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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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Cytoplasmic granule formation and cytoskeletal anchoring is mediated by KH3-KH4 of ZBP1. ZBP1 fragments fused to GFP were transiently transfected into CEFs. Localization of GFP-tagged proteins was analyzed before (A–J) and after (F'–J') extraction with Triton X-100. (A–E) Note that all protein fragments lacking the two COOH-terminal KH domains (KH3 and KH4) in their entirety were evenly distributed throughout the cytoplasm (A, 1–179; B, 1–289; C, 1–397; D, 195–522; E, 195–403) and no granular structures were observed. All of these fusion proteins were extracted from the cytoplasm by treatment with Triton X-100 before fixation (L). (F–K) CEFs transiently transfected with: F, 1–576; G, 1–556; H, 74–576; I, 84–576; J, 189–576; and K, 317–576. The NH2-terminal truncated fragments formed large granules that were resistant to detergent extraction. The left column (F–J) shows nonextracted fixed cells. Cells in the right column (F'–J') have been Triton extracted. Note that granules formed by constructs in G (1–556) and H (74–576) are enriched in the lamellae, whereas granules formed by the remaining constructs, I (84–576), J (189–576), and K (317–576), were evenly distributed in the cytoplasm. (L) To demonstrate the loss of COOH-terminal truncated GFP fusion proteins by extraction, the loss of GFP signal was visualized after the introduction of Triton X-100 (t0). The COOH-terminal truncated fragment (1–397) was initially diffusely distributed in the cytoplasm. No significant signal was detected after 20 s of Triton extraction. Images shown are at introduction of Triton X-100 (t0), 10 s post-Triton (t10), and 20 s post-Triton (t20). Bars, 10 μm.
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fig2: Cytoplasmic granule formation and cytoskeletal anchoring is mediated by KH3-KH4 of ZBP1. ZBP1 fragments fused to GFP were transiently transfected into CEFs. Localization of GFP-tagged proteins was analyzed before (A–J) and after (F'–J') extraction with Triton X-100. (A–E) Note that all protein fragments lacking the two COOH-terminal KH domains (KH3 and KH4) in their entirety were evenly distributed throughout the cytoplasm (A, 1–179; B, 1–289; C, 1–397; D, 195–522; E, 195–403) and no granular structures were observed. All of these fusion proteins were extracted from the cytoplasm by treatment with Triton X-100 before fixation (L). (F–K) CEFs transiently transfected with: F, 1–576; G, 1–556; H, 74–576; I, 84–576; J, 189–576; and K, 317–576. The NH2-terminal truncated fragments formed large granules that were resistant to detergent extraction. The left column (F–J) shows nonextracted fixed cells. Cells in the right column (F'–J') have been Triton extracted. Note that granules formed by constructs in G (1–556) and H (74–576) are enriched in the lamellae, whereas granules formed by the remaining constructs, I (84–576), J (189–576), and K (317–576), were evenly distributed in the cytoplasm. (L) To demonstrate the loss of COOH-terminal truncated GFP fusion proteins by extraction, the loss of GFP signal was visualized after the introduction of Triton X-100 (t0). The COOH-terminal truncated fragment (1–397) was initially diffusely distributed in the cytoplasm. No significant signal was detected after 20 s of Triton extraction. Images shown are at introduction of Triton X-100 (t0), 10 s post-Triton (t10), and 20 s post-Triton (t20). Bars, 10 μm.

Mentions: The localization pattern of GFP–ZBP1 was compared with endogenous ZBP1 in CEFs after transfection with GFP–ZBP1. In CEFs, overexpressed GFP–ZBP1 formed large cytoplasmic granules (Fig. 1 D) similar to those formed by endogenous ZBP1 (Fig. 1 A). Granules were enriched at cell protrusions, the perinuclear region, and the retracting tail of the cells. Like endogenous ZBP1, GFP–ZBP1 granules were resistant to cytoplasmic extraction with Triton X-100, indicating that they maintained stable interactions with the cytoskeleton (Fig. 2 F′).


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)

Cytoplasmic granule formation and cytoskeletal anchoring is mediated by KH3-KH4 of ZBP1. ZBP1 fragments fused to GFP were transiently transfected into CEFs. Localization of GFP-tagged proteins was analyzed before (A–J) and after (F'–J') extraction with Triton X-100. (A–E) Note that all protein fragments lacking the two COOH-terminal KH domains (KH3 and KH4) in their entirety were evenly distributed throughout the cytoplasm (A, 1–179; B, 1–289; C, 1–397; D, 195–522; E, 195–403) and no granular structures were observed. All of these fusion proteins were extracted from the cytoplasm by treatment with Triton X-100 before fixation (L). (F–K) CEFs transiently transfected with: F, 1–576; G, 1–556; H, 74–576; I, 84–576; J, 189–576; and K, 317–576. The NH2-terminal truncated fragments formed large granules that were resistant to detergent extraction. The left column (F–J) shows nonextracted fixed cells. Cells in the right column (F'–J') have been Triton extracted. Note that granules formed by constructs in G (1–556) and H (74–576) are enriched in the lamellae, whereas granules formed by the remaining constructs, I (84–576), J (189–576), and K (317–576), were evenly distributed in the cytoplasm. (L) To demonstrate the loss of COOH-terminal truncated GFP fusion proteins by extraction, the loss of GFP signal was visualized after the introduction of Triton X-100 (t0). The COOH-terminal truncated fragment (1–397) was initially diffusely distributed in the cytoplasm. No significant signal was detected after 20 s of Triton extraction. Images shown are at introduction of Triton X-100 (t0), 10 s post-Triton (t10), and 20 s post-Triton (t20). Bars, 10 μm.
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Related In: Results  -  Collection

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fig2: Cytoplasmic granule formation and cytoskeletal anchoring is mediated by KH3-KH4 of ZBP1. ZBP1 fragments fused to GFP were transiently transfected into CEFs. Localization of GFP-tagged proteins was analyzed before (A–J) and after (F'–J') extraction with Triton X-100. (A–E) Note that all protein fragments lacking the two COOH-terminal KH domains (KH3 and KH4) in their entirety were evenly distributed throughout the cytoplasm (A, 1–179; B, 1–289; C, 1–397; D, 195–522; E, 195–403) and no granular structures were observed. All of these fusion proteins were extracted from the cytoplasm by treatment with Triton X-100 before fixation (L). (F–K) CEFs transiently transfected with: F, 1–576; G, 1–556; H, 74–576; I, 84–576; J, 189–576; and K, 317–576. The NH2-terminal truncated fragments formed large granules that were resistant to detergent extraction. The left column (F–J) shows nonextracted fixed cells. Cells in the right column (F'–J') have been Triton extracted. Note that granules formed by constructs in G (1–556) and H (74–576) are enriched in the lamellae, whereas granules formed by the remaining constructs, I (84–576), J (189–576), and K (317–576), were evenly distributed in the cytoplasm. (L) To demonstrate the loss of COOH-terminal truncated GFP fusion proteins by extraction, the loss of GFP signal was visualized after the introduction of Triton X-100 (t0). The COOH-terminal truncated fragment (1–397) was initially diffusely distributed in the cytoplasm. No significant signal was detected after 20 s of Triton extraction. Images shown are at introduction of Triton X-100 (t0), 10 s post-Triton (t10), and 20 s post-Triton (t20). Bars, 10 μm.
Mentions: The localization pattern of GFP–ZBP1 was compared with endogenous ZBP1 in CEFs after transfection with GFP–ZBP1. In CEFs, overexpressed GFP–ZBP1 formed large cytoplasmic granules (Fig. 1 D) similar to those formed by endogenous ZBP1 (Fig. 1 A). Granules were enriched at cell protrusions, the perinuclear region, and the retracting tail of the cells. Like endogenous ZBP1, GFP–ZBP1 granules were resistant to cytoplasmic extraction with Triton X-100, indicating that they maintained stable interactions with the cytoskeleton (Fig. 2 F′).

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