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Cell Biology and Metabolism Program
The Cell Biology and Metabolism Program (CBMP) conducts studies in various areas of molecular cell biology, including the mechanisms of intracellular protein trafficking and organelle biogenesis, host-pathogen interactions, the adaptive responses to environmental stresses, the biology of small non-coding RNAs and small proteins, regulation of the cell cycle during oogenesis, and liver cell physiology. A salient feature of the CBMP is its outstanding capabilities in state-of-the-art fluorescence microscopy techniques, including imaging of cells in real time, photobleaching, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy. The CBMP’s imaging capabilities have been greatly enhanced with the development of photoactivated localization microscopy (PALM). In addition, the CBMP maintains facilities for working with many model organisms, including bacteria, yeast, Drosophila, mice, and mammalian cells. The knowledge gained from the study of basic cellular processes is applied to the elucidation of the causes of human diseases, including disorders of protein trafficking and bile acid secretion as well as neurodegeneration and microbial pathogenesis.
The Unit on Cell Polarity, headed by Irwin Arias, which focuses on mechanisms and regulation of polarization mainly in hepatocytes. Live-cell fluorescent imaging, biochemical, genetic, and molecular techniques are applied to studies of longterm non-dividing cultures of mammalian hepatocytes that retain mRNA expression and structural and functional profiles. These investigations revealed distinct intracellular pathways for trafficking of apical membrane transporter proteins. The relation of these processes to AMP and LKB kinases and the kinases’ activation by bile acids is being investigated particularly with regard to tight junction formation, cytoskeletal organization, and apical recycling of ATP-binding cassette transporters. The Unit’s goal is to identify components and regulation of these trafficking processes, their role in creating and maintaining cellular polarity, and the molecular defects responsible for heritable and acquired bile secretory failure (cholestasis).
The Section on Intracellular Protein Trafficking, led by Juan Bonifacino, focuses on the molecular machinery involved in protein sorting to endosomes, lysosomes, and related organelles. Research over the past year has shed light on the mechanisms used by the human immunodeficiency virus-1 (HIV-1) to exploit the endocytic and lysosomal targeting machinery of host cells for optimal replication. In particular, the research demonstrated that the Nef protein of HIV-1 downregulates the CD4 co-receptor through interaction with the adaptor protein-2 (AP-2) complex at the plasma membrane and the multivesicular body machinery on endosomes. In addition, the AP-4 complex was shown to play a role in the trafficking and processing of the Alzheimer’s disease amyloid precursor protein. Other studies revealed important aspects of the function of the retromer and GARP complexes in protein trafficking between endosomes and the Golgi complex and in lysosome biogenesis.
The Protein Biogenesis Section, led by Ramanujan Hegde, continues its work on the trafficking of newly synthesized secretory and membrane proteins. Notable advances this past year include the discovery of a novel factor whose depletion by mislocalized prion protein (PrP) causes neurodegeneration, identification of an altered conformation associated with disease-causing forms of PrP, and analysis of the altered trafficking pathways taken by several human disease PrP mutants. In parallel, a new membrane protein insertion pathway recently discovered by the Section is being mechanistically dissected. A significant advance this past year was the resolution of the X-ray structure and functional analysis of a central component in this pathway.
The Section on Gamete Development, led by Mary Lilly, examines cell cycle regulation during oogenesis. Over the last year the Section has continued to examine the pathways that coordinate meiotic progression with gamete differentiation. From these studies, the Section identified components of the cell cycle machinery that regulate the meiotic S phase and genomic stability in oogenesis. Additionally, progress was made in delineating the multiple interacting pathways that ensure the maintenance of mitotic quiescence during the highly conserved prophase I meiotic arrest of the oocyte. Also, the Section discovered a germ line–specific function for a highly conserved component of the nuclear pore complex in regulation of oocyte differentiation and meiotic progression.
Jennifer Lippincott-Schwartz’s Section on Organelle Biology has continued to investigate new features of numerous cellular processes by using novel fluorescence imaging approaches combined with quantitative analysis and mathematical modeling. Among the areas of investigation are (1) membrane partitioning and its role in protein sorting and transport in the Golgi apparatus, (2) mitochondrial morphology and its regulation of cell cycle progression, (3) control of primary cilia dynamics, (4) intercellular transfer between stem and niche cells, (5) origin of autophagosomes, (6) live-cell photoactivation localization microscopy (PALM) for single-particle tracking, and (7) cytoskeletal and endomembrane cross-talk in three-dimensional polarized cells and developing Drosophila syncytial blastoderm embryos.
The Unit on Microbial Pathogenesis, headed by Matthias Machner, analyzes virulence mechanisms of the bacterium Legionella pneumophila, the causative agent of a severe pneumonia known as legionnaires’ disease. The pathogen hijacks proteins and transport vesicles of infected host cells to establish a protective vacuole that supports replication. An essential aspect of L. pneumophila virulence is the delivery of a large repertoire of effector proteins into the host cell cytosol. The Unit’s main goal is to determine the molecular function of translocated L. pneumophila effectors and to identify host-cell pathways modulated by these proteins. Recent advances include the discovery of two L. pneumophila proteins, SidM and LidA, which modulate the activity of host-cell Rab1, a small GTPase regulating secretory vesicle traffic. Other studies revealed that SidM catalyzes not only release of Rab1 from its chaperone GDI but also Rab1 activation by triggering GDP/GTP exchange.
The Section on Environmental Gene Regulation, headed by Gisela Storz, studies small, regulatory RNAs in E. coli. Many of these bacterial RNAs act analogously to eukaryotic miRNA and siRNAs to regulate mRNA stability and translation. Along with identifying additional small RNAs and characterizing their functions, the Section has helped develop general tools for the study of these RNA regulators. The Section also initiated a project to identify and characterize another category of largely overlooked genes that encode small proteins of less than 50 amino acids. Systematic screens for growth conditions that lead to increased expression and for phenotypes associated with null mutations combined with the identification of copurifying proteins are yielding insights into the physiological roles of these small proteins.