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E H Kislauskis

Publications and source records attributed to E H Kislauskis.

9 recordsLinked to original sources
PubMed2000

Colony-stimulating factor-1 (CSF-1) rescues osteoblast attachment, survival and sorting of beta-actin mRNA in the toothless (tl-osteopetrotic) mutation in the rat.

We have shown that in the osteopetrotic rat mutation toothless (tl) osteoblasts are absent from older bone surfaces in mutants and that mutant osteoblasts in vivo lack the prominent stress fiber bundles polarized along bone surfaces in osteoblasts from normal littermates. Our recent data demonstrate that in normal osteoblasts in vitro beta- and gamma-actin mRNAs have different, characteristic intracellular distributions and that tl (mutant) osteoblasts fail to differentially sort these mRNAs. Because bone resorption and formation are highly interdependent and injections of CSF-1, a growth factor, increase bone resorption and growth in tl rats, we examined the effects of CSF-1 treatment on osteoblast survival and ultrastructure in vivo and ability to sort actin mRNAs in vitro. Neonatal CSF-1 treatment of mutants restores osteoblasts on older bone surfaces, normalizes the intracellular distribution of stress fibers in osteoblasts in vivo and promotes normal sorting of beta-actin mRNA in mutant osteoblasts in vitro without normalizing gamma-actin distribution. These data suggest the beta- and gamma-actin mRNAs in osteoblasts are sorted by different mechanisms and that the differential sorting of beta-actin mRNA is related to the characteristic polarization of stress fibers in osteoblasts and their survival on bone surfaces. This experimental system can be used to explore the relationships and regulation of these aspects of cell and tissue biology.

Actins↗
PubMed1998

Actin mRNA isoforms are differentially sorted in normal osteoblasts and sorting is altered in osteoblasts from a skeletal mutation in the rat.

Actin isoform sorting has been shown to occur in a variety of cell types in culture. To this list we add osteoblasts, in which we show by in situ hybridization that beta-actin is distributed primarily in cell processes and on one side of the nucleus and gamma-actin has a perinuclear distribution. Osteoblasts from the skeletal mutation toothless (tl), evaluated under identical conditions, fail to sort these actin isoforms differentially and exhibit diffuse labeling as their major manifestation. Northern analyses of actin mRNAs showed no differences between normal and mutant cultures. Shortened osteoblast life span and an inability to direct osteoclast-mediated bone resorption have recently been demonstrated in tl mutants. The present results suggest that a failure of osteoblasts to sort actin mRNAs may be related to one or both of these pathological manifestations in this mutation and represent, to our knowledge, the first correlation of an actin mRNA-sorting abnormality with a mammalian disease.

Actins↗
PubMed1997

beta-Actin messenger RNA localization and protein synthesis augment cell motility.

In chicken embryo fibroblasts (CEFs), beta-actin mRNA localizes near an actin-rich region of cytoplasm specialized for motility, the lamellipodia. This localization is mediated by isoform-specific 3'-untranslated sequences (zipcodes) and can be inhibited by antizipcode oligodeoxynucleotides (ODNs) (Kislauskis, E.H., X.-C. Zhu, and R.H. Singer. 1994. J. Cell Biol. 127: 441-451). This inhibition of beta-actin mRNA localization resulted in the disruption of fibroblast polarity and, presumably, cell motility. To investigate the role of beta-actin mRNA in motility, we correlated time-lapse images of moving CEFs with the distribution of beta-actin mRNA in these cells. CEFs with localized beta-actin mRNA moved significantly further over the same time period than did CEFs with nonlocalized mRNA. Antizipcode ODN treatment reduced this cell translocation while control ODN treatments showed no effect. The temporal relationship of beta-actin mRNA localization to cell translocation was investigated using serum addition to serum-deprived cultures. beta-actin mRNA was not localized in serum-deprived cells but became localized within minutes after serum addition (Latham, V.M., E.H. Kislauskis, R.H. Singer, and A.F. Ross. 1994. J. Cell Biol. 126:1211-1219). Cell translocation increased over the next 90 min, and actin synthesis likewise increased. Puromycin reduced this cell translocation and blocked this induction in cytosolic actin content. The serum induction of cell movement was also inhibited by antizipcode ODNs. These observations support the hypothesis that beta-actin mRNA localization and consequent protein synthesis augment cell motility.

Actins↗
PubMed1997

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

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.

Actins↗
PubMed1994

Beta-actin mRNA localization is regulated by signal transduction mechanisms.

Beta-actin mRNA is localized in the leading lamellae of chicken embryo fibroblasts (CEFs) (Lawrence, J., and R. Singer. 1986. Cell. 45:407-415), close to where actin polymerization in the lamellipodia drives cellular motility. During serum starvation beta-actin mRNA becomes diffuse and non-localized. Addition of FCS induces a rapid (within 2-5 min) redistribution of beta-actin mRNA into the leading lamellae. A similar redistribution was seen with PDGF, a fibroblast chemotactic factor. PDGF-induced beta-actin mRNA redistribution was inhibited by the tyrosine kinase inhibitor herbimycin, indicating that this process requires intact tyrosine kinase activity, similar to actin filament polymerization and chemotaxis. Lysophosphatidic acid, which has been shown to rapidly induce actin stress fiber formation (Ridley, A., and A. Hall. 1992. Cell. 790:389-399), also increases peripheral beta-actin mRNA localization within minutes. This suggests that actin polymerization and mRNA localization may be regulated by similar signaling pathways. Additionally, activators or inhibitors of kinase A or C can also delocalize steady-state beta-actin mRNA in cells grown in serum, and can inhibit the serum induction of peripherally localized beta-actin mRNA in serum-starved CEFs. These data show that physiologically relevant extracellular factors operating through a signal transduction pathway can regulate spatial sites of actin protein synthesis, which may in turn affect cellular polarity and motility.

Actins↗
PubMed1994

Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype.

We have characterized the structure and function of RNA sequences that direct beta-cytoplasmic actin mRNA to the cell periphery were mapped to two segments of 3'-untranslated region by expression of LacZ/beta-actin chimeric mRNAs in chicken embryo fibroblasts (CEFs). A 54-nt segment, the "RNA zipcode," and a homologous but less active 43-nt segment each localized beta-galactosidase activity to the leading lamellae. This zipcode contains the full activity, and mutations or deletions within it reduce, but do not eliminate, its activity, indicating that several motifs contribute to the activity. Two of these motifs, when multimerized, can regenerate almost full activity. These sequences are highly conserved in evolution, since the human beta-actin zipcode, positioned identically in the 3'UTR localizes equally well in chicken cells. Complementary phosphorothioate oligonucleotides against the zipcode delocalized endogenous beta-actin mRNA, whereas those complementary to the region just outside the zipcode, or sense oligonucleotides, did not. Actin mRNA or protein levels were unaffected by the antisense treatments, but a dramatic change in lamellipodia structure, and actin stress fiber organization was observed using the same antizipcode oligonucleotides which delocalized the mRNA. Hence, discrete 3'UTR sequences direct beta-actin isoform synthesis to the leading lamellae and affect cell morphology, presumably through the actin cytoskeleton.

Actins↗
PubMed1993

Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments.

We demonstrate that in differentiating myoblasts, the mRNAs encoding two actin isoforms, beta-cytoplasmic, and alpha-cardiac, can occupy different cytoplasmic compartments within the same cytoplasm. beta-actin mRNA is localized to the leading lamellae and alpha-actin mRNA is associated with a perinuclear compartment. This was revealed by co-hybridizing, in situ, fluorochrome-conjugated oligonucleotide probes specific for each isoform. To address the mechanism of isoform-specific mRNA localization, molecular chimeras were constructed by insertion of actin sequences between the Lac Z coding region and SV-40 3'UTR in a reporter plasmid. These constructs were transiently expressed in a mixed culture of embryonic fibroblasts, myoblasts and myotubes, beta-galactosidase activity within transfectants was revealed by a brief incubation with its substrate (X-gal). Since the blue-insoluble reaction product co-localized with the specific mRNAs expressed from each construct, it was used as a bioassay for mRNA localization. Transfectants were scored as either perinuclear, peripheral or nonlocalized with respect to the distribution of the blue product. The percentage of transfectants within those categories was quantitated as a function of the various constructs. This analysis revealed that for each actin mRNA its 3'UTR is necessary and sufficient to direct reporter transcripts to its appropriate compartment; beta-actin peripheral and alpha-actin perinuclear. In contrast, sequences from the 5'UTR through the coding region of either actin gene did not localize the blue product. Therefore, 3'UTR sequences play a key role in modulating the distribution of actin mRNAs in muscle cells. We propose that the mechanism of mRNA localization facilitates actin isoform sorting in the cytoplasm.

Actins↗
PubMed1992

Determinants of mRNA localization.

RNA localization provides a mechanism for protein targeting within developing or differentiating cells. Specific cis-acting sequences on mRNA mediate this process. Such 'localizer' or 'zipcode' nucleic acid sequences have been restricted to the 3' untranslated region of several mRNAs. The presence of genetic information denoting a spatial component of translation adds a new dimension to gene expression.

Animals↗