Figure 1.
Mammalian FCHSD1 and FCHSD2 are homologous to fly Nwk and form oligmers with each other.
(A) Schematic drawing of the domain structures of Mus musculus FCHSD1, FCHSD2 and Drosophila melanogaster Nwk. The identity of F-BAR domains and SH3 domains between FCHSD1, FCHSD2 and Nwk is indicated below each domain. (B and C) Western blots showing that FCHSD1 and FCHSD2 were co-immunoprecipitated with themselves and with each other. Expression vectors were transfected into HEK293 cells to express epitope-tagged FCHSD1 and FCHSD2, and cell lysates were subjected to immunoprecipitation. IP indicates antibody used for immunoprecipitation and WB indicates antibody used for detection.
Figure 2.
Expression analysis of mouse Fchsd1 and Fchsd2 in different tissues.
(A) Total RNA from postnatal day 5 mouse tissues was extracted and used as template for reverse transcription. PCR was performed using this cDNA as template. Upper panel, Fchsd1 mRNA is expressed more abundantly in nervous tissues. Middle panel, Fchsd2 mRNA is expressed ubiquitously in all tissues examined. Lower panel, β-actin specific primers were used as the RT-PCR template control. (B) Total proteins of postnatal day 5 mouse cochlea and vestibula were extracted and separated by PAGE and detected with antibodies against FCHSD1(Novus), FCHSD2, or SNX9.
Figure 3.
FCHSD1 and FCHSD2 immunolocalization in mouse cochlear hair cells.
Shown are single confocal sections. FCHSD1 or FCHSD2 immunoreactivity visualized with Cy5 or FITC-conjugated secondary antibody was distinctly associated with stereocilia or cuticular plate, which were visualized with rhodamine-conjugated phalloidin. (A) FCHSD1 immunoreactivity in the cuticular plate of 3-week old mouse cochlear hair cells. (B) FCHSD1 immunoreactivity in the cuticular plate of 3-week old mouse outer hair cells. (C) FCHSD2 immunoreactivity in the hair bundles of 6-week old mouse cochlear hair cells. (D) FCHSD2 immunoreactivity in the hair bundles of 7-month old mouse inner hair cells. (E) FCHSD2 immunoreactivity in the hair bundles of 2-week old mouse outer hair cells. The Novus FCHSD1 antibody was used in (A) and (B). Scale bars: 10 µm in(A-C), 5 µm in (D) and (E).
Figure 4.
FCHSD2, but not FCHSD1, binds to WASP and N-WASP and regulates WASP-Arp2/3-mediated F-actin polymerization in vitro.
(A) Western blots showing that FCHSD2, but not FCHSD1, was co-immunoprecipitated with WASP and N-WASP. Expression vectors were transfected into HEK293 cells to express epitope-tagged proteins, and cell lysates were subjected to immunoprecipitation. IP indicates antibody used for immunoprecipitation and WB indicates antibody used for detection. (B) WASP-Arp2/3-mediated F-actin polymerization was enhanced by GST-FCHSD2ΔC, but not by GST-FCHSD1ΔC (or GST). Monomeric actin (3 µM, 10% pyrene-labeled) was incubated with 1 µM GST-FCHSD1ΔC (or GST-FCHSD2ΔC, or GST as control) in the presence of 40 nM His-WASP145 and 20 nM Arp2/3 protein complex, and F-actin polymerization was tracked by monitoring the increase of pyrene fluorescence using a spectrofluorometer. a.u., absorbance units.
Figure 5.
FCHSD1, but not FCHSD2, binds to SNX9 and enhances SNX9’s activity in stimulating WASP-Arp2/3-mediated F-actin polymerization in vitro.
(A) Western blots showing that FCHSD1, but not FCHSD2, was co-immunoprecipitated with SNX9. (B) Schematic drawing of the domain structures of FCHSD1 and SNX9 that are used in the co-immunoprecipitation experiments in (C) and (D). (C) Western blots showing that the F-BAR domain of FCHSD1 was co-immunoprecipitated with SNX9. (D) Western blots showing that the SH3 domain of SNX9 was co-immunoprecipitated with FCHSD1. Expression vectors were transfected into HEK293 cells to express epitope-tagged proteins, and cell lysates were subjected to immunoprecipitation. IP indicates antibody used for immunoprecipitation and WB indicates antibody used for detection. The immunoglobulin heavy and light chains are indicated as “H” and “L”, respectively. (E) GST-SNX9 stimulated WASP-Arp2/3-mediated F-actin polymerization in vitro, which was enhanced by GST-FCHSD1ΔC. (F) Both GST-SNX9 and GST-FCHSD2ΔC stimulated WASP-Arp2/3-mediated F-actin polymerization in vitro, but there was no synergistic effect between these two proteins. Monomeric actin (3 µM, 10% pyrene-labeled) was incubated with 1 µM GST-FCHSD1ΔC (or GST-FCHSD2ΔC, or GST-SNX9) in the presence of 40 nM His-WASP145 and 20 nM Arp2/3 protein complex, and F-actin polymerization was tracked by monitoring the increase of in pyrene fluorescence using a spectrofluorometer. a.u., absorbance units.
Figure 6.
SNX9 colocalizes with FCHSD1 in the cuticular plate of mouse cochlear hair cells.
Shown are single confocal sections. SNX9 immunoreactivity visualized with FITC-conjugated secondary antibody was distinctly associated with the cuticular plate, which was visualized with rhodamine-conjugated phalloidin. (A) SNX9 immunoreactivity in the cuticular plate of 2-week old mouse cochlear hair cells. (B) SNX9 immunoreactivity in the cuticular plate of 3-week old mouse outer hair cells. (C) and (D) SNX9 immunoreactivity colocalized with FCHSD1 immunoreactivity, which was visualized with Cy5-conjugated secondary antibody, in the cuticular plate of 3 week-old mouse cochlear hair cells. The Novus FCHSD1 antibody was used in (C) and (D). Scale bars: 10 µm in (A–C), 5 µm in (D).