![Figure 2. α5β1 integrin is internalized by a clathrin- and ECM-dependent mechanism during MT-induced focal adhesion disassembly. (a) Surface α5 integrin levels in NIH3T3 cells measured by flow cytometry before (no nocodazole [noco]) and during MT-induced focal adhesion disassembly. The histogram represents mean fluorescent intensity for each sample normalized to the value at 0 min MT regrowth. Data are from three independent experiments. (b) Comparison of surface α5 and α1 integrin levels in HA1 NIH3T3 cells measured by flow cytometry during MT-induced focal adhesion disassembly. The histogram represents the mean fluorescent intensity for each sample normalized to the value at 0 min MT regrowth. Data are from two independent experiments. (c) Levels of surface α5 integrin in NIH3T3 cells depleted of either clathrin or GAPDH and stimulated for MT-induced focal adhesion disassembly. The histogram represents the mean fluorescent intensity normalized to the value at 0 min MT regrowth. Data are the mean of three independent experiments. *, P < 0.01 by Student’s t test. (d) Immunofluorescence of β1 integrin in NIH3T3 fibroblasts transiently transfected with Rab5Q79L-GFP and treated with 10 µM nocodazole (NZ) or after 30 min of MT regrowth. Arrowheads in the top panel indicate Rab5Q79L-GFP endosomes that do not contain internalized integrin; arrowheads in the bottom panel indicate Rab5Q79L-GFP endosomes that contain internalized integrin. (e) Quantification of the extent of colocalization of β1 integrin with Rab5Q79L-GFP–positive endosomes at 0 and 60 min of MT regrowth. The histogram represents two independent experiments in which 10 cells were analyzed for each condition. (f) Immunofluorescence of β1 integrin accumulation in a transferrin- and Rab11-positive compartment during MT-induced focal adhesion disassembly. The left panel shows β1 integrin in nocodazole-treated cells. Panels on the right show β1 integrin colocalization with internalized transferrin or Rab11 in a perinuclear compartment 60 min after MT regrowth. Arrows indicate β1 integrin colocalizing with transferrin (top) or Rab11 (bottom). (a–c and e) Error bars are SEM. Bars, 15 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/187/5/10.1083_jcb.200904054/6/jcb_200904054_rgb_fig2.jpeg?Expires=1767711045&Signature=aTo~vn4z~IrkUeypcH0~JLUoq-Mc7VHlOm~CXD5LMC25yGcn9-3ooLHy9UuP6ir7-LtOCNnYZ0vgfgF-mwyRNmlbT3uNPSEx6t~Z37aCMcAdWXEaza-oJPDUA64sJ347xoaO71zmQihO-grGb4r~V1buwa7~KmysMvrqG5WeBMsZ5J8pm6EZxaQm74m3mRSzUfh8oJGw6Ix5PAfkN3BjoKIIz4I0Cz04UkXSKKsxXZA2V0fglA-Qhtk8tTNVanKriE0x-IXF4tdm~ZiTlncvKHHnW4wrE26SOkRoCjSPL1mvBKVcVX6Ab2hmnq2SpWBwmS9IZ6Us-CynN8YQ58mcdg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
α5β1 integrin is internalized by a clathrin- and ECM-dependent mechanism during MT-induced focal adhesion disassembly. (a) Surface α5 integrin levels in NIH3T3 cells measured by flow cytometry before (no nocodazole [noco]) and during MT-induced focal adhesion disassembly. The histogram represents mean fluorescent intensity for each sample normalized to the value at 0 min MT regrowth. Data are from three independent experiments. (b) Comparison of surface α5 and α1 integrin levels in HA1 NIH3T3 cells measured by flow cytometry during MT-induced focal adhesion disassembly. The histogram represents the mean fluorescent intensity for each sample normalized to the value at 0 min MT regrowth. Data are from two independent experiments. (c) Levels of surface α5 integrin in NIH3T3 cells depleted of either clathrin or GAPDH and stimulated for MT-induced focal adhesion disassembly. The histogram represents the mean fluorescent intensity normalized to the value at 0 min MT regrowth. Data are the mean of three independent experiments. *, P < 0.01 by Student’s t test. (d) Immunofluorescence of β1 integrin in NIH3T3 fibroblasts transiently transfected with Rab5Q79L-GFP and treated with 10 µM nocodazole (NZ) or after 30 min of MT regrowth. Arrowheads in the top panel indicate Rab5Q79L-GFP endosomes that do not contain internalized integrin; arrowheads in the bottom panel indicate Rab5Q79L-GFP endosomes that contain internalized integrin. (e) Quantification of the extent of colocalization of β1 integrin with Rab5Q79L-GFP–positive endosomes at 0 and 60 min of MT regrowth. The histogram represents two independent experiments in which 10 cells were analyzed for each condition. (f) Immunofluorescence of β1 integrin accumulation in a transferrin- and Rab11-positive compartment during MT-induced focal adhesion disassembly. The left panel shows β1 integrin in nocodazole-treated cells. Panels on the right show β1 integrin colocalization with internalized transferrin or Rab11 in a perinuclear compartment 60 min after MT regrowth. Arrows indicate β1 integrin colocalizing with transferrin (top) or Rab11 (bottom). (a–c and e) Error bars are SEM. Bars, 15 µm.
