Limits...
Annexin VI-mediated loss of spectrin during coated pit budding is coupled to delivery of LDL to lysosomes.

Kamal A, Ying Y, Anderson RG - J. Cell Biol. (1998)

Bottom Line: Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding.The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded.Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.

ABSTRACT
Previously we reported that annexin VI is required for the budding of clathrin-coated pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding. Annexin VI-dependent budding is accompanied by the loss of approximately 50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the budding of these new coated pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of coated pits, one of which requires the annexin VI-dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.

Show MeSH

Related in: MedlinePlus

Microinjection of AnxVIΔ192 or antispectrin IgG inhibits LDL uptake. Human fibroblasts were microinjected with 3 mg/ml FITC-dextran and 100 μM AnxVIwt (A and B) or 100  μM AnxVIΔ192 (C and D) or 300 μg/ml anti-rabbit IgG (E and F) or 300 μg/ml antispectrin IgG  (G and H). The cells were allowed to recover for  1 h at 37°C and then the uptake of fluorescent  LDL (25 μg/ml) for 1 h at 37°C was measured. B,  D, F, and H show LDL uptake and A, C, E, and  G show FITC-dextran staining of the same cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132873&req=5

Figure 8: Microinjection of AnxVIΔ192 or antispectrin IgG inhibits LDL uptake. Human fibroblasts were microinjected with 3 mg/ml FITC-dextran and 100 μM AnxVIwt (A and B) or 100 μM AnxVIΔ192 (C and D) or 300 μg/ml anti-rabbit IgG (E and F) or 300 μg/ml antispectrin IgG (G and H). The cells were allowed to recover for 1 h at 37°C and then the uptake of fluorescent LDL (25 μg/ml) for 1 h at 37°C was measured. B, D, F, and H show LDL uptake and A, C, E, and G show FITC-dextran staining of the same cells.

Mentions: The dominant-negative–acting AnxVIΔ192 had the same effect on coated pit budding and spectrin loss in vitro as ALLN (refer to Fig. 1 B and Fig. 3 A). Therefore, we microinjected AnxVIwt and AnxVIΔ192 into normal human fibroblasts and compared the effect of these proteins on the uptake of fluorescent LDL (Fig. 8, A–D and Table III). Cells were injected with the indicated recombinant protein mixed with FITC-labeled dextran to mark the cell, allowed to recover for 1 h, and then incubated an additional 1 h in the presence of fluorescent LDL. Cells injected with the AnxVIwt (Fig. 8 A) took up normal amounts of pyrene- labeled LDL, compared with nearby uninjected cells, and delivered it to the perinuclear area of the cell (Fig. 8 B). Internalization of LDL in cells injected with AnxVIΔ192 (Fig. 8 C), by contrast, was markedly reduced (Fig. 8 D) compared with neighboring uninjected cells. In addition, the LDL-positive vesicles in these cells tended to be more randomly distributed. Quantification of these results (Table III) showed that 83% of the cells injected with AnxVIΔ192 had a reduced uptake of LDL and 17% did not internalize any detectable LDL.


Annexin VI-mediated loss of spectrin during coated pit budding is coupled to delivery of LDL to lysosomes.

Kamal A, Ying Y, Anderson RG - J. Cell Biol. (1998)

Microinjection of AnxVIΔ192 or antispectrin IgG inhibits LDL uptake. Human fibroblasts were microinjected with 3 mg/ml FITC-dextran and 100 μM AnxVIwt (A and B) or 100  μM AnxVIΔ192 (C and D) or 300 μg/ml anti-rabbit IgG (E and F) or 300 μg/ml antispectrin IgG  (G and H). The cells were allowed to recover for  1 h at 37°C and then the uptake of fluorescent  LDL (25 μg/ml) for 1 h at 37°C was measured. B,  D, F, and H show LDL uptake and A, C, E, and  G show FITC-dextran staining of the same cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132873&req=5

Figure 8: Microinjection of AnxVIΔ192 or antispectrin IgG inhibits LDL uptake. Human fibroblasts were microinjected with 3 mg/ml FITC-dextran and 100 μM AnxVIwt (A and B) or 100 μM AnxVIΔ192 (C and D) or 300 μg/ml anti-rabbit IgG (E and F) or 300 μg/ml antispectrin IgG (G and H). The cells were allowed to recover for 1 h at 37°C and then the uptake of fluorescent LDL (25 μg/ml) for 1 h at 37°C was measured. B, D, F, and H show LDL uptake and A, C, E, and G show FITC-dextran staining of the same cells.
Mentions: The dominant-negative–acting AnxVIΔ192 had the same effect on coated pit budding and spectrin loss in vitro as ALLN (refer to Fig. 1 B and Fig. 3 A). Therefore, we microinjected AnxVIwt and AnxVIΔ192 into normal human fibroblasts and compared the effect of these proteins on the uptake of fluorescent LDL (Fig. 8, A–D and Table III). Cells were injected with the indicated recombinant protein mixed with FITC-labeled dextran to mark the cell, allowed to recover for 1 h, and then incubated an additional 1 h in the presence of fluorescent LDL. Cells injected with the AnxVIwt (Fig. 8 A) took up normal amounts of pyrene- labeled LDL, compared with nearby uninjected cells, and delivered it to the perinuclear area of the cell (Fig. 8 B). Internalization of LDL in cells injected with AnxVIΔ192 (Fig. 8 C), by contrast, was markedly reduced (Fig. 8 D) compared with neighboring uninjected cells. In addition, the LDL-positive vesicles in these cells tended to be more randomly distributed. Quantification of these results (Table III) showed that 83% of the cells injected with AnxVIΔ192 had a reduced uptake of LDL and 17% did not internalize any detectable LDL.

Bottom Line: Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding.The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded.Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.

ABSTRACT
Previously we reported that annexin VI is required for the budding of clathrin-coated pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding. Annexin VI-dependent budding is accompanied by the loss of approximately 50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the budding of these new coated pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of coated pits, one of which requires the annexin VI-dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.

Show MeSH
Related in: MedlinePlus