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Research Scientist, Cell Biology Director of Electron Microscopy Core Facility Assistant Investigator, Ludwig Institute for Cancer Research |
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Phone: (203) 785-3681 Lab: (203) 785-5390 Fax: (203) 785-7446 e-mail: marc.pypaert@yale.edu |
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Department of Cell Biology <Courier Address> |
In addition to providing support and service in electron microscopy, we collaborate with a number of labs, looking at different aspects of membrane trafficking in yeast and mammalian cells. One such collaboration, with the laboratory of Graham Warren, is a study by immuno-electron microscopy of the mechanism of Golgi reassembly at the end of mitosis. During interphase, the mammalian Golgi apparatus exists as a single-copy organelle made of 4-5 stacked cisternae. Immunocytochemical studies have shown that the various enzymes and structural proteins of the Golgi apparatus are localized to specific cisternae, thereby creating a cis to trans polarity within the organelle (Fig. 1). To allow accurate partitioning of this organelle, the Golgi breaks down at the onset of mitosis into hundreds of clusters of small vesicles that partition randomly between the two poles of the dividing cell (Fig. 2). At the end of mitosis, these Golgi fragments coalesce to reform the stacked cisternae. Reassembly of the Golgi must be an organized event since the cis-trans polarity is restored in the newly-reassembled Golgi, yet little is known about the exact sequence of events that regenerate this polarity. One hypothesis, supported by observations both in vivo and in vitro, is that the cis Golgi rebuilds first and acts as a template on which the rest of the Golgi reassembles. Using triple immunolabeling of ultrathin cryosections of a mixed population of mitotic and interphase NRK cells, we are attempting to determine the rate at which different Golgi markers appear in the reassembling Golgi stack during telophase (Fig. 3). Identifying the order of Golgi reassembly should ultimately allow a greater understanding of the role of structural Golgi proteins during cell division, and the means by which the Golgi remains stacked during interphase.
Figure 1: Triple-labeling of an interphase Golgi stack (NRK cells).
Three Golgi markers label three separate cisternae of the Golgi stack. GM130 (15 nm gold) localizes to a fenestrated cis cisterna that is closely apposed to an ER exit site. Mannosidase II (5 nm gold; arrows) labeling is present on the next two cisternae of the stack (medial Golgi). TGN38 labeling (10 nm gold) is found on a tubular network at the opposite side of the stack – the trans-Golgi network. nm. Bar: 200
Figure 2: Triple-labeling of a mitotic Golgi cluster (Metaphase NRK cell).
Mannosidase II (5 nm gold; arrows), TGN38 (10 nm gold) and GM130 (15 nm gold) are found on distinct vesicles or small tubules in the same cluster. Bar: 250 nm.
Figure 3: A reassembling Golgi stack in a telophase NRK cell.
A small cisterna containing GM130 (15 nm gold; arrowhead) is surrounded by tubules and vesicles, some of which contain Mannosidase II (5 nm gold; arrows). Bar: 200 nm.
Selected Publications
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Seemann J, Jokitalo E, Pypaert M, Warren G. (2000) Matrix proteins can generate the higher order architecture of the Golgi apparatus. Nature 407: 1022-1026. 
Fölsch H, Pypaert M, Schu P, Mellman I. (2001) Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells. J Cell Biol. 152: 595-606.
Seemann J, Pypaert M, Taguchi T, Malsam J, Warren G. (2002) Partitioning of the matrix fraction of the Golgi apparatus during mitosis in animal cells. Science 295: 848-851.
Pelletier L, Stern C A, Pypaert M, Sheff D, Ngo HM, Roper N, He CY, Hu K, Toomre D, Coppens I, Roos DS, Joiner KA, Warren G. (2002) Golgi biogenesis in Toxoplasma gondii. Nature 418: 548-552.
Trombetta ES, Ebersold M, Garrett W, Pypaert M, Mellman I. (2003) Activation of lysosomal function during dendritic cell maturation. Science 299: 1400-1403.
Taguchi T, Pypaert M, Warren G. (2003) Biochemical sub-fractionation of the Mammalian Golgi apparatus. Traffic 4: 344-352.
Wang Y, Seemann J, Pypaert M, Shorter J, Warren G. (2003) A direct role for GRASP65 as a mitotically regulated Golgi stacking factor. EMBO J. 22: 3279-3290.
Ang AL, Fölsch H, Koivisto U-M, Pypaert M, Mellman I. (2003) The Rab8 GTPase selectively regulates AP-1B–dependent basolateral transport in polarized Madin-Darby canine kidney cells. J Cell Biol. 163: 339-350.
