RUI: Investigation of Copine Function in Dictyostelium

In the past 10 years, great strides have been made in the identification of proteins that mediate and regulate membrane vesicle trafficking in eukaryotic cells. Different proteins function in distinct steps of vesicular trafficking such as budding, transport, targeting, and fusion of vesicles. Most of these proteins are well conserved with homologs found in organisms from yeast to mammals. However, the exact roles of many of these proteins remain unclear. Furthermore, it is likely that not all proteins involved in these processes have been identified. One new candidate for a role in vesicle membrane trafficking is a family of recently discovered proteins called copines. Copines were first isolated from Paramecium in 1997 by their ability to bind phospholipids in a calcium-dependent manner. Paramecium have two closely related copine genes and analysis of current sequence databases indicates that multiple copine homologs exist not only in ciliates, but also in slime molds, green plants, nematodes, mice, and humans. The high degree of conservation of copines among diverse organisms suggests they play a fundamental role in eukaryotic cells. Structurally, copines have two C2 domains at the N-terminus and a region similar to the A domain found in integrins at the C-terminus. The C2 domain is a calcium-/phospholipid- binding motif originally identified in protein kinase C. The A domain in an intracellular soluble protein is a unique characteristic of copines because this domain is typically found in extracellular proteins or extracellular portions of membrane proteins. Following the A domain, copines have a variable length C-terminal domain that is relatively rich in prolines. This domain may confer unique characteristics to the different copine family members and a site for protein-protein interactions. Several lines of evidence suggest that copines may function in vesicular trafficking. First, antibodies raised against a human copine recognize a protein that binds to chromaffin granules indicating that copines bind secretory vesicles. In addition, several proteins thought to be involved in membrane trafficking, such as synaptotagmin, rabphilin, DOC2, and munc13, contain multiple C2 domains that confer calcium/phospholipid binding properties. The long-term research objective is to define in molecular terms the mechanisms underlying membrane vesicle trafficking. In the short term, the research goal is to determine the general role of copines in eukaryotic cells using the model genetic organism, Dictyostelium discoideum. Genetic studies in yeast have been fundamental to the identification and characterization of proteins involved in vesicular membrane trafficking. However, no copine homologs exist in yeast. Dictyostelium provides the same genetic advantages as yeast and preliminary research demonstrates the existence of two copine homologs in Dictyostelium. The two main objectives of the research are: 1. Determine the localization of copines in live cells by expressing green fluorescent protein tagged copines in Dictyostelium and examining the cells by fluorescence microscopy. This experimental approach will be used to determine transient changes in localization during different cellular behaviors. 2. Create copine gene knockout mutants in Dictyostelium with gene replacement by homologous recombination and analyze the mutant phenotypes. This experimental approach will be used to determine loss of function in membrane trafficking pathways due to loss of copine gene function. Once these two objectives are met, it will be possible to correlate the intracellular location of copines with loss-of-function phenotypes in copine mutants and produce a more specific hypothesis about the function of copines in eukaryotic cells.