Juan Bonifacino, PhD, Chief
The Cell Biology and Metabolism Branch (CBMB) conducts studies in various areas of molecular cell biology, including the mechanisms of intracellular protein trafficking and organelle biogenesis, iron metabolism, adaptive responses to environmental stresses, regulation of the cell cycle during oogenesis, and liver cell physiology. A salient feature of the CBMB is its outstanding capabilities in state-of-the-art fluorescence microscopy techniques, which include cell imaging in real time, photobleaching, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy. During 2006, the CBMB greatly enhanced its imaging capabilities with the development of photoactivated localization microscopy (PALM). In addition, the CBMB maintains facilities for work with many model organisms, including bacteria, yeast, Drosophila, mice, and mammalian cells. The CBMB applies knowledge gained from the study of basic cellular processes to elucidation of the causes of human diseases, including disorders of protein trafficking, iron overload, and bile acid secretion as well as neurodegeneration and viral pathogenesis.
Over the past year, the Unit on Cell Polarity, headed by Irwin Arias, has continued its studies on the mechanisms responsible for the selective trafficking of proteins to the apical domain of hepatocytes and other polarized cells. The goal of the studies is to identify components and regulation of these processes, their role in creating and maintaining cellular polarity, and molecular defects responsible for inheritable and acquired bile secretory failure (cholestasis). The group developed a novel system for imaging the apical surface of polarized epithelial cells, demonstrated that fenestrae in hepatic endothelial cells are defective in mice deficient in caveolin 1 expression, and performed high-resolution live-cell imaging of the recycling endosomal pathway for bile canalicular ABC transporters.
The Section on Intracellular Protein Trafficking, led by Juan Bonifacino, has continued its studies on the molecular machinery responsible for transport of proteins to intracellular compartments such as the Golgi complex, endosomes, lysosomes, and lysosome-related organelles. The Section elucidated the pathogenesis of the hereditary pigmentation and bleeding disorder Hermansky-Pudlak syndrome. One form of the syndrome results from a defect in one component of the protein transport machinery known as AP-3. The defect was found to cause altered transport of the pigment-synthesizing enzyme tyrosinase, thus explaining the basis for defective pigmentation in the disease.
Ramanujan Hegde's Unit on Protein Biogenesis discovered and is elucidating the molecular basis of a new site of cellular regulation: the translocation of secretory and membrane proteins into the mammalian ER. In one particularly noteworthy example, translocation of some proteins into the ER, is found to be selectively reduced during certain types of stress. This protective pathway, termed pre-emptive quality control, attenuates the adverse consequences of protein misfolding in the ER. Selective regulation of translocation was traced to a protein's signal sequence, modifications of which are shown to exacerbate or alleviate protein aggregation in cultured cells and modify the course of neurodegeneration in transgenic mice. The group has also discovered and purified a novel targeting factor for membrane protein insertion.
Catherine Jackson's Unit on GTPase Regulation of Membrane Traffic showed that an activator of the Arf1 GTPase (GBF1) is required for recruitment of a key lipase (ATGL ) to lipid droplets, an essential step in lipid catabolism of stored fat in cells. ATGL is a key triglyceride lipase involved in mobilization of triglycerides from lipid droplets. ATGL knockout mice become obese, demonstrating the importance of the enzyme in the regulation of fat storage in mammals and indicating that GBF1 regulation of ATGL may have important implications for understanding and treating obesity in humans.
Mary Lilly's Unit on Cell Cycle Regulation studies the developmental regulation of the cell cycle. During 2006, the Unit demonstrated that the cyclin-dependent kinase inhibitor Dacapo promotes licensing of DNA replication origins in both mitotic and endocycling cells of Drosophila. This work represents one of the first reports of a cyclin-dependent kinase inhibitor acting to promote replication licensing in a metazoan. In addition, the Unit continued its studies on regulation of highly conserved prophase I meiotic arrest of animal oocytes. Researchers determined that in Drosophila the translational inhibitor Bruno maintains prophase I meiotic arrest by inhibiting accumulation of mitotic cyclins. The Unit's studies provide a framework for understanding how meiotic progression and gamete differentiation are coordinated during oogenesis.
Jennifer Lippincott-Schwartz's Section on Organelle Biology has continued to introduce novel fluorescence imaging approaches, using them to investigate important cellular processes. In one project carried out in collaboration with Eric Betzig and Harald Hess of the Janelia Farm Research Campus, researchers developed a method termed photoactivated localization microscopy (PALM) to overcome the diffraction barrier in fluorescence microscopy. In a second project, the Section developed a fluorescence protease protection assay that makes it possible to determine the topology of a protein in a living cell. Other projects investigated the biogenesis of the Golgi apparatus, autophagosomes, and peroxisomes; cell cycle changes in mitochondria morphology and potential; the organization of endomembranes in the developing Drosophila syncitial blastoderm embryo; and the dynamics of the primary cilium.
Tracey Rouault's Section on Human Iron Metabolism studies mammalian iron metabolism by using mouse models and tissue culture. Rouault previously identified and characterized two major cytosolic iron regulatory proteins (IRPs). Targeted deletion of each IRP in mice revealed that misregulation of iron metabolism due to loss of IRP2 causes functional iron deficiency, erythropoietic protoporphyria, anemia, and neurodegeneration in animals. The Section also focuses on mammalian iron-sulfur cluster assembly because of its relevance to IRP1 regulation. Researchers characterized numerous mammalian genes involved in iron-sulfur cluster synthesis and developed in vitro and in vivo methods to assess cluster biogenesis. The Section's discoveries may promote understanding and treatment of neurodegenerative diseases, especially Parkinson's disease and Friedreich ataxia, and hematologic disorders such as refractory anemias and erythropoietic protoporphyria.
The Section on Environmental Gene Regulation, headed by Gisela Storz, has continued its studies of small, non-coding RNAs in E. coli. Many of these bacterial RNAs act analogously to eukaryotic miRNA and siRNAs to regulate mRNA stability and translation. Along with carrying out screens to identify additional non-coding RNAs and characterizing the functions of some of the RNAs, the group has helped develop tools for the study of these regulators. Investigators also initiated a project to discover the functions of small proteins (fewer than 50 amino acids) encoded by another category of genes that, along with regulatory RNAs, has largely been overlooked.
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