Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Mechanosensitive TRPC1 Channels Promote Calpain Proteolysis of Talin to Regulate Spinal Axon Outgrowth

doi: 10.1523/JNEUROSCI.2142-12.2013

Figure Lengend Snippet: Morpholino-mediated knockdown of xTRPC1 reduces the effects of MS channel blockers on neurite outgrowth. a, Western blot for TRPV4 from dorsal section lysates (see Materials and Methods) of control embryos and embryos injected with a TRPV4 morpholino (injected into 2 cells at the 4-cell stage). TRPV4 protein levels are reduced by 49.1 ± 16.1% (n = 4). As described previously in mouse, the TRPV4 protein appears as two bands of ~107 and 75 kDa, representing full-length TRPV4 and possibly a splice variant (Liedtke and Friedman, 2003). b, The rate of neurite outgrowth on FN-coated glass in response to subtracting AGAs from neurons injected with control, TRPV4, or TRPC1 morpholinos normalized to the precondition rate of outgrowth (n ≥ 42 for each condition). c, The rate of neurite outgrowth on FN-coated glass in response to the addition of 10 µm GsMTx4 from neurons injected with control, TRPV4, or TRPC1 morpholinos (n ≥ 29 for each condition). Statistical comparisons were made between control morphant neurons and TRP channel morphant neurons. d, A box-and-whisker plot shows reduced TRPC1 immunofluorescence labeling in TRPC1 morphant growth cones. e–g, A representative growth cone immunolabeled for TRPC1 (e) and phalloidin to label F-actin (f). g, The merged image shows that TRPC1 (green) localizes with F-actin (blue) at the tips and along the shaft of filopodia (arrows). Scale bar, 4 µm. *p < 0.05, **p < 0.01, and ***p < 0.001 using either a one-way ANOVA with a Tukey’s post test (b) or a Kruskal–Wallis test with a Dunn’s post test (c).

Article Snippet: GsMTx4 was chemically synthesized and folded from a linear peptide by SynPep.

Techniques: Knockdown, Western Blot, Control, Injection, Variant Assay, Whisker Assay, Immunofluorescence, Labeling, Immunolabeling

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Mechanosensitive TRPC1 Channels Promote Calpain Proteolysis of Talin to Regulate Spinal Axon Outgrowth

doi: 10.1523/JNEUROSCI.2142-12.2013

Figure Lengend Snippet: The rate of neurite outgrowth depends on MS channel activity and substratum elasticity. a, The rate of outgrowth of Xenopus spinal axons on FN-coated glass during a control media wash (with AGAs, n = 61) and during the removal of AGAs in the presence of 2 mm Ca2+ (n = 34), 0 Ca2+ (n =16), 10 µm GsMTx4 (GsM, n = 82), or 100 µm gadolinium (Gd3+, n = 44) normalized to the precondition rate of outgrowth. Note a significant decrease in the rate of neurite outgrowth when AGAs are removed in 2 mm Ca2+ (**p < 0.01), but a significant increase in the rate of neurite outgrowth with addition of the MS channel blocker, GsMTx4 (***p < 0.001). b, Dose-dependent effects of gentamicin on the rate of neurite outgrowth (n ≥ 16 for each concentration). c, The basal rate of neurite outgrowth of neurons cultured on FN coated onto rigid substrata is significantly slower compared with flexible FN (see Materials and Methods; ***p < 0.001, n ≥ 34). d, The effects of pharmacologically altering MS channel activity depends on the elasticity of the substratum. The rate of axon extension is reduced by activating MS channels (−AGAs) on rigid PDMS-FN (n = 40) and FN glass (n = 35), but not on flexible FN (n = 45). Acceleration of neurite outgrowth with the addition of GsMTx4 is most pronounced on FN glass (n = 82) and FN-rigid PDMS (n = 56), but to a lesser degree on FN-flexible PDMS (n = 45). **p < 0.01 and ***p < 0.001 compared with neurons on glass. All statistical analysis was completed with a Kruskal–Wallis test with a Dunn’s multiple-comparison test.

Article Snippet: GsMTx4 was chemically synthesized and folded from a linear peptide by SynPep.

Techniques: Activity Assay, Control, Concentration Assay, Cell Culture, Comparison

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Mechanosensitive TRPC1 Channels Promote Calpain Proteolysis of Talin to Regulate Spinal Axon Outgrowth

doi: 10.1523/JNEUROSCI.2142-12.2013

Figure Lengend Snippet: MS channel activity increases the incidence and frequency of filopodial Ca2+ transients and baseline [Ca2+]i. a, A pseudocolored Fluo-4-loaded growth cone showing regions used to measure fluorescent intensities over time. Images were captured at 10 Hz for 3 min during solution changes. Scale bar, 3 µm. b, Traces of Fluo-4 fluorescent signals measured in three filopodia over 1 min periods before and after the removal of AGAs and 1 min after the addition of GsMTx4. #, denotes a single global Ca2+ transient. c, d, The incidence (c) and frequency (d) of filopodial Ca2+ transients were determined (see Materials and Methods) in control media (n = 290), in the absence of AGAs (n = 237), and after the addition of GsMTx4 (n = 174) or Gd3+ (n = 159) on FN-glass substrata. Filopodial Ca2+ transients were also measured on flexible FN-PDMS with and without AGAs (n = 103 and 52, respectively). **p < 0.01 and ***p < 0.001 compared with control condition (+AGA on glass) using a Kruskal–Wallis test with a Dunn’s post test. e, Fura-2, a ratiometric Ca2+ indicator, was used to determine the [Ca2+]i within growth cones during AGA subtraction. There was a significant increase in the baseline [Ca2+]i of growth cones on FN glass after the removal of AGAs, but not by growth cones on FN-PDMS (***p < 0.001, two-way ANOVA).

Article Snippet: GsMTx4 was chemically synthesized and folded from a linear peptide by SynPep.

Techniques: Activity Assay, Control

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Mechanosensitive TRPC1 Channels Promote Calpain Proteolysis of Talin to Regulate Spinal Axon Outgrowth

doi: 10.1523/JNEUROSCI.2142-12.2013

Figure Lengend Snippet: Ca2+ influx through MS channels activates calpain in growth cones. a, The average rates of neurite outgrowth over 15 min periods before treatment (white bars), after the addition of inhibitors (gray bars), and after the subsequent addition of inhibitors with 10 µm GsMTx4 (black bars). Inhibitors of Ca2+ effectors included the CaMKII inhibitor KN62 (5 µm), the calcineurin inhibitor CsA (10 nm), and the calpain protease inhibitors (1 µm CPI; 10 µm ALLM). b, A schematic diagram illustrates the Ca2+ signaling pathway (black) and inhibitors (red) used throughout this figure. Dominant-negative protein inhibitors are in parentheses. c–e, A fluorogenic calpain substrate, t-BOC, was used to measure protease activity during activation and inhibition of MS channels. c, Pseudocolored fluorescence images of growth cones loaded with t-BOC for 30 min under indicated conditions. Scale bar, 10 µm. d, The intensities of t-BOC fluorescence (12-bit scale) plotted over 30 min with or without AGAs in 0 or 2 mm Ca2+ (n ≥ 83 for each condition). e, t-BOC fluorescence intensity values measured at 30 min after t-BOC addition and the baseline is normalized to control conditions in the presence of AGAs. Removing AGAs increases t-BOC fluorogenesis in the presence of 2 mm Ca2+, but this is prevented in 0 mm Ca2+ and with MS channel blockers, with inhibition of calpain and in by growing neurons on flexible FN (PDMS). f, The rate of outgrowth was measured in the presence of inhibitors of Src family kinases included PP2 (1 µm) and SU6656 (1 µm), and in neurons expressing KD-Src. Control data are transferred from a for comparison. g, The rate of outgrowth was quantified for 30 min before (black) and after (gray) the removal of AGAs in each condition. The removal of AGAs reduces the rate of outgrowth in wild-type and eGFP-Talin overexpressing neurons, but is blocked by the addition of 1 µm CPI or expression of the calpain-resistant talin mutant, eGFP-Talin-L432G. *p < 0.05 and ***p < 0.001 using a Kruskal–Wallis test with a Dunn’s post test.

Article Snippet: GsMTx4 was chemically synthesized and folded from a linear peptide by SynPep.

Techniques: Dominant Negative Mutation, Activity Assay, Activation Assay, Inhibition, Fluorescence, Control, Expressing, Comparison, Mutagenesis

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Mechanosensitive TRPC1 Channels Promote Calpain Proteolysis of Talin to Regulate Spinal Axon Outgrowth

doi: 10.1523/JNEUROSCI.2142-12.2013

Figure Lengend Snippet: TRPC1 is required for mechanically induced Ca2+ influx and calpain protease activity in growth cones. a, b, Removing AGAs increases the incidence (a) and frequency (b) of filopodial Ca2+ transients in control morphant, but not TRPC1 morphant growth cones (n ≥ 188 filopodia for each condition, ***p < 0.001, Mann–Whitney test). c, d, Fura-2 ratiometric Ca2+ imaging shows that baseline [Ca2+]i increases in wild-type, but not TRPC1 morphant growth cones during removal of AGAs. c, eGFP (top) was coinjected with the TRPC1 morpholino to identify TRPC1 knockdown (arrowheads) and wild-type (arrows) growth cones. Note that wild-type growth cones exhibit a higher fura-2 ratio (bottom) compared with TRPC1 knockdown growth cones. Scale bar, 10 µm. d, Quantification of [Ca2+]i from wild-type and TRPC1 knockdown growth cones over 25 min during removal of AGAs (n ≥ 21 growth cones for each condition, ***p < 0.001, two-way ANOVA). e, Fura-2 ratiometric measurement of growth cone [Ca2+]i during mechanical stimulation with a hypotonic solution. In control morpholino growth cones, hypotonic solution induces a rapid increase in [Ca2+]i, which is partially blocked with GsMTx4. In TRPC1 knockdown growth cones, elevation [Ca2+]i in response to hypotonic solution is reduced and GsMTx4 causes no additional change (n ≥ 26 growth cone for each condition, ***p < 0.001, two-way ANOVA). Note that there is a significant residual Ca2+ elevation in response to hypotonic solution in TRPC1-morphant neurons, suggesting additional MS channels are expressed on growth cones. f, t-BOC fluorescence intensity values measured at 30 min after t-BOC addition and AGA removal in TRPC1 knockdown and control morpholino-injected neurons (n ≥ 40 growth cone for each condition, **p < 0.01, Mann–Whitney test).

Article Snippet: GsMTx4 was chemically synthesized and folded from a linear peptide by SynPep.

Techniques: Activity Assay, Control, MANN-WHITNEY, Imaging, Knockdown, Fluorescence, Injection

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Mechanosensitive TRPC1 Channels Promote Calpain Proteolysis of Talin to Regulate Spinal Axon Outgrowth

doi: 10.1523/JNEUROSCI.2142-12.2013

Figure Lengend Snippet: Asymmetric MS channel activity induces growth cone turning on rigid substrata. a, Phase contrast images of a representative growth cone at the beginning (above) and end of a 45 min exposure to a gradient of GsMTx4 (GsM; 500 µm in pipette) in normal 2 mm Ca2+ saline solution. b–e, Identical assays were performed as in a, with 1× culture media in the pipette (b), in a 0 Ca2+ saline solution (c), with neurons plated onto a flexible FN-PDMS substratum (d), in the presence of 1 µm CPI (e), and in TRPC1 MO neurons (f). Axon trajectory traces of individual neurons shows the direction of outgrowth, with the leading edge of vertically oriented axons positioned at the plot origin at t = 0 (n ≥ 10 for all conditions). Note that in a gradient of GsMTx4, neurons orient toward the pipette (indicated by arrow) and have longer trajectories, indicating an accelerated rate of outgrowth. g, Cumulative distribution of growth cone turning angles for the conditions shown in a–f. h, The mean turning angle (±SEM) of each condition described in a–f, left. Mean rate of axon outgrowth (±SEM) for each condition described in a–f, right. *p < 0.05 and ***p < 0.001 as compared with control assays with a 1× MR gradient. All statistical analyses were completed with a Kruskal–Wallis test with a Dunn’s post test.

Article Snippet: GsMTx4 was chemically synthesized and folded from a linear peptide by SynPep.

Techniques: Activity Assay, Transferring, Saline, Control

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Mechanosensitive TRPC1 Channels Promote Calpain Proteolysis of Talin to Regulate Spinal Axon Outgrowth

doi: 10.1523/JNEUROSCI.2142-12.2013

Figure Lengend Snippet: Local modulation of MS channels drives asymmetric filopodial Ca2+ transients. a, A representative Fluo-4-loaded growth cone showing the division of proximal versus distal filopodial relative to a perpendicular gradient of GsMTx4 applied from the right. This image was generated by summing all frames of a 6 min time series captured at 1 s intervals. b, Summed frames binned over 1 min time intervals show the changes in Fluo-4 intensity (∑Δf/f0) during exposure to the GsMTx4 gradient (see Materials and Methods). Hot colors indicate that the frequency of filopodial Ca2+ transients was greater on the distal (upper) versus proximal (lower) side of this growth cone during the initial exposure to graded GsMTx4. c, The average (±SEM) frequency of Ca2+ transients in proximal versus distal filopodia of growth cones in gradients of GsMTx4 in wild-type (c) or TRPC1 knockdown neurons (d). In wild-type, but not in TRPC1 knockdown neurons, distal filopodia exhibit a significantly higher frequency of Ca2+ transients compared with proximal within 1 min of gradient production (***p < 0.001, two-way ANOVA). Scale bars: 4 µm.

Article Snippet: GsMTx4 was chemically synthesized and folded from a linear peptide by SynPep.

Techniques: Generated, Knockdown

Journal: ACS nano

Article Title: Magnetic Vortex Nanodiscs Enable Remote Magnetomechanical Neural Stimulation

doi: 10.1021/acsnano.0c00562

Figure Lengend Snippet: a, Fluorescence traces resulting from stimulation of DRG neurons incubated with 226 nm diameter MNDs (left) and 98 nm diameter MNDs with MF frequencies of 5 Hz (top) and 1 Hz (bottom). MF amplitude is sequentially increased from 7 mT to 28 mT (marked by the shaded regions). MF is applied in 4 pulses of 10 s with 30 s wait times between pulses. b, Summary of cell response rate for conditions permuted in (a). Error bars represent standard error of the mean. c, DRGs incubated with 1 μM piezo inhibitor GsMTx4 (left) and with TRPV4 antagonist HC-067047 (right) both show a decrease in activity after the first stimulation sequence. d, Confocal images of HEK-293 cells loaded with the calcium indicator Fluo-4 and transfected with the mechanosensitive TRPV4 channel labeled with mCherry. Scale bars = 100 μm. e, The response of unmodified HEK-293 cells decorated with MNDs to MF application (left), the response of HEK-293 cells expressing TRPV4 decorated with MNDs to MF application (middle), and the blocked response of HEK-293 cells decorated with MNDs, expressing TRPV4, and incubated 1 μM TRPV4 antagonist, HC-067047 (right). In experiments shown in (c) and (e), a 5 Hz, 26 mT MF is applied in 3 sequences of 10 s as shown in the shaded grey regions. The number of cells per condition is 300. Standard error is represented by shaded area in fluorescence traces.

Article Snippet: The PIEZO-specific inhibitor GsMTx4 (Smartox) was added to Tyrode’s solution at a concentration of 1 μM.

Techniques: Fluorescence, Incubation, Activity Assay, Sequencing, Transfection, Labeling, Expressing