Figure 1.
Calu-1/2 are translocated into the lumen and secreted.
(A) Western blotting analysis of endogenous calu-1/2 from total cell lysate (TCL) and cultured medium (CM) of HeLa cells. (B) Western blotting analysis of overexpressed calu-1/2-EGFP of HeLa cells. Samples of TCL and CM were separated by SDS-PAGE and probed with anti-EGFP and anti-tubulin antibodies. Coomassie Blue staining (Co.St.) was used as a loading control of CM. (C) Immunofluorescence of EGFP or Calu-1/2-EGFP overexpressing HeLa cells probed with anti-Giantin antibody. Bar, 10 µm. (D) Immunofluorescence of EGFP or Calu-2-EGFP overexpressing HEK293T cells probed with anti-Giantin antibody. Bar, 10 µm. (E) HeLa cells were immunofluorescently labeled by the indicated antibodies after being permeabilized by digitonin. Scale bar, 10 µm. (F) Subcellular localization of 11 EGFP-fusion proteins of calu-2 transcripts in HeLa cells, which were co-transfected with GRIP-mRFP and permeabilized by Triton X-100 after fixation. Numbers indicate the amino acid positions in calu-2. In the cartoon, transcripts which localized on the secretory pathway are marked by green ticks. Red crosses indicate the transcripts that are smeared in HeLa cells. Scale bar, 10 µm.
Figure 2.
Time-lapse imaging of the intracellular transport of calu-1/2-EGFP.
(A) Representative images of HeLa cells overexpressing calu-1/2-EGFP. Calu-1/2-EGFP fusion proteins localize in the Golgi apparatus and the ER networks, with the nuclei indicated. Vesicles are indicated by arrowheads. Scale bar, 10 µm. (B–C) Representative images of the intracellular transport of calu-1/2-EGFP. (B) Movements of single vesicles (indicated by one arrowhead) were tracked, and movements of rod-shaped vesicles (indicated by two arrowheads) are shown in (C). Scale bar, 1 µm.
Figure 3.
Calu-1/2-EGFP enter or exit the Golgi apparatus.
(A–D) The representative HeLa cells transfected with calu-1-EGFP (A and B) and calu-2-EGFP (C and D) visualized by the Andor spinning disk confocal microscope system. The rectangles show magnified areas. Arrowheads point to those vesicles which enter (A and C) and exit (B and D) the Golgi apparatus. The time interval between two individual frames is 0.48 seconds. Scale bar, 1 µm.
Figure 4.
Calu-1/2-EGFP are secreted in two different types.
(A) Time-lapse observation of calu-1-EGFP (left) and calu-2-EGFP (right) secretion in the “secretion after attachment” mode. Scale bar, 4 µm. (B) Vesicle tracks in calu-1-EGFP-expressing HeLa cells. Images were acquired at 0.188-second intervals. These sequential frames were overlaid as time projections to illustrate vesicle paths. The rectangles are magnified areas showing the accumulation of calu-1-EGFP in the cellular processes. Scale bar, 10 µm. (C) Time-lapse observation of calu-1-EGFP secretion in the “secretion after accumulation” mode. Note that the arrow head pointed calu-1-EGFP dots moved to the arrow pointed dot, and the arrow pointed dot vanished finally. Scale bar, 4 µm.
Figure 5.
The transport of calu-1/2-EGFP-containing vesicles are microtubule-dependent but actin-independent.
(A) Western blotting analysis in TCL and CM of the calu-2-EGFP transfected cells treated with DMSO, nocodazole or cytochalasin. (B) Immunofluorescence of calu-2-EGFP expressing HeLa cells using anti-α-tubulin antibody. Arrow heads indicate calu-2-EGFP dots spreading along the microtubules to cellular processes. The rectangle areas were amplified. Scale bar, 10 µm.
Figure 6.
Calu-1/2-EGFP-containing vesicles are transported by Kif5b and cytoplasmic dynein.
(A–B) Histograms show the distribution of velocities of calu-1/2-EGFP-containing vesicles in HeLa cells. The dashed lines indicate that most vesicles move at 0.7–1 µm/s and 1.1–1.5 µm/s. Approximately 2000 dots (≥10 cells) were measured for each group. (C) The inward and outward velocity of calu-1/2-EGFP-containing vesicles. (D) Western blotting analysis of calu-2-EGFP secretory level in HeLa cells co-transfected with the indicated dominant negative vectors. (E) Western blotting analysis of calu-2-EGFP secretory level in HEK293T cells co-transfected with the indicated dominant negative vectors. (F) Western blotting analysis of calu-2-EGFP secretory level of Kif5b or cytoplasmic dynein knockdown HeLa cells. (G) GST pull-down analysis of calu-2-EGFP transfected cells using GST or GST-Kif5b-CBD. (H) Immunoprecipitation assay of calu-2-EGFP and myc-p150 overexpressing HEK293T cells using normal IgG or anti-myc antibody.
Figure 7.
Dominant negative effects of Kif5b on the calu-1/2-EGFP-containing vesicle trafficking in HeLa cells.
(A) The representative picture shows the tracks of moving vesicles containing calu-1-EGFP in the presence of mRFP or mRFP-Kif5b-CBD. These tracks of moving vesicles are real-time projections of individual frames over 3 minutes. (B) The numbers of moving calu-1-EGFP-containing vesicles per minute corresponding to (A) were manually counted (≥10 cells in each group). (C) The representative picture shows the tracks of moving vesicles containing calu-2-EGFP in the presence of mRFP or mRFP-Kif5b-CBD. These tracks of moving vesicles are real-time projections of individual frames over 3 minutes. (D) The numbers of moving calu-2-EGFP-containing vesicles per minute corresponding to (C) were manually counted (≥10 cells in each group). Scale bar, 10 µm.
Figure 8.
The export signal for calu-1/2-EGFP.
(A) Percentage secretion of different calu-2 truncated constructs. The right column shows the percentage secretion of nine constructs showed in the left column. These secretory rates were calculated according to the Western blotting results (data not shown, three independent experiments). (B) Point mutation effects on calu-2-EGFP secretion. Western blotting analysis showed calu-2-EGFP secretory level of two point mutations. Samples were probed with anti-EGFP and anti-tubulin antibodies. Co.St. was used as a loading control of CM. (C) The percentage secretion of calu-2-EGFP mutation constructs shown in (B) (three independent experiments).
Figure 9.
Schematic diagram illustrating the intracellular transport and secretory process of calu-1/2-EGFP.
(A) In the eukaryotic cell, calu-1/2-EGFP are simultaneously synthesized and translocated into the lumen of the endoplasmic reticulum, and then exit the ER with the help of the motor protein cytoplasmic dynein, being enclosed in vesicles (step I). After entering the Golgi apparatus, calu-1/2-EGFP are glycosylated and then transported within the secretory vesicles along microtubules by Kif5b to the periphery of the cell (step II). Then, calu-1/2-EGFP are secreted to the extracellular space either directly after attaching to the cell membrane (step III) or first accumulate in cellular processes and then are released together (step IV). (B) Model for “secretion after attachment”. (C) Model for “secretion after accumulation”.