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Stoichiometry of Nck-dependent actin polymerization in living cells.

ABSTRACT: Regulation of actin dynamics through the Nck/N-WASp (neural Wiskott-Aldrich syndrome protein)/Arp2/3 pathway is essential for organogenesis, cell invasiveness, and pathogen infection. Although many of the proteins involved in this pathway are known, the detailed mechanism by which it functions remains undetermined. To examine the signaling mechanism, we used a two-pronged strategy involving computational modeling and quantitative experimentation. We developed predictions for Nck-dependent actin polymerization using the Virtual Cell software system. In addition, we used antibody-induced aggregation of membrane-targeted Nck SH3 domains to test these predictions and to determine how the number of molecules in Nck aggregates and the density of aggregates affected localized actin polymerization in living cells. Our results indicate that the density of Nck molecules in aggregates is a critical determinant of actin polymerization. Furthermore, results from both computational simulations and experimentation support a model in which the Nck/N-WASp/Arp2/3 stoichiometry is 4:2:1. These results provide new insight into activities involving localized actin polymerization, including tumor cell invasion, microbial pathogenesis, and T cell activation.

SUBMITTER: Ditlev JA

PROVIDER: S-EPMC3365498 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Stoichiometry of Nck-dependent actin polymerization in living cells.

Ditlev Jonathon A JA Michalski Paul J PJ Huber Greg G Rivera Gonzalo M GM Mohler William A WA Loew Leslie M LM Mayer Bruce J BJ

The Journal of cell biology 20120521 5

Regulation of actin dynamics through the Nck/N-WASp (neural Wiskott-Aldrich syndrome protein)/Arp2/3 pathway is essential for organogenesis, cell invasiveness, and pathogen infection. Although many of the proteins involved in this pathway are known, the detailed mechanism by which it functions remains undetermined. To examine the signaling mechanism, we used a two-pronged strategy involving computational modeling and quantitative experimentation. We developed predictions for Nck-dependent actin ...[more]

PMID: 22613834

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Project description:BackgroundThe trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actin-associated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented.ResultsUsing live imaging of transfected COS-7 cells we observed intercellular junction-associated pools of GFP-APC in addition to previously documented microtubule-associated GFP-APC and a variety of minor localisations. Although both microtubule and junction-associated populations could co-exist within individual cells, they differed in their subcellular location, dynamic behaviour and sensitivity to cytoskeletal poisons. GFP-APC deletion mutant analysis indicated that a protein truncated immediately after the APC armadillo repeat domain retained the ability to localise to adhesive membranes in transfected cells. Supporting this, we also observed junctional APC immunostaining in cultures of human colorectal cancer cell line that express truncated forms of APC.ConclusionOur data indicate that APC can be found in two spatially separate populations at the cell periphery and these populations can co-exist in the same cell. The first localisation is highly dynamic and associated with microtubules near free edges and in cell vertices, while the second is comparatively static and is closely associated with actin at sites of cell-cell contact. Our imaging confirms that human GFP-APC possesses many of the localisations and behaviours previously seen by live imaging of Xenopus GFP-APC. However, we report the novel finding that GFP-APC puncta can remain associated with the ends of shrinking microtubules. Deletion analysis indicated that the N-terminal region of the APC protein mediated its junctional localisation, consistent with our observation that truncated APC proteins in colon cancer cell lines are still capable of localising to the cell cortex. This may have implications for the development of colorectal cancer.

Dataset Information

Stoichiometry of Nck-dependent actin polymerization in living cells.

Publications

Stoichiometry of Nck-dependent actin polymerization in living cells.

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