Periodic Reporting for period 1 - NanoBioER-STM (Nanoscale Bioanalytical measurements of Exosome Release to gain insight into the initiation of Short-Term Memory)
Periodo di rendicontazione: 2022-09-01 al 2024-08-31
In NanoBioER-STM, I describe completely novel approaches aimed at completely novel goals to measure how metabolites are packaged across nanometer organelles (vesicles, stress granules), while simultaneously pinpointing their location in live cells. I will then determine how molecular species move dynamically between organelle subcompartments in and interchange between organelles. Finally, I will use these analytical methods to examine organelles in the neurons of advanced systems, develop a model of their interactions, and create a unified theory of how organelles regulate plasticity. These are unexplored areas of nanobio chemical science as the analytical methods to pursue these goals do not yet exist. I aim to develop new methods to determine metabolites and precursors including neurotransmitters such as catecholamines, but also reactive oxygen species and their interplay, all with nanometer precision. This will allow me to define the processes that are regulating the cell and plasticity as well as controlling disease at the level of chemical interactions between organelles. The objectives are as follows:
Objective 1. Determine stress granules from single cells and to confirm its electroactive content in SGs.
1-1 Determination of ex-vivo separated SGs and confirm ROS in SGs.
1-2 Determination of SG in single cells.
1-3 Evaluation of the method in Objective 1-2.
Objective 2. Determine the changes in cell membrane lipids involved in the ROS dynamics of SGs leading to STM
2-1 Determination of ROS dynamics in single cell.
2-2 Selection of effective lipid model.
2-3 Measure the localization of designated lipids.
Objective 3. Test the effects of SGs on plasticity of neuro cells.
3-1 Lipids and zinc as the chemical effectors in short-term plasticity.
3-2 Measurement of the effect of cognition enhancing drugs on SGs dynamics.
3-3 Cognition enhancing drugs on lipid composition and zinc.
3-4 Effects of SGs on vesicles in neuro cells.
Objective 1. Determine stress granules from single cells and to confirm its electroactive content in SGs.
1-1 Determination of ex-vivo separated SGs and confirm ROS in SGs.
1-2 Determination of SG in single cells.
1-3 Evaluation of the method in Objective 1-2.
Objective 2. Determine the changes in cell membrane lipids involved in the ROS dynamics of SGs leading to STM
2-1 Determination of ROS dynamics in single cell.
2-2 Selection of effective lipid model.
2-3 Measure the localization of designated lipids.
Objective 3. Test the effects of SGs on plasticity of neuro cells.
3-1 Lipids and zinc as the chemical effectors in short-term plasticity.
3-2 Measurement of the effect of cognition enhancing drugs on SGs dynamics.
3-3 Cognition enhancing drugs on lipid composition and zinc.
3-4 Effects of SGs on vesicles in neuro cells.
1) Amperometry and Electron Microscopy show Stress Granules Induce Homotypic Fusion of Catecholamine Vesicles
An overreactive stress granule (SG) pathway and long-lived, stable SGs formation are thought to participate in the progress of neurodegenerative diseases (NDs). To understand if and how SGs contribute to disorders of neurotransmitter release in NDs, we examined the interaction between extracellular isolated SGs and vesicles. Amperometry shows that the vesicular content increases and dynamics of vesicle opening slow down after vesicles are treated with SGs, suggesting larger vesicles are formed. Data from transmission electron microscopy (TEM) clearly shows that a portion of large dense-core vesicles (LDCVs) with double/multiple cores appear, thus confirming that SGs induce homotypic fusion between LDCVs. This might be a protective step to help cells to survive following high oxidative stress. A hypothetical mechanism is proposed whereby enriched mRNA in the shell of SGs is likely to bind intrinsically disordered protein (IDP) regions of vesicle associated membrane protein (VAMP) driving a disrupted membrane between two closely buddled vesicles to fuse with each other to form double-core vesicles. Our results show that SGs induce homotypic fusion of LDCVs, providing better understanding of how SGs intervene in pathological processes and opening a new direction to investigations of SGs involved neurodegenerative disease.
This work has been published on Angewandte International Edition Chemie (2024, 63, e202400422). https://doi.org/10.1002/anie.202400422(si apre in una nuova finestra)
2) Uncovering the non-enzymatic redox pathway of stress granules using an intracellular electrochemical nanosensor with single-entity resolution
The spontaneous electrochemical activities of biomolecular condensates represent a new fundamental functioning mechanism in biochemistry and cell biology. However, our understanding of the underlying molecular mechanism and the interfacial field-dependent chemical activities remain limited. This is due to the lack of technology to probe such activities in real-time and at a single-condensate level. Here we design and implement a collision-based electrochemical nanosensor that enables probing the spontaneous redox activities of stress granules (SGs) at a single-condensate level in living cells. We show that ex-vivo separated SGs drive the spontaneous redox reactions depending on their own interfacial potentials and the constituents of the solution system. Surprisingly, water molecules, instead of solvated oxygen, are the main chemical origin of the redox activities of SGs. Finally, we demonstrate the application of this electrochemical nanosensor in in-situ probing the generation of hydrogen peroxide from SGs in mammalian cells and show that the electrochemical environment of the cells can regulate the redox activity of SGs. This work uncovers the mechanisms encoding the non-enzymatic redox activities of SGs and demonstrates a key fundamental technological capability that can be highly useful in exploring the intracellular electroactive pathways of macroscale assemblies.
This work has been submitted to Nature Nanotechnology (2024 in submission).
3) Quantitative Measurement of Hydrogen Peroxide in Individual Stress Granules in Single Cells with Platinized Nanotip Electrodes
Stress granules (SGs) play a critical role in promoting stress responses and preventing the accumulation of misfolded proteins which are closely related to the pathogenesis of neurodegenerative diseases. The quantification of reactive oxygen species (ROS) content in SGs is important for studying the mechanisms of SGs-involved oxidative stress in diseases, and yet it is incredibly difficult to measure the tiny amount of ROS in the SGs in single cells, especially in nerve cells containing secretory vesicles encapsulating electroactive neurotransmitters like catecholamines. Herein, a high-density coated contiguous platinum-petal carbon nanotip electrode (cPt-CNE) with high electrocatalytic performance towards H2O2 (the main ROS in SGs) has been specifically designed for this purpose. This allows the novel amperometric measurement of ROS content in individual SGs within single living cells at low potential to distinguish them from intracellular vesicles, and differentiate molecules inside SGs from those within vesicles. We observe that the dynamics of molecule release from SGs to the electrode is much faster compared to the transmitter released from the coexisted intracellular vesicles.
This work is in progress.
An overreactive stress granule (SG) pathway and long-lived, stable SGs formation are thought to participate in the progress of neurodegenerative diseases (NDs). To understand if and how SGs contribute to disorders of neurotransmitter release in NDs, we examined the interaction between extracellular isolated SGs and vesicles. Amperometry shows that the vesicular content increases and dynamics of vesicle opening slow down after vesicles are treated with SGs, suggesting larger vesicles are formed. Data from transmission electron microscopy (TEM) clearly shows that a portion of large dense-core vesicles (LDCVs) with double/multiple cores appear, thus confirming that SGs induce homotypic fusion between LDCVs. This might be a protective step to help cells to survive following high oxidative stress. A hypothetical mechanism is proposed whereby enriched mRNA in the shell of SGs is likely to bind intrinsically disordered protein (IDP) regions of vesicle associated membrane protein (VAMP) driving a disrupted membrane between two closely buddled vesicles to fuse with each other to form double-core vesicles. Our results show that SGs induce homotypic fusion of LDCVs, providing better understanding of how SGs intervene in pathological processes and opening a new direction to investigations of SGs involved neurodegenerative disease.
This work has been published on Angewandte International Edition Chemie (2024, 63, e202400422). https://doi.org/10.1002/anie.202400422(si apre in una nuova finestra)
2) Uncovering the non-enzymatic redox pathway of stress granules using an intracellular electrochemical nanosensor with single-entity resolution
The spontaneous electrochemical activities of biomolecular condensates represent a new fundamental functioning mechanism in biochemistry and cell biology. However, our understanding of the underlying molecular mechanism and the interfacial field-dependent chemical activities remain limited. This is due to the lack of technology to probe such activities in real-time and at a single-condensate level. Here we design and implement a collision-based electrochemical nanosensor that enables probing the spontaneous redox activities of stress granules (SGs) at a single-condensate level in living cells. We show that ex-vivo separated SGs drive the spontaneous redox reactions depending on their own interfacial potentials and the constituents of the solution system. Surprisingly, water molecules, instead of solvated oxygen, are the main chemical origin of the redox activities of SGs. Finally, we demonstrate the application of this electrochemical nanosensor in in-situ probing the generation of hydrogen peroxide from SGs in mammalian cells and show that the electrochemical environment of the cells can regulate the redox activity of SGs. This work uncovers the mechanisms encoding the non-enzymatic redox activities of SGs and demonstrates a key fundamental technological capability that can be highly useful in exploring the intracellular electroactive pathways of macroscale assemblies.
This work has been submitted to Nature Nanotechnology (2024 in submission).
3) Quantitative Measurement of Hydrogen Peroxide in Individual Stress Granules in Single Cells with Platinized Nanotip Electrodes
Stress granules (SGs) play a critical role in promoting stress responses and preventing the accumulation of misfolded proteins which are closely related to the pathogenesis of neurodegenerative diseases. The quantification of reactive oxygen species (ROS) content in SGs is important for studying the mechanisms of SGs-involved oxidative stress in diseases, and yet it is incredibly difficult to measure the tiny amount of ROS in the SGs in single cells, especially in nerve cells containing secretory vesicles encapsulating electroactive neurotransmitters like catecholamines. Herein, a high-density coated contiguous platinum-petal carbon nanotip electrode (cPt-CNE) with high electrocatalytic performance towards H2O2 (the main ROS in SGs) has been specifically designed for this purpose. This allows the novel amperometric measurement of ROS content in individual SGs within single living cells at low potential to distinguish them from intracellular vesicles, and differentiate molecules inside SGs from those within vesicles. We observe that the dynamics of molecule release from SGs to the electrode is much faster compared to the transmitter released from the coexisted intracellular vesicles.
This work is in progress.
1) Amperometry and Electron Microscopy show Stress Granules Induce Homotypic Fusion of Catecholamine Vesicles
This work integrating VIEC measurements and TEM imaging, we have discovered that SGs induce homotypic fusion between LDCVs to form multi-core vesicles that include double- and multi-core vesicles. We know from previous experiments that SGs form H2O2 and combined with the unique mRNA and protein networked structure of SGs separated by stable cores and a dynamic shell, we hypothesize that SGs attach to vesicles, bring them in close proximity and the local H2O2 loosens the lipid bilayer to induce fusion. This provides yet another distinctly new function for SGs associated with neuro-cellular physiology beyond the encapsulated cargo of SGs. The discovery of SGs-induced homotypic fusion between LDCVs might suggest a mechanism for how neural cells regulate the exocytosis when impacted by oxidative stress (formation of SGs). The effect of this process provides insights into the possibility of how SGs promote neurodegeneration.
2) Uncovering the non-enzymatic redox pathway of stress granules using an intracellular electrochemical nanosensor with single-entity resolution
This work uncovers a non-enzymatic cellular pathway for the generation of reactive oxygen species with a distinct functioning electrochemical mechanism. The nanosensing electrochemical approach presented here should be generally useful to study the electrochemical activities of biomolecular condensates in living cells. Given the ubiquitous presence and significant role of condensates in diverse cellular processes, coupled with our limited understanding of their electrochemical properties, our study introduces a novel paradigm for the interface of bio-nanotechnology and biomolecular condensates.
3) Quantitative Measurement of Hydrogen Peroxide in Individual Stress Granules in Single Cells with Platinized Nanotip Electrodes
cPt-CNEs were fabricated and used to perform direct and quantitative amperometric simultaneous analysis of SGs and vesicle content inside single living PC12 cells. We developed schemes to discriminate ROS molecules, mainly H2O2, stored in SGs from catecholamines stored in vesicles by disabling vesicle rupture on cPt-CNEs at low potential. The dynamics of H2O2 oxidation at SGs is more rapid than that for catecholamine from electroporated vesicles. A larger of molecules is found in the SGs intracellularly compared to that in isolated SGs reported in previous study.4 It seems likely that a small portion of ROS is lost during the isolation process.18 It is also likely that PC12 cells respond to arsenite differently from U2OS cells since they are different cell lines. Future developments of the platform will undoubtedly provide a deeper understanding, from the quantitative perspective, of the involvement of ROS and SGs in intracellular physiological processes. Considering the crucial role SGs played in neurodegenerative diseases in terms of facilitating stress responses and preventing the accumulation of misfolded proteins, we believe that our findings will give new insights into the developmental progress and therapeutics of these SG-related diseases.
This work integrating VIEC measurements and TEM imaging, we have discovered that SGs induce homotypic fusion between LDCVs to form multi-core vesicles that include double- and multi-core vesicles. We know from previous experiments that SGs form H2O2 and combined with the unique mRNA and protein networked structure of SGs separated by stable cores and a dynamic shell, we hypothesize that SGs attach to vesicles, bring them in close proximity and the local H2O2 loosens the lipid bilayer to induce fusion. This provides yet another distinctly new function for SGs associated with neuro-cellular physiology beyond the encapsulated cargo of SGs. The discovery of SGs-induced homotypic fusion between LDCVs might suggest a mechanism for how neural cells regulate the exocytosis when impacted by oxidative stress (formation of SGs). The effect of this process provides insights into the possibility of how SGs promote neurodegeneration.
2) Uncovering the non-enzymatic redox pathway of stress granules using an intracellular electrochemical nanosensor with single-entity resolution
This work uncovers a non-enzymatic cellular pathway for the generation of reactive oxygen species with a distinct functioning electrochemical mechanism. The nanosensing electrochemical approach presented here should be generally useful to study the electrochemical activities of biomolecular condensates in living cells. Given the ubiquitous presence and significant role of condensates in diverse cellular processes, coupled with our limited understanding of their electrochemical properties, our study introduces a novel paradigm for the interface of bio-nanotechnology and biomolecular condensates.
3) Quantitative Measurement of Hydrogen Peroxide in Individual Stress Granules in Single Cells with Platinized Nanotip Electrodes
cPt-CNEs were fabricated and used to perform direct and quantitative amperometric simultaneous analysis of SGs and vesicle content inside single living PC12 cells. We developed schemes to discriminate ROS molecules, mainly H2O2, stored in SGs from catecholamines stored in vesicles by disabling vesicle rupture on cPt-CNEs at low potential. The dynamics of H2O2 oxidation at SGs is more rapid than that for catecholamine from electroporated vesicles. A larger of molecules is found in the SGs intracellularly compared to that in isolated SGs reported in previous study.4 It seems likely that a small portion of ROS is lost during the isolation process.18 It is also likely that PC12 cells respond to arsenite differently from U2OS cells since they are different cell lines. Future developments of the platform will undoubtedly provide a deeper understanding, from the quantitative perspective, of the involvement of ROS and SGs in intracellular physiological processes. Considering the crucial role SGs played in neurodegenerative diseases in terms of facilitating stress responses and preventing the accumulation of misfolded proteins, we believe that our findings will give new insights into the developmental progress and therapeutics of these SG-related diseases.