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Sensory mechanotransduction at membrane-matrix interfaces.

Poole K, Moroni M, Lewin GR - Pflugers Arch. (2014)

Bottom Line: One component of such complexes in sensory neurons is the integral membrane scaffold protein STOML3 which is a robust physiological regulator of native mechanosensitive currents.In order to better characterize such channels in transmembrane complexes, we developed a new electrophysiological method that enables the quantification of mechanosensitive current amplitude and kinetics when activated by a defined matrix movement in cultured cells.The results of such studies strongly support the idea that ion channels in transmembrane complexes are highly tuned to detect movement of the cell membrane in relation to the ECM.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Max-Delbrück Center for Molecular Medicine, Robert-Rössle Straße 10, D-13092, Berlin, Germany.

ABSTRACT
Sensory cells specialized to detect extremely small mechanical changes are common to the auditory and somatosensory systems. It is widely accepted that mechanosensitive channels form the core of the mechanoelectrical transduction in hair cells as well as the somatic sensory neurons that underlie the sense of touch and mechanical pain. Here, we will review how the activation of such channels can be measured in a meaningful physiological context. In particular, we will discuss the idea that mechanosensitive channels normally occur in transmembrane complexes that are anchored to extracellular matrix components (ECM) both in vitro and in vivo. One component of such complexes in sensory neurons is the integral membrane scaffold protein STOML3 which is a robust physiological regulator of native mechanosensitive currents. In order to better characterize such channels in transmembrane complexes, we developed a new electrophysiological method that enables the quantification of mechanosensitive current amplitude and kinetics when activated by a defined matrix movement in cultured cells. The results of such studies strongly support the idea that ion channels in transmembrane complexes are highly tuned to detect movement of the cell membrane in relation to the ECM.

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Related in: MedlinePlus

Using elastomeric pillar arrays to quantitatively measure mechanotransduction at the membrane/matrix interface. a, b Scanning electron micrographs of elastomeric pillar arrays taken perpendicular (a) and parallel (b) to the elements of the array. c Pillar arrays can be coated with EHS-laminin (magenta), and sensory neurons acutely isolated from the mouse will attach to the array and extend neurites over the tops of the pili (green, overexpressed LifeAct-GFP). d Cells can be monitored using whole-cell patch clamp, and a glass nanostimulator can be used to deflect individual pillar elements directly underneath the neurite, bright field image, cell outlined in yellow, black arrow indicates individual pilus being deflected. e Sensory neurons respond to pillar deflection with rapidly adapting (RA), intermediate-adapting (IA) and slowly adapting (SA) currents. f Stimulus-response curves indicate the higher sensitivity of mechanoreceptors (n = 8 cells) vs nociceptors (n = 13 cells), note a Boltzmann fit of typeII mechanoreceptor data indicates that a stimulus of 13 nm is required for half-maximal activation of mechanically gated currents in these cells. g The sensitivity of type II mechanoreceptors is dependent on the presence of STOML3; C57Bl/6, n = 8 cells; stoml3−/−, n = 8 cells. (h, i) In a heterologous system, HEK-293 cells, Piezo1- (h, black triangles, n = 9 cells) and Piezo2- (i, grey triangles, n = 10 cells) mediated currents are more sensitive when these channels are co-expressed with STOML3 (cyan triangles; Piezo1 + STOML3, n = 11 cells; Piezo2 + STOML3, n = 9 cells). Significance determined using a Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. Data from [85]
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Fig2: Using elastomeric pillar arrays to quantitatively measure mechanotransduction at the membrane/matrix interface. a, b Scanning electron micrographs of elastomeric pillar arrays taken perpendicular (a) and parallel (b) to the elements of the array. c Pillar arrays can be coated with EHS-laminin (magenta), and sensory neurons acutely isolated from the mouse will attach to the array and extend neurites over the tops of the pili (green, overexpressed LifeAct-GFP). d Cells can be monitored using whole-cell patch clamp, and a glass nanostimulator can be used to deflect individual pillar elements directly underneath the neurite, bright field image, cell outlined in yellow, black arrow indicates individual pilus being deflected. e Sensory neurons respond to pillar deflection with rapidly adapting (RA), intermediate-adapting (IA) and slowly adapting (SA) currents. f Stimulus-response curves indicate the higher sensitivity of mechanoreceptors (n = 8 cells) vs nociceptors (n = 13 cells), note a Boltzmann fit of typeII mechanoreceptor data indicates that a stimulus of 13 nm is required for half-maximal activation of mechanically gated currents in these cells. g The sensitivity of type II mechanoreceptors is dependent on the presence of STOML3; C57Bl/6, n = 8 cells; stoml3−/−, n = 8 cells. (h, i) In a heterologous system, HEK-293 cells, Piezo1- (h, black triangles, n = 9 cells) and Piezo2- (i, grey triangles, n = 10 cells) mediated currents are more sensitive when these channels are co-expressed with STOML3 (cyan triangles; Piezo1 + STOML3, n = 11 cells; Piezo2 + STOML3, n = 9 cells). Significance determined using a Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. Data from [85]

Mentions: It is thus clear that the cell-matrix interface is critically important for the mechanosensitive currents that we are able to activate by cell body or neurite indentation in sensory neurons. Indentation techniques cannot directly activate mechanosensitive channels present in transmembrane complexes at the plasma membrane-matrix interface. In addition, the precise stimulus resulting from the indentation of the cell soma or a neurite segment with a glass probe is unknown as the size of the probe may vary from experiment to experiment, the precise moment when the probe contacts the surface of the cell is not known and the curvature and elasticity (both variable) of the impact site will modulate the stimulus as it is propagated by the cell itself to the membrane-matrix interface. We set out to design a completely new experimental approach that enables us to apply a mechanical stimulus of known magnitude directly to defined regions of the membrane-matrix interface whilst monitoring the cellular response using whole-cell patch clamp (Figs. 2 and 3). Briefly, an elastomeric pillar array was cast from a microfabricated master, with defined dimensions and material properties. To study mechanotransduction in sensory neurons, the tops of the cylindrical elements (pili) within this array are coated with laminin to promote cellular attachment and to restrict neurite outgrowth to the defined circular area. An individual pilus to which a neurite is bound can then be deflected using a nanomotor-driven stimulator, resulting in a mechanical stimulus directly at the cell-matrix interface. By applying pillar deflections with magnitudes between 10 and 1,000 nm to the plasma membrane-matrix interface, we could use whole-cell patch clamp to measure mechanosensitive currents directly activated by defined matrix deflections [85]. When applied to cultured sensory neurons, this method revealed that pillar deflection evoked RA, IA or SA currents in all cells. Importantly, the activation and inactivation kinetics of all three types of mechanosensitive currents were virtually identical to those found with neurite indentation [85]. This finding strongly suggests that the opening of channels measured after cell indentation is, at least in part, identical with those activated by matrix deflection. The pili method allows a highly defined part of membrane (10 μm2 in area) to be interrogated with a defined stimuli; thus, for a single neuron, we could test multiple sites. We could conclude from such experiments that mechanosensitive currents with different inactivation kinetics were often present in the same neurons. What determines the inactivation kinetics of the mechanosensitive current? It is most often assumed that channel inactivation is an intrinsic property of the channel in question. Thus, currents that inactivate with dramatically different rates may represent the activation of different channel entities. However, since the molecular nature of the channel(s) that underlie fast mechanosensitive currents is unclear, there remains the possibility that mechanical elements that are part of the mechanotransduction complex govern the rate of channel inactivation. However, several groups have described differences in the pharmacological sensitivity or ion selectivity of SA, IA and RA currents [48, 62], e.g. selective sensitivity of the SA current to block the NMB-1 peptide [30], that do suggest that currents with different inactivation properties are mediated by distinct ion channel entities.Fig. 2


Sensory mechanotransduction at membrane-matrix interfaces.

Poole K, Moroni M, Lewin GR - Pflugers Arch. (2014)

Using elastomeric pillar arrays to quantitatively measure mechanotransduction at the membrane/matrix interface. a, b Scanning electron micrographs of elastomeric pillar arrays taken perpendicular (a) and parallel (b) to the elements of the array. c Pillar arrays can be coated with EHS-laminin (magenta), and sensory neurons acutely isolated from the mouse will attach to the array and extend neurites over the tops of the pili (green, overexpressed LifeAct-GFP). d Cells can be monitored using whole-cell patch clamp, and a glass nanostimulator can be used to deflect individual pillar elements directly underneath the neurite, bright field image, cell outlined in yellow, black arrow indicates individual pilus being deflected. e Sensory neurons respond to pillar deflection with rapidly adapting (RA), intermediate-adapting (IA) and slowly adapting (SA) currents. f Stimulus-response curves indicate the higher sensitivity of mechanoreceptors (n = 8 cells) vs nociceptors (n = 13 cells), note a Boltzmann fit of typeII mechanoreceptor data indicates that a stimulus of 13 nm is required for half-maximal activation of mechanically gated currents in these cells. g The sensitivity of type II mechanoreceptors is dependent on the presence of STOML3; C57Bl/6, n = 8 cells; stoml3−/−, n = 8 cells. (h, i) In a heterologous system, HEK-293 cells, Piezo1- (h, black triangles, n = 9 cells) and Piezo2- (i, grey triangles, n = 10 cells) mediated currents are more sensitive when these channels are co-expressed with STOML3 (cyan triangles; Piezo1 + STOML3, n = 11 cells; Piezo2 + STOML3, n = 9 cells). Significance determined using a Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. Data from [85]
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: Using elastomeric pillar arrays to quantitatively measure mechanotransduction at the membrane/matrix interface. a, b Scanning electron micrographs of elastomeric pillar arrays taken perpendicular (a) and parallel (b) to the elements of the array. c Pillar arrays can be coated with EHS-laminin (magenta), and sensory neurons acutely isolated from the mouse will attach to the array and extend neurites over the tops of the pili (green, overexpressed LifeAct-GFP). d Cells can be monitored using whole-cell patch clamp, and a glass nanostimulator can be used to deflect individual pillar elements directly underneath the neurite, bright field image, cell outlined in yellow, black arrow indicates individual pilus being deflected. e Sensory neurons respond to pillar deflection with rapidly adapting (RA), intermediate-adapting (IA) and slowly adapting (SA) currents. f Stimulus-response curves indicate the higher sensitivity of mechanoreceptors (n = 8 cells) vs nociceptors (n = 13 cells), note a Boltzmann fit of typeII mechanoreceptor data indicates that a stimulus of 13 nm is required for half-maximal activation of mechanically gated currents in these cells. g The sensitivity of type II mechanoreceptors is dependent on the presence of STOML3; C57Bl/6, n = 8 cells; stoml3−/−, n = 8 cells. (h, i) In a heterologous system, HEK-293 cells, Piezo1- (h, black triangles, n = 9 cells) and Piezo2- (i, grey triangles, n = 10 cells) mediated currents are more sensitive when these channels are co-expressed with STOML3 (cyan triangles; Piezo1 + STOML3, n = 11 cells; Piezo2 + STOML3, n = 9 cells). Significance determined using a Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. Data from [85]
Mentions: It is thus clear that the cell-matrix interface is critically important for the mechanosensitive currents that we are able to activate by cell body or neurite indentation in sensory neurons. Indentation techniques cannot directly activate mechanosensitive channels present in transmembrane complexes at the plasma membrane-matrix interface. In addition, the precise stimulus resulting from the indentation of the cell soma or a neurite segment with a glass probe is unknown as the size of the probe may vary from experiment to experiment, the precise moment when the probe contacts the surface of the cell is not known and the curvature and elasticity (both variable) of the impact site will modulate the stimulus as it is propagated by the cell itself to the membrane-matrix interface. We set out to design a completely new experimental approach that enables us to apply a mechanical stimulus of known magnitude directly to defined regions of the membrane-matrix interface whilst monitoring the cellular response using whole-cell patch clamp (Figs. 2 and 3). Briefly, an elastomeric pillar array was cast from a microfabricated master, with defined dimensions and material properties. To study mechanotransduction in sensory neurons, the tops of the cylindrical elements (pili) within this array are coated with laminin to promote cellular attachment and to restrict neurite outgrowth to the defined circular area. An individual pilus to which a neurite is bound can then be deflected using a nanomotor-driven stimulator, resulting in a mechanical stimulus directly at the cell-matrix interface. By applying pillar deflections with magnitudes between 10 and 1,000 nm to the plasma membrane-matrix interface, we could use whole-cell patch clamp to measure mechanosensitive currents directly activated by defined matrix deflections [85]. When applied to cultured sensory neurons, this method revealed that pillar deflection evoked RA, IA or SA currents in all cells. Importantly, the activation and inactivation kinetics of all three types of mechanosensitive currents were virtually identical to those found with neurite indentation [85]. This finding strongly suggests that the opening of channels measured after cell indentation is, at least in part, identical with those activated by matrix deflection. The pili method allows a highly defined part of membrane (10 μm2 in area) to be interrogated with a defined stimuli; thus, for a single neuron, we could test multiple sites. We could conclude from such experiments that mechanosensitive currents with different inactivation kinetics were often present in the same neurons. What determines the inactivation kinetics of the mechanosensitive current? It is most often assumed that channel inactivation is an intrinsic property of the channel in question. Thus, currents that inactivate with dramatically different rates may represent the activation of different channel entities. However, since the molecular nature of the channel(s) that underlie fast mechanosensitive currents is unclear, there remains the possibility that mechanical elements that are part of the mechanotransduction complex govern the rate of channel inactivation. However, several groups have described differences in the pharmacological sensitivity or ion selectivity of SA, IA and RA currents [48, 62], e.g. selective sensitivity of the SA current to block the NMB-1 peptide [30], that do suggest that currents with different inactivation properties are mediated by distinct ion channel entities.Fig. 2

Bottom Line: One component of such complexes in sensory neurons is the integral membrane scaffold protein STOML3 which is a robust physiological regulator of native mechanosensitive currents.In order to better characterize such channels in transmembrane complexes, we developed a new electrophysiological method that enables the quantification of mechanosensitive current amplitude and kinetics when activated by a defined matrix movement in cultured cells.The results of such studies strongly support the idea that ion channels in transmembrane complexes are highly tuned to detect movement of the cell membrane in relation to the ECM.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Max-Delbrück Center for Molecular Medicine, Robert-Rössle Straße 10, D-13092, Berlin, Germany.

ABSTRACT
Sensory cells specialized to detect extremely small mechanical changes are common to the auditory and somatosensory systems. It is widely accepted that mechanosensitive channels form the core of the mechanoelectrical transduction in hair cells as well as the somatic sensory neurons that underlie the sense of touch and mechanical pain. Here, we will review how the activation of such channels can be measured in a meaningful physiological context. In particular, we will discuss the idea that mechanosensitive channels normally occur in transmembrane complexes that are anchored to extracellular matrix components (ECM) both in vitro and in vivo. One component of such complexes in sensory neurons is the integral membrane scaffold protein STOML3 which is a robust physiological regulator of native mechanosensitive currents. In order to better characterize such channels in transmembrane complexes, we developed a new electrophysiological method that enables the quantification of mechanosensitive current amplitude and kinetics when activated by a defined matrix movement in cultured cells. The results of such studies strongly support the idea that ion channels in transmembrane complexes are highly tuned to detect movement of the cell membrane in relation to the ECM.

Show MeSH
Related in: MedlinePlus