How membrane-bound organelles of eukaryotic cells maintain their identity and subcellular localization amidst an enormous input and outflow of membrane and protein is a central question in cell biology. Studies in this group have focused on this question and have sought to define the cellular and molecular mechanisms which underlie the organization and distribution of eukaryotic organelles. Particular attention has been paid to the Golgi complex which plays a fundamental role in the processing and sorting of protein moving through the secretory pathway. The Golgi complex in higher eukaryotes consists of stacks of flattened cisternae usually localized to the perinuclear region near the microtubular organizing center (MTOC). Recent studies have suggested this organization and positioning of the Golgi are controlled by dynamic processes. Tubulovesicular structures emerging from Golgi elements along microtubules, for example, enable adjacent Golgi stacks to communicate. In addition, reversible dispersal of Golgi elements occurs during microtubule disruption, mitosis and brefeldin A (BFA)-treatment. To further understand these processes and their relationship to the three-dimensional morphology and function of the Golgi we have taken four major approaches. 1) Imaging of the Golgi complex in living cells. Using the vital dye BODIPY-ceramide to label the Golgi complex in living cells we have performed time-lapse imaging to examine the dynamics of the Golgi complex in normal and BFA-treated cells. The important role of membrane tubulation in the normal dynamics and maintenance of Golgi structure within cells is under investigation. 2) Role of microtubules in Golgi localization and traffic. We have found that microtubules are important for both localizing the Golgi complex to the MTOC and for facilitating anterograde (ER-to-Golgi) and retrograde (Golgi-to-ER) membrane traffic into and out of this central region. Our characterization of Golgi dispersal induced by microtubule-disruption has suggested a mechanism involving membrane transport of Golgi components along retrograde and anterograde membrane pathways rather than diffusion or actin/myosin based transport. The role of microtubules in facilitating and directing membrane traffic to and from the Golgi, therefore, is likely to underlie the subcellular location of this organelle. 3) Role of microtubule motors in Golgi traffic. We have found that the microtubule motor protein, kinesin, associates with the anterograde and retrograde pathways leading to and from the Golgi complex. Conditions of microtubule disruption and low temperature treatments, which slow retrograde traffic, result in a large accumulation of kinesin on Golgi membranes. Factors that might regulate kinesin motor activity as this molecule cycles through anterograde and retrograde pathways are under investigation. 4) Role of Golgi positioning in late processing events of the secretory pathway. We have found that microtubule-dependent positioning of the Golgi complex to the MTOC is likely to facilitate the interaction of components moving through secretory and endocytic pathways.
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