Figure 5.
The regulation of the Tex2–TMEM55B interaction by PI4KII activities. (A–C) Representative images of COS7 cells expressing either Halo-PI4KIIα (magenta; A), Halo-PI4KIIβ (magenta; B), or kinase-dead PI4KIIα mutant Halo-PI4KIIα-W359A (magenta; C), along with GFP-Tex2 (green), Lamp1-mCh (gray), and BFP-TMEM55B (blue) with two boxed regions shown at the bottom. Yellow arrows indicate reduced Tex2 enrichments at PI4KII-positive/TMEM55B-positive LE/lys while red arrows denote Tex2 enrichments at PI4KII-negative/TMEM55B-positive LE/lys. (D and E) Representative images of COS7 cells expressing either GFP-Tex2-NT (green; D) or GFP-Tex2-ΔPH (green; E) along with Halo-PI4KIIs (magenta), Lamp1-mCh (gray) and BFP-TMEM55B (blue) with insets. (F) Pearson’s correlation coefficient of Tex2 vs. TMEM55B in cells expressing either empty vector (10 cells), Halo-PI4KIIα (13 cells), Halo-PI4KIIβ (14 cells), or Halo-PI4KIIα-W359A (13 cells). Pearson’s correlation coefficient of Tex2-NT vs. TMEM55B in cells expressing either empty vector (14 cells), Halo-PI4KIIα (12 cells), Halo-PI4KIIβ (11 cells), or Halo-PI4KIIα-W359A (13 cells). Pearson’s correlation coefficient of Tex2-ΔPH vs. TMEM55B in cells expressing either empty vector (12 cells), Halo-PI4KIIα (10 cells), Halo-PI4KIIβ (13 cells), or Halo-PI4KIIα-W359A (12 cells) in three independent assays. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. (G) Pearson’s correlation coefficient of Tex2 vs. PI4KIIs in cells expressing either Halo-PI4KIIα (13 cells), Halo-PI4KIIβ (14 cells), or Halo-PI4KIIα-W359A (13 cells). Pearson’s correlation coefficient of Tex2-NT vs. TMEM55B in cells expressing either Halo-PI4KIIα (12 cells), Halo-PI4KIIβ (11 cells), or Halo-PI4KIIα-W359A (13 cells). Pearson’s correlation coefficient of Tex2-ΔPH vs. TMEM55B in cells expressing either Halo-PI4KIIα (10 cells), Halo-PI4KIIβ (13 cells), or Halo-PI4KIIα-W359A (12 cells) in three independent assays. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. (H) Representative images of COS7 cells expressing GFP-Tex2 (green) and Lamp1-mCh (magenta) upon scrambled (top panel), PI4KIIα (middle panel), or PI4KIIβ siRNAs (bottom panel) with insets. Yellow arrows indicated Tex2 enrichments at LE/lys. (I) qPCR assays showing the efficiency of siRNA-mediated suppression of PI4KIIs. (J) Pearson’s correlation coefficient of Tex2 vs. LE/lys in cells upon scrambled (15 cells), PI4KIIα (18 cells), or PI4KIIβ siRNAs (14 cells). Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. (K) Representative images of either control (top panel) or Tex2 KO-1(bottom panel) HeLa cells expressing Halo-PI4KIIα (magenta; left panel) or Halo-PI4KIIβ (magenta; right panel) along with Lamp1-mCh (blue) and ER-tagRFP (green) with insets. (L) Pearson’s correlation coefficient of either PI4KIIα or PI4KIIβ vs. Lamp1 in control or Tex2 KO-1 HeLa cells in K and L. For quantification of PI4KIIα, 10 control cells and 10 Tex2 KO-1 cells were analyzed, and for quantification of PI4KIIβ, 10 control cells and 10 Tex2 KO-1 cells were analyzed in three independent experiments. Two-tailed unpaired Student’s t test. Mean ± SD. Scale bar, 10 μm in the whole cell images and 2 μm in the insets in A–E and H.

The regulation of the Tex2–TMEM55B interaction by PI4KII activities. (A–C) Representative images of COS7 cells expressing either Halo-PI4KIIα (magenta; A), Halo-PI4KIIβ (magenta; B), or kinase-dead PI4KIIα mutant Halo-PI4KIIα-W359A (magenta; C), along with GFP-Tex2 (green), Lamp1-mCh (gray), and BFP-TMEM55B (blue) with two boxed regions shown at the bottom. Yellow arrows indicate reduced Tex2 enrichments at PI4KII-positive/TMEM55B-positive LE/lys while red arrows denote Tex2 enrichments at PI4KII-negative/TMEM55B-positive LE/lys. (D and E) Representative images of COS7 cells expressing either GFP-Tex2-NT (green; D) or GFP-Tex2-ΔPH (green; E) along with Halo-PI4KIIs (magenta), Lamp1-mCh (gray) and BFP-TMEM55B (blue) with insets. (F) Pearson’s correlation coefficient of Tex2 vs. TMEM55B in cells expressing either empty vector (10 cells), Halo-PI4KIIα (13 cells), Halo-PI4KIIβ (14 cells), or Halo-PI4KIIα-W359A (13 cells). Pearson’s correlation coefficient of Tex2-NT vs. TMEM55B in cells expressing either empty vector (14 cells), Halo-PI4KIIα (12 cells), Halo-PI4KIIβ (11 cells), or Halo-PI4KIIα-W359A (13 cells). Pearson’s correlation coefficient of Tex2-ΔPH vs. TMEM55B in cells expressing either empty vector (12 cells), Halo-PI4KIIα (10 cells), Halo-PI4KIIβ (13 cells), or Halo-PI4KIIα-W359A (12 cells) in three independent assays. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. (G) Pearson’s correlation coefficient of Tex2 vs. PI4KIIs in cells expressing either Halo-PI4KIIα (13 cells), Halo-PI4KIIβ (14 cells), or Halo-PI4KIIα-W359A (13 cells). Pearson’s correlation coefficient of Tex2-NT vs. TMEM55B in cells expressing either Halo-PI4KIIα (12 cells), Halo-PI4KIIβ (11 cells), or Halo-PI4KIIα-W359A (13 cells). Pearson’s correlation coefficient of Tex2-ΔPH vs. TMEM55B in cells expressing either Halo-PI4KIIα (10 cells), Halo-PI4KIIβ (13 cells), or Halo-PI4KIIα-W359A (12 cells) in three independent assays. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. (H) Representative images of COS7 cells expressing GFP-Tex2 (green) and Lamp1-mCh (magenta) upon scrambled (top panel), PI4KIIα (middle panel), or PI4KIIβ siRNAs (bottom panel) with insets. Yellow arrows indicated Tex2 enrichments at LE/lys. (I) qPCR assays showing the efficiency of siRNA-mediated suppression of PI4KIIs. (J) Pearson’s correlation coefficient of Tex2 vs. LE/lys in cells upon scrambled (15 cells), PI4KIIα (18 cells), or PI4KIIβ siRNAs (14 cells). Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. (K) Representative images of either control (top panel) or Tex2 KO-1(bottom panel) HeLa cells expressing Halo-PI4KIIα (magenta; left panel) or Halo-PI4KIIβ (magenta; right panel) along with Lamp1-mCh (blue) and ER-tagRFP (green) with insets. (L) Pearson’s correlation coefficient of either PI4KIIα or PI4KIIβ vs. Lamp1 in control or Tex2 KO-1 HeLa cells in K and L. For quantification of PI4KIIα, 10 control cells and 10 Tex2 KO-1 cells were analyzed, and for quantification of PI4KIIβ, 10 control cells and 10 Tex2 KO-1 cells were analyzed in three independent experiments. Two-tailed unpaired Student’s t test. Mean ± SD. Scale bar, 10 μm in the whole cell images and 2 μm in the insets in A–E and H.

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