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. Author manuscript; available in PMC: 2013 Feb 1.
Published in final edited form as: Nat Cell Biol. 2012 Jul 1;14(8):874–881. doi: 10.1038/ncb2533

Figure 3. N-BAR domains are dynamically recruited to local membrane sites at the leading edge of migrating 3T3 cells.

Figure 3

(a) The proteins Nadrin2 and Amphiphysin1 are enriched in the leading edge of migrating 3T3 cells. (b) Enrichment in the leading edge is dependent on the N-BAR domain. Cells expressing a cytosolic marker (CFP, green) and the isolated N-BAR domain of Nadrin2 (left, red) and Amphiphysin1 (right, red). Note the polarized localization of the isolated N-BAR domain to the leading edge. (c) Enrichment in the leading edge is dependent on the amphipatic helix. Cells expressing a cytosolic marker (CFP, green) and the isolated N-BAR domain of Nadrin2 (left, red) and Amphiphysin1 (right, red) that is lacking the amino-terminal amphipatic helix show no polarized localization to the leading edge. (d) N-BAR domain patches show significant overlap with marker for filamentous actin. 3T3 cells were transfected with a marker for filamentous actin (f-tractin, green) and the N-BAR domain of Nadrin2 (left, red) or Amphiphysin1 (right, red), respectively. (e) N-BAR domain and a PM marker only partially overlap. 3T3 cells transfected with a membrane marker (CAAX, green) and the N-BAR domain of Nadrin2 (left, red) and Amphiphysin1 (right, red) are shown. (f) Addition of the actin polymerization inhibitor LatA reversibly inhibits N-BAR domain puncta formation. 1303 individual puncta from 12 cells were analyzed for the drug washout experiments. (g) Addition of the actin polymerization inhibitor CytoD reversibly inhibits N-BAR domain puncta formation. 428 puncta from 8 cells were analyzed for the drug washout experiments. Scale bars (a-g), 10μm. Error bars represent s.e.m. of the mean value. ** P < 0.01.