The Section on Organelle Biology investigates the mechanisms underlying the behavior of subcellular organelles (i.e., endoplasmic reticulum [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 generating cell polarity.

Evidence That All Golgi Proteins Associate with the Golgi Transiently and Depend on the Integrity of ER Export for Correct Targeting
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. Contrary to the prediction of a stable organelle model, we found that all classes of Golgi components are transiently associated with this organelle. 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 Brefeldin A (BFA) treatment and mutant constructs of Arf1 and Sar1 that are blocked at different stages of the GTPase cycle were used, the Golgi structure disassembled, leaving no residual Golgi elements. The 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 disassembly 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 with a GTP-locked Arf1 mutant or the drug H89. In these cells, chromosome condensation, spindle formation, nuclear envelope breakdown, and chromosome alignment at the metaphase plate all occurred normally, despite the presence of an intact Golgi. Strikingly, Golgi stacks remained tightly associated with centrioles and maintained their equal distribution between daughter cells at cytokinesis. Therefore, the data indicate that Golgi breakdown is not necessary for ensuring Golgi partitioning between daughter cells. Moreover, the data 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 did not segregate properly, and cytokinesis was often incomplete. This phenotype could be rescued by treating cells with BFA before H89 treatment, which released Arf1-regulated peripheral proteins from the Golgi. Given the presence of numerous Arf1-regulated peripheral proteins on the Golgi with known nuclear functions and roles in cytokinesis, we are currently investigating whether mitotic Golgi disassembly, shown to be dependent on Arf1 inactivation, releases 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 have shown that cycling of COPI on and 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 generate and maintain differentiated coated membrane domains. These domains would last longer than 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 the lipid-binding B subunits of cholera and shiga toxins (CTxB and STxB, respectively). Our data show that the raft markers cycle continuously between the plasma membrane 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. The 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 the pathway’s role in sorting raft-associated signaling molecules and the generation of cell polarity.