The section on organelle biology, led by Jennifer Lippincott-Schwartz, investigates the mechanisms underlying the behavior of subcellular organelles (i.e., ER, nuclear envelope, Golgi apparatus, endosomes and lysosomes), including their biogenesis, maintenance, membrane sorting properties and dynamics within cells. Current studies are aimed at: 1) characterizing the pathways for membrane transport into and out of organelles, and their role in organelle biogenesis and maintenance; 2) defining the pathways and machinery underlying mitotic organelle disassembly and reassembly; 3) understanding the role and regulation of coat protein complexes involved in protein trafficking; 4) developing tools to analyze organelle dynamics and protein transport within live cells; and 5) characterizing lipid raft movement within cells, and its role in coordinating signaling responses and in generating cell polarity. Evidence that all Golgi proteins associate with the Golgi transiently and depend on the integrity of ER export for correct targetting To determine whether the Golgi apparatus contains stable components, we used fluorescence photobleaching techniques to investigate the manner in which GFP-tagged members of different classes of Golgi proteins, including enzymes, matrix proteins, coat proteins and itinerant proteins, associate with Golgi membranes. We found that all classes of Golgi components are transiently associated with this organelle, contrary to the prediction of a stable organelle model. Enzymes and itinerant components were found to continuously exit and re enter the Golgi apparatus by membrane trafficking pathways to and from the ER, while Golgi matrix proteins and coatomer underwent constant, rapid exchange between membrane and cytoplasm. When ER-to-Golgi transport was inhibited by BFA treatment and the use of mutant constructs of Arf1 and Sar1 that are blocked at different stages of the GTPase cycle, Golgi structure disassembled leaving no residual Golgi elements. These results reveal that the Golgi apparatus is a dynamic steady-state system, whose maintenance depends on continual input from the ER and is regulated by the activities of the Sar1-COPII and Arf1-coatomer systems. Mitotic disassembly of the Golgi apparatus and its role in nuclear and cytoplasmic division During mitosis many membrane-bound organelles, including the ER and mitochondria, remain essentially intact. The Golgi apparatus, however, reversibly disassembles. Exactly why the Golgi disassembles during mitosis is not known, but is widely assumed to be required for partitioning Golgi membranes equivalently between daughter cells. We have tested this hypothesis by inhibiting mitotic breakdown of the Golgi apparatus using a GTP-locked Arf1 mutant or the drug H89. In these cells, chromosome condensation, spindle formation, nuclear envelope breakdown and chromosomes alignment at the metaphase plate all occurred normally despite the presence of an intact Golgi. Strikingly, Golgi stacks remained tightly associated with centrioles, and distributed equally between daughter cells at cytokinesis. The data indicate, therefore, that Golgi breakdown is not necessary for ensuring Golgi partitioning between daughter cells. Moreover, they demonstrate that the cell can still progress through mitosis in the absence of Golgi breakdown. However, when the Golgi failed to disassemble during mitosis, chromosomes didn't segregate properly and cytokinesis was often incomplete. This phenotype could be rescued by treating cells with BFA prior to H89 treatment, which released Arf1 regulated peripheral proteins from the Golgi. Given the presence of numerous Arf1-regulated peripheral proteins on the Golgi that have known nuclear functions and roles in cytokinesis, we are currently investigating whether mitotic Golgi disassembly, shown to be dependent on Arf1 inactivation, serves to release Arf1-regulated proteins from the Golgi, allowing them to function in mitotic chromosome segregation and cytokinesis. Analysis of Golgi membrane coating and uncoating by COPI and Arf1 Cytosolic coat proteins that bind reversibly to membranes play a central role in membrane transport within the secretory pathway. We have used GFP-tagged COPI and Arf1 chimeras to characterize the membrane coating/uncoating cycle of COPI and its regulation by the small GTP-binding protein, Arf1. Using low temperature to block vesicle formation, we show that cycling of COPI on/off membranes and its regulation by Arf1 can be uncoupled from vesicle formation. This demonstrates that feedback from productive vesicle budding is not necessary for COPI uncoating. Based on these results and an analysis of COPI and Arf1 membrane binding/release kinetics, we propose that repeated cycles of binding and stochastic release of Arf1 and COPI serve to generate and maintain differentiated ?coated? membrane domains. These domains would last longer than the 37 sec life-time of an individual COPI complex on membranes and would allow transport intermediates to bud on a different time-scale than COPI membrane dissociation. Rapid cycling of lipid raft markers between the cell surface and Golgi apparatus We have used time-lapse fluorescence microscopy and selective photobleaching techniques to follow the intracellular itineraries of lipid raft markers, including CD59, GPI GFP, and lipid binding B subunits of cholera and shiga toxins (CTxB and STxB, respectively). Our data show that the raft markers cycle continuously between the PM and Golgi apparatus. GPI-GFP, and a proportion of CTxB and STxB, were excluded from clathrin-coated pits and reached the Golgi apparatus independently of both clathrin-interacting endocytic machinery and rab 5. The clathrin-independent pathway to the Golgi followed by raft markers was sensitive to cholesterol depletion and to 20?C. This pathway is likely to be important for Golgi retrieval of protein machinery and glycosphingolipids that are involved in protein sorting and trafficking within the Golgi. We are currently investigating its role in sorting of raft-associated signaling molecules and in the generation of cell polarity.

Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2001
Total Cost
Indirect Cost
Name
U.S. National Inst/Child Hlth/Human Dev
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Mavrakis, Manos; Rikhy, Richa; Lippincott-Schwartz, Jennifer (2009) Cells within a cell: Insights into cellular architecture and polarization from the organization of the early fly embryo. Commun Integr Biol 2:313-4
Hailey, Dale W; Lippincott-Schwartz, Jennifer (2009) Using photoactivatable proteins to monitor autophagosome lifetime. Methods Enzymol 452:25-45
Gillette, Jennifer M; Lippincott-Schwartz, Jennifer (2009) Hematopoietic progenitor cells regulate their niche microenvironment through a novel mechanism of cell-cell communication. Commun Integr Biol 2:305-7
Shtengel, Gleb; Galbraith, James A; Galbraith, Catherine G et al. (2009) Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci U S A 106:3125-30
Patterson, George H; Hirschberg, Koret; Polishchuk, Roman S et al. (2008) Transport through the Golgi apparatus by rapid partitioning within a two-phase membrane system. Cell 133:1055-67
Belov, George A; Altan-Bonnet, Nihal; Kovtunovych, Gennadiy et al. (2007) Hijacking components of the cellular secretory pathway for replication of poliovirus RNA. J Virol 81:558-67
Mavrakis, Manos; Lippincott-Schwartz, Jennifer; Stratakis, Constantine A et al. (2007) mTOR kinase and the regulatory subunit of protein kinase A (PRKAR1A) spatially and functionally interact during autophagosome maturation. Autophagy 3:151-3
Wakabayashi, Yoshiyuki; Chua, Jennifer; Larkin, Janet M et al. (2007) Four-dimensional imaging of filter-grown polarized epithelial cells. Histochem Cell Biol 127:463-72
Altan-Bonnet, Nihal; Sougrat, Rachid; Liu, Wei et al. (2006) Golgi inheritance in mammalian cells is mediated through endoplasmic reticulum export activities. Mol Biol Cell 17:990-1005
Rey, Osvaldo; Papazyan, Romeo; Waldron, Richard T et al. (2006) The nuclear import of protein kinase D3 requires its catalytic activity. J Biol Chem 281:5149-57

Showing the most recent 10 out of 48 publications