supplementary figure 1 - nature research · merlin–iso2 (sirna nf2 iso2) (0.58 ± 0.09) p <...

Supplementary Figure 1

a Immunohistochemistry on mouse cerebellum sections

merlin-iso2

merlin-iso2

Calbindin

MBP

merged

merged

merlin-iso2

Calbindin

merlin-iso2

MBP

Nature Neuroscience: doi:10.1038/nn.3348

Suppl. Figure 1: Merlin–iso2 localizes to Purkinje cell axons (a) Merlin–iso2 immunolabeling in the adult cerebellum (postnatal day 60). Upper panel: Merlin–iso2 (green) co–localizes with the Purkinje cell (PC) marker Calbindin (red) in PC somata (arrowhead) and various axons (arrow). Lower panel: merlin–iso2 shows co–staining the myelin basic protein (MBP; red) indicating merlin–iso2´s appearance in myelinated axons (arrow). White boxes are magnifications. Scale bar 75 µm.



pNF-H

a Immunohistochemistry on mouse sciatic nerve sections

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merged

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merlin-iso2 pNF-H merged

wild type

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Suppl. Figure 2: Merlin–iso2 localizes to sciatic nerve axons (a) Longitudinal sciatic nerve sections of wildtype mice were stained either against merlin isoform1 (merlin–iso1) or merlin isoform2 (merlin–iso2) as well as phosphorylated neurofilaments (pNF–H; red) in order to track axonal processes. Only merlin–iso2 co–localizes with the axonal marker (arrow). Sections taken from merlin–iso2 knockout mice (nf2 iso2–/–) were stained in order to prove specificity of merlin–iso2 antibody (lower panel). White boxes are magnifications. Scale bar 100 µm.



pNF-H GAP-43 merged

a primary cerebellar neurons b

c d

e

f

g h


Suppl. Figure 3: Merlin mediates neurofilament phosphorylation through RhoA signaling. (a) Representative picture of primary granule cell axons. Axons of cultured neurons were identified by double staining for the following markers: phosphorylated neurofilaments (pNF–H; red) and Growth–associated protein 43 (GAP–43; red). Subsequently, axon calibers could be quantified using ImageJ software (see methods). (b) Full–length Immunoblot of Fig. 1f: Immunoblot of primary cerebellar neurons after specific knockdown of both merlin isoforms (siRNA nf2), merlin–iso1 (siRNA nf2 iso1), merlin–iso2 (siRNA nf2 iso2) or scrambled control (scr nf2). Immunoblot quantitations: Merlin–iso2: scr nf2 (1); siRNA nf2 (0.25 ± 0.05); siRNA nf2 iso1 (1.03 ± 0.07); siRNA nf2 iso2 (0.23 ± 0.05). Merlin–iso1: scr nf2 (1); siRNA nf2 (0.19 ± 0.03); siRNA nf2 iso1 (0.10 ± 0.02); siRNA nf2 iso2 (0.73 ± 0.07); n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (c) Full–length Immunoblot of Fig. 1g: Immunoblot of neuronally differentiated P19 cells following overexpression of an empty vector control (vc), merlin–iso1 (iso1) or merlin–iso2 (iso2). Immunoblot quantitations: Merlin: vc (1); iso1 (58.09 ± 9.57); iso2 (84.63 ± 14.32); n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (d) Full–length Immunoblot of Fig. 2a: Merlin specific knockdown in cerebellar neurons, either both isoforms (siRNA nf2), merlin–iso1 (siRNA nf2 iso1), merlin–iso2 (siRNA nf2 iso2) compared to scrambled control (scr nf2). Immunoblot for phosphorylated neurofilaments (pNF–H), merlin and actin. Immunoblot quantitations: pNF–H: Scrambled duplex control (scr nf2) (1); both isoforms (siRNA nf2) (0.73 ± 0.06) p < 0.05; merlin–iso1 (siRNA nf2 iso1) (0.92 ± 0.07) not significant; merlin–iso2 (siRNA nf2 iso2) (0.45 ± 0.05) p < 0.05. merlin: scr nf2 (1); siRNA nf2 (0.42 ± 0.04); siRNA nf2 iso1 (0.34 ± 0.07); siRNA nf2 iso2 (0.42 ± 0.07); n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (e) Full–length Immunoblot of Fig. 2b: Merlin overexpression in neuronally differentiated P19 cells with empty vector control (vc) compared to merlin–iso1 (iso1) and merlin–iso2 (iso2). Immunoblot quantitations: pNF–H: empty vector control (vc) (1); merlin–iso1 (iso1) (65.88 ± 8.07); merlin–iso2 (iso2) (175.75 ± 13.96). merlin: vc (1); iso1 (3.70 ± 0.73); iso2 (3.09 ± 0.58); n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (f) Full–length Immunoblot of Fig. 2c: Immunoblot for merlin and actin confirms effective knockdown and transfection levels (n = 3 blots). (g) Full–length Immunoblot of Fig. 2d: Rho–associated kinase (ROCK) knockdown prevents merlin–iso2–induced neurofilament phosphorylation. P19 cells treated with scrambled control duplexes (scr) and empty vector control


(vc), scr and merlin–iso2 overexpression (iso2), siRNA ROCK and merlin–iso2 overexpression (ROCK1/iso2). Immunoblot for phosphorylated neurofilaments (pNF–H), merlin and actin. Immunoblot quantitations: pNF–H: scrambled control duplexes (scr) and empty vector control (vc) (1); scr and merlin–iso2 overexpression (iso2) (3.60 ± 0.47) p < 0.01; siRNA ROCK and merlin–iso2 overexpression (ROCK1/iso2) (0.96 ± 0.16) not significant; Merlin: scr/vc (1); scr/iso2 (19.57 ± 1.36); ROCK1/iso2 (16.41 ± 2.36); n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (h) Full–length Immunoblot of Fig. 2e: P19 cells measured for RhoA–GTP level after merlin–iso2 (iso2) and merlin–iso1 (iso1) overexpression compared to empty vector control (vc). Immunoblot for pNF–H, actin, RhoA from cells lysates and Rho–GTP indicates activation. Immunoblot quantitations: Merlin: vector control (vc) (1); merlin–iso1 (iso1) (15.42 ± 2.37); merlin–iso2 (iso2) (26.56 ± 6.76). RhoA: vc (1); iso1 (0.70 ± 0.13) not significant; iso2 (1.23 ± 0.09) not significant. RhoA–GTP: vc (1); iso1 (1.95 ± 0.21) p < 0.05; iso2 (16.04 ± 2.39) p < 0.001; n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control).



***

d

a b c

e

f primary DRG cells

RhoGDI pNF-H merged

RhoGDIpFN-H


Suppl. Figure 4: Axonal merlin–iso2 regulates RhoA activity (a) Full–length Immunoblot of Fig. 2f: RhoA–GTP level measured in cerebellar neurons after treatment with Rho activator (CN01) or overexpression with merlin–iso2 (iso2) compared to vector control (vc). Immunoblot quantitations: pNF–H: vc/–CN01 (1); vc/+CN01 (2.93 ± 0.28) p < 0.01; iso2/–CN01 (3.41 ± 0.40) p < 0.01. merlin: vc/–CN01 (1); vc/+CN01 (1.11 ± 0.17); iso2/–CN01 (42.30 ± 5.26). RhoA–GTP: vc/–CN01 (1); vc/+CN01 (5.98. ± 0.47); iso2/–CN01 (4.56 ± 0.31); n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (b) Full–length Immunoblot of Fig. 3a: Primary dorsal root ganglion (DRG) neurons after merlin–iso2–specific knockdown (siRNA nf2 iso2) compared to scrambled duplex control (scr nf2). Immunoblot for phosphorylated neurofilaments (pNF–H), merlin–iso2 and actin. Immunoblot quantitations: pNF–H: Scrambled duplex control (scr nf2) (1); merlin–iso2 (siRNA nf2 iso2) (0.58 ± 0.09) p < 0.05. merlin: scr nf2 (1); siRNA nf2 iso2 (0.24 ± 0.08); n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (c) Total forebrain and hindbrain lysates were used to confirm isoform–specific knockout of merlin in the mice used for this study. Phosphorylation of NF–H appears mainly reduced in isoform 2 deficient mice (nf2 iso2–/–). (d) Full–length Immunoblot of Fig. 3c: Primary dorsal root ganglion cells prepared from nf2 iso2–/– mice show axon caliber growth after merlin–iso2 re–expression (*** p < 0.001; t = 3.953; df = 70; n = 172 cells / 3 mice; mean + s.e.m.). Immunoblot quantitations: pNF–H: vc (1); iso2 (1.98 ± 0.09) p < 0.05. merlin: vc (1); iso2 (13.72 ± 1.04). RhoA–GTP: vc (1); iso2 (3.25 ± 0.53) p < 0.01; n = 3 blots (density values normalized to actin and appropriate transfection control). (e) Full–length Immunoblot of Fig. 3d: Wildtype DRG cells transfected with constitutively active RhoA mutant (RhoA 63L) compared to wildtype RhoA (RhoA wt) and empty vector control (vc). Immunoblot for pNF–H, actin, RhoA and activated RhoA (RhoA–GTP). Immunoblot quantitations: pNF–H: vc (1); RhoA wt (6.94 ± 0.78) p < 0.05; RhoA 63L (11.07 ± 1.23) p < 0.01. RhoA: vc (1); RhoA wt (5.62 ± 0.71); RhoA 63L (4.85 ± 0.36). RhoA–GTP: vc (1); RhoA wt (3.37 ± 0.25); RhoA 63L (15.29 ± 1.39); n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (f) RhoGDI immunolabeling in primary DRG cells. Co–staining with phosphorylated neurofilaments indicates expression in axonal processes as well as growth cones (arrow). Scale bar 50 µm.



merlin-iso2 p190RhoGap merged

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p190RhoGap

a primary DRG cells b

c primary cerebellar neurons

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d primary DRG cells

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Suppl. Figure 5: Both RhoGDI and p190RhoGap impact on neurofilament phosphorylation and axon diameter growth in vitro (a) p190RhoGap immunolabeling in primary DRG cells. Co–staining with merlin–iso2 indicates co–localization of both proteins in axons as well as growth cones (arrow). Scale bar 50 µm. (b) Full–length Immunoblot of Fig. 3e: Knockdown of RhoGDI (siRNA arhgdia) compared to scrambled control duplexes (scr arhgdia) in merlin–iso2–deficient DRGs (nf2 iso2–/–). Immunoblot for pNF–H, RhoGDI and actin. (c) Knockdown of RhoGDI (siRNA arhgdia) compared to scrambled control duplexes (scr arhgdia) in primary granule cells results in RhoA activation, neurofilament phosphorylation and axon caliber growth (** p <0.01; t = –2.171; df = 74; n = 153 cells / 2 mice; mean + s.e.m.). Immunoblot quantitations: pNF–H: scr arhgdia (1); siRNA arhgdia (4.05 ± 0.31) p < 0.01. RhoA–GTP: scr arhgdia (1); siRNA arhgdia (2.39 ± 0.17) p < 0.05. RhoGDI: scr arhgdia (1); siRNA arhgdia (0.67 ± 0.14); n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (d) Knockdown of p190RhoGap (shRNA arhgap35) compared to vector control (vc) in primary DRG cells provokes increased pNF–H levels and radial axon outgrowth (** p < 0.01; t = –3.577; df = 70; n = 100 cells / 3 mice; mean + s.e.m.). Immunoblot quantitations: pNF–H: vc (1); shRNA arhgap35 (4.02 ± 0.18) p < 0.01. p190RhoGap: vc (1); shRNA arhgap35 (0.56 ± 0.08); n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (e–g) Immunoblotting of sciatic nerve lysates taken from wild type animals of different developmental stages from postnatal day 0 (P0) until adulthood (P60). Immunoblot for phosphorylated neurofilaments (pNF–H), merlin–iso2, merlin–iso1 RhoGDI, p190RhoGAP, RhoA, merlin and actin.



a b

c d


Suppl. Figure 6: Merlin assembles a multi–protein complex relevant for Rho activation (a) Full–length Immunoblot of Fig. 3f: Sciatic nerve lysates from merlin–iso2 knockout mice (nf2 iso2–/–) compared to merlin–iso1 knockout (nf2 iso1–/–) and wildtype (wt) mice. Immunoblot for pNF–H, neurofilaments (panNF–H), actin, RhoA and RhoGDI from cell lysates and Rho–GTP indicates activation. Immunoblot quantitations: p190RhoGap: wt (1); nf2 iso1–/– (0.75 ± 0.16) not significant; nf2 iso2–/– (0.91 ± 0.11) not significant. panNF–H: wt (1); nf2 iso1–

/– (1.19 ± 0.21) not significant; nf2 iso2–/– (0.97 ± 0.08) not significant. pNF–H: wt (1); nf2 iso1–/– (1.03 ± 0.07) not significant; nf2 iso2–/– (0.43 ± 0.08) p < 0.01. RhoA: wt (1); nf2 iso1–/– (0.89 ± 0.10) not significant; nf2 iso2–/– (0.64 ± 0.12) p < 0.05. RhoA–GTP: wt (1); nf2 iso1–/– (0.92 ± 0.05) not significant; nf2 iso2–/– (0.27 ± 0.05) p < 0.01. RhoGDI: wt (1); nf2 iso1–/– (1.16 ± 0.11) not significant; nf2 iso2–/– (1.02 ± 0.08) not significant; n = 4 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (b) Full–length Immunoblot of Fig. 3g: Immunoprecipitation (IP) of endogenous RhoGDI from primary neurons compared to IgG control. Immunoblot for p190RhoGAP, merlin–iso2, merlin–iso1 and RhoGDI. (c) Full–length Immunoblot of Fig. 3h: Immunoprecipitation (IP) studies from sciatic nerve lysates using merlin specific antibodies (merlin–iso1 and merlin–iso2) compared to IgG control. Immunoblot for p190RhoGAP and merlin. (d) Full–length Immunoblot of Fig. 3i: Flag–tagged merlin N–terminal fragments (N–term), C–terminal (iso2–C–term and iso1–C–term) and empty vector control (vc) were transfected into P19 cells and immunoprecipitated (IP) with Flag antibodies. Immunoblot for merlin (anti–Flag), pNF–H and RhoGDI.



a b

c


Suppl. Figure 7: Merlin is not a replacement factor for RhoGDI at least in vitro (a) E. coli overexpressing His–tagged full–length version of merlin–iso1 (FL iso1) and merlin–iso2 (FL iso2) as well as FERM domain (N–terminus – shared by the two isoforms) were lysed in 0.5% CHAPS lysis buffer and used for GST–RhoGDI pull downs with a GST control. 1% of lysate input is shown as input control. (b) Purified His–RhoGDI (amino acid 24–204) and BSA were applied point wise onto nitrocellulose membrane. Dried membranes were blocked and incubated with merlin FERM domain (+FERM) or BSA (–FERM) respectively. Bound FERM domain was analyzed via Immunoblot with antibody against merlin (Merlin). Protein input was visualized by ponceau staining (Input). (c) Mant–GTP was incubated with indicated combinations of RhoA, RhoGDI (GDI), FERM domain and Dbs DH/PH domain (GEF). GDP/GTP exchange rate represented by relative fluorescence of mant–GTP bound to RhoA was measured over time.



b sciatic nerve sections

pNF-H

nf2 iso2 +/+ nf2 iso2 -/-

APPpNF-H

a myelinated axons

c

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Suppl. Figure 8: nf2 iso2–/– mice show comparable axon sizes and numbers (a) Sciatic nerves of isoform 2 knockout (nf2 iso2–/–) and wild type (nf2 iso2+/+) mice show comparable axonal diameters (n = 1,627 axons / 3 mice for nf2 iso1–/–; n = 3,754 axons / 3 mice for nf2 iso2–/–; n.s., not significant; mean + s.e.m.; axon calibers stated in µm). Furthermore, sciatic nerves of isoform 2 knockout and wild type mice show comparable axon numbers (n = 1,627 axons / 3 mice for nf2 iso1–/–; n = 3,754 axons / 3 mice for nf2 iso2–/–; n.s., not significant; mean + s.e.m.). Axons were categorized according to their diameter (stated in µm). (b) Longitudinal sciatic nerve sections of wild type and merlin–iso2 knock out animals were stained against APP (green) as marker for neurodegeneration and phosphorylated neurofilaments (red) as axonal marker. No APP staining can be co–localized to axons. Magnifications show unspecific staining in erythrocytes of blood vessels. Scale bar 100 µm. (c) Sciatic nerve lysates of mice bearing conditional knockout of merlin in Schwann cells (P0–Cre;Nf2floxfl/fl) show slightly elevated levels of neurofilament phosphorylation (upper panel). Consistently, interfilament distances are increased compared to wild type (P0–Cre;Nf2flox+/+) littermates (lower panel; *** p < 0.001; t = –8.806; df = 2199; n = 2201 disatnces / 3 mice; mean + s.e.m.). Immunoblot quantitations: pNF–H: P0–Cre;Nf2flox+/+ (1); P0–Cre;Nf2floxfl/fl (1.17 ± 0.07) not significant merlin: P0–Cre;Nf2flox+/+ (1); P0–Cre;Nf2floxfl/fl (0.54 ± 0.04); n = 4 blots; mean + s.e.m. (density values normalized to actin and genotype control).



a sural nerve biopsies

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b sural nerve biopsies

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c d


Suppl. Figure 9: NF2 patient biopsies display neurofilament hypophosphorylation. (a) Sural nerve biopsies of NF2 patients and healthy controls were stained against phosphorylated neurofilament H (see Fig. 7a). NF–H phosphorylation in NF2 patient sural nerve biopsies is reduced, reflected by the mean immunohistochemical signal per vital neurofilament–positive axon (*** p < 0.001; t = 5.944; df = 27; n = 261 axons / 3 biopsies; mean + s.e.m.; the values are stated in pixel2 per µm2). (b) Interfilament distances of neurofilaments were quantified from myelinated axons of NF2 patient sural nerve biopsies compared to healthy controls (*** p < 0.001; t = –4.828; df = 1165; n = 1566 distances / 2 biopsies per group; mean + s.e.m.). (c) Full–length Immunoblot of Fig. 7e: P19 cells measured for RhoA–GTP level after transfection with patient–derived non–sense merlin mutation C784T (iso2 C784T), merlin–iso2 (iso 2 wt) and empty vector control (vc). Immunoblot for pNF–H, RhoA, merlin, actin and active RhoA (RhoA–GTP). Immunoblot quantitations: pNF–H: vc (1); iso2 wt (8.59 ± 0.38) p < 0.001; iso2 C784T (2.38 ± 0.27) p < 0.05. RhoA: vc (1); iso2 wt (1.48 ± 0.28) not significant; iso2 C784T (1.74 ± 0.19) not significant. RhoA–GTP: vc (1); iso2 wt (8.97 ± 0.68) p < 0.01; iso2 C784T (2.98 ± 0.35) p < 0.05; n = 3 blots; mean + s.e.m. (density values normalized to actin and appropriate transfection control). (d) Full–length Immunoblot of Fig. 7f: P19 cells transfected with Flag–tagged merlin–iso2 (iso 2 wt) and C784T mutant (iso 2 C784T). Immunoprecipitation using FLAG–tag antibody (Flag–IP). Immunoblot for p190RhoGAP, merlin and RhoGDI.


Supplemental Table

1 kinase candidates


Suppl. Table: Kinase screening assay List of selected serine/threonine kinases used for the screening assay to identify the merlin–associated kinase effecting neurofilament phosphorylation.


supplementary figure 1 - nature research · merlin–iso2 (sirna nf2 iso2) (0.58 ± 0.09) p <...

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