Every biochemical process that happens in a eukaryotic cell relies upon a molecular information flow that leads from receptors that inform the cell about its environment all the way to the molecular effectors that determine the appropriate cellular response. A proper information transmission requires a high degree of organization where the molecular players are organized into different cellular compartments so that the specificity of the cellular response can be properly maintained. Breakdown of this organization is the ultimate cause of all human diseases even if the affected molecular pathways differ according to the kind of disease, such as cancer, diabetes or neurodegenerative diseases just to name a few. Research described in this report has focused on the question of how cells organize their internal membranes to provide a structural framework on which molecular signaling complexes assemble to ensure proper information processing. These cellular processes are often targeted by cellular pathogens such as viruses to force the cells to produce the pathogen instead of performing the cells normal functions. Better understanding of these processes not only can provide new strategies to fight various human diseases but also to intercept the life cycle of cellular pathogens offering an alternative to antimicrobial drugs. During this period, we studied the molecular events that control the entry of Ca2+ into mammalian cells. Ca2+ is one of the most important signaling molecules in the cell and its concentration is kept at very low levels ( 10-7 M) in the cytoplasm. Cells elevate Ca2+ from this low level to trigger a variety of cellular pathways by releasing Ca2+ from intracellular storage organelles such as the endoplasmic reticulum (ER) or open specific gates at the plasma membrane (PM) for Ca2+ entry from the outside of the cell. One important Ca2+ entry pathway, called Store-Operated Calcium Entry (SOCE) is the PM Orai1 channels that are activated by ER-localized STIM1molecules that respond to depletion of the luminal Ca2+ content of the ER. Mutations in Orai1 and STIM1 have been detected in humans causing various diseases, most prominently severe immune-deficiencies. Upon ER Ca2+ store depletion, STIM1 molecules cluster and activate the PM Orai1 channels via their SOAR (Stim1-Orai-Activation Region) domain. It is poorly understood how the information on ER luminal Ca2+ decrease is transmitted to the cytoplasmic part of STIM1 through the single transmembrane segment. A key step in STIM1 activation is the release of its SOAR domain from an intramolecular clamp formed with the part of the STIM1 molecule that immediately follows the transmembrane region in the cytosolic side, called coiled-coil (CC1) region because of its predicted secondary structure. We used STIM1molecules that were truncated right after the CC1 domain to show that they are capable of capturing or releasing the isolated SOAR domain depending on luminal ER Ca2+ concentrations. Using this limited STIM1 construct we analyzed the very early molecular events that control the intramolecular clamp formed between the CC1 and SOAR domains. We found that STIM1 molecules form constitutive dimers even in resting state and their CC1 domain can also bind SOAR of another STIM1 molecule in trans. We developed a unique approach to oligomerize STIM1 molecules by targeting multiples of a small protein module (called FRB) to the ER lumen. These FRB multimers could oligomerize STIM1 molecules engineered by adding of a small protein module (called FKBP12) to their ER luminal side after adding rapamycin that induces binding of FRBs to FKBP modules. Such artificial oligomerization, however, failed to liberate the SOAR domain, or activate STIM1 molecules, whereas decreasing ER luminal Ca2+ was still capable of activation. These data suggested that oligomerization alone cannot trigger STIM1 activation. We propose that the release of SOAR from the STIM1 CC1 interaction is controlled by changes in the orientation of the two CC1 domains in STIM1 dimers upon Ca2+ unbinding. The importance of these studies is that it shed new light on the STIM1 activation process that could help us better understand how these molecules work and how the process is affected by mutations that cause human diseases due to SOCE dysfunction. These studies can also identify new ways to pharmacologically manipulate this process for the benefit of patients suffering from either hyperactivation or defective activation of this pathway. Another study performed in collaboration with the group of Dr. Sergio Grinstein at the Hospital for Sick Children in Toronto made use of our recently developed tool to visualize the small regulatory lipid, phosphatidylinositol 4-phosphate (PI4P) in living phagocytic cells. Dr. Grinsteins group has been studying the molecular events that govern the various steps during phagocytosis in macrophages. Phagocytosis is one of the most ancient defense mechanisms against foreign organisms (bacteria or cells) that also plays critical roles in clearing up cellular debris following cell death or tissue damage. Phosphoinositide lipids play an important role at all steps of phagocytosis but PI4P changes and their importance has not been explored because of the lack of appropriate tools to visualize this lipid during the process. Using our PI4P reporter, the Grinstein group has shown that PI4P has a biphasic change during phagocytosis and this change is localized to the phagocytic cup membrane during its maturation. PI4P, which is present in the plasma membrane (PM) before engagement of the target particle, is transiently enriched in the phagosomal cup. After the phagosome seals, i.e. it closes around the particle, P4P levels rapidly drop due to the hydrolytic activity of Sac2, a PI4P phosphatase and phospholipase C, the enzyme that hydrolyzes PI4P and PI(4,5)P2. PI4P disappearance coincides with the emergence of phagosomal PtdIns3P, another inositol lipid that only differs from PI4P in the position of the phosphate on the inositol ring and which is formed by a different enzyme. Conversely, the disappearance of PI3P that signals the transition from early to late phagosomes is accompanied by resurgence of PI4P, which is associated with the recruitment of phosphatidylinositol 4-kinase 2A (PI4K2A). This secondary appearance of PI4P can be prevented by silencing PI4K2A or by eliminating PI4P by a recruitable form of another PI4P 4-phosphatase, Sac1 using the FRB-FKBP heterodimerization system. Importantly, the secondary accumulation of PI4P was found to be necessary for proper phagosomal acidification. These results showed complex dynamics of P4P during phagocytosis and suggested that this phosphoinositide plays important roles during the maturation of the phagosome. The significance of these studies is that they highlighted a hitherto unrecognized and important role of PI4P in phagocytosis. Better understanding the molecular players in the phagocytic process will enhance our ability to counter the processes that parasitic bacteria developed to evade degradation by lysosomes. Lastly, this year we also expanded our repertoire of biological sensors for quantitative assessment of the changes of specific inositol and other lipid species in well-defined cellular compartments. These sensors were engineered so that they can be used in a single plasmid transfection system to monitor specific lipid changes in a particular intracellular compartment, such as the ER, the PM or the mitochondria. This system is based either on Frster resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) principles for single or cell population measurements. We described these tools as well as our experience with their use in a comprehensive review article.
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