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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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Tether force in  taxol or vinblastine arrested  cells. (a) Photomicrograph of  HeLa cells incubated for one  cell cycle with vinblastine  (cell cycle was blocked in  a stage which resembled  prometaphase or metaphase  since chromosomes had not  undergone anaphase segregation). (b) Photomicrograph  of HeLa cells blocked by  taxol at the metaphase-anaphase boundary. Bar, 5  μm. (c) 30% increase in  tether force in vinblastine arrested cells and twofold increase in tether force in taxol  arrested cells with respect to  tether force of untreated interphase cells. (d) Endocytosis was determined by  FACS® analysis. The data are  expressed as the relative fluorescence index (RFI), relative to control cells, and represents the mean of triplicate  determination ± SD.
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Figure 5: Tether force in taxol or vinblastine arrested cells. (a) Photomicrograph of HeLa cells incubated for one cell cycle with vinblastine (cell cycle was blocked in a stage which resembled prometaphase or metaphase since chromosomes had not undergone anaphase segregation). (b) Photomicrograph of HeLa cells blocked by taxol at the metaphase-anaphase boundary. Bar, 5 μm. (c) 30% increase in tether force in vinblastine arrested cells and twofold increase in tether force in taxol arrested cells with respect to tether force of untreated interphase cells. (d) Endocytosis was determined by FACS® analysis. The data are expressed as the relative fluorescence index (RFI), relative to control cells, and represents the mean of triplicate determination ± SD.

Mentions: To check whether the rise in tension is a result of a transitory modification or if it persists over longer periods when the cells are locked at the prometaphase/metaphase boundary, we examined membrane tension in mitotic HeLa cells arrested by vinblastine or taxol. Since, membrane tension is sensitive to drugs that affect microtubule polymerization (data not shown; Dai and Sheetz, 1995a), it is essential to inhibit mitosis and cell proliferation without causing changes in microtubule polymer mass. Addition of very low concentrations of vinblastine or taxol to the growth media of HeLa cell cultures induces significant cell cycle arrest, without causing net depolymerization or polymerization of microtubules (Wendell, 1993). As shown in Fig. 5 a, the anti-tumor drug taxol inhibited HeLa cell proliferation by inducing a mitotic block at the metaphase/ anaphase boundary (Jordan et al., 1993). Similarly in vinblastine-treated cells, chromosomes had not undergone anaphase segregation, and the cell cycle was blocked in a stage that resembled prometaphase or metaphase (Fig. 5 b). Interestingly, static tether force almost doubled in taxol-arrested cells and increased 40% in vinblastine-treated cells, with respect to interphase cells (Fig. 5 c). The uptake of FM1-43 in taxol and vinblastine arrested mitotic cells was determined using a FACScan® cytometer. The increase in tether force was accompanied by a 60% decrease in uptake of FM1-43 in taxol-arrested cells and a 35% decrease in vinblastine-treated cells (Fig. 5 d). These results suggest that in cells locked in mitosis the endocytosis rate is decreased and membrane tension is increased without alteration in microtubule polymer mass.


Membrane expansion increases endocytosis rate during mitosis.

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

Tether force in  taxol or vinblastine arrested  cells. (a) Photomicrograph of  HeLa cells incubated for one  cell cycle with vinblastine  (cell cycle was blocked in  a stage which resembled  prometaphase or metaphase  since chromosomes had not  undergone anaphase segregation). (b) Photomicrograph  of HeLa cells blocked by  taxol at the metaphase-anaphase boundary. Bar, 5  μm. (c) 30% increase in  tether force in vinblastine arrested cells and twofold increase in tether force in taxol  arrested cells with respect to  tether force of untreated interphase cells. (d) Endocytosis was determined by  FACS® analysis. The data are  expressed as the relative fluorescence index (RFI), relative to control cells, and represents the mean of triplicate  determination ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132908&req=5

Figure 5: Tether force in taxol or vinblastine arrested cells. (a) Photomicrograph of HeLa cells incubated for one cell cycle with vinblastine (cell cycle was blocked in a stage which resembled prometaphase or metaphase since chromosomes had not undergone anaphase segregation). (b) Photomicrograph of HeLa cells blocked by taxol at the metaphase-anaphase boundary. Bar, 5 μm. (c) 30% increase in tether force in vinblastine arrested cells and twofold increase in tether force in taxol arrested cells with respect to tether force of untreated interphase cells. (d) Endocytosis was determined by FACS® analysis. The data are expressed as the relative fluorescence index (RFI), relative to control cells, and represents the mean of triplicate determination ± SD.
Mentions: To check whether the rise in tension is a result of a transitory modification or if it persists over longer periods when the cells are locked at the prometaphase/metaphase boundary, we examined membrane tension in mitotic HeLa cells arrested by vinblastine or taxol. Since, membrane tension is sensitive to drugs that affect microtubule polymerization (data not shown; Dai and Sheetz, 1995a), it is essential to inhibit mitosis and cell proliferation without causing changes in microtubule polymer mass. Addition of very low concentrations of vinblastine or taxol to the growth media of HeLa cell cultures induces significant cell cycle arrest, without causing net depolymerization or polymerization of microtubules (Wendell, 1993). As shown in Fig. 5 a, the anti-tumor drug taxol inhibited HeLa cell proliferation by inducing a mitotic block at the metaphase/ anaphase boundary (Jordan et al., 1993). Similarly in vinblastine-treated cells, chromosomes had not undergone anaphase segregation, and the cell cycle was blocked in a stage that resembled prometaphase or metaphase (Fig. 5 b). Interestingly, static tether force almost doubled in taxol-arrested cells and increased 40% in vinblastine-treated cells, with respect to interphase cells (Fig. 5 c). The uptake of FM1-43 in taxol and vinblastine arrested mitotic cells was determined using a FACScan® cytometer. The increase in tether force was accompanied by a 60% decrease in uptake of FM1-43 in taxol-arrested cells and a 35% decrease in vinblastine-treated cells (Fig. 5 d). These results suggest that in cells locked in mitosis the endocytosis rate is decreased and membrane tension is increased without alteration in microtubule polymer mass.

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