Fig 2: Myo10 reporter knockout (Myo10tm2/tm2) mice lack full-length (motorized) Myo10, but express the brain-specific headless isoform. (A) Schematic diagram showing the reporter knockout (tm2) targeting strategy. Insertion of the targeting sequence by homologous recombination causes loss of 9594 bp, including exon 19 and part of intron 19. Notably, the headless Myo10 isoform begins at exon 20. The gene trap, polyadenylation (pA) signal, is harbored in the IRES:lacZ cassette (IRES stands for internal ribosome entry site). (B) Southern blot analysis. DNA was fragmented using the restriction enzyme EcoRI or BamHI. The position of the radiolabeled hybridization probe at the 3'-end is indicated. Labeled DNA fragments were detected using X-ray film. (C) Western blot analysis. Lysates of HEK293T cells overexpressing full-length mouse (m) Myo10 (plasmid, pCMV-Tag2B-mMyo10) or headless (Hdl) mouse Myo10 (plasmid, pCMV-Tag2B-Hdl-mMyo10) were used as positive controls (blot on the left). Whole brain lysates obtained from P10 mice were used to screen for expression of full-length and Hdl-Myo10 (blot on the right). (D) Level of Hdl-Myo10 protein expressed in Myo10tm2/tm2 mouse brain (n = 3) relative to wild-type (WT) brains (n = 3), and mouse Hdl-Myo10 transcripts obtained from the National Center for Biotechnology Information (NCBI), National Institutes of Health (NIH). The accession prefix NM_ denotes confirmed protein-coding transcripts, whereas the prefix XM_ indicates predicted protein transcripts. Green (vertical) bars are exons (ranging from 1 to 41; indicated above) and red bars are 5'-UTRs (5' untranslated regions), preceding the coding sequence. Notably, the 5'-UTR of Hdl-Myo10 transcript NM_001353142.1, which lacks exon 20 (labeled blue) and exon 21, is disrupted by insertion of the cassette (L1L2_Bact_P) used to generate the Myo10tm2 allele.

Fig 3: ß-TrCP1 regulates MYO10 ubiquitination and degradation.(A) HEK293T cells were transfected with GFP, GFP-MYO10, and FLAG–ß-TrCP1 or FLAG-Cul1 for 48 hours, IPed with the anti-GFP antibody, and blotted with indicated antibodies. Ten percent of input was run to show protein expression. (B) HEK293T cells were transfected with GFP, GFP-MYO10, and FLAG-ß–TrCP1–FL (full-length) or FLAG-ß–TrCP1–?F (F-box deleted) for 48 hours; IPed with the anti-GFP antibody; and blotted with indicated antibodies. Five percent of input was run to show protein expression. (C) A549 control or UbcH7-depleted cells were IPed with anti-MYO10 and blotted with antibodies for endogenous proteins. Input (5%) was also run for loading control. (D) HEK293T cells were transfected with FLAG–ß-TrCP1 or FLAG-ß–TrCP1–?F for 48 hours and treated with 320 µM CHX for indicated times, and protein expression was examined by specific antibodies. (E) The band intensity of MYO10 in (D) was quantitated using the ImageJ software and normalized to that of the 0-hours group. (F) A549 cells were transfected with siRNA control or targeting ß-TrCP1 for 48 hours and treated with 320 µM CHX for indicated times, and protein expression was examined by specific antibodies. (G) Quantitation of MYO10 band intensity in (F) from three replicates. (H) HEK293T cells were transfected with FLAG–ß-TrCP1 or FLAG-ß–TrCP1–?F with His-Ub for 48 hours, treated with 320 µM CHX and 15 µM MG132 for 6 hours, lysed cells under denaturing conditions, and proceeded to detect MYO10 ubiquitination as described previously (75). Ten percent of input was run for loading control. The band intensity of ubiquitinated MYO10 in the IP was quantitated by ImageJ from three blots and presented as average relative intensity (numbers below), which showed a statistical significance (P = 0.0042) between lanes 2 and 4. Quantitation data are presented as average and SD from at least three experiments. **P < 0.0001.

Fig 4: Homozygous Myo10 reporter knockout (Myo10tm2/tm2) mouse embryos develop exencephaly. (A) Pigmentation defects in homozygous mutants. Image of an adult Myo10tm2/tm2 mouse showing white belly spots. (B) Genotype frequency of offspring derived from HET (heterozygous) x HET and HET x HOM (homozygous) matings. HOM offspring were produced at less than expected frequency, indicated by dashed lines and asterices. (C) Two examples of X-gal stained, homozygous Myo10 mutant embryos at E14.5, with (left) and without (right) exencephalus, caused by failure of the cranial neural tube to close. The white arrow on the left indicates everted cranial neural folds, a hallmark of exencephalus. About 1 in 4 (24%) of homozygous Myo10 mutant (Myo10tm2/tm2) embryos developed exencephalus. Neural tube defect is abbreviated NTD. (D) Enlarged view from panel C (yellow square) and skin histological section showing Myo10 expression in the skin and hair placodes (blue spots). (E) Whole-mount X-gal staining. Myo10 is expressed in the head and the first and second branchial arches (labeled 1 and 2, respectively) of the developing embryo (E8.5 and E9.5). (F) X-gal staining and histology (E10.5) reveals expression of Myo10 in the ectoderm and dorsal regions, but not in the neural tube. ht, heart; ov, otic vesicle; s, somite; nt, neural tube; D, dorsal; V, ventral; L, lateral.

Fig 5: Headless mouse Myo10 (Hdl-mMyo10) strongly localizes to the cell periphery independent of the MyTH4-FERM domain. (A) Schematic representations of the domain structures of N-terminal EGFP-tagged mouse Myo10 (EGFP-mMyo10), Hdl-mMyo10 (EGFP- Hdl-mMyo10) and truncated variants thereof. (B) Images of fixed HEK293T cells after transfection with EGFP-mMyo10 or various EGFP-tagged Hdl-mMyo10 constructs. Cells were counterstained with Alexa Fluor 594-conjugated phalloidin, a red fluorescent F-actin probe, and imaged via a Zeiss Plan Apo 63/1.4 (oil-immersion) objective lens by superresolution structured illumination microscopy. Scale bars: 10 µm.