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Two ZBP1 KH domains facilitate beta-actin mRNA localization, granule formation, and cytoskeletal attachment.

Farina KL, Huttelmaier S, Musunuru K, Darnell R, Singer RH - J. Cell Biol. (2002)

Bottom Line: When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination.RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA.Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Chicken embryo fibroblasts (CEFs) localize beta-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of beta-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.

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Distribution of β-actin mRNA and ZBP1 in CEFs. ZBP1 localizes in granular structures (arrow) that, along with β-actin mRNA, are enriched in cell lamellae. Immunofluorescence of ZBP1 (A) and in situ hybridization of β-actin mRNA (B) in serum-stimulated CEFs. (C) Overlap of A and C. CEFs were transiently transfected with GFP-tagged ZBP1 (D) and subsequently hybridized with β-actin mRNA fluorescent oligonucleotide (E). GFP–ZBP1 forms granules that are enriched at the cell periphery. Partial masking of granules is occurring due to high signal intensities. The enlargements (3×) (E–G) of indicated region in D (arrows) demonstrate a spatial overlap (G) of β-actin probe (E) and GFP–ZBP1 (F) within the cell protrusion. Bars, 10 μm.
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fig1: Distribution of β-actin mRNA and ZBP1 in CEFs. ZBP1 localizes in granular structures (arrow) that, along with β-actin mRNA, are enriched in cell lamellae. Immunofluorescence of ZBP1 (A) and in situ hybridization of β-actin mRNA (B) in serum-stimulated CEFs. (C) Overlap of A and C. CEFs were transiently transfected with GFP-tagged ZBP1 (D) and subsequently hybridized with β-actin mRNA fluorescent oligonucleotide (E). GFP–ZBP1 forms granules that are enriched at the cell periphery. Partial masking of granules is occurring due to high signal intensities. The enlargements (3×) (E–G) of indicated region in D (arrows) demonstrate a spatial overlap (G) of β-actin probe (E) and GFP–ZBP1 (F) within the cell protrusion. Bars, 10 μm.

Mentions: The subcellular distribution of ZBP1 was analyzed in CEFs using rabbit antiserum prepared against recombinant full-length ZBP1. In CEFs, endogenous ZBP1 localizes in granular structures that are enriched within lamellipodia, the perinucleus, and the retracting tail (Fig. 1 A). The lamellar distribution of ZBP1 mimics that of β-actin mRNA (Fig. 1, A–C).


Two ZBP1 KH domains facilitate beta-actin mRNA localization, granule formation, and cytoskeletal attachment.

Farina KL, Huttelmaier S, Musunuru K, Darnell R, Singer RH - J. Cell Biol. (2002)

Distribution of β-actin mRNA and ZBP1 in CEFs. ZBP1 localizes in granular structures (arrow) that, along with β-actin mRNA, are enriched in cell lamellae. Immunofluorescence of ZBP1 (A) and in situ hybridization of β-actin mRNA (B) in serum-stimulated CEFs. (C) Overlap of A and C. CEFs were transiently transfected with GFP-tagged ZBP1 (D) and subsequently hybridized with β-actin mRNA fluorescent oligonucleotide (E). GFP–ZBP1 forms granules that are enriched at the cell periphery. Partial masking of granules is occurring due to high signal intensities. The enlargements (3×) (E–G) of indicated region in D (arrows) demonstrate a spatial overlap (G) of β-actin probe (E) and GFP–ZBP1 (F) within the cell protrusion. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172732&req=5

fig1: Distribution of β-actin mRNA and ZBP1 in CEFs. ZBP1 localizes in granular structures (arrow) that, along with β-actin mRNA, are enriched in cell lamellae. Immunofluorescence of ZBP1 (A) and in situ hybridization of β-actin mRNA (B) in serum-stimulated CEFs. (C) Overlap of A and C. CEFs were transiently transfected with GFP-tagged ZBP1 (D) and subsequently hybridized with β-actin mRNA fluorescent oligonucleotide (E). GFP–ZBP1 forms granules that are enriched at the cell periphery. Partial masking of granules is occurring due to high signal intensities. The enlargements (3×) (E–G) of indicated region in D (arrows) demonstrate a spatial overlap (G) of β-actin probe (E) and GFP–ZBP1 (F) within the cell protrusion. Bars, 10 μm.
Mentions: The subcellular distribution of ZBP1 was analyzed in CEFs using rabbit antiserum prepared against recombinant full-length ZBP1. In CEFs, endogenous ZBP1 localizes in granular structures that are enriched within lamellipodia, the perinucleus, and the retracting tail (Fig. 1 A). The lamellar distribution of ZBP1 mimics that of β-actin mRNA (Fig. 1, A–C).

Bottom Line: When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination.RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA.Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Chicken embryo fibroblasts (CEFs) localize beta-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of beta-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.

Show MeSH
Related in: MedlinePlus