Mechanical Stretch Inhibition Sensitizes Proprioceptors to Compressive Stresses

A repetitive gait cycle is an archetypical component within the behavioural repertoire of many if not all animals including humans. It originates from mechanical feedback within proprioceptors to adjust the motorprogram during locomotion and thus leads to a periodic orbit in a low dimensional space. Here, we investigate the mechanics, molecules and neurons responsible for proprioception in Caenorhabditis (C.) elegans to gain insight into how mechanosensation shapes the orbital trajectory to a well-defined limit cycle. We used genome editing, force spectroscopy and multiscale modeling and found that alternating tension and compression with the spectrin network of a single proprioceptor encodes body posture and informs TRP-4/NOMPC and TWK-16/TREK2 homologs of mechanosensitive ion channels during locomotion. In contrast to a widely accepted model of proprioceptive ‘stretch’ reception, we found that proprioceptors activated under compressive stresses in vivo and in vitro, and speculate that this property is conserved across function and species.


Introduction
. β-spectrin constrains the limit cycles during forward locomotion A,B Representative still image of (A) wildtype animals and (B) unc-70(e524) animals and the 3D density estimate for the joint probability distribution (equivalent to a discrete 3D histogram) of the two forward and turning modes in the eigenworm space. Color scale = (brown, low density; blue, high density) C Violin plots of the p-value distributions for 1000 independent tests of a bootstrapped population estimate of wt 3D probability density function (ctrl) tested against itself or a bootstrapped population estimate of unc-70(e524). Orange line indicates α=0.05 level of significance for the hypothesis H 0 that bootstrapped density functions derived from a and b are equal (see Methods). D Color-coded, statistically significant differences in the local probability density functions shown in (A) and (B).  Table S2). Importantly, neither the CRE TRP-4 is not essential to elicit curvature-dependent calcium activity We next analyzed whether 143 compression induced Ca 2+ signals depend on the TRP-4/NOMPC ion channel. Indeed, spontaneous 144 activity in immobile trp-4 mutant animals was strongly reduced, with occasional decreases in Ca 2+ 145 signals (Fig. S4B). However, when we performed Ca 2+ imaging in flexing animals, we still observed 146 DVA Ca 2+ activity during ventral body bends in trp-4(sy695) mutant animals, even though, these DVA 147 signals were less modulated by swings in body curvature (Fig. 4F). This analysis shows that TRP-4 148 expression is dispensable for compression induced Ca 2+ signal during body bending. We were thus 149 wondering if TRP-4 is sufficient to sensitize otherwise motion-insensitive neurons lacking endogenous 150 TRP-4 expression [34], to activate during dorso-ventral body bends. We hence expressed full-length 151 trp-4 cDNA and an N-terminally truncated contruct in TRNs, the gentle body touch mechanoreceptors. 152 In wildtype animals and transgenics expressing the truncated isoform, basal Ca 2+ levels measured in 153 PLM rarely changed and are seemingly uncorrelated with body curvature in slowly moving animals 154 (Fig. S4C,G). However, upon expression of full-length TRP-4 in TRNs, we observed a strong periodic 155 signal in PLM during body bending that correlated well with body bends (Fig. 4H). Interestingly, we 156 also frequently observed increases in PLM activity when the animal bent towards both, the dorsal

11/54
GCaMP6s robustly activated in response to substrate deformation (Fig. 5A). Clearly, cultured DVA 162 neurons repetitively elicited high Ca 2+ transients (Fig. 5B,C Video S4), that depended on functional 163 TRP-4 expression and could be partially blocked by non-specific cation channel inhibitor GdCl 3 (Fig.   164   5D). Most notable, we frequently found that DVA neuron became active during the force offset ( Fig.   165 5B,C) and buckled during indentation, suggesting that they sensed negative tension gradients or 166 compression. Because PDMS substrate deformation induces compressive and tensile stresses along 167 the axon, we specifically applied positive and negative tension gradients to isolated axons (Fig. 5E,F) 168 and visualized their resultant Ca 2+ transients using confocal microscopy (Fig. S7A). To do so, we   Figure 5 (preceding page). Opposing membrane tension gradients modulate DVA activity in vitro through TRP-4 and TWK-16 A Schematic of the PDMS stretching experiments. B Three successive images of the Ca 2+ -sensitive (left) and Ca 2+ -insensitive dye (right) of an isolated DVA subjected to substrate deformations. C PDMS substrate deformation D (black line) and GCaMP/mKate ratio (R/R 0 , green line) plotted against time. D Violin plot of the Ca 2+ activity derived from wt, wt treated with Gd 3+ and trp-4(sy695) mutant DVA neurons. E Schematics of the dynamic tether extrusion experiments. F Representative still images of the GCaMP signal from an isolated DVA neuron before and during tether extrusion. Dotted line indicates the location of the kymograph displayed below showing the temporal evolution of the Ca 2+ signal at the tether neck. G Representative displacement (d), force and normalized Ca 2+ signal extracted from the experiment in (F). H Bubble plot of the probability showing that a tether extrusion or relaxation leads to an increase or decrease in GCaMP activity in wt DVA neurons. ∆T indicates increases or decreases in measured tension gradient. N=48 cells. I Representative force trace and normalized Ca 2+ transients for trp-4 (orange) mutant cells. J Bubble plot of the probability showing that a tether extrusion or relaxation leads to an increase or decrease in GCaMP activity in DVA neurons mutant for trp-4, N=38, mutant cells. K In silico study of the optical trapping experiment. i) Simulated force (black trace) and temporal tension gradients (light blue). ii,iii) Open probability of the hypothetical Na + and K + channel. iv) Sum of a current through a linear combination of its average open probability x single channel conductance. Note the double peak in the purple trace becomes suppressed due to simultaneous activation of the inhibitory signal, giving rise to the experimentally observed behavior.
answer this, we first repeated the Ca 2+ imaging of DVA in moving twk-16 mutant animals. Compared 212 to wt animals, we found that the deletion of twk-16 caused a subtle increase in Ca 2+ activity in 213 DVA during spontaneous body bending ( Fig. 6C) with an overall unchanged ventral preference.  Together, this suggests that TWK-16 is able to suppress Ca 2+ transient at dorsal posture, when DVA 218 experiences mechanical tension, similar to how tension suppresses calcium activity in our in-vitro 219 experiment (Fig. 6A). 220 We then asked if TWK-16 expression in DVA is required for animal locomotion. In contrast to trp-4 221 animals, twk-16 null mutants, we observed a subtle, but significant difference between the wt and 222 mutant in 3D densities in the eigenworm space, as an indicator for a smaller body posture (Fig. 6D, 223 S8A-C). TWK-16 is also expressed in other neurons, including AVK that counteracts DVA activity [21].  Bubble plot of the probability showing that a tether extrusion or relaxation leads to an increase or decrease in GCaMP activity in DVA neurons mutant for twk-16, N=31, mutant cells. C DVA Ca 2+ data of twk-16(mir31) animal lacking functional TWK-16. (i) Video stills of DVA cell body during ventral, neutral and dorsal bends. (ii) Time series of the normalized GCaMP/mKate ratio (left axis, green) and body curvature (right axis, black). (iii) Average GCaMP/mKate ratio ± SE plotted against the phase angle of body curvature. D Representative image of a twk-16(mir31) animal and the color-coded, statistically significant differences in the local probability density functions between N2 and twk-16. Scale bar = 300µm. E,F 3D kernel density function (histogram) in the three dimensional eigenworm space for TWK-16::AID::wScarlet animals expressing TIR in DVA in (E) absence and (F) presence of auxin. G Color-coded, statistically significant differences in the local probability density functions between (E) and (F). Blue voxels indicate higher local density for untreated, beige voxels indicate higher density for auxin-treated animals on the α=0.01 level.

313
In summary, our data revealed that compressive and tensile stresses in the spectrin network 314 modulate two opposing, excitatory and inhibitory ion channels of DVA proprioceptors, TRP-4 and 315 TWK-16 respectively, that are critical to confine the full, deep modulation of Ca 2+ activity in moving 316 animals. We suggest that this may be a general mechanism by which long mechanosensory dendrites 317 achieve local computation through mechanical compartmentalization. Future experiments will need 318 to directly address how this mechanism transcends the animals kingdom and human gait adaptation.

319
Acknowledgments We would like to thank the NMSB and SLN labs for discussions and suggestion    significantly expression in the hypodermis and muscles (not shown). In short, animals were either immobilized on agar pads [71] or in microfluidic chips with varying curvature as described above.

472
Imaging was carried out on a Leica DMI6000 SP5 confocal microscope through a 63x/1.4 NA oil 473 immersion lens. Three images were acquired, the direct donor (mTFP2) excitation and emission, 474 donor excitation and acceptor emission and the direct acceptor (mVenus) excitation and acceptor 475 emission. mTFP2 was excited using the 458nm, while mVenus was excited with the 514nm line of an 476 Argon ion laser at 80% and 11% transmission respectively (with 25% of its full available power). The and the FRET efficiency calculated using  1 , a 2 , a 3 ).  software (Impetux Optics) as follows (Fig. 5G): first, bead was contacted the axon and a pulled a 747 distance of 10 µm, reaching a peak in the tether tension value; second, the bead stopped for 10 s, 748 letting the tether tension to relax down to a static value arising from the stored stress. Third, the bead 749 was brought back to the initial position, releasing F tether to almost zero; finally, the bead stopped into GCaMP-increasing (∆I > 5%) and GCaMP-decreasing (∆I < 5%). To rule out that the Ca 2+ Evan's modification for a finite loading rates was implemented to capture the strain-rate dependence 817 of the Calcium channel (TRP-4), known to respond to the change in force.
with γ k B T as the force scale. 819 We implemented the simulation in R, with a timestep of 1e-5 s and the following kinetic constant.

820
Inhibitory Channel: γ=2.93, The physical representation of the values k 0 o , k 0 C correspond to the spontaneous opening constants.
where S scales the effective stretch receptor activation function. This may be understood as decreas- test for the null hypothesis that the two kernel density functions are similar, we resampled the highly 881 oversampled population by bootstrapping to avoid spurious significance due to long tailed outliers.

882
The resampling and testing was performed 1000 times to yield a distribution of p-values which is assigned and plotted as two different colors in a 3D voxelgram (e.g. Fig. 1D  Strains and their properties used as recombination reporter to study the efficiency and specificity of the cell-type specific CRE recombination.