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PubMed1998

Dystonin is essential for maintaining neuronal cytoskeleton organization.

The mouse neurological mutant dystonia musculorum (dt) suffers from a hereditary sensory neuropathy. We have previously described the cloning and characterization of the dt gene, which we named dystonin (Dst). We had shown that dystonin is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin-binding domain. It has been shown previously that dystonin is a cytoskeletal linker protein, forming a bridge between F-actin and intermediate filaments. Here, we have used two different antibody preparations against dystonin and detected a high-molecular-weight protein in immunoblot analysis of spinal cord extracts. We also show that this high-molecular-weight protein was not detectable in the nervous system of all dt alleles tested. Immunohistochemical analysis revealed that dystonin was present in different compartments of neurons--cell bodies, dendrites, and axons, regions which are rich in the three elements of the cytoskeleton (F-actin, neurofilaments, and microtubules). Ultrastructural analysis of dt dorsal root axons revealed disorganization of the neurofilament network and surprisingly also of the microtubule network. In this context it is of interest that we observed altered levels of the microtubule-associated proteins MAP2 and tau in spinal cord neurons of different dt alleles. Finally, dt dorsal root ganglion neurons formed neurites in culture, but the cytoskeleton was disorganized within these neurites. Our results demonstrate that dystonin is essential for maintaining neuronal cytoskeleton integrity but is not required for establishing neuronal morphology.

Animals↗

PubMed2005

Disruption of nuclear maturation and rearrangement of cytoskeletal elements in bovine oocytes exposed to heat shock during maturation.

Meiotic maturation in mammalian oocytes is a complex process which involves extensive rearrangement of microtubules, actin filaments and chromosomes. Since cytoskeletal elements are sensitive to disruption by heat shock, a series of experiments were performed to determine whether physiologically relevant heat shock disrupts the progression of the oocyte through meiosis, fertilization and zygote formation. Cumulus-oocyte complexes were cultured at 38.5, 40.0 or 41.0 degrees C for the first 12 h of maturation. Incubation during the last 10 h of maturation and 18 h after fertilization was at 38.5 degrees C and in 5% (v/v) CO2 for both treatments. Examination of the cytoskeleton and the chromosome organization in matured oocytes revealed that oocytes matured at 38.5 degrees C were mostly at metaphase II (MII) stage, while the majority of heat-shocked oocytes were blocked at the first metaphase (MI), first anaphase or first telophase stages. A subset of heat-shocked oocytes possessed misshapen MI spindles with disorganized microtubules and unaligned chromosomes. A higher percentage of TUNEL-positive oocytes was noted for oocytes matured at 41.0 degrees C. Addition of 50 nmol/l sphingosine 1-phosphate to maturation medium blocked the effect of heat shock on progression through meiosis and apoptosis and increased the proportion of oocytes matured at 41.0 degrees C that were at MII. Following insemination, a high percentage of heat-shocked oocytes were unfertilized, while the majority of the control zygotes were fertilized and had two visible pronuclei. In conclusion, heat shock disrupts nuclear maturation and induces apoptosis. These alterations are likely to be involved in the mechanism underlying heat-shock-induced disruption of oocyte capacity for fertilization and subsequent development.

Actin Cytoskeleton↗

PubMed2006

Expression profile of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis: identification of essential genes in osteoclasts.

UNLABELLED: RhoGTPases regulate actin cytoskeleton dynamics, a key element in osteoclast biology. We identified three novel genes induced during RANKL-stimulated osteoclastogenesis among RhoGTPases and their exchange factors that are essential in osteoclast biology. INTRODUCTION: During the process of differentiation, adhesion to the bone matrix or osteolysis, the actin cytoskeleton of osteoclasts undergoes profound reorganization. RhoGTPases are key regulators of actin dynamics. They control cell adhesion, migration, and morphology through their action on actin cytoskeleton. In mice, there are 18 low molecular weight RhoGTPases. They are activated by guanine nucleotide exchange factors: the RhoGEFs. There are 76 RhoGEFs in mice: 65 belong to the Dbl family and 11 to the CZH family. To identify novel genes among RhoGTPases and RhoGEFs important in osteoclasts, we established the expression profiles of the complete families of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis. MATERIALS AND METHODS: The RAW264.7 cell line, mouse bone marrow macrophages, and hematopoietic stem cells were used as precursors for RANKL-induced osteoclastogenesis. Gene arrays and real-time quantitative PCR analyses were performed to establish the transcription profiles of RhoGTPase and RhoGEF genes during differentiation. Small hairpin RNA was used to knock down genes of interest. RESULTS: Of the 18 RhoGTPases and 76 RhoGEFs, the expression of three genes was upregulated by RANKL: the RhoGTPase RhoU/Wrch1, the Dbl family exchange factor Arhgef8/Net1, and the CZH family exchange factor Dock5. The inductions were observed in gene array and real-time quantitative PCR experiments performed in RAW264.7 cells. They were further confirmed in bone marrow macrophages and hematopoietic stem cells. Silencing of Wrch1 and Arhgef8 expression severely inhibited differentiation and affected osteoclast morphology. Dock5 suppression was lethal in osteoclast precursors while having no effect in fibroblasts. CONCLUSIONS: We identified three genes among RhoGTPase signaling pathways that are upregulated during RANKL-induced osteoclastogenesis. These genes are novel essential actors in osteoclasts, most likely through the control of actin cytoskeleton dynamics.

Adaptor Proteins, Signal Transducing↗

PubMed1988

Membrane IgM: interactions with the cortical cytoskeleton in the human lymphoblastoid cell line WiL2.

Cell-surface IgM (antigen receptor) sediments with the membrane fraction following osmotic lysis and homogenization of cells of the human lymphoblastoid cell line WiL2. In nonreducing buffers, SDS PAGE analysis of membrane pellets demonstrates that "native" membrane IgM exists as a dimer. In contrast to osmotic lysis, lysis of cells with the nonionic detergent Triton X-100 releases approximately 90% of the membrane-bound IgM into the supernatant; approximately 10% of the IgM pellets with the cytoskeletal fraction on centrifugation. Ligand challenge with either mu-chain-specific antibodies or concanavalin A induces a change in the state of membrane IgM making it refractory to detergent extraction, such that 43% of the IgM pellets during centrifugation. This ligand-induced retention of IgM is significantly diminished by the microfilament-disrupting agent cytochalasin D, whereas pretreatment of cells with sodium azide or colchicine results in no significant change in the percentage of membrane IgM retained by Triton X-100 residues. These results indicate that retention of IgM involves an association with the cortical actin-based cytoskeleton. Investigation of the structural basis for ligand-induced Triton X-100 retention of membrane IgM by using ferritin-conjugated antibodies, myosin subfragment S1, and stereo-imaging electron microscopy has revealed linkages between ligand-receptor (antigen-IgM) complexes and elements of the cortical actin-based cytoskeleton.

Cell Line↗

PubMed1990

The same target sequences are differentially important for activation of the interleukin 2 receptor alpha-chain gene in two distinct T-cell lines.

High-affinity receptors for interleukin 2 (IL-2) are expressed on T cells following activation. These receptors are composed of both alpha and beta chains. Expression of alpha chains and, therefore, expression of high-affinity receptors are critically regulated at the level of transcription initiation. We have further dissected the regulatory elements involved in controlling transcription of the IL-2 receptor alpha-chain (IL-2R alpha) gene. The IL-2R alpha promoter contains a kappa B site and binding sites for additional nuclear factors within a 50-base-pair region (positions -290 to -240 relative to the major transcription start site). These include one upstream of the kappa B site and one similar to the c-fos serum response element (SRE), which is downstream of the kappa B site. Mutation of the kappa B site decreases IL-2R alpha promoter activity in MT-2 cells (a T-cell line that has been transformed with human T-cell lymphotropic virus type I), but not in Jurkat cells (a T-cell leukemia line) that have been activated by phorbol 12-myristate 13-acetate (PMA). In contrast, mutation of a region upstream of the kappa B site decreases activity in PMA-induced Jurkat cells but increases activity in MT-2 cells. Mutation of the SRE-like site decreases activity in both cell types but the effect in PMA-induced Jurkat is more pronounced. Thus, these distinct cis-acting elements play different physiological roles in IL-2R alpha gene activation in MT-2 cells and PMA-induced Jurkat T cells. These studies provide direct evidence for a functionally significant SRE-like sequence in a gene other than c-fos and the actin genes and identify other elements that are critical for IL-2R alpha gene expression.

Base Sequence↗

PubMed1988

The cytoskeleton of the cultured human trabecular cell. Characterization and drug responses.

To determine the organization of the three major cytoskeletal elements of cultured human trabecular meshwork cells (actin filaments, microtubules and intermediate filaments), we employed fluorescence microscopy and stereo-transmission electron microscopy of extracted, S-1 labeled and critical-point dried cells. Morphologic changes resulting from treatment with cytochalasin B, colchicine, taxol and nocodozole were also characterized. Compared with the cynomolgus monkey trabecular cell, morphologic differences in overall cell shape and orientation of both actin filaments and microtubules were noted. However, the responses to cytoskeletal active drugs were quite similar. Taxol, nocodozole and colchicine had a marked effect on microtubule organization, while nocodozole and colchicine had a marked effect on vimentin filament organization. None of these drugs produced marked changes in human trabecular cell shape. In contrast, treatment with the anti-actin drug cytochalasin B resulted in both a marked change in cell shape associated with organizational changes in all three cytoskeletal elements. These studies suggest a central role of actin filaments in determining overall cell shape and cytoskeletal organization in the cultured human trabecular cell.

Alkaloids↗

PubMed1986

DNA sequences required for expression of a Dictyostelium actin gene.

A 2.8-kb fragment of 5' non-coding DNA from the Dictyostelium actin 15 gene has previously been shown to contain all of the cis-acting DNA sequence elements required for normal developmentally-regulated transcription of actin gene fusion RNAs when reintroduced into the genome by DNA-mediated transformation. Deletion analysis of this promoter fragment indicates that all of the necessary information is contained within a 270-bp fragment of actin 15 5' non-coding DNA. This fragment contains four short G/C-rich repeated sequences that are also found in other co-regulated Dictyostelium actin genes. A 12-bp consensus sequence, AAAAATGGGG/ATT, is present in the regions essential for expression of two different Dictyostelium actin genes, actin 6 and actin 15, but is absent from an actin gene showing a different temporal pattern of developmental regulation. Deletion analysis and DNase I footprinting implicate this sequence as a functional cis-acting element required for transcription of the actin 15 gene.

Actins↗

PubMed1999

Phenotype-dependent expression of alpha-smooth muscle actin in visceral smooth muscle cells.

Alpha-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, alpha-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (-238 to -219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of alpha-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors.

Actins↗

PubMed1983

Preparation and properties of a complex from rat liver of polyribosomes with components of the cytoskeleton.

Gel filtration with 1% agarose (Bio-Gel A-150m) separates polyribosomes bound to microsomal membranes from 'free' polyribosomes when these fractions are prepared by standard centrifugal techniques. However, when polyribosomes contained in an unfractionated postmitochondrial supernatant are run on an identical column, over 90% of the total polyribosomes are present as aggregates, designated 'membrane-cytomatrix', which are eluted in the column void volume. Polyribosomes are not released from these aggregates on removal of microsomal phospholipids by treatment of postmitochondrial supernatant with 1% Triton X-100, a neutral detergent. The aggregates are disrupted by the usual ultracentrifugation techniques used in subcellular fractionation. After treatment of membrane-cytomatrix with Triton X-100 to remove phospholipids and membrane proteins, 58% of the polyribosomes still remain associated with protein-containing complexes in the form of a cytomatrix and are not 'free'. Preparations of both membrane-cytomatrix and cytomatrix are capable of sustained protein synthesis. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the cytoskeletal proteins actin and myosin are present in the cytomatrix. Incubation of cytomatrix preparations with the actin-depolymerizing agent deoxyribonuclease I caused release of the polyribosomes. Polyribosome release by deoxyribonuclease I was prevented by prior incubation with phalloidin, which is known to stabilize F-actin. Thus polyribosomes are associated with cytoskeletal elements in rat liver, and this association is dependent on polymeric forms of actin.

Animals↗

PubMed1998

Characterization of glial filament-cytoskeletal interactions in human astrocytomas: an immuno-ultrastructural analysis.

The role that glial filaments play in cells and tumors of glial origin is not well understood. We therefore undertook the present study to determine the relationships between glial and vimentin intermediate filaments (IFs), actin microfilaments, and CD44, a cell surface glycoprotein important in cell migration and invasion, in human astrocytoma cells. Three astrocytoma cell lines, U343 MG-A (U343), U251 MG (U251), and antisense GFAP-transfected U251 (asU251) were studied using immunofluorescence confocal and immunoelectron microscopy. Furthermore, we studied the phenotypic behaviour of these astrocytoma cell lines by analyzing their migration through Matrigel in vitro. U343 astrocytoma cells had the highest expression levels of glial fibrillary acidic protein (GFAP), whereas asU251 had virtually no expression of GFAP. Parental U251 cells had intermediate expression levels of GFAP. The elimination of GFAP expression in as U251 cells was accompanied by a marked increase in vimentin, actin microfilaments and CD44 levels. Gold labeling density counts of cytoskeletal and cell surface elements demonstrated that the differences between GFAP, actin, CD44 and vimentin levels in the different astrocytoma cell lines were statistically significant (p < 0.05). Results from the in vitro invasion assay revealed that U343 cells demonstrated the least invasive potential, whereas asU251 astrocytoma cells demonstrated the most. Our results show that elimination of GFAP expression by antisense leads to marked alterations in cell morphology and phenotypic behaviour. These data imply that GFAP may be linked spatially and functionally to cytoskeletal elements which may be altered when this IF is deleted in astrocytomas.

Astrocytoma↗

PubMed2006

Permselective dysfunction of podocyte-podocyte contact upon angiotensin II unravels the molecular target for renoprotective intervention.

Ameliorating the function of the glomerular barrier to circulating proteins by blocking angiotensin II (Ang II) translates into less risk of progression toward end-stage renal failure in diabetic and nondiabetic nephropathies. However, the mechanisms underlying this barrier protection are not clear. Specialized contacts between adjacent podocytes are major candidate targets, and the actin cytoskeleton is emerging as a regulatory element. Here, we present data demonstrating that Ang II induced reorganization of F-actin fibers and redistribution of zonula occludens-1 (ZO-1) that is physically associated with actin in murine podocytes. These effects were paralleled by increased albumin permeability across podocyte monolayers. The F-actin stabilizer jasplakinolide prevented both ZO-1 redistribution and albumin leakage, suggesting that actin cytoskeleton rearrangement is instrumental to podocyte permselective dysfunction induced by Ang II. Changes in both F-actin and ZO-1 patterns were confirmed in glomeruli of rat isolated perfused kidneys on short infusion of Ang II, leading to increased protein excretion. Podocyte dysfunction was mediated by Ang II type 1 receptor and was partly dependent on Src kinase-phospholipase C activation. These data demonstrate that strategies aimed at stabilizing podocyte-podocyte contacts and targeting the relevant intracellular signal transduction are crucial to renoprotection.

Actins↗

PubMed1975

An ultrastructural study of the microfilaments in rat brain by means of heavy meromyosin labeling. I. The perikaryon, the dendrites and the axon.

Nervous tissue pieces from the caudate nucleus and the substantia nigra of the rat were incubated in cold glycerol solutions of decreasing concentrations and then transferred into standard phosphate buffer (pH 7.0) or into tris-K+-Mg++-Ca++ buffer (pH 7.9) containing HMM, prepared from rabbit skeletal muscle by tryptic digestion. As controls, pieces were immersed for an identical period in the same buffers (1) without HMM or (2) with HMM to which had been added 2.5 mM Na+ pyrophosphate or 5 mM ATP. In control neurons smooth-surfaced microfilaments, about 50 A in diameter, were observed. After reaction with HMM, the microfilaments were increased in number and density and in width to 180-200 A. A meshwork was formed. Arrowheads pointing in the same direction were spaced at regular intervals (300-350 A) among short segments of the surfaces of the microfilaments, depending upon the plane of section. More often, however, typical arrowheads were not observed, and the surfaces of the microfilaments were seen coated with polarized side-arms cross-bridging the spaces between adjacent elements at more or less regular intervals. When cross-sectioned, the microfilaments appeared as dense dots from which a material of lesser electron density radiated. Following incubation in HMM solutions containing Na+ pyrophosphate or ATP, no arrowhead structures were seen. Of particular interest was the structural relation of the actin-like filaments with occasional, tapered myosin-like filaments, and with the plasma membrane, which served as anchor points. Mitochondria and smooth ER membranes were observed to be attached to the actin-like filaments or enmeshed in the network. The microtubules, as well as most of the neurofilaments, were disrupted by the glycerination procedure at 4 degrees, and thus no precision about the structural relationship of the actin-like filaments with the latter elements could be added. The role of the actin-like filaments in the transport of material, by a mechanism of chemomechanical transduction, throughout the neuron from sites of synthesis to functional locations, and between several functional locations, is discussed.

Actins↗

PubMed1998

Activation of the skeletal alpha-actin promoter during muscle regeneration.

Little is known concerning promoter regulation of genes in regenerating skeletal muscles. In young rats, recovery of muscle mass and protein content is complete within 21 days. During the initial 5-10 days of regeneration, mRNA abundance for IGF-I, myogenin and MyoD have been shown to be dramatically increased. The skeletal alpha-actin promoter contains E box and serum response element (SRE) regulatory regions which are directly or indirectly activated by myogenin (or MyoD) and IGF-I proteins, respectively. We hypothesized that the skeletal alpha-actin promoter activity would increase during muscle regeneration, and that this induction would occur before muscle protein content returned to normal. Total protein content and the percentage content of skeletal alpha-actin protein was diminished at 4 and 8 days and re-accumulation had largely occurred by 16 days post-bupivacaine injection. Skeletal alpha-actin mRNA per whole muscle was decreased at day 8, and thereafter returned to control values. During regeneration at day 8, luciferase activity (a reporter of promoter activity) directed by -424 skeletal alpha-actin and -99 skeletal alpha-actin promoter constructs was increased by 700% and 250% respectively; however, at day 16, skeletal alpha-actin promoter activities were similar to control values. Thus, initial activation of the skeletal alpha-actin promoter is associated with regeneration of skeletal muscle, despite not being sustained during the later stages of regrowth. The proximal SRE of the skeletal alpha-actin promoter was not sufficient to confer a regeneration-induced promoter activation, despite increased serum response factor protein binding to this regulatory element in electrophoretic mobility shift assays. Skeletal alpha-actin promoter induction during regeneration is due to a combination of regulatory elements, at least including the SRE and E box.

Actins↗

PubMed1995

Single-molecule mechanics of heavy meromyosin and S1 interacting with rabbit or Drosophila actins using optical tweezers.

Single-molecule mechanical interactions between rabbit heavy meromyosin (HMM) or subfragment 1 (S1) and rabbit actin were measured with an optical tweezers piconewton, nanometer transducer. Similar intermittent interactions were observed with HMM and S1. The mean magnitude of the single interaction isotonic displacements was 20 nm for HMM and 15 nm with S1. The mean value of the force of single-molecule interactions was 1.8 pN for HMM and 1.7 pN with S1. The stiffness of myosin S1 was determined by applying a sinusoidal length change to the thin filament and measuring the corresponding force; the mean stiffness was 0.13 pN nm-1. By moving an actin filament over a long distance past an isolated S1 head, we found that cross-bridge attachment occurred preferentially at a periodicity of about 40 nm, similar to that of the actin helical repeat. Rate constants for the probability of detachment of HMM from actin were determined from histograms of the lifetime of the attached state. This gave a value of 8 s-1 or 0.8 x 10(6) M-1 s-1 for binding of ATP to the rigor complex. We conclude (1) that our HMM-actin interactions involve just one head, (2) that compliance of the cross-bridge is not in myosin subfragment 2, although we cannot say to what extent contributions arise from myosin S1 or actin, and (3) that the elemental movement can be caused by a change of shape of the S1 head, but that this would have to be much greater than the movements suggested from structural studies of S1 (Rayment et al., 1993).

Actins↗

PubMed1986

Thymic B lymphocyte clones from patients with myasthenia gravis secrete monoclonal striational autoantibodies reacting with myosin, alpha actinin, or actin.

Striational autoantibodies (StrAb), which react with elements of skeletal muscle cross-striations, occur frequently in patients with thymoma associated with myasthenia gravis (MG). Dissociated thymic lymphocytes from 22 of 72 MG patients secreted StrAb when cultured with PWM. A high yield of EBV-transformed B cell lines was established from thymus, thymoma, and peripheral blood of seven patients with MG, but clones secreting StrAb arose only from the three patients who had StrAb in their sera. The monoclonal StrAb bound to A bands or I bands in skeletal muscle of human, rat, and frog. One bound to mitochondria in addition to myofibrillar I bands. None bound to nuclei, smooth muscle, or gastric mucosal cells. In immunoblot analyses and ELISAs the monoclonal StrAb bound to muscle and nonmuscle isotypes of myosin, alpha actinin, and/or actin. All bound to contractile proteins common to thymus and muscle, and one selectively immunostained epithelial cells of the thymic medulla. From these antigenic specificities we suggest that StrAb might arise as an immune response directed against the cytoskeletal anchoring proteins associated with nicotinic acetylcholine receptors in thymic epithelial cells undergoing neoplastic transformation to thymoma.

Actinin↗

PubMed1998

The role of cadherin endocytosis in endothelial barrier regulation: involvement of protein kinase C and actin-cadherin interactions.

We have previously reported that exposure of endothelial monolayers to low (0.12 mM) extracellular calcium significantly decreased the endothelial solute barrier, and that this effect was reversed by restoring 'normal' (1.2 mM) calcium (1). This effect was shown to be dependent on cadherins, however the molecular mechanisms through which barrier was altered by low calcium were not characterized. Here we investigated the mechanism of increased endothelial permeability produced by low calcium exposure. Endothelial permeability was significantly increased by exposure to low (0.12 mM) calcium; this effect was attenuated by pre-treatment with the protein kinase C (PKC) inhibitor, staurosporine (2 x 10(-7) M) for 30 min. Cell border retraction and gap formation produced by low calcium was also prevented by staurosporine. Treatment of monolayers with 0.12 mM calcium also stimulated the endocytosis of endothelial cadherins. This low calcium mediated cadherin endocytosis was also prevented by pretreatment with staurosporine. Low calcium mediated endocytosis was also prevented by the actin filament toxin, cytochalasin D (1 ug/ml, 30 min). We conclude that the mechanism of low calcium mediated loss of endothelial barrier function is mediated in part by a PKC dependent endocytosis of endothelial cadherins, which may involve interactions with the actin cytoskeleton. Physiological regulation of the in vivo endothelial barrier may also involve PKC dependent-actin mediated endocytosis of cadherin junctional elements.

Actins↗

PubMed1989

Functional serum response elements upstream of the growth factor-inducible gene zif268.

The zif268 gene, which encodes a protein with three typical zinc finger sequences, is induced in mouse 3T3 cells by serum, phorbol 12-myristate 13-acetate platelet-derived growth factor, and fibroblast growth factor. The induction is coordinate with that of c-fos. The 5'-flanking region of zif268 contains sequences that resemble known regulatory elements, including four CC(A or T)6GG sequences similar to the core serum response elements (SREs) found upstream of c-fos and actin genes. To determine whether the zif268 SRE-like elements mediate induction, CAT (chloramphenicol acetyltransferase) plasmids with different lengths of zif268 upstream sequences were tested for inducibility in 3T3 cells by serum, platelet-derived growth factor, or phorbol 12-myristate 13-acetate. In addition, double-stranded oligonucleotides corresponding to each of the four zif268 putative SREs were tested individually for responsiveness when placed upstream of a thymidine kinase gene promoter. Each of the four SREs conferred inducibility by the agents tested, and multiple SREs resulted in greater inducibility than did a single element. Each of the zif268 SREs also competed with the c-fos SRE for binding by serum response factor present in HeLa cell nuclear extract. We conclude that the zif268 SRE-like sequences are functional and probably account for the coordinate induction of zif268 and c-fos.

Animals↗

PubMed1998

Osteocyte shape is dependent on actin filaments and osteocyte processes are unique actin-rich projections.

Osteocytes are derived from a select group of osteoblasts that have undergone a final differentiation. Due to their inaccessibility when embedded in the bone matrix, very little is known about the osteocyte cytoskeleton. This study provides an extensive analysis of the osteocyte cytoskeleton, based on the successful isolation of osteocytes from 16-day embryonic chick calvariae. We used OB7.3, a chicken osteocyte-specific monoclonal antibody, to confirm the osteocytic phenotype of the isolated cells and established culture conditions to promote growth of cells that most resemble osteocytes in vivo. Immunofluorescence staining with antitubulin, antivimentin, and antiactin showed the relative distribution of the microtubules, intermediate filaments, and actin filaments in both osteocyte cell body and processes. Field emission scanning electron microscopy revealed the three-dimensional relationships of the cytoskeletal elements and a unique organization of actin bundles that spanned the cell body and osteocyte processes. When combined with drug studies, these experiments demonstrate that actin filaments are crucial for the maintenance of osteocyte shape. Furthermore, we identified two actin-bundling proteins, alpha-actinin and fimbrin, in osteocyte processes. The prominence and unique distribution of fimbrin in osteocyte processes provides the possibility of its use as an intracellular marker to distinguish osteocytes from osteoblasts.

Actinin↗