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The scaffolding protein IQGAP1 co-localizes with actin at the cytoplasmic face of the nuclear envelope: implications for cytoskeletal regulation.

Johnson MA, Henderson BR - Bioarchitecture (2012)

Bottom Line: The IQGAP1 and F-actin staining overlapped that of microtubules at the nuclear envelope, revealing a pattern strikingly similar to that observed at the plasma membrane.In detergent-extracted cells IQGAP1 was retained at cytoskeletal structures at the nuclear envelope.This finding has new implications for involvement of IQGAP1 in cell polarization and migration events and potentially in cell cycle-associated nuclear envelope assembly/disassembly.

View Article: PubMed Central - PubMed

Affiliation: Westmead Institute for Cancer Research; University of Sydney; Westmead Millennium Institute at Westmead Hospital; Westmead, Australia.

ABSTRACT
IQGAP1 is an important cytoskeletal regulator, known to act at the plasma membrane to bundle and cap actin filaments, and to tether the cortical actin meshwork to microtubules via plus-end binding proteins. Here we describe the novel subcellular localization of IQGAP1 at the cytoplasmic face of the nuclear envelope, where it co-located with F-actin. The IQGAP1 and F-actin staining overlapped that of microtubules at the nuclear envelope, revealing a pattern strikingly similar to that observed at the plasma membrane. In detergent-extracted cells IQGAP1 was retained at cytoskeletal structures at the nuclear envelope. This finding has new implications for involvement of IQGAP1 in cell polarization and migration events and potentially in cell cycle-associated nuclear envelope assembly/disassembly.

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Figure 1. Novel nuclear envelope localization of IQGAP1. (A) Nuclear halo staining of IQGAP1 occurs in several cell lines. Deconvolution fluorescence cell images of human epithelial-derived MCF-7 and HT29 cancer cells and mouse fibroblast NIH 3T3 cells. Cells were immunolabelled and stained for IQGAP1 (H-109, Santa-Cruz; red), F-actin (phalloidin-FITC; green) and DNA (Hoechst; blue). (B) IQGAP1 locates to the cytoplasmic face of the outer nuclear membrane. Deconvolution microscopy fluorescence cell images of MCF-7 cells stained with IQGAP1 (red) and FxFG-repeat nucleoporins (mAb414; green). The graph depicts pixel intensity of IQGAP1 (red) and nucleoporin (green) staining from the indicated cross-section in the enlarged micrograph (toward cytoplasm on right). (C) High resolution scoring of IQGAP1 co-localization with nuclear envelope in MCF-7 cells (n = 62). (D) Electron micrographs of ultrathin cryosections of MCF-7 cells immunolabelled with IQGAP1 pAb. Cells with no primary antibody showed no specific staining pattern (not shown). Thin closed arrow indicates nuclear rim; broad open arrow indicates immunogold-labeling of IQGAP1. White bar, 200 nm.
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Figure 1: Figure 1. Novel nuclear envelope localization of IQGAP1. (A) Nuclear halo staining of IQGAP1 occurs in several cell lines. Deconvolution fluorescence cell images of human epithelial-derived MCF-7 and HT29 cancer cells and mouse fibroblast NIH 3T3 cells. Cells were immunolabelled and stained for IQGAP1 (H-109, Santa-Cruz; red), F-actin (phalloidin-FITC; green) and DNA (Hoechst; blue). (B) IQGAP1 locates to the cytoplasmic face of the outer nuclear membrane. Deconvolution microscopy fluorescence cell images of MCF-7 cells stained with IQGAP1 (red) and FxFG-repeat nucleoporins (mAb414; green). The graph depicts pixel intensity of IQGAP1 (red) and nucleoporin (green) staining from the indicated cross-section in the enlarged micrograph (toward cytoplasm on right). (C) High resolution scoring of IQGAP1 co-localization with nuclear envelope in MCF-7 cells (n = 62). (D) Electron micrographs of ultrathin cryosections of MCF-7 cells immunolabelled with IQGAP1 pAb. Cells with no primary antibody showed no specific staining pattern (not shown). Thin closed arrow indicates nuclear rim; broad open arrow indicates immunogold-labeling of IQGAP1. White bar, 200 nm.

Mentions: IQGAP1 is generally regarded as a plasma membrane-associated scaffolding protein as it is most commonly detected at cell:cell membrane junctions and membrane ruffles.19 We previously confirmed the specificity of IQGAP1 polyclonal antibody H-109 (Santa Cruz Biotech, CA) by siRNA-mediated knockdown, and showed that this antibody detects similar endogenous IQGAP1 staining patterns to that of ectopically-expressed GFP-tagged IQGAP1.21 A careful analysis of IQGAP1 subcellular localization with this antibody has revealed a ‘nuclear halo’ pattern of IQGAP1 by immunofluorescence microscopy, reminiscent of that previously reported for actin in interphase NIH 3T3 cells.25 IQGAP1 is inextricably linked to F-actin,14 therefore we co-stained for F-actin with FITC-conjugated phalloidin to determine the degree of co-localization. Human epithelial-derived breast (MCF-7) and colon (HT29) cancer cells, as well as immortalized non-tumorigenic mouse fibroblasts (NIH 3T3), each showed IQGAP1 nuclear halos that co-localized with F-actin (Fig. 1A). The nuclear halo staining was also detected in SW480 (colon cancer), U2OS (osteosarcoma) and HeLa (cervical) cancer cells, but not in HCT116 colon cancer cells, and the staining pattern was abolished by IQGAP1 knockdown (data not shown).


The scaffolding protein IQGAP1 co-localizes with actin at the cytoplasmic face of the nuclear envelope: implications for cytoskeletal regulation.

Johnson MA, Henderson BR - Bioarchitecture (2012)

Figure 1. Novel nuclear envelope localization of IQGAP1. (A) Nuclear halo staining of IQGAP1 occurs in several cell lines. Deconvolution fluorescence cell images of human epithelial-derived MCF-7 and HT29 cancer cells and mouse fibroblast NIH 3T3 cells. Cells were immunolabelled and stained for IQGAP1 (H-109, Santa-Cruz; red), F-actin (phalloidin-FITC; green) and DNA (Hoechst; blue). (B) IQGAP1 locates to the cytoplasmic face of the outer nuclear membrane. Deconvolution microscopy fluorescence cell images of MCF-7 cells stained with IQGAP1 (red) and FxFG-repeat nucleoporins (mAb414; green). The graph depicts pixel intensity of IQGAP1 (red) and nucleoporin (green) staining from the indicated cross-section in the enlarged micrograph (toward cytoplasm on right). (C) High resolution scoring of IQGAP1 co-localization with nuclear envelope in MCF-7 cells (n = 62). (D) Electron micrographs of ultrathin cryosections of MCF-7 cells immunolabelled with IQGAP1 pAb. Cells with no primary antibody showed no specific staining pattern (not shown). Thin closed arrow indicates nuclear rim; broad open arrow indicates immunogold-labeling of IQGAP1. White bar, 200 nm.
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Related In: Results  -  Collection

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Figure 1: Figure 1. Novel nuclear envelope localization of IQGAP1. (A) Nuclear halo staining of IQGAP1 occurs in several cell lines. Deconvolution fluorescence cell images of human epithelial-derived MCF-7 and HT29 cancer cells and mouse fibroblast NIH 3T3 cells. Cells were immunolabelled and stained for IQGAP1 (H-109, Santa-Cruz; red), F-actin (phalloidin-FITC; green) and DNA (Hoechst; blue). (B) IQGAP1 locates to the cytoplasmic face of the outer nuclear membrane. Deconvolution microscopy fluorescence cell images of MCF-7 cells stained with IQGAP1 (red) and FxFG-repeat nucleoporins (mAb414; green). The graph depicts pixel intensity of IQGAP1 (red) and nucleoporin (green) staining from the indicated cross-section in the enlarged micrograph (toward cytoplasm on right). (C) High resolution scoring of IQGAP1 co-localization with nuclear envelope in MCF-7 cells (n = 62). (D) Electron micrographs of ultrathin cryosections of MCF-7 cells immunolabelled with IQGAP1 pAb. Cells with no primary antibody showed no specific staining pattern (not shown). Thin closed arrow indicates nuclear rim; broad open arrow indicates immunogold-labeling of IQGAP1. White bar, 200 nm.
Mentions: IQGAP1 is generally regarded as a plasma membrane-associated scaffolding protein as it is most commonly detected at cell:cell membrane junctions and membrane ruffles.19 We previously confirmed the specificity of IQGAP1 polyclonal antibody H-109 (Santa Cruz Biotech, CA) by siRNA-mediated knockdown, and showed that this antibody detects similar endogenous IQGAP1 staining patterns to that of ectopically-expressed GFP-tagged IQGAP1.21 A careful analysis of IQGAP1 subcellular localization with this antibody has revealed a ‘nuclear halo’ pattern of IQGAP1 by immunofluorescence microscopy, reminiscent of that previously reported for actin in interphase NIH 3T3 cells.25 IQGAP1 is inextricably linked to F-actin,14 therefore we co-stained for F-actin with FITC-conjugated phalloidin to determine the degree of co-localization. Human epithelial-derived breast (MCF-7) and colon (HT29) cancer cells, as well as immortalized non-tumorigenic mouse fibroblasts (NIH 3T3), each showed IQGAP1 nuclear halos that co-localized with F-actin (Fig. 1A). The nuclear halo staining was also detected in SW480 (colon cancer), U2OS (osteosarcoma) and HeLa (cervical) cancer cells, but not in HCT116 colon cancer cells, and the staining pattern was abolished by IQGAP1 knockdown (data not shown).

Bottom Line: The IQGAP1 and F-actin staining overlapped that of microtubules at the nuclear envelope, revealing a pattern strikingly similar to that observed at the plasma membrane.In detergent-extracted cells IQGAP1 was retained at cytoskeletal structures at the nuclear envelope.This finding has new implications for involvement of IQGAP1 in cell polarization and migration events and potentially in cell cycle-associated nuclear envelope assembly/disassembly.

View Article: PubMed Central - PubMed

Affiliation: Westmead Institute for Cancer Research; University of Sydney; Westmead Millennium Institute at Westmead Hospital; Westmead, Australia.

ABSTRACT
IQGAP1 is an important cytoskeletal regulator, known to act at the plasma membrane to bundle and cap actin filaments, and to tether the cortical actin meshwork to microtubules via plus-end binding proteins. Here we describe the novel subcellular localization of IQGAP1 at the cytoplasmic face of the nuclear envelope, where it co-located with F-actin. The IQGAP1 and F-actin staining overlapped that of microtubules at the nuclear envelope, revealing a pattern strikingly similar to that observed at the plasma membrane. In detergent-extracted cells IQGAP1 was retained at cytoskeletal structures at the nuclear envelope. This finding has new implications for involvement of IQGAP1 in cell polarization and migration events and potentially in cell cycle-associated nuclear envelope assembly/disassembly.

Show MeSH
Related in: MedlinePlus