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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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Anchoring of ZBP1 granules to the actin cytoskeleton is mediated by KH domains. CEF cells were transfected with GFP-tagged ZBP1 (A–C) or GFP-tagged KH1-KH4 (189–576) (D–F). After treatment with either cytochalasin D or colchicine, cells were Triton extracted, fixed, labeled, and viewed. Localization of GFP proteins on the microfilament (Phalloidin) or microtubule (anti-tubulin) systems was visualized and compared. In colchicine-treated cells, the microtubule system was significantly diminished; however, both GFP-tagged proteins were still associated with granular structures and retained their typical localization compared with untreated cells. In contrast, treatment with cytochalasin D destroyed the microfilament system and caused a significant loss of protein after extraction. Residual granular staining was only observed in the perinuclear region, indicating that granule formation and specific subcellular distribution mediated by the KH domains is mainly based on the actin cytoskeleton.
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fig4: Anchoring of ZBP1 granules to the actin cytoskeleton is mediated by KH domains. CEF cells were transfected with GFP-tagged ZBP1 (A–C) or GFP-tagged KH1-KH4 (189–576) (D–F). After treatment with either cytochalasin D or colchicine, cells were Triton extracted, fixed, labeled, and viewed. Localization of GFP proteins on the microfilament (Phalloidin) or microtubule (anti-tubulin) systems was visualized and compared. In colchicine-treated cells, the microtubule system was significantly diminished; however, both GFP-tagged proteins were still associated with granular structures and retained their typical localization compared with untreated cells. In contrast, treatment with cytochalasin D destroyed the microfilament system and caused a significant loss of protein after extraction. Residual granular staining was only observed in the perinuclear region, indicating that granule formation and specific subcellular distribution mediated by the KH domains is mainly based on the actin cytoskeleton.

Mentions: Cytoskeleton disruption experiments revealed that the cytoskeletal association of ZBP1 was primarily mediated through the actin cytoskeleton. We observed a dramatic loss of ZBP1-containing granules in Triton X-100–extracted cells after treatment with cytochalasin D (Fig. 4, A–C). However, treatment with colchicine resulted in little difference in appearance. In some cases, after microtubule disruption there seemed to be a loss of granules from the perinuclear region of the cytoplasm, whereas granules at the periphery were retained. In contrast, in cells treated with cytochalasin D there were residual granules surrounding the nucleus. Granules could sometimes be detected along microtubule tracks extending out toward the periphery of the cell; ΔGFP–KH1-KH4 (189–576) gave results similar to full-length ZBP1 (Fig. 4, D–F). It remained sensitive to cytochalasin D treatment, indicating that microfilament association is mainly mediated by the COOH-terminal KH domains.


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)

Anchoring of ZBP1 granules to the actin cytoskeleton is mediated by KH domains. CEF cells were transfected with GFP-tagged ZBP1 (A–C) or GFP-tagged KH1-KH4 (189–576) (D–F). After treatment with either cytochalasin D or colchicine, cells were Triton extracted, fixed, labeled, and viewed. Localization of GFP proteins on the microfilament (Phalloidin) or microtubule (anti-tubulin) systems was visualized and compared. In colchicine-treated cells, the microtubule system was significantly diminished; however, both GFP-tagged proteins were still associated with granular structures and retained their typical localization compared with untreated cells. In contrast, treatment with cytochalasin D destroyed the microfilament system and caused a significant loss of protein after extraction. Residual granular staining was only observed in the perinuclear region, indicating that granule formation and specific subcellular distribution mediated by the KH domains is mainly based on the actin cytoskeleton.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172732&req=5

fig4: Anchoring of ZBP1 granules to the actin cytoskeleton is mediated by KH domains. CEF cells were transfected with GFP-tagged ZBP1 (A–C) or GFP-tagged KH1-KH4 (189–576) (D–F). After treatment with either cytochalasin D or colchicine, cells were Triton extracted, fixed, labeled, and viewed. Localization of GFP proteins on the microfilament (Phalloidin) or microtubule (anti-tubulin) systems was visualized and compared. In colchicine-treated cells, the microtubule system was significantly diminished; however, both GFP-tagged proteins were still associated with granular structures and retained their typical localization compared with untreated cells. In contrast, treatment with cytochalasin D destroyed the microfilament system and caused a significant loss of protein after extraction. Residual granular staining was only observed in the perinuclear region, indicating that granule formation and specific subcellular distribution mediated by the KH domains is mainly based on the actin cytoskeleton.
Mentions: Cytoskeleton disruption experiments revealed that the cytoskeletal association of ZBP1 was primarily mediated through the actin cytoskeleton. We observed a dramatic loss of ZBP1-containing granules in Triton X-100–extracted cells after treatment with cytochalasin D (Fig. 4, A–C). However, treatment with colchicine resulted in little difference in appearance. In some cases, after microtubule disruption there seemed to be a loss of granules from the perinuclear region of the cytoplasm, whereas granules at the periphery were retained. In contrast, in cells treated with cytochalasin D there were residual granules surrounding the nucleus. Granules could sometimes be detected along microtubule tracks extending out toward the periphery of the cell; ΔGFP–KH1-KH4 (189–576) gave results similar to full-length ZBP1 (Fig. 4, D–F). It remained sensitive to cytochalasin D treatment, indicating that microfilament association is mainly mediated by the COOH-terminal KH domains.

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