in assembling signalling complexes that are important for cell motility and cell proliferation.     

Nucleotide hydrolysis in cytoskeletal assembly.

Two major polymers of the cytoskeleton, actin filaments and microtubules, are assembled with expenditure of energy: the ATP/GTP tightly bound to actin/tubulin is irreversibly hydrolyzed to ADP/GTP during the assembly process, and liberation of Pi in the medium occurs subsequent to the incorporation of subunits in the polymer. Pi release acts as a switch, causing the destabilization of protein-protein interactions in the polymer, therefore regulating the dynamics of these fibres. An understanding of this regulation in vivo requires that progress be made in four areas: the chemistry of the NTPase reaction; the structure of the intermediates in nucleotide hydrolysis and the nature of the conformational switch; the regulation of parameters involved in dynamic instability of microtubules; and the possible involvement of nucleotide hydrolysis in the macroscopic organization of these polymers in highly concentrated solutions, compared with the simple case of a equilibrium polymers. Progress made along these lines will define trends for future investigation.     

Optimal orientation in branched cytoskeletal networks

Actin cytoskeletal protrusions in crawling cells, or lamellipodia, exhibit various morphological properties such as two characteristic peaks in the distribution of filament orientation with respect to the leading edge. To understand these properties, using the dendritic nucleation model as a basis for cytoskeletal restructuring, a kinetic-population model with orientational-dependent branching (birth) and capping (death) is constructed and analyzed. Optimizing for growth yields a relation between the branch angle and filament orientation that explains the two characteristic peaks. The model also exhibits a subdominant population that allows for more accurate modeling of recent measurements of filamentous actin density along the leading edge of lamellipodia in keratocytes. Finally, we explore the relationship between orientational and spatial organization of filamentous actin in lamellipodia and address recent observations of a prevalence of overlapping filaments to branched filaments-a finding that is claimed to be in contradiction with the dendritic nucleation model.     

A mathematical approach to cytoskeletal assembly

The cytoskeleton is a fundamental and important part of cell's structure, and is known to play a large role in controlling the shape, function, division, and motility of the cell. In recent years, the traditional biological and biophysical experimental work on the cytoskeleton has been enhanced by a variety of theoretical, physical and mathematical approaches. Many of these approaches have been developed in the traditional frameworks of physico-chemical and statistical mechanics or equilibrium thermodynamic principles. An alternative is to use kinetic modelling and couch the analysis in terms of differential equations which describe mean field properties of cytoskeletal networks or assemblies. This paper describes two such recent efforts. In the first part of the paper, a summary of work on the kinetics of polymerization, fragmentation, and dynamics of actin and polymers in the presence of gelsolin (which nulceates, fragments, and caps the filaments) is given. In the second part, some of the kinetic models aimed at elucidating the spatio-angular density distribution of actin filaments interacting via crosslinks is described. This model given insight into effects that govern the formation of clusters and bundles of actin filaments, and their spatial distribution. Received: 7 January 1998 / Revised version: 4 March 1998 / Accepted: 7 March 1998     

Collective dynamics of active cytoskeletal networks

Self organization mechanisms are essential for the cytoskeleton to adapt to the requirements of living cells. They rely on the intricate interplay of cytoskeletal filaments, crosslinking proteins and molecular motors. Here we present an in vitro minimal model system consisting of actin filaments, fascin and myosin-II filaments exhibiting pulsatile collective dynamics and superdiffusive transport properties. Both phenomena rely on the complex competition of crosslinking molecules and motor filaments in the network. They are only observed if the relative strength of the binding of myosin-II filaments to the actin network allows exerting high enough forces to unbind actin/fascin crosslinks. This is shown by varying the binding strength of the acto-myosin bond and by combining the experiments with phenomenological simulations based on simple interaction rules.     

Caldesmon phosphorylation in actin cytoskeletal remodeling

Caldesmon is an actin-binding protein that is capable of stabilizing actin filaments against actin-severing proteins, inhibiting actomyosin ATPase activity, and inhibiting Arp2/3-mediated actin polymerization in vitro. Caldesmon is a substrate of cdc2 kinase and Erk1/2 MAPK, and phosphorylation by either of these kinases reverses the inhibitory effects of caldesmon. Cdc2-mediated caldesmon phosphorylation and the resulting dissociation of caldesmon from actin filaments are essential for M-phase progression during mitosis. Cells overexpressing the actin-binding carboxyterminal fragment of caldesmon fail to release the fragment completely from actin filaments during mitosis, resulting in a higher frequency of multinucleated cells. PKC-mediated MEK/Erk/caldesmon phosphorylation is an important signaling cascade in the regulation of smooth muscle contraction. Furthermore, PKC activation has been shown to remodel actin stress fibers into F-actin-enriched podosome columns in cultured vascular smooth muscle cells. Podosomes are cytoskeletal adhesion structures associated with the release of metalloproteases and degradation of extracellular matrix during cell invasion. Interestingly, caldesmon is one of the few actin-binding proteins that is associated with podosomes but excluded from focal adhesions. Caldesmon also inhibits the function of gelsolin and Arp2/3 complex that are essential for the formation of podosomes. Thus, caldesmon appears to be well positioned for playing a modulatory role in the formation of podosomes. Defining the roles of actin filament-stabilizing proteins such as caldesmon and tropomyosin in the formation of podosomes should provide a more complete understanding of molecular systems that regulate the remodeling of the actin cytoskeleton in cell transformation and invasion.     

The beta integrins and cytoskeletal nanoimprinting

"Nanoimprinting", whereby topographical features are directly imprinted on or in cells, has recently been documented. The mechanism(s) underlying this may explain the cause of cell behavioural alterations as a result of contact with nanotopography. Integrin-mediated cell-substrate adhesions are likely to play a key role in this phenomenon due to their involvement in bidirectional signalling between extra- and intracellular environments. We describe the effects of blocking beta1 and beta3 integrin subunits on the ability of the cytoskeleton to conform to colloidal-derived nanotopography. Scanning electron and atomic force microscopy were used to characterise substrate nanofeatures. Nanofeature circularity was calculated relative to substrate topography and the cytoskeleton of cells cultured on patterned and planar surfaces in the presence/absence of beta1/beta3 integrin antibody. Cross-correlation investigations were similarly conducted by producing a target image relative to individual topographical nanofeatures. This was then compared with cytoskeletal patterning in the presence/absence of beta integrin antibodies. Inhibiting the beta1 subunits increased the ability of fibroblasts to nanoimprint, while inhibiting the beta3 subunit reduced nanoimprinting of the topography to the cell. Fibroblasts cultured on planar substrates also expressed some features sharing similarities with those observed in cells on the nanotopography, indicating an inherent cytoskeletal nanopatterning at high resolution.