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Mechanical control of the sense of touch by β-spectrin

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Abstract

The ability to sense and respond to mechanical stimuli emanates from sensory neurons and is shared by most, if not all, animals. Exactly how such neurons receive and distribute mechanical signals during touch sensation remains mysterious. Here, we show that sensation of mechanical forces depends on a continuous, pre-stressed spectrin cytoskeleton inside neurons. Mutations in the tetramerization domain of Caenorhabditis elegans β-spectrin (UNC-70), an actin-membrane crosslinker, cause defects in sensory neuron morphology under compressive stress in moving animals. Through atomic force spectroscopy experiments on isolated neurons, in vivo laser axotomy and fluorescence resonance energy transfer imaging to measure force across single cells and molecules, we show that spectrin is held under constitutive tension in living animals, which contributes to elevated pre-stress in touch receptor neurons. Genetic manipulations that decrease such spectrin-dependent tension also selectively impair touch sensation, suggesting that such pre-tension is essential for efficient responses to external mechanical stimuli.

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Figure 1: The shape of the AVM sensory neuron during body movement.
Figure 2: Loss of unc-70 β-spectrin function causes buckling in TRNs during ventral bending.
Figure 3: Loss of β-spectrin function decreases TRN tension.
Figure 4: unc-70(e524) TRNs fail to retract after laser axotomy.
Figure 5: UNC-70 is under constitutive tension in neurons of living animals.
Figure 6: Loss of unc-70 function impairs touch sensitivity, but not synthetic light sensitivity.

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Acknowledgements

We thank J. Shaw, A. Slade and S. Minne of Bruker Instruments for generous loan of AFM; Z. Liao for worm injections; D. Ramallo, C. Buckley, A. Olson, J. Mulholland for assistance with microscopy; K. Shen for loan of equipment for worm injection and strain integration; C.P. Heisenberg, K.C. Huang and W.J. Nelson for comments. Confocal microscopy and laser axotomy conducted in the Neuroscience Microscopy Service (NMS, partially supported by NS06973) and the Cell Science Imaging Facility (CSIF) at Stanford. Some strains provided by M. Chalfie, E. Jorgensen, W. Schaefer and the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (partially supported by S10RR05574). Work supported by a Stanford Bio-X IIP award (A.R.D., M.B.G.), the National Institutes of Health (DP2OD007078 to A.R.D., RO1NS047715 and RO1EB006745 to M.B.G.), the National Science Foundation (No. 1136790, A.R.D.), Stanford Cardiovascular Institute Seed Grant (A.R.D.), Burroughs-Wellcome Career Award at the Scientific Interface (A.R.D.) and a Long-term fellowship from HFSP (M.K.).

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M.K., A.R.D. and M.B.G. conceived the research and designed experiments; M.K. carried out and analysed experiments; M.K., A.R.D. and M.B.G. wrote the paper.

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Correspondence to Miriam B. Goodman.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 unc-70(e524) and unc-70(n493) encode missense mutations of conserved residues in UNC-70 β-spectrin.

a, Chromatogram of the unc-70 locus in wild type (top) and unc-70 mutant (bottom) worms near the codon encoding E2008. b, Chromatogram of the unc-70 locus in wild type (top) and unc-70 mutant (bottom) worms the codon encoding L2044P. c, Schematic domain architecture of C. elegans UNC-70 spectrin, consisting of an N-terminal actin binding domain (ABD), 17 spectrin repeats and a C-terminal pleckstrin homology domain. Yellow star indicates the position of the premature stop codon encoded by unc-70(s1502); red stars show the position of the missense mutations encoded by unc-70(n493) and unc-70(e524). Protein sequence alignment of human erythrocyte β-spectrin and non-erythrocyte β-spectrin from seven species: Homo sapiens, Mus musculus, Gallus gallus, Danio rerio, C. elegans, C. briggsae, D. melanogaster. Color scale represents the degree of conservation; red = 100% and blue = 20%. d, Close-up view of the sequence alignment of spectrin repeat 17. Highlighted in red are residues that are 100% conserved in all species; green boxes are the two missense mutations used in this study. e, 3-D model of the α–β tetramerization domain. Orange indicates β-spectrin repeats R16 and R17; green indicates β-spectrin repeats R0 and R1. The two mutated residues, E2008K in unc-70(e524) and L2044P in unc-70(n493) are shown in space-filling mode. Figure assembled in PyMol from PDB 3LBX (Ref.1).

Supplementary Figure 2 Disrupting the TRN-epidermis attachment is not sufficient to cause buckling and attachments are intact in unc-70 mutants.

a, AVM shape during dorsal (top) and ventral (bottom) bending in him-4(e1267) mutants lacking full hypodermal attachment. b, Neuron curvature vs. body curvature in him-4(e1267) mutants. N = number of animals and n = number of still images; collected during n = 4 imaging sessions. c, UNC-70 is not required for TRN attachment. (c) Schematic showing the position of the ALML and AVM. The slab visualizes the arrangement of muscles (blue) and TRNs (green) in respect to the body axis. Individual attachment sites linking body wall muscles and TRNs to the hypodermis are shown in magenta. d, f, h, Representative micrographs anti-myotactin showing attachment sites in (d) control, (f) unc-70(e524) and (h) unc-70(n493) mutant animals. e, g, i, Distribution of interpunctum intervals for attachment sites stained in (e) control, (g) unc-70(e524) and (i) unc-70(n493). Distributions were fitted to a Gamma-distribution with the median location and the residuals from each fit are shown above each graph. N = number of interpunctum intervals analysed for each genotype; more than 25 images analysed for each genotype during n = 3 imaging sessions.

Supplementary Figure 3 Expressing wild-type UNC-70 in the epidermis reduces buckling observed in unc-70 mutants.

a, Representative images of AVM shape during ventral (top) and dorsal (bottom) bending in unc70(s1502);oxIs95, unc-70(e524);oxIs95, unc-70(n493);oxIs95 mutant animals. The oxIs95 transgene drives expression of full-length, wild-type UNC70 in the epidermis. The bright fluorescence in the pharynx is the co-transformation marker used to construct transgenic animals expressing wild-type unc-70 in the epidermis b, AVM shape during ventral (top) and dorsal (bottom) bending in transgenic worms expressing a dominant negative-spectrin fragment in TRNs only. Scale bar in b applies to a and is 50 μm. c, Average neuron curvature vs. average body curvature of the same genotypes as in a. Each point is an average of 10–30 curvature measurements along the body in each frame (Methods). The shaded area (beige) is a moving average of the standard deviation for 15–20 points. r is the Pearsons correlation coefficient of neuron and body curvature. Statistical difference of the correlation coefficient r was tested using a Fisher z-transform of r; p values relative to controls are indicated in the upper right. n still images collected from N animals during three imaging sessions. d, Average neuron curvature vs. average body curvature of the same genotypes as in b. Each point is an average of 10–30 curvature measurements along the body in each frame (Methods). The shaded area (beige) is a moving average of the standard deviation for 15–20 points. r is the Pearsons correlation coefficient of neuron and body curvature. Statistical difference of the correlation coefficient r was tested using a Fisher z-transform of r. Significant differences to control worms are indicated by a p value indicated in the upper right. n still images collected from N animals during three imaging sessions. e, f, Maximum off-axis deformation (buckling), umax, of AVM as a function of body strain derived from the data in c and d. Each point is the maximum deformation detected at a given body posture. Solid black line is a running average of 15 images; beige curve is a fit of the buckling deformation as function of (see Supplementary Note 1). Exact p values (upper left) are relative to wild type, with a significance level of α = 0.01. Tensile strain is positive and increases to the left, compressive strain is negative and increases to the right of the zero strain.

Supplementary Figure 4 Laser axotomy of TRNs in him-4(+) worms.

a, Schematic diagram of the dual-femtosecond laser axotomy set-up. b, Strain rate of control and unc70 mutant TRNs after laser cutting. Thick horizontal bars are the median and the number of axotomies is indicated in each column. Axotomies were performed in n = 16 control,n = 5 unc-70(e524), and n = 7 unc-70(n493) animals during four imaging sessions/genotype. Negative values occur either due to relaxation of an initially compressed neurite, relaxation after initial expansion during ablation or diffusion of fluorophore into the initially bleached ends proximal to the cut site. Median values were significantly different (U-test); p values (inset) show no significant difference as a function of genotype. c, Retracted distance, D0/2, in control and unc70 mutant worms after laser cutting. Bars are the median retracted distance for and p-values derived from a U-test are shown in the inset.

Supplementary Figure 5 UNC-70(TSMod) and UNC-70(N-TSMod) are less mobile that cytoTSMod.

a, Representative fluorescence recovery after photobleaching (FRAP) trace for cytoplasmic TSMod. Red curve is a single-exponential fit to the data points. Similar results obtained in n=17 neurites analysed during two imaging sessions. Note, recovery was complete after bleaching. b, Representative FRAP traces of UNC-70(TSMod) and UNC-70(N-TSMod). Red curves are exponential fits to the data. Similar results were obtained in n=13 neurites for both UNC-70(TSMod) and UNC-70(N-TSMod) analysed during two imaging sessions. c, Diffusion coefficients estimated from rate of fluorescence recovery for UNC70(TSMod), N-TSMod, and cytoTSMod. Box plots show the median, quartiles and 0.1 and 0.9 percentiles. d, Mobile fraction of UNC-70(TSMod), N-TSMod and cytoTSMod expressing neurites. Box plots show the median, quartiles and 0.1 and 0.9 percentiles.

Supplementary Figure 6 Intermolecular FRET assessment and fluorescence lifetime measurements.

a, b, Number of photons emitted by UNC-70(5aa)/High FRET worms in the (a) acceptor channel after direct illumination and (b) the corresponding FRET efficiency image based in the number of photons collected for each bleed-through corrected pixel. c Distribution of photons undergoing FRET (FRET efficiency) after donor excitation vs. number of acceptor photon after direct excitation of the acceptor. Each dot indicates a measurement derived from a pixel in (b). Black line is a linear fit to the 200 points with the highest acceptor photon counts. r is the correlation coefficient of the fit. df Same representation as above for UNC-70(TSMod) worms. g, Representative fluorescence lifetime image (FLIM) of a UNC-70(TSMod) expressing worm. Lookup table corresponds to the fluorescence lifetime at each pixel derived from the photon arrival histogram after fitting with a double exponential decay. Similar images were obtained in more than 6 animals. h, Fluorescence lifetime histogram of a representative pixel showing the number of photons collected as a function of arrival time. Black solid line indicates a double exponential fit to the data convolved with a synthetic instrument response function. i, Fluorescence lifetime for nine ROIs in n=6 worms analysed during two imaging sessions. j, Correlation between FLIM-FRET efficiency and sensitized emission FRET efficiency derived from intensity based measurements (meanSD). Black solid line indicates perfect correlation of the two quantities. Gray dots are values taken from published studies of constructs using the same genetically-encoded flurophores. High and low FRET values were taken from Day, et al.(Ref.3).

Supplementary Table 1 C. elegans strains and transgenes.
Supplementary Table 2 Primers used to build novel transgenes.
Supplementary Table 3 Number of tethers analyzed by AFM force spectroscopy as a function of genotype, velocity, and treatment condition

Supplementary information

Supplementary Information

Supplementary Information (PDF 1837 kb)

Control worm expressing GFP in touch receptor neurons.

Movie shows AVM morphology during ventral (compressive) and dorsal (tensile) bending. Scale bar: 50 μm. Frame rate: 0.5/s. (AVI 1900 kb)

unc-70(s1502) mutant worm expressing GFP in touch receptor neurons.

Movie shows AVM morphology during ventral (compressive) and dorsal (tensile) bending. Scale bar: 50 μm. Frame rate: 0.5/s. (AVI 2684 kb)

unc-70(e524) mutant worm expressing GFP in touch receptor neurons.

Movie shows AVM morphology during ventral (compressive) and dorsal (tensile) bending. Scale bar: 50 μm. Frame rate: 0.5/s. (AVI 1897 kb)

unc-70(n493) mutant worm expressing GFP in touch receptor neurons.

Movie shows AVM morphology during ventral (compressive) and dorsal (tensile) bending. Scale bar: 50 μm. Frame rate: 0.5/s. (AVI 1896 kb)

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Krieg, M., Dunn, A. & Goodman, M. Mechanical control of the sense of touch by β-spectrin. Nat Cell Biol 16, 224–233 (2014). https://doi.org/10.1038/ncb2915

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