Visualization and Molecular Analysis of Actin Assembly in Living Cells

Schafer, Dorothy A.; Welch, Matthew D.; Machesky, Laura M.; Bridgman, Paul C.; Meyer, Shelley M.; Cooper, John A.

doi:10.1083/jcb.143.7.1919

Cited by 161 publications

(137 citation statements)

References 76 publications

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“…erns the supramolecular organization of actin filaments in motile cells and has a profound effect on motility and migration (13)(14)(15)(16)(17)(18)(19)(20). With EGFP-CALI, we recapitulate the loss-of-function phenotypes of Capping protein and Mena reported previously: CALI of capping protein creates a local increase in barbed ends and F-actin resulting in numerous protrusive structures (21), whereas CALI of Mena induces larger and more stable lamellipodial protrusions (17).…”

mentioning

confidence: 69%

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Vitriol

Uetrecht

Shen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Chromophore-assisted laser inactivation (CALI) is a light-mediatedtechnique that offers precise spatiotemporal control of protein inactivation, enabling better understanding of the protein's role in cell function. EGFP has been used effectively as a CALI chromophore, and its cotranslational attachment to the target protein avoids having to use exogenously added labeling reagents. A potential drawback to EGFP-CALI is that the CALI phenotype can be obscured by the endogenous, unlabeled protein that is not susceptible to light inactivation. Performing EGFP-CALI experiments in deficient cells rescued with functional EGFP-fusion proteins permits more complete loss of function to be achieved. Here, we present a modified lentiviral system for rapid and efficient generation of knockdown cell lines complemented with physiological levels of EGFP-fusion proteins. We demonstrate that CALI of EGFP-CapZ␤ increases uncapped actin filaments, resulting in enhanced filament growth and the formation of numerous protrusive structures. We show that these effects are completely dependent upon knocking down the endogenous protein. We also demonstrate that CALI of EGFP-Mena in Mena/VASP-deficient cells stabilizes lamellipodial protrusions.capping protein ͉ Mena/VASP ͉ spatiotemporal ͉ lentivirus

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mentioning

confidence: 69%

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Vitriol

Uetrecht

Shen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…To more precisely characterize the localization of S100A4 at the leading edge, we compared the localization of S100A4 to two proteins known to localize to the leading edge, N-WASP and Arp3 (48,49). We found the wild type S100A4 to almost perfectly colocalize with N-WASP in MDA-MB-231 cells at the very tip of the leading edge of lamellipodia (Fig.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the Metastasis-associated Protein, S100A4

Kim

Helfman

2003

Journal of Biological Chemistry

104

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Elevated S100A4 protein expression is associated with metastatic tumor progression and appears to be a strong molecular marker for clinical prognosis. S100A4 is a calcium-binding protein that is known to form homodimersandinteractswithseveralproteinsinacalciumdependent manner. Here we show that S100A4 localizes to lamellipodia structures in a migrating breast cancerderived cell line and colocalizes with a known S100A4-interacting protein, myosin heavy chain IIA, at the leading edge. We demonstrate that S100A4 mutants that are defective in either their ability to dimerize or in calcium binding are unable to interact with myosin heavy chain IIA. An S100A4 mutant that is deficient for calcium binding retains the ability to form homodimers, suggesting that S100A4 can exist as calcium-free or calcium-bound dimers in vivo. However, a calcium-bound S100A4 monomer only interacts with another calcium-bound monomer and not with an S100A4 mutant that does not bind calcium. Interestingly, despite the calcium dependence for interaction with known protein partners, calcium binding is not necessary for localization to lamellipodia. Both wild type and a mutant that is deficient for calcium binding colocalize with known markers of actively forming leading edges of lamellipodia, Arp3 and neuronal Wiskott-Aldrich syndrome protein. These data suggest that S100A4 localizes to the leading edge in a calciumindependent manner, and identification of the proteins that are involved in localizing S100A4 to the lamellipodial structures may provide novel insight into the mechanism by which S100A4 regulates metastasis.Cancer mortality most often results directly from metastatic spread to distal, vital organs. The isolation and characterization of molecular markers that can identify patients at a high risk for metastatic spread and consequently a poor prognosis is of obvious importance. There is increasing evidence that a member of the S100 family of proteins, S100A4 (also known as mts1, p9Ka, CAPL, calvasculin, and FspI), is one such molecular marker for metastatic potential with high prognostic significance. An increase in S100A4 protein expression has been correlated with a worse prognosis for patients with different types of cancer including colorectal, gallbladder, bladder, esophageal, breast, and nonsmall lung cancer (1-7). S100A4 has also been found to be more highly expressed in malignant tumor samples than in either benign tumor cells or normal tissue (3, 8 -11). Causal evidence for the role of S100A4 in metastasis comes from studies that show a metastatic phenotype can be either induced by ectopic expression (12, 13) or inhibited by reduced expression (14, 15). However, transgenic mice overexpressing S100A4 do not develop tumors, suggesting that S100A4 is not tumorigenic per se (16). Instead, S100A4 can more appropriately be categorized as a potential inducer of metastasis in a given tumorigenic background. This categorization is supported by the observation that the progeny from mice overexpressing S100A4 crossed with mice overexpr...

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“…2,3 The Arp2/3 complex rapidly catalyzes actin filament nucleation and elongation, and is also essential for efficient remodeling of actin filaments by ATP hydrolysis. [4][5][6] The Arp2/3 complex is activated by binding to the Wiskott-Aldrich syndrome protein (WASP) family proteins. There are five WASP family proteins; WASP, neural WASP (N-WASP), and WASP family verprolin-homologous protein (WAVE)1-3, known to be a suppressor of cAMP-receptors (SCAR1-3).…”

mentioning

confidence: 99%

Coexpression of Arp2 and WAVE2 predicts poor outcome in invasive breast carcinoma

2007

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Breast carcinoma with a high histologic grade is highly invasive and metastatic. One reason for its irregular morphology is the formation of excessive protrusions due to assemblages of branched actin filament networks. In mammalian cells, the actin-related protein 2 and 3 (Arp2/3) complex initiates actin assembly to form lamellipodial protrusions by binding to the Wiskott-Aldrich syndrome (WASP)/WASP family verprolinehomologous protein2 (WAVE2), a member of the WASP protein family. In this study, the localization Arp2 and WAVE2 in breast carcinoma was investigated to clarify whether coexpression of the two proteins is associated with histologic grade or patient outcome. Immunohistochemical staining of Arp2 and WAVE2 was performed on mirror specimens of 197 breast carcinomas, and the association between coexpression of the two proteins and clinicopathologic factors was examined. Kaplan-Meier disease-free survival and overall survival curves were analyzed to determine the prognostic significance of Arp2 and WAVE2 coexpression in breast carcinoma. Coexpression of Arp2 and WAVE2 was detected in 64 (36%) of 179 invasive ductal carcinomas and in 2 (11%) of 18 ductal carcinomas in situ, but was not detected in any adjacent non-cancerous tissue. The proportion of cancer cells expressing both Arp2 and WAVE2 was significantly higher in cases with high histologic grade (Po0.0001), and cases with lymph node metastasis (P ¼ 0.0150). The patients whose cancer cells showed such coexpression had shorter disease-free (P ¼ 0.0002) and overall survival (P ¼ 0.0122) than patients whose cancer cells expressed only one or none of Arp2 and WAVE2. Multivariate Cox regression analysis revealed that coexpression of Arp2 and WAVE2 is an independent factor for both tumor recurrence (P ¼ 0.0308) and death (P ¼ 0.0455). These results indicate that coexpression of Arp2 and WAVE2 is a significant prognostic factor that is closely associated with aggressive morphology of invasive ductal carcinoma of the breast.

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Visualization and Molecular Analysis of Actin Assembly in Living Cells

Cited by 161 publications

References 76 publications

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Characterization of the Metastasis-associated Protein, S100A4

Coexpression of Arp2 and WAVE2 predicts poor outcome in invasive breast carcinoma

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