Intracellular distribution of beta-actin mRNA is polarized in embryonic corneal epithelia

1994 ◽  
Vol 107 (1) ◽  
pp. 105-115
Author(s):  
B. Yeh ◽  
K.K. Svoboda
Keyword(s):  
Cell Nucleus ◽  
Basal Cell ◽  
Cell Membranes ◽  
Intracellular Distribution ◽  
Protein Distribution ◽  
Filamentous Actin ◽  
Beta Actin ◽  
Whole Mount ◽  
Actin Mrna ◽  
Actin Protein

The intracellular distribution of filamentous actin (F-actin), all actin isoforms and beta-actin mRNA were analyzed in whole-mount preparations of freshly isolated corneal epithelia. Filamentous actin distribution was analyzed with fluorescently tagged phalloidin. An antibody that recognizes an epitope on both globular (G-actin) and F-actin was used in an immunohistochemical analysis of actin protein distribution. Whole-mount epithelial tissues were examined with a confocal laser scanning microscope (CLSM). Biotinylated oligonucleotide probes specific for the beta-actin mRNA were used, and visualized with avidin-FITC. The intracellular localization of the beta-actin mRNA was similar to the F-actin protein distribution. In the most apical optical sections of embryonic cornea, actin staining delineated the cell borders and microvilli of the periderm cells. The actin is also detected as an organized network at the interface between the basal and periderm cells. At the level of the basal cell nucleus, F-actin is sparse, associating only with the lateral cell membranes. However, at the optical plane below the nuclei, the actin forms an elaborate actin cortical mat. Actin mRNA staining was visualized as discrete punctate areas. The beta-actin mRNA was positive at the optical plane just below the periderm cell apical membrane surface, similar to actin in microvilli. These cells also contained punctate staining near the cell membranes and in the periderm-basal cell junction area. At the level of the basal cell nucleus the actin mRNA was present in a punctate pattern along the cell membranes. Below the basal cell nuclei the actin mRNA staining increased at the level of the actin cortical mat. These experiments are the first demonstration that actin mRNA is polarized in embryonic corneal epithelia and co-localized with actin protein in an intact tissue.

scholarly journals Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

1992 ◽  
Vol 117 (4) ◽  
pp. 787-797 ◽  
Author(s):  
C Lloyd ◽  
G Schevzov ◽  
P Gunning
Keyword(s):  
Actin Cytoskeleton ◽  
Feedback Regulation ◽  
Cytochalasin D ◽  
Actin Gene ◽  
Down Regulation ◽  
Actin Genes ◽  
Beta Actin ◽  
Actin Mrna ◽  
Actin Protein

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.

Polyamine deficiency causes reorganization of F-actin and tropomyosin in IEC-6 cells

1994 ◽  
Vol 267 (3) ◽  
pp. C715-C722 ◽  
Author(s):  
S. A. McCormack ◽  
J. Y. Wang ◽  
L. R. Johnson
Keyword(s):  
Actin Filaments ◽  
Cytoskeletal Proteins ◽  
Stress Fibers ◽  
Filamentous Actin ◽  
Rhodamine Phalloidin ◽  
Tropomyosin Isoforms ◽  
Actin Cortex ◽  
Beta Actin ◽  
Actin Mrna ◽  
As Stress

In earlier work we have shown that polyamine-deficient IEC-6 cells lose most of their ability to migrate. In this report we describe the effect of polyamine deficiency on the cytoskeleton of migrating IEC-6 cells. Cells were grown on cover slips for 4 days. One-third of the monolayer was removed, and the remainder was incubated for 6 h. The monolayers were fixed and stained with rhodamine phalloidin for actin filaments and by immunocytochemistry for tropomyosin. In control cells, actin filaments were found as stress fibers traversing the cell, in a thin actin cortex often visible on only one edge of the cell, and in fine fibers extending into the lamellipodia. Tropomyosin was found in the same distribution. A Western blot showed that tropomyosin was present as 35- and 37-kDa isoforms. In polyamine-deficient cells, actin stress fibers were less dense, whereas the actin cortex was greatly increased in density and lamellipodia were less extensive. Tropomyosin distribution was similar and included a 30-kDa isoform not seen previously. In spite of the obvious changes in the distribution of these cytoskeletal proteins, the concentrations of filamentous actin, beta-actin mRNA, and the higher molecular weight tropomyosin isoforms did not change. In all cases the addition of putrescine to polyamine-deficient cells prevented the changes described. We conclude that polyamines are essential for migration in this system because of their effects on the organization of cytoskeletal actin, tropomyosin, and perhaps other proteins as well.

scholarly journals Beta and gamma actin mRNAs are differentially located within myoblasts

1993 ◽  
Vol 122 (4) ◽  
pp. 825-832 ◽  
Author(s):  
MA Hill ◽  
P Gunning
Keyword(s):  
Steady State ◽  
Protein Function ◽  
Intracellular Localization ◽  
Cell Movement ◽  
Cell Periphery ◽  
Amino Terminal ◽  
Beta Actin ◽  
Nonmuscle Cells ◽  
Actin Mrna ◽  
Actin Protein

Actin is of fundamental importance to all eukaryotic cells. Of the six mammalian actins, beta (beta) and gamma (gamma) cytoplasmic are the isoforms found in all nonmuscle cells and differ by only four amino acids at the amino-terminal region. Both genes are regulated temporally and spatially, though no differences in protein function have been described. Using fluorescent double in situ hybridization we describe the simultaneous intracellular localization of both beta and gamma actin mRNA. This study shows that myoblasts differentially segregate the beta and gamma actin mRNAs. The distribution of gamma actin mRNA, only to perinuclear and nearby cytoplasm, suggests a distribution based on diffusion or restriction to nearby cytoplasm. The distribution of beta actin mRNA, perinuclear and at the cell periphery, implicates a peripheral localizing signal which is unique to the beta isoform. The peripheral beta actin mRNA corresponded to cellular morphologies, extending processes, and ruffling edges that reflect cell movement. Total actin and gamma actin protein steady-state distributions were identified by specific antibodies. gamma actin protein was found in both stress fibers and at the cell plasma membrane and does not correspond to its mRNA distribution. We suggest that localized protein synthesis rather than steady-state distribution functionally differentiates between the actin isoforms.

scholarly journals Characterization of a beta-actin mRNA zipcode-binding protein.

1997 ◽  
Vol 17 (4) ◽  
pp. 2158-2165 ◽  
Author(s):  
A F Ross ◽  
Y Oleynikov ◽  
E H Kislauskis ◽  
K L Taneja ◽  
R H Singer
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Affinity Purification ◽  
Nuclear Export Signal ◽  
Degenerate Primers ◽  
Band Shift ◽  
Beta Actin ◽  
Actin Mrna

Localization of beta-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' untranslated region of the mRNA, including a 54-nucleotide element which has been termed the "zipcode" (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize beta-actin mRNA.

Novel chicken actin gene: third cytoplasmic isoform

1985 ◽  
Vol 5 (5) ◽  
pp. 1151-1162
Author(s):  
D J Bergsma ◽  
K S Chang ◽  
R J Schwartz
Keyword(s):  
Nucleotide Sequence ◽  
Single Copy ◽  
Actin Gene ◽  
Single Copy Gene ◽  
Gene Diversification ◽  
Mrna Transcripts ◽  
Copy Gene ◽  
Actin Mrna ◽  
Distinct Member ◽  
Actin Protein

We identified a novel chicken actin gene. The actin protein deduced from its nucleotide sequence very closely resembles the vertebrate cytoplasmic actins; accordingly, we classified this gene as a nonmuscle type. We adopted the convention for indicating the nonmuscle actins of the class Amphibia (Vandekerckhove et al., J. Mol. Biol. 152:413-426) and denoted this gene as type 5. RNA blot analysis demonstrated that the type 5 actin mRNA transcripts accumulate in adult tissues in a pattern indicative of a nonmuscle actin gene. Genomic DNA blots indicated that the type 5 actin is a single copy gene and a distinct member of the chicken actin multigene family. Inspection of the nucleotide sequence revealed many features that distinguished the type 5 gene from all other vertebrate actin genes examined to date. These unique characteristics include: (i) an initiation Met codon preceding an Ala codon, a feature previously known only in plant actins, (ii) a single intron within the 5' untranslated region, with no interruptions in the coding portion of the gene, and (iii) an atypical Goldberg-Hogness box (ATAGAA) preceding the mRNA initiation terminus. These unusual features have interesting implications for actin gene diversification during evolution.

An amphibian cytoskeletal-type actin gene is expressed exclusively in muscle tissue

Development ◽  
1987 ◽  
Vol 101 (2) ◽  
pp. 393-402 ◽  
Author(s):  
T.J. Mohun ◽  
N. Garrett
Keyword(s):  
Nucleotide Sequence ◽  
Protein Isoforms ◽  
Actin Gene ◽  
Promoter Regions ◽  
Conserved Sequence ◽  
Beta Actin ◽  
Genes Encoding ◽  
Actin Mrna ◽  
Ccaat Box ◽  
Equivalent Region

The complete nucleotide sequence of two Xenopus actin genes encoding cytoskeletal protein isoforms has been determined. Transcripts from these genes are remarkably similar in nucleotide sequence throughout their length and code for type-5 and type-8 cytoskeletal actins. Both share some sequence homology with human gamma-actin mRNA within the 3′ untranslated region but none with the equivalent region of any vertebrate beta-actin transcript. The promoter regions of the two Xenopus genes are virtually identical from the cap site to the CCAAT box and show extensive homology further upstream. Despite such similarity, the two genes are divergently expressed during embryonic development. The type-5 actin gene is expressed in all regions of the developing embryo whilst the type-8 gene is coregulated with the muscle-specific skeletal actin gene. In common with mammalian and avian cytoskeletal actin counterparts, the Xenopus genes possess a conserved sequence within their promoter that has previously been identified as a transcription-factor-binding site.

Molecular cloning and characterization of mutant and wild-type human beta-actin genes

1984 ◽  
Vol 4 (10) ◽  
pp. 1961-1969
Author(s):  
J Leavitt ◽  
P Gunning ◽  
P Porreca ◽  
S Y Ng ◽  
C S Lin ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Human Fibroblasts ◽  
Actin Gene ◽  
Actin Genes ◽  
Beta Actin ◽  
Gene Libraries ◽  
Actin Mrna ◽  
Dna Fragment ◽  
Specific Sequences

There are more than 20 beta-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional beta-actin genes, we used the new method of B. Seed (Nucleic Acids Res. 11:2427-2446, 1983) for selecting genomic clones by homologous recombination. A derivative of the pi VX miniplasmid, pi AN7 beta 1, was constructed by insertion of the 600-base-pair 3' untranslated region of the beta-actin mRNA expressed in human fibroblasts. Five clones containing beta-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete beta-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then used to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant beta-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived clones verified the identity of the beta-actin gene expressed in human fibroblasts.

scholarly journals Simultaneous in situ detection of two mRNAs in the same cell using riboprobes labeled with biotin and 35S.

1990 ◽  
Vol 38 (7) ◽  
pp. 917-922 ◽  
Author(s):  
S Ozden ◽  
C Aubert ◽  
D Gonzalez-Dunia ◽  
M Brahic
Keyword(s):  
In Situ Hybridization ◽  
Theiler's Virus ◽  
Bhk Cells ◽  
Beta Actin ◽  
Actin Mrna ◽  
High Level ◽  
Radiolabeled Probe ◽  
In Situ Detection

We used 35S-labeled and biotinylated cRNAs (riboprobes) to detect simultaneously two different mRNAs by in situ hybridization. In a first step we established the conditions under which each type of probe achieved the same high level of sensitivity. We then used these conditions to hybridize BHK cells infected with Theiler's virus, a murine picornavirus, with a mixture of a virus-specific biotinylated riboprobe and a 35S-labeled riboprobe specific for beta-actin mRNA. Both mRNAs could be detected in the same cell, although the sensitivity achieved by the radiolabeled probe was reduced by about 40% by the simultaneous hybridization with the biotinylated probe.

scholarly journals Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.

1985 ◽  
Vol 5 (7) ◽  
pp. 1649-1654 ◽  
Author(s):  
S H Waters ◽  
R J Distel ◽  
N B Hecht
Keyword(s):  
Untranslated Region ◽  
Cell Types ◽  
Dot Blot ◽  
Size Classes ◽  
Cytoplasmic Actin ◽  
Beta Actin ◽  
Actin Mrna ◽  
Residual Bodies ◽  
Pachytene Spermatocytes ◽  
Sexually Mature

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.

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