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From Force to Function - Investigating Mechanosensitive Piezo Receptors by AFM
Wednesday, February 15, 2017
Mechanosensation 2628-Pos Board B235 From Force to Function - Investigating Mechanosensitive Piezo Receptors by AFM Benjamin M. Gaub, Daniel J. Mueller. D-BSSE, ETH Z€ urich, Basel, Switzerland. Mechanosensation is the process by which cells sense mechanical forces and translate them into electrical and chemical signals. Important physiological functions including sensation of touch, sensation of sound and proprioception are based on mechanosensation. Recent studies identified the molecular identity of a highly conserved group of mechanosensitive receptors, called Piezo receptors, which are both necessary and sufficient for cells to sense force. Currently, structure-function information of Piezo receptors is limited, and the mechanism how these receptors sense force, which conformational changes they undergo, and which precise mechanical stimuli cause these channels to open, remains poorly understood. Studies to date have used rather brute forces reaching several hundrets of nanoNewton to mechanically activate Piezo channels, which are likely too high to be of specific nature. To manipulate Piezo receptors at high precision and accuracy in a cellular context, new techniques have to be applied. We are currently developing assays to investigate Piezo receptors at the molecular level with delicate force control. Using a combination of high-resolution atomic force microscopy (AFM) and time-lapse fluorescent calcium imaging, we aim to find more efficient ways to functionally control Piezo receptors by externally applied forces. Importantly, the tip of the AFM cantilever can be conjugated with small molecules, including ligands and antibodies, which enables manipulation of membrane proteins at nanometer precision with picoNewton sensitivity. We will use this assay to i) get a more quantitative understanding of Piezo gating by mechanical forces, ii) directly compare Piezo receptor responses to different modes of mechanical stimulation including pressure, stretch and vibration, and iii) screen native extracellular matrix (ECM) derived ligands for their ability to bind and functionally modulate the Piezo channel. Our results will help shed light on the force sensing mechanism of mammalian mechanosensitive receptors. 2629-Pos Board B236 Molecular Dynamics Analysis on the Force Transmission Pathway via Inter-Subunit Pathway for Mechano-Gating of Bacterial Mechanosensitive Channel MscL Yasuyuki Sawada1, Takeshi Nomura2, Masahiro Sokabe1. 1 Mechanobiology Lab, Nagoya University Graduate School of Medicine, Nagoya, Japan, 2Physical Therapy, Grad Sch Health Sciences Kyushu Nutrition Welfare Univ, Kitakyushu, Japan. The bacterial mechanosensitive channel MscL is constituted of homopentamer of a subunit with TM1 inner and TM2 outer transmembrane helix. The major issue on MscL is to understand the gating mechanism driven by membrane tension. Upon membrane stretch, the helices are dragged by lipids at the tension sensor F78 and tilted, accompanied by outward sliding, leading to a gate expansion. To get insights into the relationship between F78 and the gate including G22, we performed MD simulations of G22N GOF and F78N LOF MscLs and G22N showed spontaneous opening without membrane stretch, while F78N could not be opened even under strong membrane tension. To assess the role of Asn22 for the spontaneous opening, the double mutant G22N/ F78N MscL simulation was performed with and without membrane stretch and found that G22N/F78N MscL did not begin channel opening upon membrane stretch under the both conditions. Furthermore, we found that the closed MscL can transmit resting tension to the gate via the interaction between F78 in TM2 and I32-L36-I40 in the neighboring TM1 and the transmitted force can lead to slight opening of the pore. It is suggested that the substitution of F78 with Asn loses the transmission pathway, leading to be harder to open than G22N even though G22N/F78N has Asn22 with the hydrophilic side chain. 2630-Pos Board B237 Human Piezo1 Membrane Localization and Gating Kinetics are Modulated by Cholesetrol Levels Pietro Ridone1, Charles Cox1, Massimo Vassalli2, Elvis Pandzic3, Philip Gottlieb4, Boris Martinac1. 1 Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Institute, Sydney, Australia, 2Institute of Biophysics IBF, Genova, Italy, 3 Biomedical Imaging Facility (BMIF), University of New South Wales, Sydney, Australia, 4State University of New York, Buffalo, NY, USA. The human mechanosensitive ion channel Piezo1 gates in response to membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently little is known
533a
regarding theplasma membrane localization and organization of Piezo1, but previous work suggests that membrane cholesterol content is a key determinant of Piezo1 function. Using a previously characterised Piezo1-GFP fusion protein (hP1-1591-GFP[1]), we investigated the effect of the cholesterol depleting agent methyl-b-Cyclodextrin (mbCD) on the membrane organization and the response of Piezo1 to mechanical force in HEK-293 cells. STORM superresolution imaging revealed at the nanoscale that Piezo channels associate in the membrane as clusters, as previously inferred by electrophysiological data [2]. Both cluster size and diffusion rates, as determined using TIRF microscopy, were modulated by treatment with mbCD (5 mM). In addition, electrophysiological recordings in the cell-attached configuration revealed that mbCD caused a right-ward shift in the Piezo1 pressure-response curve and a delay in the initial response (i.e. increased latency). We suggest that cholesterol rich micro-domains host Piezo1 clusters and that this nanoscale membrane organization is essential for efficient Piezo1-mediated mechanotransduction. This is consistent with cholesterol rich domains acting as ‘‘membrane force foci’’. [1] Cox et al., (2016) Nat Commun 20;7:10366 [2] Bae et al., (2013) PNAS 110(12): E1162-E1168 2631-Pos Board B238 Enantiomeric Forms of Abeta Peptides Inhibit the Shear Stress Response of PIEZO1 Philip A. Gottlieb1, Mohammad M. Maneshi2, Radhakrishnan Gnanasambandam1, Susan Z. Hua2, Frederick Sachs1. 1 Physiology and Bophysics, SUNY at Buffalo, Buffalo, NY, USA, 2 Department of Mechanical & Aerospace Engineering, SUNY at Buffalo, Buffalo, NY, USA. PIEZO1 is a eukaryotic mechanosensitive ion channel that is cation selective and rapidly inactivates. Using fluid shear stress as a stimulus generated in a microfluidic chamber, we have monitored PIEZO activity in the presence of various peptides. To do so, we first developed a cell line that overexpresses the PIEZO1 channel in HEK293T cells. We estimate that there are about 5000 channels per cell. These cells are seeded in the microfluidic chamber and loaded with calcium indicator. A pressure servo is fitted to the chamber and allows control of the applied shear stress stimulus. The calcium change is monitored as an increase in fluorescence. Applying a 10 ms pulse at 13 dyn/cm2, we observed a robust response for the PIEZO1 cell line well above the background observed for HEK293T cells only. Peak response is achieved within a few seconds and is followed by adaptation. Disrupters of the cytoskeleton applied to the cell line, such as cytochalasinD and cholchicine, rendered the PIEZO1 channel unresponsive to the stimulus. The peptide GsMTx4 inhibits PIEZO1 currents. We measured the effect of GsMTx4 on PIEZO1 activity as a way to compare the microfluidic approach to previous work. We applied various concentrations and measure the peak response. The inhibition curve showed a Ki of around 250 nM similar to the value measured previously by patch clamp. Next, we examined the effect of Abeta peptides on PIEZO1 activity. A known attribute of these peptides is their ability to bind membranes. Given PIEZO1 is modulated by membrane tension we wanted to determine how the channel behaved in the presence of Abeta peptides. We prepared the monomer form of the peptide and measured the peak response as a function of peptide concentration. At the same time we prepared the D form of these peptides and measured them as well. Our data show that both the L and D form of Abeta 1-40 and 1-42 inhibit channel function in the fM to pM range. The enantiomeric forms of each peptide were similar in their ability to inhibit PIEZO1 activity. The lack of stereospecific interaction indicates that these peptides modulate activity of the channel by changing the properties of the membrane. This work was funded by NIHLBI and NINDS. 2632-Pos Board B239 Mechanosensitivity of Coupled Active Hair-Cell Bundles Tracy-Ying Zhang1, Seung Ji2, Dolores Bozovic1. 1 Department of Physics and Astronomy, University of California Los Angeles, Los Angeles, CA, USA, 2Department of Physical Science, Los Angeles Mission College, Sylmar, CA, USA. The sensitivity of the auditory system depends in part on the active response of hair cells in the inner ear. Individual hair bundles display frequency selectivity and compressive nonlinearity in response to stimuli. In many auditory and vestibular end organs, the hair bundles are coupled by overlying structures. This motivates our study on how coupling affects the hair bundle sensitivity. We couple two to four spontaneously oscillating bundles with a microbead, and apply mechanical stimuli to the group. Prior work showed that under these coupling conditions, innate oscillations synchronize. Here, we show that the synchronized bundles exhibit broad frequency selectivity, over a bandwidth encompassing each bundle’s natural frequency. The amplitude of the response shows a nonlinear dependence on the applied stimulus. Experimental data will be presented, as well as a simple numerical model, to explain how the
Mechanosensation 2628-Pos Board B235 From Force to Function - Investigating Mechanosensitive Piezo Receptors by AFM Benjamin M. Gaub, Daniel J. Mueller. D-BSSE, ETH Z€ urich, Basel, Switzerland. Mechanosensation is the process by which cells sense mechanical forces and translate them into electrical and chemical signals. Important physiological functions including sensation of touch, sensation of sound and proprioception are based on mechanosensation. Recent studies identified the molecular identity of a highly conserved group of mechanosensitive receptors, called Piezo receptors, which are both necessary and sufficient for cells to sense force. Currently, structure-function information of Piezo receptors is limited, and the mechanism how these receptors sense force, which conformational changes they undergo, and which precise mechanical stimuli cause these channels to open, remains poorly understood. Studies to date have used rather brute forces reaching several hundrets of nanoNewton to mechanically activate Piezo channels, which are likely too high to be of specific nature. To manipulate Piezo receptors at high precision and accuracy in a cellular context, new techniques have to be applied. We are currently developing assays to investigate Piezo receptors at the molecular level with delicate force control. Using a combination of high-resolution atomic force microscopy (AFM) and time-lapse fluorescent calcium imaging, we aim to find more efficient ways to functionally control Piezo receptors by externally applied forces. Importantly, the tip of the AFM cantilever can be conjugated with small molecules, including ligands and antibodies, which enables manipulation of membrane proteins at nanometer precision with picoNewton sensitivity. We will use this assay to i) get a more quantitative understanding of Piezo gating by mechanical forces, ii) directly compare Piezo receptor responses to different modes of mechanical stimulation including pressure, stretch and vibration, and iii) screen native extracellular matrix (ECM) derived ligands for their ability to bind and functionally modulate the Piezo channel. Our results will help shed light on the force sensing mechanism of mammalian mechanosensitive receptors. 2629-Pos Board B236 Molecular Dynamics Analysis on the Force Transmission Pathway via Inter-Subunit Pathway for Mechano-Gating of Bacterial Mechanosensitive Channel MscL Yasuyuki Sawada1, Takeshi Nomura2, Masahiro Sokabe1. 1 Mechanobiology Lab, Nagoya University Graduate School of Medicine, Nagoya, Japan, 2Physical Therapy, Grad Sch Health Sciences Kyushu Nutrition Welfare Univ, Kitakyushu, Japan. The bacterial mechanosensitive channel MscL is constituted of homopentamer of a subunit with TM1 inner and TM2 outer transmembrane helix. The major issue on MscL is to understand the gating mechanism driven by membrane tension. Upon membrane stretch, the helices are dragged by lipids at the tension sensor F78 and tilted, accompanied by outward sliding, leading to a gate expansion. To get insights into the relationship between F78 and the gate including G22, we performed MD simulations of G22N GOF and F78N LOF MscLs and G22N showed spontaneous opening without membrane stretch, while F78N could not be opened even under strong membrane tension. To assess the role of Asn22 for the spontaneous opening, the double mutant G22N/ F78N MscL simulation was performed with and without membrane stretch and found that G22N/F78N MscL did not begin channel opening upon membrane stretch under the both conditions. Furthermore, we found that the closed MscL can transmit resting tension to the gate via the interaction between F78 in TM2 and I32-L36-I40 in the neighboring TM1 and the transmitted force can lead to slight opening of the pore. It is suggested that the substitution of F78 with Asn loses the transmission pathway, leading to be harder to open than G22N even though G22N/F78N has Asn22 with the hydrophilic side chain. 2630-Pos Board B237 Human Piezo1 Membrane Localization and Gating Kinetics are Modulated by Cholesetrol Levels Pietro Ridone1, Charles Cox1, Massimo Vassalli2, Elvis Pandzic3, Philip Gottlieb4, Boris Martinac1. 1 Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Institute, Sydney, Australia, 2Institute of Biophysics IBF, Genova, Italy, 3 Biomedical Imaging Facility (BMIF), University of New South Wales, Sydney, Australia, 4State University of New York, Buffalo, NY, USA. The human mechanosensitive ion channel Piezo1 gates in response to membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently little is known
533a
regarding theplasma membrane localization and organization of Piezo1, but previous work suggests that membrane cholesterol content is a key determinant of Piezo1 function. Using a previously characterised Piezo1-GFP fusion protein (hP1-1591-GFP[1]), we investigated the effect of the cholesterol depleting agent methyl-b-Cyclodextrin (mbCD) on the membrane organization and the response of Piezo1 to mechanical force in HEK-293 cells. STORM superresolution imaging revealed at the nanoscale that Piezo channels associate in the membrane as clusters, as previously inferred by electrophysiological data [2]. Both cluster size and diffusion rates, as determined using TIRF microscopy, were modulated by treatment with mbCD (5 mM). In addition, electrophysiological recordings in the cell-attached configuration revealed that mbCD caused a right-ward shift in the Piezo1 pressure-response curve and a delay in the initial response (i.e. increased latency). We suggest that cholesterol rich micro-domains host Piezo1 clusters and that this nanoscale membrane organization is essential for efficient Piezo1-mediated mechanotransduction. This is consistent with cholesterol rich domains acting as ‘‘membrane force foci’’. [1] Cox et al., (2016) Nat Commun 20;7:10366 [2] Bae et al., (2013) PNAS 110(12): E1162-E1168 2631-Pos Board B238 Enantiomeric Forms of Abeta Peptides Inhibit the Shear Stress Response of PIEZO1 Philip A. Gottlieb1, Mohammad M. Maneshi2, Radhakrishnan Gnanasambandam1, Susan Z. Hua2, Frederick Sachs1. 1 Physiology and Bophysics, SUNY at Buffalo, Buffalo, NY, USA, 2 Department of Mechanical & Aerospace Engineering, SUNY at Buffalo, Buffalo, NY, USA. PIEZO1 is a eukaryotic mechanosensitive ion channel that is cation selective and rapidly inactivates. Using fluid shear stress as a stimulus generated in a microfluidic chamber, we have monitored PIEZO activity in the presence of various peptides. To do so, we first developed a cell line that overexpresses the PIEZO1 channel in HEK293T cells. We estimate that there are about 5000 channels per cell. These cells are seeded in the microfluidic chamber and loaded with calcium indicator. A pressure servo is fitted to the chamber and allows control of the applied shear stress stimulus. The calcium change is monitored as an increase in fluorescence. Applying a 10 ms pulse at 13 dyn/cm2, we observed a robust response for the PIEZO1 cell line well above the background observed for HEK293T cells only. Peak response is achieved within a few seconds and is followed by adaptation. Disrupters of the cytoskeleton applied to the cell line, such as cytochalasinD and cholchicine, rendered the PIEZO1 channel unresponsive to the stimulus. The peptide GsMTx4 inhibits PIEZO1 currents. We measured the effect of GsMTx4 on PIEZO1 activity as a way to compare the microfluidic approach to previous work. We applied various concentrations and measure the peak response. The inhibition curve showed a Ki of around 250 nM similar to the value measured previously by patch clamp. Next, we examined the effect of Abeta peptides on PIEZO1 activity. A known attribute of these peptides is their ability to bind membranes. Given PIEZO1 is modulated by membrane tension we wanted to determine how the channel behaved in the presence of Abeta peptides. We prepared the monomer form of the peptide and measured the peak response as a function of peptide concentration. At the same time we prepared the D form of these peptides and measured them as well. Our data show that both the L and D form of Abeta 1-40 and 1-42 inhibit channel function in the fM to pM range. The enantiomeric forms of each peptide were similar in their ability to inhibit PIEZO1 activity. The lack of stereospecific interaction indicates that these peptides modulate activity of the channel by changing the properties of the membrane. This work was funded by NIHLBI and NINDS. 2632-Pos Board B239 Mechanosensitivity of Coupled Active Hair-Cell Bundles Tracy-Ying Zhang1, Seung Ji2, Dolores Bozovic1. 1 Department of Physics and Astronomy, University of California Los Angeles, Los Angeles, CA, USA, 2Department of Physical Science, Los Angeles Mission College, Sylmar, CA, USA. The sensitivity of the auditory system depends in part on the active response of hair cells in the inner ear. Individual hair bundles display frequency selectivity and compressive nonlinearity in response to stimuli. In many auditory and vestibular end organs, the hair bundles are coupled by overlying structures. This motivates our study on how coupling affects the hair bundle sensitivity. We couple two to four spontaneously oscillating bundles with a microbead, and apply mechanical stimuli to the group. Prior work showed that under these coupling conditions, innate oscillations synchronize. Here, we show that the synchronized bundles exhibit broad frequency selectivity, over a bandwidth encompassing each bundle’s natural frequency. The amplitude of the response shows a nonlinear dependence on the applied stimulus. Experimental data will be presented, as well as a simple numerical model, to explain how the