The Journal of Neuroscience, May 24, 2006, ():
Ca2+ Influx through Mechanosensitive Channels Inhibits Neurite Outgrowth in Opposition to Other Influx Pathways and Release from Intracellular Stores
J. Neurosci. Jacques-Fricke et al.
26: 5656
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1. Spontaneous Ca2+ transients increase in Xenopus spinal neurons cultured on FN upon removal of aminoglycoside antibiotics. a. Three representative examples of Fluo-4 fluorescent Ca2+ signals measured within growth cones during washout of antibiotics. Images were collected at 5 sec intervals 5 min before and after washout of all antibiotics (100 units/ml penicillin, 0.1 mg/ml streptomycin, 100 µM gentamicin) at time points indicated by the arrowheads. b. The average frequency of Ca2+ transients in growth cones with and without antibiotics. Fluorescence changes of 20% above baseline were scored as a Ca2+ transient. Only growth cones that exhibited at least one Ca2+ transient during the 10 min imaging period were included in this figure. n = 121 growth cones. **p<0.001.
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Supplementary Figure 2. GsMTx4 does not cause a significant change in global intracellular Ca2+ concentration. Neurons loaded with the cytosolic Ca2+ indicator Fura-2 (a) or the near-membrane Ca2+ indicator FFP-18 (b) were imaged for 10 min before and over 25 min after the elimination of extracellular Ca2+ or the addition of 5 µM GsMTx4 in 2 mM extracellular Ca2+. Data were normalized to the fluorescence ratio immediately before elimination of extracellular Ca2+ or the addition of 5 µM GsMTx4. a. Using the cytosolic Ca2+ indicator Fura-2, the Ca2+i falls significantly below the average baseline Ca2+i in 2 mM extracellular Ca2+ after 8 min in 0 Ca2+ MR (asterisk). On the other hand, blocking SACs with GsMTx4 does not cause significant change in baseline Ca2+i even after a 25 min exposure. b. Using the near-membrane Ca2+ indicator FFP-18, an immediate drop in the fluorescence ratio is record upon shifting to 0 Ca2+ MR, suggesting distinct Ca2+ domains are detected with this indicator. In this domain, detectable Ca2+i falls significantly below the average Ca2+i in 2 mM extracellular Ca2+ after 5.5 min of Ca2+ removal (asterisk). However, GsMTx4 still does not produce a significant reduction in baseline Ca2+i after a 25 min exposure. n≥14 for all treatments, *p<0.05.
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Supplementary Figure 3. Model for Ca2+ channel-specific effects on neurite extension. Schematic drawing illustrating the functional effects of different channel activities on neurite outgrowth. SACs can be gated directly by mechanical forces from the lipid bilayer (yellow arrows), or indirectly through mechanically activated phospholipase A2 (PLA¬2¬). For example, signaling intermediates downstream of PLA¬2¬ and arachodonic acid (AA) have been shown to directly activate mechanosensitive TrpV4 channels (Vriens et al., 2004). Since non-specific blockers of Ca2+ influx across the plasma membrane (PM) like Gd3+, gentamicin (Gent) and ruthenium red (RR) stimulate neurite outgrowth similar to 0 Ca2+, these blockers appear to effectively reduce Ca2+ influx through multiple channel types linked to distinct effectors of motility. However, the specific channel blocker GsMTx4 more exclusively targets SACs linked to effector pathways that negatively regulate outgrowth, producing a larger growth-promoting effect. Other blockers, such as SKF-96365 and Cd2+, produce opposite effects on outgrowth by reducing influx through TRPC and other channels that positively regulate outgrowth. In addition, Ca2+ release from the endoplasmic reticulum (ER) through ryanodine and IP3 receptors is also linked to positive effector pathways. The slow Ca2+ chelator EGTA can quench global, but not fast local Ca2+ changes, whereas BAPTA binds Ca2+ fast enough to quench all Ca2+ changes, making these chelators useful tools for dissecting the function of Ca2+ microdomains in neurite outgrowth.
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Supplementary Video 1. Reducing Ca2+ influx through SACs with GsMTx4 increases neurite growth rates. Time-lapse phase-contrast image sequence of elongating axons from a spinal cord explant (lower right) before and after addition of 5 µM GsMTx4. This image sequence includes the neurite depicted in Fig. 1b (arrow), although all neurites accelerate to a similar extent after addition of GsMTx4 in this movie. Images were captured at 1 min intervals for 30 min before and after application of GsMTx4. The time of addition of GsMTx4 is indicated in the time-lapse movie. Playback is at 10 frames/sec. Scale bar, 20 µm.
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Supplementary Video 2. Reducing Ca2+ influx through SACs with GsMTx4 increases neurite growth rates in vivo. Time-lapse fluorescence image sequence of a Xenopus spinal neuron expressing GFP in the developing spinal cord. Confocal images were captured at 15 sec intervals for 15 min before and after application of GsMTx4. The time of addition of GsMTx4 is labeled in the time-lapse movie. The frame position was manually adjusted in this time-lapse sequence to account for image drift. Dorsal is up and anterior to the right. Playback is at 15 frames/sec. Scale bar, 20 µm.
