Supplementary Figure 1-10 with Figure legends

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1 332 coordinates mechanotransduction and growth cone bifurcation in sensory neurons Li Yang Chiang 1, Kate Poole, Beatriz E. Oliveira, Neuza Duarte,Yinth Andrea Bernal Sierra, Leena Bruckner Tuderman, Manuel Koch, Jing Hu and Gary R. Lewin Supplementary Figure 1-10 with Figure legends

2 Supplementary Figure 1 Original scanned Western blot for Figure 2A

a b NF200 TRPV1-332 c 100 NF200-positive (137) (123) d 100 TRPV1-positive (137) (123) % Total 75 50 %Total 75 50 25 25 0-332 0-332 Supplementary Figure 2 Neurochemistry of sensory neurons on laminin

Mechanoreceptors make up most myelinated afferents and stained positive for heavy neurofilament NF200 (green). Nociceptors often express the capsaicin receptor TRPV1 (red).

3 a b NF200 TRPV1-332 c 100 NF200-positive (137) (123) d 100 TRPV1-positive (137) (123) % Total %Total Supplementary Figure 2 Neurochemistry of sensory neurons on laminin and laminin-332 substrates. Both (a) and laminin-332 (b) support robust neurite outgrowth for mechanoreceptors and nociceptors in culture (Scale bar is 20ìm). Mechanoreceptors make up most myelinated afferents and stained positive for heavy neurofilament NF200 (green). Nociceptors often express the capsaicin receptor TRPV1 (red). (c,d) Proportion of NF200-positive neurons and TRPV1-positive cells in each culture condition. Stacked histograms show that the number of each neuron subtype was not altered on laminin-332 substrates compared to the laminin control. The number of neurons measured is indicated on the top of each stacked histogram (not significantly different, Chisquared test). Scale bar is 20µm.

a 100 (56) (21) (18) (16) b % Total 75 50 SA IA -111 â1 VI V IV III II á1 VI V IVb IIIb IVa IIIa III IV V VI ã1-332 á3 â3 IIIa VI III-V III IV V II ã2 25 RA I I c 0 Soluble L-332 L + â 3 L + ã 2

2 ** ** L+â 3 : L L+ã 2 : L L+L-332 +CM6 : L Supplementary Figure 3 Mechanisms of laminin-332 action on mechanotransduction suppression and neurite growth.

(abbreviated L+ã2) (number of neurons indicated in parentheses) (not significantly different Chi-square test). (b) Cartoon representing the structure of laminin proteins.

4 a 100 (56) (21) (18) (16) b % Total SA IA -111 â1 VI V IV III II á1 VI V IVb IIIb IVa IIIa III IV V VI ã1-332 á3 â3 IIIa VI III-V III IV V II ã2 25 RA I I c 0 Soluble L-332 L + â 3 L + ã 2 G-domain 100 nm G-domain +â 3 +ã 2 L+L-332+CM6 Ratio of neurite lengths ** ** L+â 3 : L L+ã 2 : L L+L-332 +CM6 : L Supplementary Figure 3 Mechanisms of laminin-332 action on mechanotransduction suppression and neurite growth. (a) Stacked histograms showing the proportion of mechanically activated currents on a laminin-332 substrate compared to laminin mixed with laminin-332 sub-domains: â3 (abbreviated L+â3) or ã2 (abbreviated L+ã2) (number of neurons indicated in parentheses) (not significantly different Chi-square test). (b) Cartoon representing the structure of laminin proteins. The first three repeats of the G-domain contain binding sites for integrin receptors as compared to laminin-111 contains truncated globular domains on the á3, â3 and ã2 chains that comprise the short arms of the laminin molecule, bars indicate the approximate region of the molecule corresponding to the â3 and ã2 recombinant proteins (right panel, cartoon modified from Marinkovic 2007). (c) Mixture of laminin with laminin-332 sub-domain â3 or ã2 does not locally alter neurite morphology in comparison with the laminin substrate, showing that the suppressive activity does not reside in the free parts of the â3 or ã2 chain. In the presence of the CM6 antibody, neurite morphology is still altered on a laminin/laminin-332 mixture substrate, suggesting that CM6 does not rescue laminin-332 modulation of neurite outgrowth. Note that the o ratio of neuritic length on laminin/laminin-332 in presence of CM6 versus laminin at 90 is significantly reduced in comparison with laminin+â3 versus laminin or laminin+ã2 versus laminin (p<0.01 unpaired t-test, scale bar is 25 ìm).

5 100 (16) (15) stimulus % of total SA IA RA 0 laminin laminin: laminin :1 100 ms 25 pa Supplementary Figure 4: Mechanosensitive currents and the suppressive effect of laminin-332 are not changed with QX-314 in the recording pipette. To confirm that the effect of laminin-332 reflects inhibition of mechanically-gated currents, rather than of TTX-resistant voltage gated sodium channels, a subset of experiments were repeated in which voltage-gated channels were blocked by the addition of 10 mm QX-314 to the intracellular pipette solution. Mechanical responses of cells cultured on either laminin or laminin:laminin-332 (15:1) were indistinguishable from those where recorded from neurons superperfused with TTX.

A B C 10 µm Supplementary Figure 5: The CM6 antibody recognizes laminin-332

Microcontact printing was used to pattern glass substrates with laminin

The CM6 antibody (which blocks the integrin binding 3 erugif y tanmelp us

6 A B C 10 µm Supplementary Figure 5: The CM6 antibody recognizes laminin-332 containing matrices but not EHS derived laminin. Microcontact printing was used to pattern glass substrates with laminin (A), laminin-332 (B) or laminin:laminin-332 (15:1) (C). The CM6 antibody (which blocks the integrin binding 3 erugif y tanmelp us site on laminin-332) did not label EHS-laminin, but did label laminin-332 when present alone, or in a mixture with EHS-laminin.

7 (n=2) Thickess of stimulated neurite (µm) (n=10) (n=2) (n=2) (n=8) (n=9) RA IA SA Supplementary Figure 6 Thick neurites are not necessary for evoking mechanically activated currents. Each of the three types of mechanosensitive currents can be evoked when stimulating thinner as well as larger neuritic profiles (**p<0.01, ***p<0.001 unpaired t-test). The data for this Figure was taken from Figure 4F in the main MS.

8 a 1.2 * * ** b (140) (45) (n=30) Neurite outgrowth (µm/min) Neurite outgrowth (µm/min) laminin/ laminin laminin laminin laminin/ laminin laminin laminin-332 Substrate Substrate Supplementary Figure 7 Quantification of neurite outgrowth speed. (a) Analysis of the speed of neurite outgrowth on various substrates. The specific substrate is indicated under each data set, with green lines indicating stripes of laminin and purple lines indicating stripes of laminin mixed with laminin-332. In each case the bold lines indicate the substrate over which growth was analysed. All individual data points from panel (a) are displayed in panel (b) to indicate the extent of variability in growth cone speed.

9 epid. epid. derm. derm. * * Supplementary Figure 8 Immunocytochemical characterization of skin biopsies from a control and a patient with JEB (A:) Positive immunofluorescence labeling of laminin ß3 chain in normal human skin (antibody6f12), labeling decorates the dermo-epidermal border (arrows). (B) No laminin ß3 chain labeling was observed in the skin of a JEB patient using the same antibody. The arrows depict the position of the dermo-epidermal border. (C) Positive staining of laminin a3 chain in normal human skin (antibody Bm165), again labeling decorates the dermo-epidermal border (arrows). (D) No labeling for laminin a3 chain in the skin of the same JEB patient using the same antibody. The skin specimen contains a blister (star), the epidermis forms the blister roof. The arrows depict the position of the dermo-epidermal border on the blister floor. (E) Positive immunofluoresence labeling of the dermo-epidermal border (arrows) with an antibody against collagen VII in normal human skin (antibody LH-7.2). (F) Essentially the same pattern of collagen VII immunofluorescence was observed in the skin of the JEB patient using the same antibody. The skin specimen contains a blister (star), the epidermis forms the blister roof. The arrows depict the position of the dermo-epidermal border on the blister floor. epid.: epidermis; derm.: dermis; arrows: dermo-epidermal border ; star: blister due to dermo-epidermal separation; asterisk: non-specific antibody reactivity with the horny layer; scale bar: 100 µm

a Control b 30 JEB Branch point distance (µm) 25 20 15 10 5 25µm 0 Control matrix JEB matrix Supplementary Figure 9.

Acutely isolated DRG neurons from 4 week old mice were cultured for 24 hours on coverslips coated with a keratinocyte-derived matrix from control human and

10 a Control b 30 JEB Branch point distance (µm) µm 0 Control matrix JEB matrix Supplementary Figure 9. Neurite branching is not significantly different on keratinocyte-derived matrix from JEB patients, compared with control human samples. Acutely isolated DRG neurons from 4 week old mice were cultured for 24 hours on coverslips coated with a keratinocyte-derived matrix from control human and JEB patient keratinocytes. Example images of cells labeled with antibodies against PGP 9.5 (a). There was no significant difference in the distance between branch points on neurites for cells cultured on either substrate (b).

A EHS laminin EHS laminin: laminin 332, 30:1

number of neurites 15 10 5 EHS laminin EHS

laminin 332, 15:1 laminin 332 0 0 100 200

secondary neurites 52 48 53 47 84 89 102 91

A spectrum of neurite morphologies on the

substrate were analyzed using a Sholl analysis

(C), Neurite widths of randomly selected

11 A EHS laminin EHS laminin: laminin 332, 30:1 EHS laminin: laminin 332, 15:1 laminin 332 B number of neurites EHS laminin EHS laminin: laminin 332, 30:1 EHS laminin: laminin 332, 15:1 laminin distance from soma ( m) neurite width ( m) primary neurites * laminin 30:1 15:1 L-332 secondary neurites laminin 30:1 15:1 L-332 Supplementary figure 10: Substrates inhibitory for mechanotransduction do not effect neurite tree formation or structure. (A), Representative epifluorescence images of PGP-9.5 labeled DRG neuronal morphologies are shown. A spectrum of neurite morphologies on the tested substrates was observed (3 independent cultures). (B), Neurite trees, from 42 cells for each substrate were analyzed using a Sholl analysis (Sholl, 1953 J. Anat. 87, ). (C), Neurite widths of randomly selected primary and secondary neurites were calculated from these images. Only the neurite trees of cells grown on laminin-332 were significantly different to those grown on EHS-laminin (two-way ANOVA analysis). Scale bar is 50 µm. C

12 Supplementary Table 1 Substrates ( EHS laminin) Keratinocytes Keratinocytederived matrix Keratinocyteconditioned medium 2 n=43 n=20 n=14 RA cells n=32 n=4 n=1 -latency (ms) -mean current amplitude (pa) -activation τ 1 (ms) -inactivation τ 2 (ms) 32.2 ± ± ± ± ± ± ± ± 80,8 0.6±0.1 2,3±0, ± ± ± ± ±0.3 SA cells n=24 n=15 -latency (ms) -mean current amplitude (pa) -activation τ 1 (ms) 27.8 ± ± ± ± ± ± 1.2 *** ± ± 2.6 ** 29.9 ± ± 2.4 *** 77 ± ± 16 *** 23.1 ± ± ± ± 0.3 IA cells n=9 n=5 n=0 n=2 -latency (ms) -mean current amplitude (pa) -activation τ 1 (ms) -inactivation τ 2 (ms) 29.0 ± ± ± ± ± ± ± 1.5 ** ± ± ± ± ± 2.6 ** ± ± ± 5.3 No response n=19 n=12 n= ± ± ± 1.5 Physiological properties of recorded neurons possessing an RA, SA or IA current on different substrates. For each group the mean cell soma diameter, mechanical latency, mechanosensitive current amplitude and activation time constant (τ 1 the time constant calculated from a mono-exponential fit of the current activation) or inactivation time constant (τ 2 the inactivation time constant calculated from a mono-exponential fit of the current inactivation, when relevant) is shown. The significant increase in mechanical latency and τ 1 are noted for cells with an SA or IA current on keratinocytes and keratinocyte-derived matrix (unpaired t-test; **p<0.01; ***p<0.001).

13 Supplementary Table 2 Substrates SCC25- derived matrix L : L : 1 L : L : 1 L : L : 1 L : L : 1 L : denatured L : 1 L : L-332: CM6 15 : 1 : 2 Soluble laminin- 332 L : 3 10:1 L : 2 10:1 Pattern L vs L Vertical Pattern L vs L horizontal Pattern L vs L- 332 On Pattern L vs L- 332 On n=45 n=25 n=30 n=25 n=18 n=18 n=13 n= 27 n=21 n=18 n=16 n=32 n=32 n=21 n=21 RA cells n=8 n=8 n=3 n=8 n=8 n=9 n=14 n=14 n=10 n=2 -latency (ms) -mean current amplitude (pa) -activation τ1 (ms) -inactivation τ2 (ms) 27.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.4 SA cells n=16 n=13 n=5 n=4 n=11 n=11 n=10 n=8 n=9 -latency (ms) -mean current amplitude (pa) -activation τ1 (ms) 26.3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.2 IA cells n=5 n=3 n=0 n=2 n=0 n=3 n=2 n=3 n=3 n=2 n=1 n=3 n=4 n=1 n=1 -latency (ms) -mean current amplitude (pa) -activation τ1 (ms) -inactivation τ2 (ms) 27.7 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1 0.4± ± ± ± ± ± ± ± ± ± ± ± ± ± No response n=16 n=14 n=9 n=3 n=1 n=3 n=1 n=2 n=4 n=4 n=2 n=10 30± ± ± ± ± ± ± ± ± ± ± ± ± 1.8 Physiological properties of recorded neurons possessing an RA, SA or IA current on different substrates. For each group the mean cell soma diameter, mechanical latency, mechanosensitive current amplitude and activation time constant (τ 1 the time constant calculated from a mono-exponential fit of the current activation) or inactivation time constant (τ 2 the inactivation time constant calculated from a mono-exponential fit of the current inactivation) is shown.

14 Supplementary Table 3 Substrates Total Measured Area(µm 2 ) Density of attachments <75nm (unit/ µm 2 ) Density of attachments >75nm (unit/ µm 2 ) Average length of attachment <75nm(nm) Average length of attachment >75nm(nm) (4 preparations) 7.7 µm ± 7.5 (N=1143) 4.9 ± 1.4 (N=33) 22.8 ± 0.6 (N=1143) ± 2.8 (N=33) -111 (3 preparations) 8.3 µm ± 7.8 (N=1235) 4.7 ± 1.2 (N=36) 23.2 ± 0.8 (N=1235) ± 3.1 (N=36) -332 (3 preparations) 9.2 µm ± 5.1 (N=1372) 0.4 ± 0.2** (N=4) 21.3 ± 0.4 (N=1372) 99.4 ± 8.7 (N=4) L:L332 = 30:1 (2 preparations) 9.1 µm ± 5.1 (N=1360) 1 ± 0.5** (N=8) 20.7 ± 0.4 (N=1360) ± 7.9 (N=8) Quantification of electron microscopic data shows results from DRG neurons cultured on different substrates: on laminin (prep=4), on laminin-111 (prep=3) laminin-332 (prep=3), and on a laminin:laminin :1 mixture (n=2). For each experiment, the total measured area was summed up. Density and average length of short (<75 nm) and long links (>75 nm) were calculated respectively. No significant differences in density of short attachments were noted on a laminin-111 and on a laminin-332 substrate as compared with neurons on a laminin substrate. The number of long links on a laminin-332 substrate is significantly reduced. N= number of identified electron dense attachments. P<0.01; unpaired t-test.

Mechanosensitive currents in the neurites of cultured mouse sensory neurones

J Physiol 577.3 (2006) pp 815 828 815 Mechanosensitive currents in the neurites of cultured mouse sensory neurones Jing Hu and Gary R. Lewin Department of Neuroscience, Max-Delbrück Center for Molecular