Cell Biology and Metabolism Program
Juan S. Bonifacino, PhD, Program Director
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, the adaptive responses to environmental stresses, the regulation of the cell cycle during oogenesis, liver cell physiology, and the biology of small non-coding RNAs and small proteins. A salient feature of the CBMP is its outstanding capabilities in state-of-the-art fluorescence microscopy techniques, including the 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 viral pathogenesis.
The Unit on Cell Polarity, headed by Irwin Arias, uses live-cell fluorescent imaging, biochemical, genetic, and molecular techniques to study mechanisms responsible for trafficking of proteins selectively to the apical domain of hepatocytes and other polarized cells. The Unit’s goal is to identify the components and regulation of these trafficking processes, their role in creating and maintaining cellular polarity, and the molecular defects responsible for inheritable and acquired bile secretory failure (cholestasis).
The Section on Intracellular Protein Trafficking, led by Juan Bonifacino, has continued its work on the molecular machinery involved in protein sorting to endosomes and lysosomes. Research over the past year has shed light on the mechanisms used by 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 coreceptor from the cell surface of host cells through interaction with the adaptor protein-2 (AP-2) complex at the plasma membrane and the multivesicular body machinery on endosomes. Other studies revealed the atomic structure of another sorting complex named retromer and showed that it is recruited to membranes through interaction with the small GTPase Rab7. The Section showed that an additional sorting complex named GARP participates in the sorting of mannose-6-phosphate receptors from endosomes to the Golgi complex.
The Protein Biogenesis Section, led by Ramanujan Hegde, has continued its investigations into the trafficking of newly synthesized secretory and membrane proteins. Notable advances this past year include the discovery of a new pathway for neurodegeneration caused by mislocalization of prion protein (PrP), identification of a novel interacting partner for mislocalized PrP, and identification of an altered conformation associated with a disease-causing form of PrP. In addition, the Section is studying the pathways of normal PrP trafficking and degradation and how disease-causing mutations in PrP influence such events. The Section continues its progress in identifying additional factors in a novel membrane protein insertion pathway for tail-anchored proteins which was discovered during the previous year.
Headed by Mary Lilly, the Unit on Cell Cycle Regulation studies the developmental regulation of the cell cycle. The Unit continued its examination of the cell-cycle forces that define developmentally programmed endocycles, a common variant cell cycle in which DNA replication occurs without an intervening mitosis. Highly metabolically active cells, such as Drosophila ovarian nurse cells and the giant trophoblast of the mammalian placenta, often grow via endoreplication. The Unit demonstrated that the cyclic activation of the anaphase-promoting complex/cyclosome (APC/C) facilitates the licensing of DNA replication origins and cell-cycle progression during the endocycle. Based on these studies, the Unit formulated and tested a model for the minimum cell-cycle inputs needed to construct a G-/S-phase oscillator. In addition, the Unit has worked to define the pathways that coordinate meiotic progression with gamete differentiation during oogenesis and identified a novel role for components of the nup107-133 nucleoporin complex in the maintenance of the meiotic cycle and oocyte identity in developing ovarian cysts.
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) biogenesis and turnover of peroxisomes, (3) mitochondrial morphology and its regulation of cellcycle progression, (4) control of primary cilia dynamics, (5) intercellular transfer between stem and niche cells, (6) origin of autophagosomes, (7) live-cell photoactivation localization microscopy (PALM) for single-particle tracking, and (8) cytoskeletal and endomembrane cross-talk in three-dimensional polarized cells and developing Drosophila syncytial blastoderm embryos.
The Section on Environmental Gene Regulation, headed by Gisela Storz, studies 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 identifying additional non-coding RNAs and characterizing their functions, the Section has helped develop general tools for the study of these regulators. The Section also initiated a project to identify and characterize a category of largely overlooked genes that encode small proteins of less than 50 amino acids, along with their regulatory RNAs. Systematic screens for growth conditions that lead to increased expression and for phenotypes associated with null mutations are yielding insights into the physiological roles of these small proteins.