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Membrane expansion increases endocytosis rate during mitosis.

Raucher D, Sheetz MP - J. Cell Biol. (1999)

Bottom Line: Mitosis in mammalian cells is accompanied by a dramatic inhibition of endocytosis.We have found that the addition of amphyphilic compounds to metaphase cells increases the endocytosis rate even to interphase levels.Detergents and solvents all increased endocytosis rate, and the extent of increase was in direct proportion to the concentration added.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Mitosis in mammalian cells is accompanied by a dramatic inhibition of endocytosis. We have found that the addition of amphyphilic compounds to metaphase cells increases the endocytosis rate even to interphase levels. Detergents and solvents all increased endocytosis rate, and the extent of increase was in direct proportion to the concentration added. Although the compounds could produce a variety of different effects, we have found a strong correlation with a physical alteration in the membrane tension as measured by the laser tweezers. Plasma membrane tethers formed by latex beads pull back on the beads with a force that was related to the in-plane bilayer tension and membrane- cytoskeletal adhesion. We found that as cells enter mitosis, the membrane tension rises as the endocytosis rate decreases; and as cells exited mitosis, the endocytosis rate increased as the membrane tension decreased. The addition of amphyphilic compounds decreased membrane tension and increased the endocytosis rate. With the detergent, deoxycholate, the endocytosis rate was restored to interphase levels when the membrane tension was restored to interphase levels. Although biochemical factors are clearly involved in the alterations in mitosis, we suggest that endocytosis is blocked primarily by the increase in apparent plasma membrane tension. Higher tensions inhibit both the binding of the endocytic complex to the membrane and mechanical deformation of the membrane during invagination. We suggest that membrane tension is an important regulator of the endocytosis rate and alteration of tension is sufficient to modify endocytosis rates during mitosis. Further, we postulate that the rise in membrane tension causes cell rounding and the inhibition of motility, characteristic of mitosis.

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(a) Quantitation of  uptake of fluorescein-dextran  by interphase and mitotic  cells in different phases. Endocytic events in the various  phases of mitosis are illustrated with paired fluorescence micrographs of FM1-43  (top) and DAPI-labeled  HeLa cells (bottom). (b)  Prometaphase; (c) interphase (left) and metaphase  (right); (d) anaphase; (e)  telophase; (f) quantitation of  uptake of FM1-43 labeled endocytic vesicles by interphase and mitotic cells in different phases. The data given are average from seven  different experiments and the number of labeled endocytic vesicles was counted in 10–60 cells at each mitotic phase. Bars indicate SEM.
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Figure 1: (a) Quantitation of uptake of fluorescein-dextran by interphase and mitotic cells in different phases. Endocytic events in the various phases of mitosis are illustrated with paired fluorescence micrographs of FM1-43 (top) and DAPI-labeled HeLa cells (bottom). (b) Prometaphase; (c) interphase (left) and metaphase (right); (d) anaphase; (e) telophase; (f) quantitation of uptake of FM1-43 labeled endocytic vesicles by interphase and mitotic cells in different phases. The data given are average from seven different experiments and the number of labeled endocytic vesicles was counted in 10–60 cells at each mitotic phase. Bars indicate SEM.

Mentions: In mitosis, a dramatic decrease in endocytosis rate has been reported (Berlin et al., 1978). We repeated those observations (Fig. 1) using either fluorescent dextran or FM1-43 uptake as a measure of endocytosis rate by measuring the number of vesicles per cell and the total fluorescence intensity. With fluorescent dextran, the endocytosis rate normally decreased to ∼10% of interphase level in metaphase. FM1-43 is a water-soluble dye that is membrane impermeable, but it is readily incorporated into endocytic vesicles and is retained after cell fixation (Betz and Bewick, 1992; Terasaki, 1995). Fig. 1, b–e shows FM1-43 labeling of HeLa cells in various stages of mitosis in parallel with the DAPI staining patterns that enable rapid identification of mitotic phases in HeLa cells (Fig. 1, b–e, bottom). While interphase cells were brightly stained with FM1-43-labeled endocytic vesicles spread throughout the cytoplasm (low numerical aperture objectives were used to detect even out of focus vesicles), many fewer labeled vesicles were found in mitotic cells (Fig. 1, b–e, top). The number of endocytic events in each image frame was counted for each mitotic phase and the results are summarized in Fig. 1 e. In prometaphase, uptake of endocytic vesicles decreased to 40% of interphase value. Metaphase cells had the lowest endocytosis rate (20% of interphase). In progressing to anaphase and cytokinesis, endocytosis increased, corresponding to 60% of the interphase value. It should be noted that the value for endocytosis rate in cytokinesis is most likely higher than shown in Fig. 1, a or f. This is because during the incubation period before observation the cells probably moved from the short anaphase period into cytokinesis. We have also quantified endocytosis by measuring the sum of the fluorescence intensities of the FM1-43-labeled endocytic vesicles. Results obtained by this method were similar to results obtained by counting the number of fluorescent spots. A similar decrease in FM1-43 uptake, as compared with interphase cells, was evident from prometaphase through cytokinesis reaching the lowest value in metaphase (Fig. 1 f). Since quantification of endocytosis by counting the number of fluorescent vesicles is simple and reproducible and the data are consistent with that obtained by measuring the sum of the fluorescence intensities, all endocytosis measurements are presented in this form. The smaller decrease in FM1-43 uptake to 20% of interphase compared with 10% for dextran could be result of a decrease in average vesicle size, since the ratio of vesicle membrane area to volume increases in smaller vesicles.


Membrane expansion increases endocytosis rate during mitosis.

Raucher D, Sheetz MP - J. Cell Biol. (1999)

(a) Quantitation of  uptake of fluorescein-dextran  by interphase and mitotic  cells in different phases. Endocytic events in the various  phases of mitosis are illustrated with paired fluorescence micrographs of FM1-43  (top) and DAPI-labeled  HeLa cells (bottom). (b)  Prometaphase; (c) interphase (left) and metaphase  (right); (d) anaphase; (e)  telophase; (f) quantitation of  uptake of FM1-43 labeled endocytic vesicles by interphase and mitotic cells in different phases. The data given are average from seven  different experiments and the number of labeled endocytic vesicles was counted in 10–60 cells at each mitotic phase. Bars indicate SEM.
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Related In: Results  -  Collection

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Figure 1: (a) Quantitation of uptake of fluorescein-dextran by interphase and mitotic cells in different phases. Endocytic events in the various phases of mitosis are illustrated with paired fluorescence micrographs of FM1-43 (top) and DAPI-labeled HeLa cells (bottom). (b) Prometaphase; (c) interphase (left) and metaphase (right); (d) anaphase; (e) telophase; (f) quantitation of uptake of FM1-43 labeled endocytic vesicles by interphase and mitotic cells in different phases. The data given are average from seven different experiments and the number of labeled endocytic vesicles was counted in 10–60 cells at each mitotic phase. Bars indicate SEM.
Mentions: In mitosis, a dramatic decrease in endocytosis rate has been reported (Berlin et al., 1978). We repeated those observations (Fig. 1) using either fluorescent dextran or FM1-43 uptake as a measure of endocytosis rate by measuring the number of vesicles per cell and the total fluorescence intensity. With fluorescent dextran, the endocytosis rate normally decreased to ∼10% of interphase level in metaphase. FM1-43 is a water-soluble dye that is membrane impermeable, but it is readily incorporated into endocytic vesicles and is retained after cell fixation (Betz and Bewick, 1992; Terasaki, 1995). Fig. 1, b–e shows FM1-43 labeling of HeLa cells in various stages of mitosis in parallel with the DAPI staining patterns that enable rapid identification of mitotic phases in HeLa cells (Fig. 1, b–e, bottom). While interphase cells were brightly stained with FM1-43-labeled endocytic vesicles spread throughout the cytoplasm (low numerical aperture objectives were used to detect even out of focus vesicles), many fewer labeled vesicles were found in mitotic cells (Fig. 1, b–e, top). The number of endocytic events in each image frame was counted for each mitotic phase and the results are summarized in Fig. 1 e. In prometaphase, uptake of endocytic vesicles decreased to 40% of interphase value. Metaphase cells had the lowest endocytosis rate (20% of interphase). In progressing to anaphase and cytokinesis, endocytosis increased, corresponding to 60% of the interphase value. It should be noted that the value for endocytosis rate in cytokinesis is most likely higher than shown in Fig. 1, a or f. This is because during the incubation period before observation the cells probably moved from the short anaphase period into cytokinesis. We have also quantified endocytosis by measuring the sum of the fluorescence intensities of the FM1-43-labeled endocytic vesicles. Results obtained by this method were similar to results obtained by counting the number of fluorescent spots. A similar decrease in FM1-43 uptake, as compared with interphase cells, was evident from prometaphase through cytokinesis reaching the lowest value in metaphase (Fig. 1 f). Since quantification of endocytosis by counting the number of fluorescent vesicles is simple and reproducible and the data are consistent with that obtained by measuring the sum of the fluorescence intensities, all endocytosis measurements are presented in this form. The smaller decrease in FM1-43 uptake to 20% of interphase compared with 10% for dextran could be result of a decrease in average vesicle size, since the ratio of vesicle membrane area to volume increases in smaller vesicles.

Bottom Line: Mitosis in mammalian cells is accompanied by a dramatic inhibition of endocytosis.We have found that the addition of amphyphilic compounds to metaphase cells increases the endocytosis rate even to interphase levels.Detergents and solvents all increased endocytosis rate, and the extent of increase was in direct proportion to the concentration added.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Mitosis in mammalian cells is accompanied by a dramatic inhibition of endocytosis. We have found that the addition of amphyphilic compounds to metaphase cells increases the endocytosis rate even to interphase levels. Detergents and solvents all increased endocytosis rate, and the extent of increase was in direct proportion to the concentration added. Although the compounds could produce a variety of different effects, we have found a strong correlation with a physical alteration in the membrane tension as measured by the laser tweezers. Plasma membrane tethers formed by latex beads pull back on the beads with a force that was related to the in-plane bilayer tension and membrane- cytoskeletal adhesion. We found that as cells enter mitosis, the membrane tension rises as the endocytosis rate decreases; and as cells exited mitosis, the endocytosis rate increased as the membrane tension decreased. The addition of amphyphilic compounds decreased membrane tension and increased the endocytosis rate. With the detergent, deoxycholate, the endocytosis rate was restored to interphase levels when the membrane tension was restored to interphase levels. Although biochemical factors are clearly involved in the alterations in mitosis, we suggest that endocytosis is blocked primarily by the increase in apparent plasma membrane tension. Higher tensions inhibit both the binding of the endocytic complex to the membrane and mechanical deformation of the membrane during invagination. We suggest that membrane tension is an important regulator of the endocytosis rate and alteration of tension is sufficient to modify endocytosis rates during mitosis. Further, we postulate that the rise in membrane tension causes cell rounding and the inhibition of motility, characteristic of mitosis.

Show MeSH
Related in: MedlinePlus