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. 2020 Mar 13;3:117. doi: 10.1038/s42003-020-0843-2

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Interference reflection microscopy (IRM) image of REF52 cell on RGD-membrane. b Intensity profiles of IRM, F-actin, and YFP-paxillin along the dashed line in a. The region of podosome core (F-actin, labeled by CF594-phalloidin) exhibits the lowest intensity in the IRM channel and represents the close contact and protrusive F-actin polymerization towards the substrate. c Kymograph of plasma membrane (PM, labeled by mCherry-KRas-CT), F-actin (labeled by BFP2-UtrCH), and RGD-NA680 during the podosome formation in MEF cell. Plasma membrane becomes enriched and encircles the protrusive F-actin (arrowheads) (see Supplementary Fig. 7c). d Intensity profiles at t₁ and t₂ in d. The intensities of plasma membrane gradually increase around the F-actin (arrowheads). e Kymograph of BIN1-mCherry, plasma membrane (PM, labeled by PM-GFP), and F-actin (labeled by BFP2-UtrCH) during the podosome formation in MEF cell. BIN1 localizes at the site of plasma membrane invagination and encircles the protrusive F-actin (see Supplementary Fig. 7d and Supplementary Movie 4). f BIN1-mCherry specifically colocalizes with integrin-β3-GFP at the podosome ring and surrounds dot-like F-actin assembly of the podosome core. F-actin is labeled by BPF2-UtrCH in REF52 cell. Inset: the boxed region (5 × 5 μm²). g, h N-BAR-GFP (aa 1–267), not BIN1∆N-BAR-GFP (aa 268–476) colocalizes with BIN1-mCherry around BFP2-UtrCH labeled podosome core in REF52 cell. Inset: the boxed region (5 × 5 μm²). Scale bars represent 5 µm.