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PubMed2005

Bone turnover mediates preferential localization of prostate cancer in the skeleton.

Bone metastasis is a common untreatable complication associated with prostate cancer. Metastatic cells seed in skeletal sites under active turnover containing dense marrow cellularity. We hypothesized that differences in these skeletal-specific processes are among the critical factors that facilitate the preferential localization of metastatic prostate cancer in bone. To test this, athymic mice were administered PTH to induce bone turnover and increase marrow cellularity daily 1 wk before and after intracardiac inoculation of luciferase-tagged PC-3 cells. Tumor localization was monitored by bioluminescence imaging weekly for 5 wk. At the time of tumor inoculation, PTH-treated mice demonstrated significant increases in serum levels of bone turnover markers such as osteocalcin and tartrate-resistant acid phosphatase 5b and in the number of tartrate-resistant acid phosphatase-positive osteoclasts per millimeter of bone when compared with the other groups. Likewise, PTH treatment stimulated a qualitative increase in marrow cellular proliferation as determined by 5-bromo-2'-deoxyuridine immunostaining. Skeletal metastases formed in the hind limb and craniofacial regions of young mice with no difference between groups. In adult mice, however, bioluminescent signals in the hind limb and craniofacial regions were 3-fold higher in PTH-treated mice vs. controls. Fluorochrome labeling revealed increased bone formation activity in trabecular bone adjacent to tumors. When zoledronic acid, a nitrogen-containing bisphosphonate that inhibits osteoclast-mediated bone resorption, was administered concurrently with PTH, a significant reduction in the incidence of bone tumors was observed. Overall, these studies provide new evidence that skeletal sites rich in marrow cellularity under active turnover offer a more congenial microenvironment to facilitate cancer localization in the skeleton.

Acid Phosphatase↗
PubMed1984

Aging and dietary modulation of rat skeleton and parathyroid hormone.

Studies were carried out on SPF F344 male rats to evaluate the effects of aging and life-prolonging food restriction, without malnutrition, on rat skeleton and circulating PTH. Six-week-old F344 rats were divided into five groups. Group 1 rats were fed ad libitum a diet that contained 21% protein. Group 2 rats were fed 60% of the mean food intake of group 1 rats from 6 weeks of age for the rest of their lives. Group 3 rats were fed 60% of the ad libitum food intake until 6 months of age and then switched to ad libitum feeding. Group 4 rats were fed ad libitum until 6 months of age, and then switched to 60% of the ad libitum food intake. Group 5 rats were fed ad libitum a diet that contained only 12.6% protein so that these animals ingested the same amount of protein per day as the group 2 rats. In group 1 animals, bone length, weight, density, and calcium content increased rapidly with age and plateaued at about 12 months of age. There was no evidence of bone loss in these animals until about 24 months of age, but by 27 months, the animals had lost appreciable amounts of bone. The circulating immunoreactive PTH levels of the animals increased with advancing age, with a marked rise at 27 months. The age-related changes in bone and serum PTH levels of rats in groups 3 and 5 were similar to those of group 1 animals, except that a terminal increase in serum PTH did not occur in group 5 rats. In the groups 2 and 4 animals which were food restricted for the longest period, bone growth and maturation were slowed down, but the animals did not experience senile bone loss or marked terminal increase in circulating PTH. The salutary effects of food restriction were, therefore, not due specifically to the restriction of protein intake or to restricting food intake only during the period of rapid growth.

Aging↗
PubMed1993

The effect of recombinant human (1-84) or synthetic human (1-34) parathyroid hormone on the skeleton of adult osteopenic ovariectomized rats.

The purpose of this study was to evaluate the dose-effect relationship of recombinant human PTH [hPTH-(1-84)] and synthetic human PTH [sPTH-(1-34)] for the skeleton of estrogen-deplete osteopenic rats. Ex vivo densitometry of regionalized whole femurs and histomorphometry of proximal tibial cancellous bone were the end points. Retired breeder female rats, aged 6-7 months, were used. Ten were killed at baseline, and the rest were ovariectomized (OVX). On day 42, a pre-PTH treatment OVX group was killed. The rest were then treated by daily sc injection with hPTH (0, 1.55, 15.5, or 155 micrograms/kg BW.day) or sPTH (0.55, 5.5, or 55 micrograms/kg BW.day) and killed on day 70. The level of cancellous bone mineral was lower in pretreatment OVX rats than at baseline. It was higher in rats treated with 15.5-155 micrograms/kg.day hPTH and 5.5-55 micrograms/kg.day sPTH than in pretreatment and vehicle-treated OVX rats. Cancellous bone volume was also lower both 42 and 70 days after OVX. Although hPTH did not affect cancellous bone volume, treatment with 5.5-55 micrograms/kg.day sPTH caused higher bone volume than in either pretreatment or vehicle-treated OVX rats. Trabecular number declined after OVX and did not change with PTH treatment. In contrast, trabecular thickness declined after OVX, but was higher after 15.5-155 micrograms/kg.day hPTH and 5.5-55 micrograms/kg.day sPTH treatment. In OVX rats, the amount of mineralizing surface was greater by day 42 and fell toward control levels by day 70. It was greater in rats treated with 15.5-155 micrograms/kg.day hPTH or 5.5-55 micrograms/kg.day sPTH than in vehicle-treated OVX rats. On a molar basis, sPTH was modestly more potent than hPTH. Osteoclast surface was not affected by PTH treatment. Treating estrogen-deplete osteopenic adult rats for 28 days with 15.5-155 micrograms/kg.day hPTH or 5.5-55 micrograms/kg.day sPTH increases trabecular thickness, but not trabecular number, to cause a rise in bone mass. The extent of mineralizing surface rises without a change in resorption surface. The marked rise in mineralizing surfaces suggests that extended in vivo treatment with PTH activates osteogenic precursor cells near once quiescent surfaces to become osteoblasts.

Animals↗
PubMed2004

In boys with abnormal developmental tempo, maturation of the skeleton and the hypothalamic-pituitary-gonadal axis remains synchronous.

The primary mechanism that initiates puberty is unknown. One possible clue is that pubertal maturation often parallels skeletal maturation. Conditions that delay skeletal maturation also tend to delay the onset of puberty, whereas conditions that accelerate skeletal maturation tend to hasten the onset of puberty. To examine this relationship, we studied boys with congenital adrenal hyperplasia (n = 13) and familial male-limited precocious puberty (n = 22), two conditions that accelerate maturational tempo, and boys with idiopathic short stature (n = 18) in which maturational tempo is sometimes delayed. In all three conditions, the onset of central puberty generally occurred at an abnormal chronological age but a normal bone age. Boys with the greatest skeletal advancement began central puberty at the earliest age, whereas boys with the greatest skeletal delay began puberty at the latest age. Furthermore, the magnitude of the skeletal advancement or delay matched the magnitude of the pubertal advancement or delay. This synchrony between skeletal maturation and hypothalamic-pituitary-gonadal axis maturation was observed among patients within each condition and also between conditions. In contrast, the maturation of the hypothalamic-pituitary-gonadal axis did not remain synchronous with other maturational processes including weight, height, or body mass index. We conclude that in boys with abnormal developmental tempo, maturation of the skeleton and the hypothalamic-pituitary-gonadal axis remains synchronous. This synchrony is consistent with the hypothesis that in boys, skeletal maturation influences hypothalamic-pituitary-gonadal axis maturation.

Adolescent↗
PubMed2005

A densitometric and morphometric analysis of the skeleton in adults with varying degrees of growth hormone deficiency.

CONTEXT: Low bone mass is a characteristic feature of the adult GH deficiency (GHD) syndrome, but recent dual-energy x-ray absorptiometry (DXA) studies in patients with GH-receptor and GHRH-receptor gene mutations suggest that the situation is more complex. OBJECTIVE: The objective was to define bone areal and volumetric densities and morphometry in hypopituitary adults. DESIGN: The study was a cross-sectional case-controlled study performed between 1999 and 2001. SETTING: The study was undertaken at an endocrine tertiary referral center. PATIENTS: Thirty patients with GHD, 24 with GH insufficiency (GHI) [peak GH, 3-7 microg/liter (9-21 mU/liter)], and 30 age- and sex-matched controls were included for study. MAIN OUTCOME MEASURES: DXA and peripheral quantitative computed tomography (pQCT) derived bone density and morphometry were measured. RESULTS: No densitometric or morphometric abnormalities were detected in GHD patients who acquired their deficiency during adult life. GHD adults of childhood-onset (CO-GHD) showed decreased bone mineral density at the lumbar spine and hip on DXA. pQCT of the radius showed that CO-GHD patients have normal trabecular bone mineral density and only a 2% decrease in cortical density. Radial bone area was reduced 14.5%, cortical thickness 20%, and cortical cross-sectional area 23%, culminating in a reduction in cortical bone of 25%. The "apparent" low DXA bone density in CO-GHD adults therefore relates primarily to reduced cortical thickness and smaller bone area. DXA and pQCT data derived from adults with GHI revealed no evidence of densitometric or morphometric abnormalities. CONCLUSIONS: 1) Adult-onset GHD patients have normal bone density and size. 2) CO-GHD adults have marginally reduced cortical density but significantly reduced cortical bone as a result of reduced cortical thickness and bone size. 3) GHI has no measurable impact on the skeleton.

Absorptiometry, Photon↗
PubMed2004

Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton.

Indian hedgehog (Ihh) is indispensable for development of the osteoblast lineage in the endochondral skeleton. In order to determine whether Ihh is directly required for osteoblast differentiation, we have genetically manipulated smoothened (Smo), which encodes a transmembrane protein that is essential for transducing all Hedgehog (Hh) signals. Removal of Smo from perichondrial cells by the Cre-LoxP approach prevents formation of a normal bone collar and also abolishes development of the primary spongiosa. Analysis of chimeric embryos composed of wild-type and Smo(n/n) cells indicates that Smo(n/n) cells fail to contribute to osteoblasts in either the bone collar or the primary spongiosa but generate ectopic chondrocytes. In order to assess whether Ihh is sufficient to induce bone formation in vivo, we have analyzed the bone collar in the long bones of embryos in which Ihh was artificially expressed in all chondrocytes by the UAS-GAL4 bigenic system. Although ectopic Ihh does not induce overt ossification along the entire cartilage anlage, it promotes progression of the bone collar toward the epiphysis, suggesting a synergistic effect between ectopic Ihh and endogenous factors such as the bone morphogenetic proteins (BMPs). In keeping with this model, Hh signaling is further found to be required in BMP-induced osteogenesis in cultures of a limb-bud cell line. Taken together, these results demonstrate that Ihh signaling is directly required for the osteoblast lineage in the developing long bones and that Ihh functions in conjunction with other factors such as BMPs to induce osteoblast differentiation. We suggest that Ihh acts in vivo on a potential progenitor cell to promote osteoblast and prevent chondrocyte differentiation.

Animals↗
PubMed2005

A novel G protein-coupled receptor, related to GPR4, is required for assembly of the cortical actin skeleton in early Xenopus embryos.

As the fertilized Xenopus egg undergoes sequential cell divisions to form a blastula, each cell develops a network of cortical actin that provides shape and skeletal support for the whole embryo. Disruption of this network causes loss of shape and rigidity of the embryo, and disrupts gastrulation movements. We previously showed that lysophosphatidic acid (LPA) signaling controls the change in cortical actin density that occurs at different stages of the cell cycle. Here, we use a gain-of-function screen, using an egg cDNA expression library, to identify an orphan G protein-coupled cell-surface receptor (XFlop) that controls the overall amount of cortical F-actin. Overexpression of XFlop increases the amount of cortical actin, as well as embryo rigidity and wound healing, whereas depletion of maternal XFlop mRNA does the reverse. Both overexpression and depletion of XFlop perturb gastrulation movements. Reciprocal rescue experiments, and comparison of the effects of their depletion in early embryos, show that the XLPA and XFlop signaling pathways play independent roles in cortical actin assembly, and thus that multiple signaling pathways control the actin skeleton in the blastula.

Actins↗
PubMed1994

The role of Pax-1 in axial skeleton development.

Previous studies have identified a single amino-acid substitution in the transcriptional regulator Pax-1 as the cause of the mouse skeletal mutant undulated (un). To evaluate the role of Pax-1 in the formation of the axial skeleton we have studied Pax-1 protein expression in early sclerotome cells and during subsequent embryonic development, and we have characterized the phenotype of three different Pax-1 mouse mutants, un, undulated-extensive (unex) and Undulated short-tail (Uns). In the Uns mutation the whole Pax-1 locus is deleted, resulting in the complete absence of Pax-1 protein in these mice. The other two genotypes are interpreted as hypomorphs. We conclude that Pax-1 is necessary for normal vertebral column formation along the entire axis, although the severity of the phenotype is strongest in the lumbar region and the tail. Pax-1-deficient mice lack vertebral bodies and intervertebral discs. The proximal part of the ribs and the rib homologues are also missing or severely malformed, whereas neural arches are nearly normal. Pax-1 is thus required for the development of the ventral parts of vertebrae. Embryonic analyses reveal that although sclerotomes are formed in mutant embryos, abnormalities can be detected from day 10.5 p.c. onwards. The phenotypic analyses also suggest that the notochord still influences vertebral body formation some days after the sclerotomes are formed. Furthermore, the notochord diameter is larger in mutant embryos from day 12 p.c., due to increased cell proliferation. In the strongly affected genotypes the notochord persists as a rod-like structure and the nucleus pulposus is never properly formed. Since the notochord is Pax-1-negative these findings suggest a bidirectional interaction between notochord and paraxial mesoderm. The availability of these Pax-1 mutant alleles permitted us to define an early role for Pax-1 in sclerotome patterning as well as a late role in intervertebral disc development. Our observations suggest that Pax-1 function is required for essential steps in ventral sclerotome differentiation, i.e. for the transition from the mesenchymal stage to the onset of chondrogenesis.

Animals↗
PubMed1996

A role for mel-18, a Polycomb group-related vertebrate gene, during theanteroposterior specification of the axial skeleton.

Segment identity in both invertebrates and vertebrates is conferred by spatially restricted distribution of homeotic gene products. In Drosophila, the expression of Homeobox genes during embryogenesis is initially induced by segmentation gene products and then maintained by Polycomb group and Trithorax group gene products. Polycomb group gene homologs are conserved in vertebrates. Murine mel-18 and closely related bmi-1 are homologous to posterior sex combs and suppressor two of zeste. Mel-18 protein mediates a transcriptional repression via direct binding to specific DNA sequences. To gain further insight into the function of Mel-18, we have inactivated the mel-18 locus by homologous recombination. Mice lacking mel-18 survive to birth and die around 4 weeks after birth after exhibiting strong growth retardation. Similar to the Drosophila posterior sex combs mutant, posterior transformations of the axial skeleton were reproducibly observed in mel-18 mutants. The homeotic transformations were correlated with ectopic expression of Homeobox cluster genes along the anteroposterior axis in the developing paraxial mesoderm. Surprisingly, mel-18-deficient phenotypes are reminiscent of bmi-1 mutants. These results indicate that the vertebrate Polycomb group genes mel-18 and bmi-1, like Drosophila Polycomb group gene products, might play a crucial role in maintaining the silent state of Homeobox gene expression during paraxial mesoderm development.

Animals↗
PubMed2000

Early myotome specification regulates PDGFA expression and axial skeleton development.

Reciprocal defects in signaling between the myotome and the sclerotome compartments of the somites in PDGFRalpha and Myf5 mutant embryos lead to alterations in the formation of the vertebrae and the ribs. To investigate the significance of these observations, we have examined the role of PDGF signaling in the developing somite. PDGFA ligand expression was not detected in the myotome of Myf5 null mutant embryos and PDGFA promoter activity was regulated by Myf5 in vitro. PDGFA stimulated chondrogenesis in somite micromass cultures as well as in embryos when PDGFA was knocked into the Myf5 locus, resulting in increased vertebral and rib development. PDGFA expression in the myotome was fully restored in embryos in which MyoD has been introduced at the Myf5 locus but to a lesser extent in similar myogenin knock-in embryos. These results underscore the importance of growth factor signaling within the developing somite and suggest an important role for myogenic determination factors in orchestrating normal development of the axial skeleton.

Alleles↗
PubMed2001

Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation.

Indian hedgehog (Ihh), one of the three mammalian hedgehog (Hh) proteins, coordinates proliferation and differentiation of chondrocytes during endochondral bone development. Smoothened (Smo) is a transmembrane protein that transduces all Hh signals. In order to discern the direct versus indirect roles of Ihh in cartilage development, we have used the Cre-loxP approach to remove Smo activity specifically in chondrocytes. Animals generated by this means develop shorter long bones when compared to wild-type littermates. In contrast to Ihh mutants (Ihh(n)/Ihh(n)), chondrocyte differentiation proceeds normally. However, like Ihh(n)/Ihh(n) mice, proliferation of chondrocytes is reduced by about 50%, supporting a direct role for Ihh in the regulation of chondrocyte proliferation. Moreover, by overexpressing either Ihh or a constitutively active Smo allele (Smo*) specifically in the cartilage using the bigenic UAS-Gal4 system, we demonstrate that activation of the Ihh signaling pathway is sufficient to promote chondrocyte proliferation. Finally, expression of cyclin D1 is markedly downregulated when either Ihh or Smo activity is removed from chondrocytes, indicating that Ihh regulates chondrocyte proliferation at least in part by modulating the transcription of cyclin D1. Taken together, the present study establishes Ihh as a key mitogen in the endochondral skeleton.

Animals↗
PubMed2002

Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head.

The vertebrate face contains bones that differentiate from mesenchymal cells of neural crest origin, which colonize the median nasofrontal bud and the first branchial arches. The patterning of individual facial bones and their relative positions occurs through mechanisms that remained elusive. During the early stages of head morphogenesis, an endodermal cul-de-sac, destined to become Sessel's pouch, underlies the nasofrontal bud. Reiterative outpocketings of the foregut then form the branchial pouches. We have tested the capacity of endoderm of the avian neurula to specify the facial skeleton by performing ablations or grafts of defined endodermal regions. Neural crest cells that do not express Hox genes respond to patterning cues produced regionally in the anterior endoderm to yield distinct skeletal components of the upper face and jaws. However, Hox-expressing neural crest cells do not respond to these cues. Bone orientation is likewise dependent on the position of the endoderm relative to the embryonic axes. Our findings thus indicate that the endoderm instructs neural crest cells as to the size, shape and position of all the facial skeletal elements, whether they are cartilage or membrane bones.

Animals↗
PubMed2003

Archvillin, a muscle-specific isoform of supervillin, is an early expressed component of the costameric membrane skeleton.

The membrane skeleton protein supervillin binds tightly to both F-actin and membranes and can potentiate androgen receptor activity in non-muscle cells. We report that muscle, which constitutes the principal tissue source for supervillin sequences, contains a approximately 250 kDa isoform of supervillin that localizes within nuclei and with dystrophin at costameres, regions of F-actin membrane attachment in skeletal muscle. The gene encoding this protein, 'archvillin' (Latin, archi; Greek, árchos; 'principal' or 'chief'), contains an evolutionarily conserved, muscle-specific 5' leader sequence. Archvillin cDNAs also contain four exons that encode approximately 47 kDa of additional muscle-specific protein sequence in the form of two inserts within the function-rich N-terminus of supervillin. The first of these muscle-specific inserts contains two conserved nuclear targeting signals in addition to those found in sequences shared with supervillin. Archvillin, like supervillin, binds directly to radiolabeled F-actin and co-fractionates with plasma membranes. Colocalization of archvillin with membrane-associated actin filaments, non-muscle myosin II, and--to a lesser extent--vinculin was observed in myoblasts. Striking localizations of archvillin protein and mRNA were observed at the tips of differentiating myotubes. Transfected protein chimeras containing archvillin insert sequences inhibited myotube formation, consistent with a dominant-negative effect during early myogenesis. These data suggest that archvillin is among the first costameric proteins to assemble during myogenesis and that it contributes to myogenic membrane structure and differentiation.

Amino Acid Sequence↗
PubMed1998

Characterisation of two articulins, the major epiplasmic proteins comprising the membrane skeleton of the ciliate Pseudomicrothorax.

Most protists possess a unique membrane skeleton, the epiplasm, which is involved in pattern forming processes of the cell cortex and functions in maintaining cell shape. Articulins, a novel class of cytoskeletal proteins, are major constituents of the epiplasm. We have isolated cDNAs encoding the two major articulins of the ciliate Pseudomicrothorax dubius. Peptide sequence data confirm the identity of the cloned cDNAs encoding articulins 1 and 4. With the data presented here sequence information for all major articulins of ciliates as well as the distantly related euglenoids is available. Sequence comparison of the two newly characterised ciliate articulins with the previously determined sequences of p60, a minor articulin of the same species, and the two euglenoid articulins reveals general sequence principles and uncovers new features of this protein family. The hallmark of articulins is a central core domain of repetitive motifs of alternating valine and proline residues, the VPV-motif. These VPV-motif repeats are either 12-residues, or in some places, six residues long. Positively and negatively charged residues segregate in register with valine and proline positions. The VPV-motif is unique to articulins. The terminal domains flanking the core are generally hydrophobic and contain a series of hexa- or heptapeptide repeats rich in glycine and hydrophobic residues. The sequences of these short repeats are very similar in articulins of the same species but are not conserved between euglenoids and ciliates.

Amino Acid Sequence↗
PubMed1998

Articulins and epiplasmins: two distinct classes of cytoskeletal proteins of the membrane skeleton in protists.

The cortex of ciliates, dinoflagellates and euglenoids comprises a unique structure called the epiplasm, implicated in pattern-forming processes of the cell cortex and in maintaining cell shape. Despite significant variation in the structural organization of their epiplasm and cortex, a novel type of cytoskeletal protein named articulin is the principal constituent of the epiplasm in the euglenoid Euglena and the ciliate Pseudomicrothorax. For another ciliate, Paramecium, epiplasmins, a group of polypeptides with common biochemical properties, are the major constituents of the epiplasm. Using molecular tools and affinity purification we have selected polyclonal antibodies and identified epitopes of monoclonal antibodies that identify epitopes characteristic of articulins and epiplasmins. With these antibodies we have analysed the occurrence of the two types of cytoskeletal proteins in a dinoflagellate, a euglenoid and several ciliates. Our results indicate that both articulins and epiplasmins are present in these organisms, suggesting that both contribute to the organization of the membrane skeleton in protists. Articulins and epiplasmins represent two distinct classes of cytoskeletal proteins, since different polypeptides were labeled by articulin core domain-specific or epiplasmin epitope-specific antibodies in each organism studied. In one case, a polypeptide in Pseudomicrothorax was identified that reacts with both articulin core domain-specific and with anti-epiplasmin monoclonal antibodies; however, the epiplasmin monoclonal antibody epitope was mapped to the C terminus of the polypeptide, well outside the central VPV-repeat core domain that contains the articulin monoclonal antibody epitope and that is the hallmark of the articulins.

Amino Acid Sequence↗
PubMed1986

Proteins of the membrane skeleton in rat Sertoli cells.

Analogues of the alpha, beta and gamma subunits of human spectrin and erythroid protein 4.1 have been detected in rat Sertoli cell primary cultures. Immunofluorescence of permeabilized cells showed that erythroid type spectrin, protein 4.1 and actin co-distribute within the cells as filamentous structures. Fodrin-like molecules were distributed in a diffuse manner, mostly associated with the plasma membrane. Immunoprecipitation and immunoblotting experiments indicated that the polypeptides present in rat Sertoli cells are immunologically related and display molecular weights similar to their analogues in the human erythroid and non-erythroid membrane skeleton.

Actins↗
PubMed1996

Mutually exclusive distribution of the focal adhesion associated proteins and the erythrocyte membrane skeleton proteins in the human fibroblast plasma membrane undercoat.

The ends of stress fibers in the basal portion of cells (basal stress fibers) are anchored to focal adhesions, and stress fibers in the apical part of cells (apical stress fibers) are attached to the apical membrane, forming a structure (the apical plaque; KATOH, K. et al. (1995). Cell Motil. Cytoskel., 31: 177-195) resembling the focal adhesion. In addition to these two sites, stress fibers also make lateral contact with the plasma membrane but little is known about the molecular composition of this type of stress fiber-membrane interaction sites. Several actin-membrane association types are known, each employing a different set of proteins, and the focal adhesion and the erythrocyte membrane skeleton are the best characterized systems. We investigated by immunofluorescence microscopy if there is any morphological basis for the involvement of the erythrocyte membrane proteins in the stress fiber-plasma membrane association sites in cultured human fibroblasts. Our results indicated that fodrin (nonerythrocyte type spectrin) and ankyrin were generally associated with the plasma membrane, but that they were clearly excluded from the focal adhesion, the apical plaque and the stress fiber. Thus, it appears that the spectrin and the integrin based actin-membrane association systems are mutually excluded in the fibroblast membrane undercoat. Protein localization at the lateral stress fiber-membrane association site was also studied. Our data indicated that, while talin, vinculin, paxillin, fibronectin receptor and integrin beta 1 were present at the three stress fiber-membrane association sites, vitronectin receptor and integrin alpha v were absent from the apical plaque and the lateral association site. While the plasma membrane at the focal adhesion adheres tightly to a solid substrate, the cell surface of the apical plaque is free. Although the lateral association site faces the substrate, the molecular composition of this site is similar to that of the apical plaque.

Actin Cytoskeleton↗
PubMed1984

Co-localization of SV40 T antigen and p53 with immunological analogues of microtubule-associated protein-1 on the nuclear skeleton.

Rat SV-3Y1 cells were stained with double immunofluorescence. Treatment of cells with detergent and salt removed about 80% of the antigens and revealed immunofluorescent flecks of the SV40 large T antigen and p53 bound to the nuclear skeleton. These flecks exactly corresponded to the fluorescent spots produced by antibodies against microtubule associated protein-1. The MAP-1 analogues may function in the initiation of DNA synthesis through the interaction with T-antigen and p53.

Animals↗