Journal: Molecular & Cellular Proteomics : MCP

Article Title: Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes *

doi: 10.1074/mcp.RA119.001450

Figure Lengend Snippet: CLASP2 and G2L1 colocalize in 3T3-L1 adipocytes. A–B, Live-cell imaging of adipocytes cultured in complete media coexpressing mRuby2-Tubulin (magenta) and GFP-CLASP2-HA (A, green) or mCherry-G2L1-myc (B, green). Live cells were imaged using TIRFM on a 2-s acquisition interval. Time series images to the right of the whole cell image are used to highlight +TIP dynamics within the indicated ROI. C, Immunofluorescence images of adipocytes cultured in complete media cooverexpressing GFP-CLASP2-HA (green) and mCherry-G2L1-myc (magenta). Cells were fixed and immunolabeled for tubulin (inverted white). Bottom row is magnified ROI. ROI, region of interest. Scale bar = 20 μm.

Article Snippet: To generate pLenti-iRFP670-Tubulin, tubulin was first subcloned out of the mRuby2-Tubulin-6 vector and into piRFP670-N1 (Addgene #45457, a gift from Vladislav Verkhusha). iRFP670-Tubulin was then subcloned out of the piRFP670-Tubulin vector and into the pLenti backbone to generate pLenti-iRFP670-Tubulin.

Techniques: Live Cell Imaging, Cell Culture, Immunofluorescence, Immunolabeling

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes *

doi: 10.1074/mcp.RA119.001450

Figure Lengend Snippet: The effect of insulin on CLASP2 +TIP dynamics. Live-cell imaging of adipocytes serum-starved for one hour and subsequently stimulated with 100 nm insulin. Live cells were imaged using TIRFM on a two-second acquisition interval. A, Single frame of live-cell imaging of adipocytes coexpressing GFP-CLASP2 (green) and mRuby2-Tubulin (magenta) at basal state (top row) or 10 mins (bottom row) following insulin stimulation. CLASP2 is displayed in inverted white to highlight +TIP density. B, Quantification of CLASP2-containing +TIP density per unit area (μm2) in adipocytes at basal state or 10 mins following insulin stimulation. Percent increase in CLASP2-containing +TIP density is indicated to the right. Statistical comparison made by paired parametric t test, n = 5 cells. C, Temporally color-coded projection of GFP-CLASP2 localization during a 30 s live-cell imaging interval. The length and extent of color overlap of time-projected CLASP2 localization indicates reduced displacement and hence lower velocity of CLASP2-containing +TIPs during the imaging interval in adipocytes after 10 mins of insulin stimulation. D, Quantification of CLASP2-containing +TIP velocity in adipocytes at basal state or 10 mins following insulin stimulation indicates reduced velocity of CLASP2-containing +TIPS after insulin treatment. Statistical comparison made by unpaired parametric t test, n = 123–125 CLASP2-containing +TIPS from five cells. Scale bars = 5 μm. E, Image of entire cells in the basal state (left panel) or 8 mins post insulin treatment (right panel) extracted from supplemental Video 2. Time series images under the whole cell image are used to highlight insulin-stimulated +TIP dynamics within the indicated ROI. F, Time series extracted from supplemental Video 2 of the indicated ROIs at either the basal state or at 4 mins post insulin treatment to present an example of changing CLASP2 microtubule plus-end dynamics with insulin stimulation. G, Live cell CLASP2-containing +TIP dynamics of the ROI extracted from supplemental Video 2 were captured in the basal state followed by stimulation with insulin. Each insulin-stimulated CLASP2-containing +TIP trail length time point was compared against the basal CLASP2-containing +TIP trail length to test for significant differences. t test; *p ≤ 0.05, **p ≤ 0.01. Red circles represent outlier data points. ROI, region of interest. BAS, basal. INS, insulin. Scale bar = 10 μm.

Article Snippet: To generate pLenti-iRFP670-Tubulin, tubulin was first subcloned out of the mRuby2-Tubulin-6 vector and into piRFP670-N1 (Addgene #45457, a gift from Vladislav Verkhusha). iRFP670-Tubulin was then subcloned out of the piRFP670-Tubulin vector and into the pLenti backbone to generate pLenti-iRFP670-Tubulin.

Techniques: Live Cell Imaging, Imaging