Figure 1.
Depletion of SEPT6, SEPT7, BORG4, AP-3μ and Rab7 affect transport from early to late endosomes.
(A) Hela expressing Gag-GFP cells were transfected with indicated siRNAs. Culture supernatants (VLP) and cell lysates prepared as described in Materials and Methods were probed by western blotting with anti GFP and anti LAMP1 antibodies and then quantified (B). Values were normalized to tubulin. The secreted GAP-GFP represented 59±4, 46±1, 14±7, 5.7±3, 9.8±4.5, 8.2±5, 11±5.8, 21.8±7, 10.8±7.6, 15±8.9, 14±6.5% of the total GAG-GFP. (C) Cell surface receptor bound Alexa 564-transferrin was endocytosed for the indicated period of time in HeLa cells treated with the indicated siRNAs. Fluorescence intensities were then quantified. (D) Anti GFP antibodies pre-bound to the cell surface GFP-MPR of GFP-MPR expressing cells treated with the indicated siRNAs were endocytosed for indicated periods of time as indicated in Materials and Methods. Co-localization between the endocytosed anti GFP antibody and GFP-MPR was quantified. (E) Cell surface receptor bound Alexa-EGF (green) was endocytosed for the indicated periods of time in HeLa cells treated with the indicated siRNAs as indicated in Materials and Methods. Fluorescence intensities were quantified. (F) Similarly treated cells were stained with antibodies against EEA1 and the extent of co-localization of Alexa-EGF with EEA1 was calculated. (G) Cell surface receptor bound EGF was endocytosed for the indicated periods of time in siRNA-treated HeLa cells. The cells were stained with antibodies against the activated EGF receptor phosphorylated on Tyr1068 (pEGFR) and EEA1. The fluorescence associated with the pEGFR was quantified. (H) Lysates from similarly treated cells were probed by western blotting using antibodies against the pEGFR or the total EGF receptor. The values are means ± SD of 3 independent experiments.
Figure 2.
SEPT6 and SEPT7 bind F-actin and AP-3-positive structures.
HeLa cells stably expressing GFP-SEPT6 (green) were labeled with anti SEPT7 (red) and anti tubulin (blue) antibodies (A). HeLa cells stably expressing GFP-AP3 (white) were untreated (B) or treated with siRNA targeting Borg4 (C) and then labeled with anti SEPT7 antibodies (red). F-actin and microtubules were labeled with phalloidin or anti tubulin antibodies (green) (Bar 10 µm). (D) AP-3 was immunoprecipitated from a HeLa cell extract with anti AP-3 antibodies and the immunoprecipitates were probed by western blotting using antibodies against AP-3 and SEPT7.
Figure 3.
Depletion of SEPT6, SEPT7 affects the motility of endosomes and the dynamic association of AP-3.
(A) Time-lapse video microscopy of HeLa cells stably expressing GFP-AP-3d and transiently expressing Cherry-SEPT6, Cherry-SEPT7 or mRFP-Lifeact (acquisition: 200 ms/frame, intervals between frames: 475 ms) (Bar 5 µm). (B) HeLa cells stably expressing GFP-AP-3δ were depleted or not from SEPT6 or SEPT7 and observed by video-microscopy. The upper panels shows examples of trajectories of GFP-AP-3δ-positive objects (300 objects per condition). Cell surface receptor-bound Alexa-EGF was endocytosed for 5 min. in control and SEPT6- or SEPT7-depleted Hela cells and Alexa-EGF-positive objects were observed by video-microscopy. The lower panels show examples of trajectories (300 objects per condition). (C) Cell surface bound Alexa-EGF was internalized in GFP-AP-3δ expressing HeLa cells depleted or not from SEPT6 or SEPT7 and followed by videomicrosopy. The extend of colocalization between GFP-AP-3 and Alexa-EGF was estimated. (D) The interaction of GFP-AP-3 with individual Alexa-EGF-positive structures was recorded and quantified (25 structures per condition) (E). The values are means ± SD of 3 independent experiments.
Figure 4.
Depletion of SEPT6, SEPT7 affects the dynamic association of ESCRT sub-complexes.
(A) Cell surface receptor-bound Alexa-EGF was endocytosed for the indicated periods of time in HeLa cells treated with the indicated siRNAs. Cells were then stained with anti Hrs antibodies. Extent of colocalization between Alexa-EGF and Hrs was quantified. (B) Similarly treated cells were stained with anti CHMP2B (Vps 2), an ESCRT-III subunit. Extent of colocalization between Alexa-EGF and ESCRTIII was determined. (C) Control and siRNA treated HeLa cells were stained with antibodies against Hrs and Tsg101. (D) The extent of colocalization between Hrs and Tsg101 was quantified. (E) Co-localization of LRSAM1 with Tsg101 and AP-3 and quantification (G). (F) AP-3 was immunuprecipitated from HeLa cell extracts with anti AP-3δ antibodies. The immunoprecipitates were probed by western blotting using antibodies against AP-3d and LRSAM1. The values are means ± SD of at least 3 independent experiments.
Figure 5.
Morphology of early endosomes in SEPT7 and AP-3 depleted cells.
(A) HeLa cells depleted or not in SEPT7 or AP-3 were allowed to fluid phase endocytose HRP for 7 min and then processed for HRP detection. (B). Similarly, HeLa cells were allowed to internalize for 7 min Alexa-EGF pre-bound to its cell surface receptors. Cryosections were labeled with primary antibodies against LAMP1, Alexa and secondary antibodies (LAMP-1, 10 nm gold; Alexa-EGF, 15 nm gold, arrows, Bars 200 nm). (C) Model of SEPT6 and SEPT7 function in MVB biogenesis.
Table 1.
Quantification of MVBs and enlarged endosomes in control, SEPT7- or AP-3-depleted HeLa cells.