Analysis of transferrin recycling in mitotic and interphase hela cells by quantitative fluorescence microscopy

Sager, Polly R.; Brown, Paul A.J.; Berlin, Richard D.

doi:10.1016/0092-8674(84)90005-9

Cited by 84 publications

(66 citation statements)

References 21 publications

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“…As expected, the receptor, which contains a YXXΦ sorting motif was regulated in the same way as the CD8-YAAL reporter. Our transferrin uptake experiments confirmed many previous reports of reduced transferrin uptake during mitosis (7,11,15,22,36,37). Staining of surface TfR indicated that it was abundant at the cell surface during all stages of the cell cycle in HeLa cells.…”

Section: Discussionsupporting

confidence: 80%

“…Further studies demonstrated that uptake of fluorescent dextran ceases during mitosis and also confirmed the timing of shutdown to be in early prophase (7). Many other studies went on to confirm and further develop the theory of mitotic shutdown of endocytosis, showing that it is temporary and resumes in telophase (8)(9)(10)(11)(12)(13)(14)(15)(16)(17). The reason for shutdown is unclear (18,19), but one recent idea is that "moonlighting" functions for endocytic proteins during mitosis may contribute to the inhibition (20,21).…”

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confidence: 95%

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Clathrin-mediated endocytosis is inhibited during mitosis

Fielding

Willox

Okeke

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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A long-standing paradigm in cell biology is the shutdown of endocytosis during mitosis. There is consensus that transferrin uptake is inhibited after entry into prophase and that it resumes in telophase. A recent study proposed that endocytosis is continuous throughout the cell cycle and that the observed inhibition of transferrin uptake is due to a decrease in available transferrin receptor at the cell surface, and not to a shutdown of endocytosis. This challenge to the established view is gradually becoming accepted. Because of this controversy, we revisited the question of endocytic activity during mitosis. Using an antibody uptake assay and controlling for potential changes in surface receptor density, we demonstrate the strong inhibition of endocytosis in mitosis of CD8 chimeras containing any of the three major internalization motifs for clathrin-mediated endocytosis (YXXΦ, [DE]XXXL[LI], or FXNPXY) or a CD8 protein with the cytoplasmic tail of the cationindependent mannose 6-phosphate receptor. The shutdown is not gradual: We describe a binary switch from endocytosis being "on" in interphase to "off" in mitosis as cells traverse the G 2 /M checkpoint. In addition, we show that the inhibition of transferrin uptake in mitosis occurs despite abundant transferrin receptor at the surface of HeLa cells. Our study finds no support for the recent idea that endocytosis continues during mitosis, and we conclude that endocytosis is temporarily shutdown during early mitosis.

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Section: Discussionsupporting

confidence: 80%

mentioning

confidence: 95%

Clathrin-mediated endocytosis is inhibited during mitosis

Fielding

Willox

Okeke

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Multiple steps in the transfer of the growing GAG chains through the Golgi apparatus are probably affected. Such transfers are believed to occur by membrane vesicles, and a defect in their capacity to fuse with Golgi membrane, as Warren points out (42), would represent the functional equivalent of the failure to form endocytic vesicles (1, 2), to discharge secretory vesicles (9), or recycle receptors (35,41). All vesicle-mediated transport would appear to be inhibited during mitosis.…”

Section: Discussionmentioning

confidence: 99%

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1.

Preston¹,

Regula²,

Sager³

et al. 1985

The Journal of Cell Biology

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[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two-to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate.Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.In their pioneering work on glycosaminoglycan (GAG) ~ synthesis by cultured cells, Kraemer and Tobey (14) showed that a significant fraction of trypsin-releasable (i.e., surface) GAGs was shed during mitosis. Although the Chinese hamster ovary (CHO) cells analyzed were grown in suspension, viewed from the perspective of recent studies of cell adhesion (e.g., reference 32), it seemed likely that the shedding of GAGs was related to the familiar ease with which mitotic cells are detached from monolayers. We hypothesized that any effect of shedding would be reinforced by a coordinate reduction in GAG synthesis that would delay replacement of surface GAGs, and that a rapid resumption of GAG synthesis in early G i would be required for cell reattachment.A parallel motivation for our studies arose from the accumulating evidence that membrane vesicle-mediated transport is depressed during mitosis. This is shown clearly in work from our laboratory demonstrating depression of endocytosis ( 1, 2) and transferrin recycling (35) and from that of Warren and colleagues showing a decrease in surface transferrin receptors (41), and in depression of histamine secretion (9). These studies focus primarily on fusion and budding events at the plasma membrane. In addition, the insertion of the G t Abbreviations used in this paper. CHO, Chinese hamster ovary; GAG, glycosaminoglycan.protein of vesicular stomatitis virus is also depressed during mitosis (42), which suggests a defect in its processing through the Golgi apparatus. Since carbohydrate chains of GAGs are largely assembled within the Golgi apparatus by multiple enzymes (31), a general defect in processing should affect GAG synthesis. Moreover, the apparent localization of sulfation to the trans-Gol~ apparatus (7, 44) would serve to focus on later steps of the processing sequence.We describe here the rates of labeled sulfate incorporation into CHO cells during mitosis, in their subsequent movement into G1, and in late G2. We followed incorporat...

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“…There is also a general cessation of membrane traffic (Warren, 1985, including inhibition of the endocytic pathway as first described by Fawcett (1965). Both invagination of coated pits (Berlin et al, 1978;Pypaert et al, 1987) and recycling of receptors to the cell surface (Sager et al, 1984;Warren et al, 1984) are blocked. From these observations it can be inferred that fusion between endocytic vesicles is also inhibited in mitotic cells.…”

Section: Introductionmentioning

confidence: 99%

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

Woodman

Adamczewski

Hunt

et al. 1993

MBoC

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Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.

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Analysis of transferrin recycling in mitotic and interphase hela cells by quantitative fluorescence microscopy

Cited by 84 publications

References 21 publications

Clathrin-mediated endocytosis is inhibited during mitosis

Clathrin-mediated endocytosis is inhibited during mitosis

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1.

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

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