Novel keratins identified by quantitative proteomic analysis as the major cytoskeletal proteins of equine (Equus caballus) hoof lamellar tissue

The dermo-epidermal interface that connects the equine distal phalanx to the cornified hoof wall withstands great biomechanical demands, but is also a region where structural failure often ensues as a result of laminitis. The cytoskeleton in this region maintains cell structure and facilitates intercellular adhesion, making it likely to be involved in laminitis pathogenesis, although it is poorly characterized in the equine hoof lamellae. The objective of the present study was to identify and quantify the cytoskeletal proteins present in the epidermal and dermal lamellae of the equine hoof by proteomic techniques. Protein was extracted from the mid-dorsal epidermal and dermal lamellae from the front feet of 5 Standardbred geldings and 1 Thoroughbred stallion. Mass spectrometry-based spectral counting techniques, PAGE, and immunoblotting were used to identify and quantify cytoskeletal proteins, and indirect immunofluorescence was used for cellular localization of K14 and K124 (where K refers to keratin). Proteins identified by spectral counting analysis included 3 actin microfilament proteins; 30 keratin proteins along with vimentin, desmin, peripherin, internexin, and 2 lamin intermediate filament proteins; and 6 tubulin microtubule proteins. Two novel keratins, K42 and K124, were identified as the most abundant cytoskeletal proteins (22.0 ± 3.2% and 23.3 ± 4.2% of cytoskeletal proteins, respectively) in equine hoof lamellae. Immunoreactivity to K14 was localized to the basal cell layer, and that to K124 was localized to basal and suprabasal cells in the secondary epidermal lamellae. Abundant proteins K124, K42, K14, K5, and α1-actin were identified on 1- and 2-dimensional polyacrylamide gels and aligned with the results of previous studies. Results of the present study provide the first comprehensive analysis of cytoskeletal proteins present in the equine lamellae by using mass spectrometry-based techniques for protein quantification and identification.

Interaction of metal salts with cytoskeletal motor protein systems

Interactions of chemicals with the microtubular network of cells may lead to genotoxicity. Micronuclei (MN) might be caused by interaction of metals with tubulin and/or kinesin. The genotoxic effects of inorganic lead and mercury salts were studied using the MN assay and the CREST analysis in V79 Chinese hamster fibroblasts. Effects on the functional activity of motor protein systems were examined by measurement of tubulin assembly and kinesin-driven motility. Lead and mercury salts induced MN dose-dependently. The no-effect-concentration for MN induction was 1.1 μM PbCl2, 0.05 μM Pb(OAc)2 and 0.01 μM HgCl2. The in vitro results obtained for PbCl2 correspond to reported MN induction in workers occupationally exposed to lead, starting at 1.2 μM Hg(II) (Vaglenov et al., 2001, Environ. Health Perspect. 109, 295-298). The CREST Analysis indicate aneugenic effects of Pb(II) and aneugenic and additionally clastogenic effects of Hg(II). Lead (chloride, acetate, and nitrate) and mercury (chloride and nitrate) interfered dose-dependently with tubulin assembly in vitro. The no-effect-concentration for lead salts in this assay was 10 μM. Inhibition of tubulin assembly by mercury started at 2 μM. The gliding velocity of microtubules along immobilised kinesin molecules was affected by 25 μM Pb(NO3)2 and 0.1 μM HgCl2 in a dose-dependent manner. Our data support the hypothesis that lead and mercury genotoxicity may result, at least in part, via disturbance of chromosome segregation via interaction with cytoskeletal proteins.

Target Cell Topography and Cytoskeletal Reorganization in Natural Killer Cell Activity

The immune system has to recognize and destroy abnormal or infected cells to maintain homeostasis. Natural killer (NK) cells directly recognize and kill transformed or virus-infected cells without prior sensitization. We have studied both virus-infected and tumor cells in order to identify the target structures involved in triggering NK activity. Mouse/human cell hybrids containing various human chromosomes were used as targets. The human chromosome responsible for activating NK cell killing was identified to chromosome number 6. The results suggest that activated NK cells recognize ligands that are encoded on human chromosome 6. We showed that the ligand on the target cell side was intercellular adhesion molecule 2 (ICAM-2). There was no difference in the level of expression of ICAM-2, however, but a drastic difference was seen in the distribution of the molecule: ICAM-2 was evenly distributed on the surface of the NK-resistant cells, but almost totally redistributed to the tip of uropods, bud-like extensions, which were absent from the parental cells. Interestingly, the gene coding for cytoskeletal linker protein ezrin has been localized to human chromosome 6, and there was a colocalization of ezrin and ICAM-2 in the uropods. Furthermore, the transfected human ezrin into NK cell-resistant cells induced uropod formation, ICAM-2 and ezrin redistribution to newly formed uropods, and sensitized target cells to NK cell killing. These data reveal a novel form of NK cell recognition: target structures are already present on normal cells; they become detectable only after abnormal redistribution into hot spots on the target cell membrane. NK cells are central players in the defence against virus infections. They inhibit the spread of infection, allowing time for specific immune responses to develop. The virus-proteins that directly activate human NK cell killing are largely unknown. We studied the sensitivity of virus-specific early proteins of Semliki Forest virus (SFV) to NK killing. The viral non-structural proteins (nsP1-4) translated early in the virus cycle were transfected in NK-resistant cells. Viral early gene nsP1 alone efficiently sensitized target cells to NK activity, and the tight membrane association of nsP1 seems to be critical in the triggering of NK killing. NsP1 protein colocalized with (redistributed) ezrin in filopodia-like structures to which the NK cells were bound. The results suggest that also in viral infections NK cells react to rapid changes in membrane topography. Based on the results of this thesis, a new model of target cell recognition of NK cells can be suggested: reorganization of the cytoskeleton induces alterations in cell surface topography, and this new pattern of surface molecules is recognized as "altered-self".

Structural basis of cytoskeletal regulation by twinfilin

The actin cytoskeleton is required, in all eukaryotic organisms, for several key cellular functions such as cell motility, cytokinesis, and endocytosis. In cells, actin exists either in a monomeric state (G-actin) or in a filamentous form (F-actin). F-actin is the functional form, which can assemble into various structures and produce direct pushing forces that are required for different motile processes. The assembly of actin monomers into complicated three-dimensional structures is tightly regulated by a large number of actin regulating proteins. One central actin regulating protein is twinfilin. Twinfilin consists of two actin depolymerizing-factor homology (ADF-H) domains, which are capable of binding actin, and is conserved from yeast to mammals. Previously it has been shown that twinfilin binds to and sequesters G-actin, and interacts with the heterodimeric capping protein. More recently it has been found that twinfilin also binds to the fast growing actin filament ends and prevents their growth. However, the cellular role of twinfilin and the molecular mechanisms of these interactions have remained unclear. In this study we characterized the molecular mechanisms behind the functions of twinfilin. We demonstrated that twinfilin forms a high-affinity complex with ADP-bound actin monomers (ADP-G-actin). Both ADF-H domains are capable of binding G-actin, but the C-terminal domain contains the high-affinity binding site. Our biochemical analyses identified twinfilin s C-terminal tail region as the interaction site for capping protein. Contrary to G-actin binding, both ADF-H domains of twinfilin are required for the actin filament barbed end capping activity. The C-terminal domain is structurally homologous to ADF/cofilin and binds to filament sides in a similar manner, providing the main affinity for F-actin during barbed end capping. The structure of the N-terminal domain is more distant from ADF/cofilin, and thus it can only associate with G-actin or the terminal actin monomer at the filament barbed end, where it regulates twinfilin s affinity for barbed ends. These data suggest that the mechanism of barbed end capping is similar for twinfilin and gelsolin family proteins. Taken together, these studies revealed how twinfilin interacts with G-actin, filament barbed ends, and capping protein, and also provide a model for how these activities evolved through a duplication of an ancient ADF/cofilin-like domain.

Involvement of cytoskeletal structures in nerve-growth-factor-mediated induction of ornithine decarboxylase.

Induction of ornithine decarboxylase elicited in response to nerve-growth factor in target organs is greatly decreased by preincubation of these tissues with cytoskeletal poisons such as vinblastine, diamide, cytochalasin B and colchicine. These results are interpreted as evidence for the involvement of receptor-associated cytoskeletal structures in mediating the nerve-growth-factor-specific induction of ornithine decarboxylase.

Involvement of cytoskeletal structures in nerve-growth-factor-mediated induction of ornithine decarboxylase

Induction of ornithine decarboxylase elicited in response to nerve-growth factor in target organs is greatly decreased by preincubation of these tissues with cytoskeletal poisons such as vinblastine, diamide, cytochalasin B and colchicine. These results are interpreted as evidence for the involvement of receptor-associated cytoskeletal structures in mediating the nerve-growth-factor-specific induction of ornithine decarboxylase.

Corticosterone Treatment Results in Enhanced Release of Peptidergic Vesicles in Astrocytes via Cytoskeletal Rearrangements

While the effect of stress on neuronal physiology is widely studied, its effect on the functionality of astrocytes is not well understood. We studied the effect of high doses of stress hormone corticosterone, on two physiological properties of astrocytes, i.e., gliotransmission and interastrocytic calcium waves. To study the release of peptidergic vesicles from astrocytes, hippocampal astrocyte cultures were transfected with a plasmid to express pro-atrial natriuretic peptide (ANP) fused with the emerald green fluorescent protein (ANP.emd). The rate of decrease in fluorescence of ANP.emd on application of ionomycin, a calcium ionophore was monitored. Significant increase in the rate of calcium-dependent exocytosis of ANP.emd was observed with the 100 nM and 1 M corticosterone treatments for 3 h, which depended on the activation of the glucocorticoid receptor. ANP.emd tagged vesicles exhibited increased mobility in astrocyte culture upon corticosterone treatment. Increasing corticosterone concentrations also resulted in concomitant increase in the calcium wave propagation velocity, initiated by focal ATP application. Corticosterone treatment also resulted in increased GFAP expression and F-actin rearrangements. FITC-Phalloidin immunostaining revealed increased formation of cross linked F-actin networks with the 100 nM and 1 M corticosterone treatment. Alternatively, blockade of actin polymerization and disruption of microtubules prevented the corticosterone-mediated increase in ANP.emd release kinetics. This study reports for the first time the effect of corticosterone on gliotransmission via modulation of cytoskeletal elements. As ANP acts on both neurons and blood vessels, modulation of its release could have functional implications in neurovascular coupling under pathophysiological conditions of stress.

A pseudo-spin model for the wall of cytoskeletal microtubule

A pseudo-spin model is intended to describe the physical dynamics of unbound electrons in the wall of cytoskeletal microtubule (MT). Due to the inherent symmetry of the structure and the electric properties in the MT, one may treat it as a one-dimensional ferroelectric system, and describe the nonlinear dynamics of dimer electric dipoles in one protofilament of the MT by virtue of the double-well potential. Consequently, the physical problem has been mapped onto the pseudo-spin system, and the mean-field approximation has been taken to get some physical results.

Quantum information processing in the wall of cytoskeletal microtubules

Microtubules (MT) are composed of 13 protofilaments, each of which is a series of two-state tubulin dimers. In the MT wall, these dimers can be pictured as "lattice" sites similar to crystal lattices. Based on the pseudo-spin model, two different location states of the mobile electron in each dimer are proposed. Accordingly, the MT wall is described as an anisotropic two-dimensional (2D) pseudo-spin system considering a periodic triangular "lattice". Because three different "spin-spin" interactions in each cell exist periodically in the whole MT wall, the system may be shown to be an array of three types of two-pseudo-spin-state dimers. For the above-mentioned condition, the processing of quantum information is presented by using the scheme developed by Lloyd.

Perspectives on the origin of microfilaments, microtubules, the relevant chaperonin system and cytoskeletal motors - a commentary on the spirochaete origin of flagella

The origin of cytoskeleton and the origin of relevant intracellular transportation system are big problems for understanding the emergence of eukaryotic cells. The present article summarized relevant information of evidences and molecular traces on the origin of actin, tubulin, the chaperonin system for folding them, myosins, kinesins, axonemal dyneins and cytoplasmic dyneins. On this basis the authors proposed a series of works, which should be done in the future, and indicated the ways for reaching the targets. These targets are mainly: 1) the reconstruction of evolutionary path from MreB protein of archaeal ancestor of eukaryotic cells to typical actin; 2) the finding of the MreB or MreB-related proteins in crenarchaea and using them to examine J. A. Lake's hypothesis on the origin of eukaryote from "eocytes" (crenarchaea); 3) the examinations of the existence and distribution of cytoskeleton made of MreB-related protein within coccoid archaea, especially in amoeboid archaeon Thermoplasm acidophilum; 4) using Thermoplasma as a model of archaeal ancestor of eukaryotic cells; 5) the searching for the homolog of ancestral dynein in present-day living archaea. During the writing of this article, Margulis' famous spirochaete hypothesis on the origin of flagella and cilia was unexpectedly involved and analyzed from aspects of tubulins, dyneins and spirochaetes. Actually, spirochaete cannot be reasonably assumed as the ectosymbiotic ancestor of eukaryotic flagella and cilia, since their swing depends upon large amount of bacterial flagella beneath the flexible outer wall, but not depends upon their intracellular tubules and the assumed dyneins. In this case, if they had "evolved" into cilia and lost their bacterial flagella, they would immediately become immobile! In fact, tubulin and dynein-like proteins have not been found in any spirochaete.

Quasi-3D Cytoskeletal Dynamics of Osteocytes under Fluid Flow

Osteocytes respond to dynamic fluid shear loading by activating various biochemical pathways, mediating a dynamic process of bone formation and resorption. Whole-cell deformation and regional deformation of the cytoskeleton may be able to directly regulate this process. Attempts to image cellular deformation by conventional microscopy techniques have been hindered by low temporal or spatial resolution. In this study, we developed a quasi-three-dimensional microscopy technique that enabled us to simultaneously visualize an osteocyte's traditional bottom-view profile and a side-view profile at high temporal resolution. Quantitative analysis of the plasma membrane and either the intracellular actin or microtubule (MT) cytoskeletal networks provided characterization of their deformations over time. Although no volumetric dilatation of the whole cell was observed under flow, both the actin and MT networks experienced primarily tensile strains in all measured strain components. Regional heterogeneity in the strain field of normal strains was observed in the actin networks, especially in the leading edge to flow, but not in the MT networks. In contrast, side-view shear strains exhibited similar subcellular distribution patterns in both networks. Disruption of MT networks caused actin normal strains to decrease, whereas actin disruption had little effect on the MT network strains, highlighting the networks' mechanical interactions in osteocytes.

Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells.

The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Young's moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs.

Novel interaction between RET and membrane cytoskeletal-linker protein ezrin

RET is a receptor tyrosine kinase that mediates key signaling events, and promotes cell survival, development, and migration. Activation of RET requires a ligand from the glial cell line-derived neurotrophic factor (GDNF) family and a co-receptor from the GDNF family receptor α (GFRα). Alternative splicing of RET leads to two major isoforms, RET9 and RET51, that contain distinct C-terminal amino acids. Differences in their cytoplasmic tails confer differential binding to adaptor proteins, and in this study, the membrane cytoskeletal-linker protein ezrin was shown in an interaction with RET51, but not RET9, in a ligand- and kinase-dependent manner. Results indicated that Y1096 on RET51 is the ezrin recruitment site, and the adaptor protein Grb2 may mediate this interaction. These results suggest that ezrin may play a role in the downstream signaling and recycling pathways of RET51. Thus, the identified novel interaction may provide insight in the longer term into how ezrin and RET51 contribute together to functional processes such as cell migration and invasion.