tie inverted microscope stand  (Nikon)

Journal: The Journal of Cell Biology

Article Title: Single-molecule lipid biosensors mitigate inhibition of endogenous effector proteins

doi: 10.1083/jcb.202412026

Figure Lengend Snippet: Fluorescent tagging of AKT1 at its genomic locus. (A) CRISPR/Cas9 directed cutting at the 3′ end of the AKT1 ORF is coupled to homology-directed repair to integrate an in-frame NG2 11 tag, encoding the 11th strand of neonGreen2. The edit is made in a HEK293A cell line stably expressing NG2 1–10 , the remainder of the neonGreen protein. The two neonGreen2 protein fragments assemble in the cell to generate fluorescent NG2 protein. (B) Western blot of AKT protein from a sorted, polyclonal population of edited cells shows the appearance of a second molecular weight band, consistent with the 81.9 kDa complex between AKT1 (55.7 kDa) and NG2 (26.2 kDa). (C) TIRF imaging of AKT1-NG2 cells exhibit discrete fluorescent spots at the cell surface, which increase in density after PI3K activation with 10 ng/ml EGF. The light lines are data points from images recorded at 20 Hz; the thicker, darker line is the 5 s rolling average. (D and E) AKT1-NG2 spots are single molecules: (D) The intensity of fluorescent AKT1 spots pooled from a representative experiment shows a mono-modal lognormal distribution (green). The data are fit with a model assuming the intensity is derived from a mixture of monomeric, dimeric or trimeric fluorescent proteins calibrated against a known monomeric mNeonGreen fluorescent protein distribution. The fit predicts 98.1% monomers with a reduced χ 2 of 1.06. (E) The results of this analysis pooled across six cells from three independent experiments yields consistent results with mean χ 2 of 1.67 ± 0.40 (s.e.). (F) Extended TIRF imaging of AKT-NG2 cells with reduced duty cycle to minimize photobleaching reveals robust recruitment of AKT1 after EGF stimulation. Data are means ± s.e. of 20 cells pooled from two independent experiments. (G) PI3K activation is sufficient to recruit AKT1 to the plasma membrane. AKT1-NG2 cells were transfected with PM-targeted Lyn N-terminal 11 residues fused to FRB and the PI3K p110 catalytic subunit-binding iSH2 fused to FKBP and mCherry. 1 µM rapamycin was added to cells to induce dimerization of FRB and FKBP and hence recruitment of iSH2/p110 to the plasma membrane, inducing PIP 3 synthesis. AKT1-NG2 is further increased on the PM by this maneuver. Data are means ± s.e. of 32 cells pooled from three independent experiments. Insets in F and G show zoomed view indicated in the center of the cell. Source data are available for this figure: .

Article Snippet: Imaging was performed on a Nikon TiE inverted microscope stand with motorized TIRF illuminator (Nikon) fiber-coupled to a four line Oxxius laser launch equipped with 405-, 488-, 561-, and 638-nm laser lines.

Techniques: CRISPR, Stable Transfection, Expressing, Western Blot, Molecular Weight, Imaging, Activation Assay, Derivative Assay, Clinical Proteomics, Membrane, Transfection, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Single-molecule lipid biosensors mitigate inhibition of endogenous effector proteins

doi: 10.1083/jcb.202412026

Figure Lengend Snippet: PH-AKT1 PIP 3 biosensor abolishes endogenous AKT recruitment to the PM. (A) Hypothesized competition for PIP 3 between the overexpressed PH domain and endogenous AKT1-NG2. (B) Raw 16-bit intensity levels of mCherry fluorescence in HEK293A cells transiently transfected with the indicated mass of PH-AKT1-mCherry for 24 h. Small data points represent measurements of individual cells, whereas large points are the means of each of three independent experiments. Points are color matched by experiment. Grand means ± s.e. are also indicated. (C) TIRF imaging of AKT1-NG2 cells from (B) stimulated with 10 ng/ml EGF. Insets show zoomed view indicated in the center of the cell. (D) Relative PM fluorescence of PH-AKT1-mCherry during time lapse imaging and stimulation with 10 ng/ml EGF. (E) As in D, except the density of endogenous AKT1-NG2 molecules are counted. Data in D and E are grand means of three experiments ± s.e. imaging 8–10 cell each. (F) Data from E were pooled from all three experiments, and raw 12-bit mCherry fluorescence intensity data (constant laser power and camera gain between experiments) was used to bin cellular measurements as indicated on the x-axis. Small points represent individual cell measurements, whereas the large symbols are means ± 95% confidence interval. Data were normal by Kolmogorov-Smirnov test, and results of a Holm-Šídák’s multiple comparisons test are indicated comparing to control cells (“0” cohort); *** P = 0.002, **** P < 0.0001, performed post-hoc to an ordinary one-way ANOVA (F = 36.73, P < 0.0001).

Article Snippet: Imaging was performed on a Nikon TiE inverted microscope stand with motorized TIRF illuminator (Nikon) fiber-coupled to a four line Oxxius laser launch equipped with 405-, 488-, 561-, and 638-nm laser lines.

Techniques: Fluorescence, Transfection, Imaging, Control