Final report
i. Determine if partial exocytotic release is ubiquitous in nature. We have carried out experiments with both pancreatic beta cells and at an octopamine-containing neuron in the fly larva to show that partial release is the predominant form of exocytosis for insulin/serotonin and octopamine, respectively. We published this in two papers in Angewandte Chemie. See figure 1. We published another paper in Angewandte Chemie examining the release of serotonin from gut BON cells, which is partial. We recently examined the serotonergic fly neurons in Drosophila and found some vesicles release about 30% of their cargo and some essentially 100%. The general theme has been that the majority of exocytosis is partial.
ii. Vesicle measurements and nanoscale subvesicular measurements. We have developed two types of nanopore electrodes that can be used to measure vesicle content and size, one for extracellular (see figure 2) and the other for intracellular experiments. These works have both been published in the Journal of the American Chemical Society. We used nanoscale secondary ion mass spectrometry (NanoSIMS) to quantify the content of transmitter vesicles and to view the difference between the dense core and the halo of the vesicle. We then used it for the analysis of partial release from vesicles by showing that labeled drug was taken up during exocytotic release and transmitter was decreased by 60% after stimulation of PC12 cells. These works were both published in ACS Nano. We then used NanoSIMS to quantitatively probe the halo and dense core in a paper in Int J Molec Sci. We used correlative transmission electron microscopy (TEM) and NanoSIMS imaging to examine the relation between fraction of partial exocytotic release and vesicle diameter to test the hypothesis that they are dependent. We found they are not (Angewandte Chemie). We also made great progress with electrochemistry showing that the Hofmeister effect can be used to regulate exocytosis and to develop compartment-selective sensors for vesicle content. We published these works in the J Am Chem Soc and two in Angewandte Chemie. We have investigated the pore opening of single vesicles at electrified interfaces, showing that vesicles open via electroporation towards the electrode surface (ACS Nano) and used combined STED and NanoSIMS to examine stress granule turnover in neuronal progenitor cells (Int J Mol Sci). Furthermore, we developed a method to measure the contents of exosomes and found that those from adrenal chromaffin cells contain catecholamine neurotransmitters (J Am Chem Soc). We also used our methods to discover that reactive oxygen species are formed by stress granules and went on to show that this release apparently controls homotypic fusion of vesicles in vitro, both works published in Angewandte Chemie.
iii. Lipids in signaling plasticity, nanoimaging. We have developed a paradigm for plasticity in exocytotic release from a cell model by use of repeated stimulations and shown a key parameter is partial release (published in Proceedings of the National Academy of Sciences, USA) and followed this with mass spectrometry imaging of model cells with similar paradigms. This comparison has been published in the International Journal of Chemical Sciences. We have also developed NanoSIMS strategies to quantitatively examine the contents of single nanometer vesicles across their structure, published in ACS Nano. We have also developed combined electrochemistry and NanoSIMS strategies to examine the effects of iron homeostasis on partial release (Angewandte Chemie). Finally, we found that omega-3 and-6 fatty acids alter the lipid composition of membranes and vesicle size thereby regulating exocytotic release and catecholamine storage (ACS Chem Neurosci). We used combined confocal fluorescence microscopy and electrochemistry to develop a method to examine the dynamics of vesicle opening on the electrode surface (ACS Measurement Science Gold). This is shown in Figure 3. This led to a new hypothesis of the surface reaction chemistry required for the vesicle to undergo electroporation.
iv. Drugs, zinc, lipids, and learning/memory. We have been highly successful here showing that drugs that affect cognitive ability (cocaine, methylphenidate, zinc, barbiturates, lidocaine, chemo treatment drugs, etc) all affect exocytosis and several have now been shown to affect the lipid membrane structure providing a potential mechanism for understanding the initiation of plasticity. These studies have been published in numerous recent papers. We showed that the drug modafinil changes the lipid composition of the brain (Angewandte Chemie) and then proposed a mechanism for the action of modafinil on cognition (QRB Discovery, see Figure 4). We have found that on our model systems, all drugs that are known or suspected to diminish cognitive ability in humans cause a decrease in the partial release fraction in our model cell systems. In contrast, to date, all drugs that are known or suspected to increase cognitive ability in humans result in an increase in the fraction of exocytotic release. This forms the basis for a preliminary model of the initiation of plasticity and we feel could be important in the mechanism of short-term memory.
