Figure 5.
EEA1- and RILP-positive endosomes are more extensively bound to septin-coated microtubules than LBPA- and Rab27a-containing MVBs. (A and B) Maximum intensity projections of confocal microscopy z-series sections of subconfluent MDCK cells, which were transfected with GFP-RILP (inverted forest green) and stained for endogenous SEPT7 (inverted blue) and EEA1 (A; inverted magenta) or LPBA (B; inverted magenta). Scale bars, 10 and 5 μm (insets). (C) Mean percentage (±SEM, error bars) of GFP-RILP endosomes that overlap (SEPT7-bound) and do not overlap with SEPT7 (SEPT7-free). Quantification was performed in subconfluent MDCK cells (n = 21) which were transfected with GFP-RILP and stained for endogenous SEPT7. Data were analyzed with a Mann-Whitney test. **** P < 0.0001. (D and E) Mean percentage (±SEM, error bars) of total (D) and SEPT7-bound (E) GFP-RILP endosomes that contain EEA1 (EEA1+) and LBPA (LBPA+). Quantification was performed in subconfluent MDCK cells, which were transfected with GFP-RILP and stained for endogenous SEPT7, EEA1 (n = 10 cells), and LBPA (n = 11 cells). Data were analyzed with an unpaired t test with Welch’s correction. n.s., not significant, * P < 0.05. (F) Mean ratios (±SEM, error bars) of SEPT7-bound to SEPT7-free GFP-RILP endosomes with or without EEA1. Quantification was performed in subconfluent MDCK cells (n = 10), which were transfected with GFP-RILP and stained for endogenous SEPT7 and EEA1. Data were analyzed with a Mann-Whitney test. ** P < 0.01. (G) Mean ratios (±SEM, error bars) of SEPT7-bound to SEPT7-free GFP-RILP endosomes with or without LBPA. Quantification was performed in subconfluent MDCK cells (n = 11), which were transfected with GFP-RILP and stained for endogenous SEPT7 and LBPA. Data were analyzed with a Mann-Whitney test. n.s., not significant. (H) Maximum intensity projections of confocal microscopy z-series sections of subconfluent MDCK-GFP-CD63 cells (inverted forest green), which were transfected with mRuby3-Rab27a (inverted magenta) and stained for endogenous SEPT7 (inverted blue). Scale bars, 10 and 5 μm (insets). (I) Mean percentage (±SEM, error bars) of total mRuby3-Rab27a endosomes with or without overlap with SEPT7 filaments (n = 11 cells). Data were analyzed with a Mann-Whitney test. n.s., not significant. (J) Mean percentage (±SEM, error bars) of SEPT7-bound GFP-CD63 with or without overlap with mRuby3-Rab27a endosomes (n = 11 cells). Data were analyzed with a Mann-Whitney test. n.s., not significant. (K) Mean ratios (±SEM, error bars) of SEPT7-bound to SEPT7-free GFP-CD63 endosomes with or without mRuby3-Rab27a. Quantification was performed in subconfluent MDCK-GFP-CD63 cells, which were transfected with mRuby3-Rab27a (n = 11 cells). Data were analyzed with an unpaired t test with Welch’s correction. n.s., not significant.

EEA1- and RILP-positive endosomes are more extensively bound to septin-coated microtubules than LBPA- and Rab27a-containing MVBs. (A and B) Maximum intensity projections of confocal microscopy z-series sections of subconfluent MDCK cells, which were transfected with GFP-RILP (inverted forest green) and stained for endogenous SEPT7 (inverted blue) and EEA1 (A; inverted magenta) or LPBA (B; inverted magenta). Scale bars, 10 and 5 μm (insets). (C) Mean percentage (±SEM, error bars) of GFP-RILP endosomes that overlap (SEPT7-bound) and do not overlap with SEPT7 (SEPT7-free). Quantification was performed in subconfluent MDCK cells (n = 21) which were transfected with GFP-RILP and stained for endogenous SEPT7. Data were analyzed with a Mann-Whitney test. **** P < 0.0001. (D and E) Mean percentage (±SEM, error bars) of total (D) and SEPT7-bound (E) GFP-RILP endosomes that contain EEA1 (EEA1+) and LBPA (LBPA+). Quantification was performed in subconfluent MDCK cells, which were transfected with GFP-RILP and stained for endogenous SEPT7, EEA1 (n = 10 cells), and LBPA (n = 11 cells). Data were analyzed with an unpaired t test with Welch’s correction. n.s., not significant, * P < 0.05. (F) Mean ratios (±SEM, error bars) of SEPT7-bound to SEPT7-free GFP-RILP endosomes with or without EEA1. Quantification was performed in subconfluent MDCK cells (n = 10), which were transfected with GFP-RILP and stained for endogenous SEPT7 and EEA1. Data were analyzed with a Mann-Whitney test. ** P < 0.01. (G) Mean ratios (±SEM, error bars) of SEPT7-bound to SEPT7-free GFP-RILP endosomes with or without LBPA. Quantification was performed in subconfluent MDCK cells (n = 11), which were transfected with GFP-RILP and stained for endogenous SEPT7 and LBPA. Data were analyzed with a Mann-Whitney test. n.s., not significant. (H) Maximum intensity projections of confocal microscopy z-series sections of subconfluent MDCK-GFP-CD63 cells (inverted forest green), which were transfected with mRuby3-Rab27a (inverted magenta) and stained for endogenous SEPT7 (inverted blue). Scale bars, 10 and 5 μm (insets). (I) Mean percentage (±SEM, error bars) of total mRuby3-Rab27a endosomes with or without overlap with SEPT7 filaments (n = 11 cells). Data were analyzed with a Mann-Whitney test. n.s., not significant. (J) Mean percentage (±SEM, error bars) of SEPT7-bound GFP-CD63 with or without overlap with mRuby3-Rab27a endosomes (n = 11 cells). Data were analyzed with a Mann-Whitney test. n.s., not significant. (K) Mean ratios (±SEM, error bars) of SEPT7-bound to SEPT7-free GFP-CD63 endosomes with or without mRuby3-Rab27a. Quantification was performed in subconfluent MDCK-GFP-CD63 cells, which were transfected with mRuby3-Rab27a (n = 11 cells). Data were analyzed with an unpaired t test with Welch’s correction. n.s., not significant.

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