Wolfhard Almers - US grants

The aim is to determine how action potentials cause contraction in amphibian skeletal muscle. Various voltage-clamp techniques will be used to study: 1) ionic channels contributing to electrical excitability; 2) the potential dependence and kinetics of contractile activation, and 3) the role of dielectric displacement currents in the regulation of contraction. Pharmacological modifications of the excitation-contraction coupling mechanism and of calcium channels will be compared, combining electrical recording and fluoresence microscopy.

The physiologic activity of many cells, including muscle, neurons and several secretory cells, is strongly influenced by cytoplasmic Ca. To regulate Ca, cells possess intracellular organelles that sequester and release Ca, and Ca transport proteins in the cell membrane, that extrude Ca or allow Ca to enter. This grant has three aims. The first is to explore the mechanism of Ca release from internal stores of a striated muscle (the myotomal lamellae of the chordate Amphioxus) and a secretory cell (the rat peritoneal mast cell). Cytoplasmic Ca will be monitored by a fluorescent Ca indicator, using fluorescence microscopy, microfluorimetry and patch voltage clamp. The preparations were chosen because they offer uniquely favorable conditions for the experiments proposed; the insights gained are almost certain to apply also to mammalian muscle and other secretory cells. The second aim is to characterize Ca channels, which regulate Ca entry into muscle, Ca channels will be studied in adult and embryonic skeletal muscle, and in oocytes that have inserted muscle-type Ca channels into their cell membranes under direction of previously injected mRNA. Special attention will be given to (a) the mechanism of ion selectivity, which is of crucial significance for a transport protein with the task of selectively transporting an ion present at low concentration, and (b) the interaction of Ca channels with dihydropyridines, a clinically used Ca antagonist that is regarded as a specific, high-affinity marker for Ca channels and has become fundamental for biochemical studies of Ca channels. A third aim is to test whether Ca-influx (e.g. through an antigen-activated Ca channel) contributes to the increase in cytoplasmic Ca in mast cells. If an influx-mediated component is found, attempts will be made to record antigen-activated Ca currents.

Fusion and fission of membrane-bounded cells and organelles are fundamental events governing the compartmentalization of biological space. It is proposed to use video microscopy and an ultra-sensitive, electrical assay of the cell surface to study, in living cells, the mechanism of membrane fusion during exocytosis of single secretory vesicles. In particular we will explore a novel hypothesis: that the first step in membrane fusion is the formation of an ion channel (the fusion pore) that connects the inside of the vesicle to the cell exterior much like a gap junction would connect two adjacent cells, and that subsequent event in exocytosis are a consequence of electrolyte fluxes through the fusion pore. Patch clamp studies of single secretory vesicles will reveal the type of ion channels present in the vesicle membrane. We will also explore "fusion pores" possibly formed by virus fusion proteins stably expressed in a fibroblast-derived cell line, in order to test whether there are similarities between fusion events induced by viruses and fusion events during exocytosis. Lastly, we plan to explore the role of cytosolic (Ca++) and other cytoplasmic messengers in the control of pinocytosis and membrane turnover.

Fusion and fission of membrane-bounded cells and organelles are fundamental events governing the compartmentalization of biological space. It is proposed to use video microscopy and an ultra-sensitive, electrical assay of the cell surface to study, in living cells, the mechanism of membrane fusion during exocytosis of single secretory vesicles. In particular we will explore a novel hypothesis: that the first step in membrane fusion is the formation of an ion channel (the fusion pore) that connects the inside of the vesicle to the cell exterior much like a gap junction would connect two adjacent cells, and that subsequent event in exocytosis are a consequence of electrolyte fluxes through the fusion pore. Patch clamp studies of single secretory vesicles will reveal the type of ion channels present in the vesicle membrane. We will also explore "fusion pores" possibly formed by virus fusion proteins stably expressed in a fibroblast-derived cell line, in order to test whether there are similarities between fusion events induced by viruses and fusion events during exocytosis. Lastly, we plan to explore the role of cytosolic (Ca++) and other cytoplasmic messengers in the control of pinocytosis and membrane turnover.

DESCRIPTION (provided by applicant): Neurons and endocrine cells secrete transmitter or hormones in Ca- triggered bursts of exocytosis. In a broad sense, exocytosis is how neurons and neuroendocrine cells "talk". Secretion involves four steps: (i) Making the secretory vesicle and filling it with cargo, (ii) transporting the vesicle to a site of exocytosis, (iii) membrane fusion or exocytosis, and finally (iv) the retrieval of the vesicle's membrane by mechanisms broadly called endocytosis. In synaptic vesicles, a new synaptic vesicle is made from the membrane thus retrieved, and the four events form a cycle. All four steps involve multiple proteins and multiple layers of regulation. All four can fail in disease, and most or all neurologic diseases ultimately result in the failure of one or more steps in the synaptic vesicle cycle. This grant proposes functional studies of exo- and endocytosis at the level of single secretory vesicles in intact neurons and endocrine cells. Using an unconventional light microscopy we helped to adapt for this purpose, we will specifically and simultaneously mark proteins in exo- and endocytic vesicles in different fluorescent colors, and then observe the recruitment and release of these proteins to vesicles, and the recruitment and release of vesicles from the plasma membrane. Through observations in living cells, we focus on the following questions. (1) How are synaptic vesicles transported and recruited to their docking sites at presynaptic terminals? (2) How completely do secretory vesicles fuse with the plasma membrane? (3) How do neurons and endocrine cells deal with the material that exocytosis has inserted into the plasma membrane, and is such material inserted in the first place? The planned experiments will generate direct evidence in a way not possible with other known methods, and will address fundamental questions in cellular neurobiology.

Cells continually engage in clathrin-mediated endocytosis to internalize bits of plasma membrane along with their receptors and signaling molecules. Some two dozen different proteins are though to participate in clathrin-mediated endocytosis, along them actin. Despite intense study in several labs, the role of actin in clathrin-mediated endocytosis remains unclear. This application proposes to test the idea that this actin polymerization provides force, (a) to invaginate coated pits and separate them from the plasma membrane (scission) and/or (b) to propel freshly formed vesicles from the plasma membrane into the cytosol. A specially constructed evanescent field fluorescence microscope will be used to view the plasma membrane and the adjacent 100 nm of cytosol and organelles contained therein. Fibroblasts expressing red fluorescent clathrin will be transfected with GFP-actin and observed in two colors. Single clathrin-coated pits are imaged in living fibroblasts to observe directly the minute movements of coated pits as they invaginate, to detect their separation from the plasma membrane as they become coated vesicles, and to image small assemblies of actin filaments that may form at endocytic sites. Fibroblasts expressing red clathrin will be transfected also with GFP conjugates of other essential proteins in endocytic sites. Fibroblasts expressing red clathrin will be transfected also with GFP conjugates of other essential proteins in endocytosis, in order to determine the sequence with which the proteins are first recruited to endocytic sites and then released. Experiments with inhibitory mutants and microinjected inhibitory peptides will reveal which proteins are required to initiate actin polymerization at endocytic sites, and what happens to a coated pit or vesicle if actin polymerization is prevented. The proposed work will clarify the role of an actin mediated event that is directly related to endocytosis. Clathrin-mediated endocytosis influences multiple cell functions, and actin polymerization is controlled by one of the most extensive regulatory networks used in cells. Defects in either can lead to disease. The proposed work will contribute insights on a fundamental activity performed by all cells.

DESCRIPTION (provided by applicant): When sending chemical messages, cells release transmitters or hormones by Ca-triggered exocytosis. During exocytosis, the membrane of a secretory vesicle fuses with the plasma membrane. Fusion is mediated by three SNARE proteins and the proteins Munc18 and Synaptotagmin, and regulated by accessory proteins including Munc13, CAPS, complexin, granulophilin and rabphilin. Partial or complete structures exist for the SNAREs, for Munc18 and complexin, and our biochemical and structural knowledge about all the above proteins is substantial. In live cells, exocytosis has been explored exclusively by assays that, in one way or another, report membrane fusion. Foremost among them is electrophysiology. However, electrophysiology cannot directly report steps that precede fusion. In absence of direct assay, it is still unknown how the proteins mentioned above interact with each other or with secretory vesicles in living cells. We plan to use high-resolution light microscopy to observe single secretory granules in endocrine cells, as well as the exocytosis-related proteins recruited and released by them. We focus on how secretory vesicles dock at the plasma membrane. Single docking sites are observed simultaneously with the docking vesicle and its exocytosis. We use a microscope that has been calibrated by single-molecule imaging in terms of molecules per granule or molecules per cell surface are. If necessary, imaging is combined with electrophysiology. The goal is to understand the molecular steps in docking, and to determine which proteins form the molecular bridge connecting the membranes of a granule with that of the cell when docking has occurred. This work will provide a basic understanding of the role of each of the proteins of interest. More broadly, the work will explore an important and novel avenue to explore the action of proteins in living cells, at the level of single organelles and single proteins. Because most messages between cells are sent by exocytosis, exocytosis is an important cellular process. When exocytosis is defective in pancreatic beta cells then diabetes is the result. When exocytosis is defective or altered in neurons, then so is the basic mechanism of neural communication in the brain, and cognitive malfunctions result. Through increasing the basic mechanisms of exocytosis and its control, this work will be helpful in finding treatment strategies for diseases where cells fail to send messages.