Periodic Reporting for period 3 - NanoBioNext (Nanoscale Biomeasurements of Nerve Cells and Vesicles: Molecular Substructure and the Nature of Exocytosis)
Período documentado: 2021-08-01 hasta 2023-01-31
Problem/issue addressed. It was proposed to develop novel analytical tools at the micro to nanoscale level that are needed to understand critically important parts of neurotransmitter release in the brain. These include, 1) the vesicle closing during partial release, 2) the effect of substructure of transmitter vesicles on communication and plasticity, and 3) cell membrane lipid changes that might strengthen or weaken cell-to-cell communication via changing the rate of exocytosis or the fraction released in partial exocytosis (plasticity). Several completely new electrochemical methods, a completely new combination of electrochemistry with STED super-resolution microscopy, and a model for the initial plasticity in short-term memory has been examined.
Importance to society. The combined methods open new doorways to study protein-lipid interactions, drug sequestration in endosomes, and the analysis of organelles in living systems. The nanoscale biomeasurement paradigms developed here make it possible to quantify and localize lipids and metals in cell models, neurons, and vesicles with a spatial resolution of 40 nm. The combination of electrochemistry with STED and NanoSIMS open new directions in nanobio analytical chemistry. This will provide a unique possibility to understand chemical communication and the plasticity of neurotransmitter release that is the likely starting point or initiator of short-term memory. Short-term memory is a key component lost in aging and neurodegenerative disease so the initiation of this process is highly important. The concept that drugs might affect lipid distribution in the brain as part of their mechanism of action could be very important, leading to new strategies for medicine.
Objectives of the Project
i. Determine if partial exocytotic release is ubiquitous in nature. The plan was to examine the concept of partial release during exocytosis for generality in biology by addressing a completely different, but independently important, cell system – insulin secretion from pancreatic beta cells. Insulin exocytosis is an intense area of study, but partial release has not been considered.
ii. Vesicle measurements and nanoscale subvesicular measurements. The plan was to develop a nanopore electrode capable of placement in a cell and dynamically measuring the catecholamine (or other electroactive transmitter) in the protein dense core of a nanometer vesicle. Another part of the plan is to continue to develop schemes to combine electrochemical measurements in living cells with the intracellular vesicle impact cytometry (IVIEC) method my group has developed and NanoSIMS imaging of the same cells after fixing. Finally, a plan is to combine the IVIEC with STED imaging of live cells.
iii. Lipids in signaling plasticity, nanoimaging. The plan was to develop methodological paradigms to examine, first, cell models, and then later, simple animal models to examine the membrane lipid chemistry and vesicle structural chemistry associated with dynamic changes in exocytosis. Electrochemical measurements of neurotransmitter release, alone and combined with protein/lipid measurements with STED super-resolution microscopy and NanoSIMS, will be used to determine protocols to find plastic responses (changes in release amount, fraction, or dynamics).
iv. Drugs, zinc, lipids, and learning/memory. The cell and animal protocols from aim iii will then be used for analysis of lipid or zinc changes using nanoscale mass spectrometry and super-resolution optical imaging methods in concert with electrochemical measurements of exocytosis dynamics. I will test the effects of cognition-enhancing drugs like methylphenidate, modafinil, cocaine (and possibly zinc or lipids in the diet, or glutamate receptor antagonists; e.g. MK-801, CNQX) on the amount and distribution of lipids in the brain.
Importance to society. The combined methods open new doorways to study protein-lipid interactions, drug sequestration in endosomes, and the analysis of organelles in living systems. The nanoscale biomeasurement paradigms developed here make it possible to quantify and localize lipids and metals in cell models, neurons, and vesicles with a spatial resolution of 40 nm. The combination of electrochemistry with STED and NanoSIMS open new directions in nanobio analytical chemistry. This will provide a unique possibility to understand chemical communication and the plasticity of neurotransmitter release that is the likely starting point or initiator of short-term memory. Short-term memory is a key component lost in aging and neurodegenerative disease so the initiation of this process is highly important. The concept that drugs might affect lipid distribution in the brain as part of their mechanism of action could be very important, leading to new strategies for medicine.
Objectives of the Project
i. Determine if partial exocytotic release is ubiquitous in nature. The plan was to examine the concept of partial release during exocytosis for generality in biology by addressing a completely different, but independently important, cell system – insulin secretion from pancreatic beta cells. Insulin exocytosis is an intense area of study, but partial release has not been considered.
ii. Vesicle measurements and nanoscale subvesicular measurements. The plan was to develop a nanopore electrode capable of placement in a cell and dynamically measuring the catecholamine (or other electroactive transmitter) in the protein dense core of a nanometer vesicle. Another part of the plan is to continue to develop schemes to combine electrochemical measurements in living cells with the intracellular vesicle impact cytometry (IVIEC) method my group has developed and NanoSIMS imaging of the same cells after fixing. Finally, a plan is to combine the IVIEC with STED imaging of live cells.
iii. Lipids in signaling plasticity, nanoimaging. The plan was to develop methodological paradigms to examine, first, cell models, and then later, simple animal models to examine the membrane lipid chemistry and vesicle structural chemistry associated with dynamic changes in exocytosis. Electrochemical measurements of neurotransmitter release, alone and combined with protein/lipid measurements with STED super-resolution microscopy and NanoSIMS, will be used to determine protocols to find plastic responses (changes in release amount, fraction, or dynamics).
iv. Drugs, zinc, lipids, and learning/memory. The cell and animal protocols from aim iii will then be used for analysis of lipid or zinc changes using nanoscale mass spectrometry and super-resolution optical imaging methods in concert with electrochemical measurements of exocytosis dynamics. I will test the effects of cognition-enhancing drugs like methylphenidate, modafinil, cocaine (and possibly zinc or lipids in the diet, or glutamate receptor antagonists; e.g. MK-801, CNQX) on the amount and distribution of lipids in the brain.
Progress to date.
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.
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 are preparing a publication on NanoSIMS analysis of partial release.
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, pubished in ACS Nano.
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.
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.
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 are preparing a publication on NanoSIMS analysis of partial release.
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, pubished in ACS Nano.
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.
Progress to date.
i. Determine if partial exocytotic release is ubiquitous in nature. Work continues on cells derived from human stem cells to make dopamine neurons as well as nanogap electrodes to examine vesicles in large numbers.
ii. Vesicle measurements and nanoscale subvesicular measurements. Work continues on the means to discriminate the vesicular halo from the dense core and combined electrochemistry and STED analysis where significant progress has been made.
iii. Lipids in signaling plasticity, nanoimaging. Work with fly brains/cells and STED combined with nanoelectrochemistry is ongoing. We also have a serious of experiments planned with the NanoSIMS to examine the changes in vesicle structure over time.
iv. Drugs, zinc, lipids, and learning/memory. We continue to work to understand the interaction between drug and chemical effectors and plasticity in exocytosis. Work with modafinil is nearing publication and studies on diet and glutamate receptor antagonists are in the planning stages.
i. Determine if partial exocytotic release is ubiquitous in nature. Work continues on cells derived from human stem cells to make dopamine neurons as well as nanogap electrodes to examine vesicles in large numbers.
ii. Vesicle measurements and nanoscale subvesicular measurements. Work continues on the means to discriminate the vesicular halo from the dense core and combined electrochemistry and STED analysis where significant progress has been made.
iii. Lipids in signaling plasticity, nanoimaging. Work with fly brains/cells and STED combined with nanoelectrochemistry is ongoing. We also have a serious of experiments planned with the NanoSIMS to examine the changes in vesicle structure over time.
iv. Drugs, zinc, lipids, and learning/memory. We continue to work to understand the interaction between drug and chemical effectors and plasticity in exocytosis. Work with modafinil is nearing publication and studies on diet and glutamate receptor antagonists are in the planning stages.