|Coordinate||80,484,332 bp (GRCm38)|
|Base Change||A ⇒ G (forward strand)|
|Gene Name||myosin ID|
|Chromosomal Location||80,482,126-80,780,025 bp (-)|
|Amino Acid Change||Leucine changed to Proline|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000037819]|
AA Change: L972P
|Predicted Effect||probably damaging
PolyPhen 2 Score 0.986 (Sensitivity: 0.74; Specificity: 0.96)
|Meta Mutation Damage Score||0.9132|
|Is this an essential gene?||Non Essential (E-score: 0.000)|
|Candidate Explorer Status||CE: excellent candidate; Verification probability: 0.968; ML prob: 0.9692; human score: 6.5|
Linkage Analysis Data
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice|
|Last Updated||2018-10-11 1:56 PM by Anne Murray|
|Record Created||2013-03-02 4:41 PM by Emre Turer|
The whisper phenotype was identified among G3 mice of the pedigree R0096, some of which showed susceptibility to dextran sulfate sodium (DSS)-induced colitis exhibiting . diarrhea and rectal bleeding as well as weight loss by seven days after exposure to DSS (Figure 1); weight loss continued to progress through day 10 post-DSS treatment (Figure 2).
|Nature of Mutation|
Gene validated by crossing to 2nd ENU allele. Horton/Whisper compound heterozygous mice are DSS sensitive.
Whole exome HiSeq sequencing of the G1 grandsire identified 69 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Myo1d: a T to C transition at base pair 80,484,332 (v38) on chromosome 11 corresponding to base pair 295,724 in the GenBank genomic region NC_000077 encoding Myo1d. The strongest association was found with a recessive model of linkage to the DSS-induced weight loss at day 10, wherein 10 affected variant homozygotes departed phenotypically from 16 homozygous reference mice and 24 heterozygous mice with a P value of 1.384 x 10-13 (Figure 3). The mutation corresponds to residue 3,148 in the mRNA sequence NM_177390 within exon 22 of 22 total exons.
The mutated nucleotide is indicated in red. The mutation results in a leucine (L) to proline (P) substitution at position 972 (L972P) in the Myo1d protein, and is strongly predicted by Polyphen-2 to cause loss of function (probably damaging; score = 1.00).
|Illustration of Mutations in
Gene & Protein
Myo1d encodes myosin 1D (Myo1d; alternatively, Myr4), a member of the class I family of unconventional myosins (Figure 4). The class I myosins are molecular motors that control the mechanical properties of cell membranes by mediating membrane/cytoskeleton adhesion (1). The whisper mutation (L972P) is within the TH1 domain of Myo1d. The TH1 domain in class I myosins mediates myosin dimerization, targets each myosin to its subcellular location, binds directly to acidic phospholipids, and specifies the function(s) (e.g., cargo binding and enzymatic activities) of a myosin (2;3).
For more information on Myo1d, please see the record for horton.
The apical brush border of the intestinal epithelial cells that line the small intestine is comprised of tightly packed microvilli (4). A core actin bundle and associated actin-binding proteins are essential to maintain the stability of each microvillus (4). Unconventional myosins in the intestine have been studied. Myosin-1a (Myo1a) connects the microvillar membrane to the actin bundle underneath (5-7). Myosins from classes I, II, V, VI, and VII also target to the actin-rich domain in the brush border (8;9). Myo1a has several functions within the enterocyte including the organization of apical membrane domains (10), controlling apical membrane tension (1), and the shedding of vesicles from the tips of the microvilli (11). Knockout of Myo1a (Myo1a-/-) in mice resulted in defects in the brush border membrane composition and apical membrane herniations in some enterocyte brush borders (12), but the knockout mice exhibited no noticeable physiological symptoms indicating that other myosins may compensate for Myo1a. In the wild-type microvillus, Myo1a is excluded from the distal tip compartment where Myo1d localized, indicating that Myo1a and Myo1d have different functions within the microvillus (4). In Myo1a-/- mice, the levels of Myo1d in the brush border are upregulated and redistributed along the length of the microvillus (4). The redistribution of Myo1d upon the loss of Myo1a expression indicates that Myo1d can compensate for the function of Myo1a in the brush border (4). Benesh et al. propose that Myo1d functions in the early stages of vesicle formation at the tips of the microvilli and that the redistribution of Myo1d upon loss of Myo1a expression indicates that Myo1d and Myo1a may compete for a shared binding site within the microvillus (4). The localization of Myo1d to the terminal web, a filamentous structure at the apical surface of epithelial cells that possess microvilli, indicates that Myo1d may function in the short-range transport, docking, and/or fusion of apically directed vesicles derived from the Golgi (13). The Myo1d at the tips of the microvilli may function to control actin dynamics or to transport components along the microvillar axis (4). Alternatively, Myo1d may also function in the formation and/or release of vesicles from the microvillar tips (14).
Whisper genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transition.
Whisper(F): 5’- CATTAGAGGGCAGACAGCGATTAGC -3’
Whisper(R): 5’- CGGATTGAACCCTGAAACTGCCAC -3’
Whisper_seq(F): 5’- CCTGGGATGCTGATCGAG -3’
Whisper_seq(F): 5’- TGCCACCCTACAGGAGC -3’
1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40X
6) 72°C 10:00
7) 4°C ∞
The following sequence of 440 nucleotides is amplified (Chr.11: 80484074-80484513, GRCm38):
cattagaggg cagacagcga ttagctgagc tcctgggatg ctgatcgagg tggaacgagt
gtggggatgc agaggacgag tgagggcctg cctgcctttt gtgccaggct caggctcttg
ctcccctggg ccaggcctct gatgtgcagc agtcagttcc cgggcacact gaggatgaag
cccgagcggt tcttggtgaa gtcaggctgt ggctgattga gccgggtctc cacagagacg
gtgcatttct tcccgtgcag gctgcactgc accgggttgg tgacgttcac ttgaaggtgg
cgcttctcac tgcaagagta gaacagacag gccaagcggc cagtgagtgg gcagacacag
tgctgtggtt tccttggcaa ccctcagggc cctgccattt gccaggctcc tgtagggtgg
Primer binding sites are underlined and the sequencing primer is highlighted; the mutated nucleotide is shown in red text (A>G, Chr. (+) strand; T>C, sense strand).
1. Nambiar, R., McConnell, R. E., and Tyska, M. J. (2009) Control of Cell Membrane Tension by Myosin-I. Proc Natl Acad Sci U S A. 106, 11972-11977.
2. Hasson, T., Skowron, J. F., Gilbert, D. J., Avraham, K. B., Perry, W. L., Bement, W. M., Anderson, B. L., Sherr, E. H., Chen, Z. Y., Greene, L. A., Ward, D. C., Corey, D. P., Mooseker, M. S., Copeland, N. G., and Jenkins, N. A. (1996) Mapping of Unconventional Myosins in Mouse and Human. Genomics. 36, 431-439.
3. McConnell, R. E., and Tyska, M. J. (2010) Leveraging the Membrane - Cytoskeleton Interface with Myosin-1. Trends Cell Biol. 20, 418-426.
4. Benesh, A. E., Nambiar, R., McConnell, R. E., Mao, S., Tabb, D. L., and Tyska, M. J. (2010) Differential Localization and Dynamics of Class I Myosins in the Enterocyte Microvillus. Mol Biol Cell. 21, 970-978.
5. Mooseker, M. S., and Tilney, L. G. (1975) Organization of an Actin Filament-Membrane Complex. Filament Polarity and Membrane Attachment in the Microvilli of Intestinal Epithelial Cells. J Cell Biol. 67, 725-743.
6. Matsudaira, P. T., and Burgess, D. R. (1979) Identification and Organization of the Components in the Isolated Microvillus Cytoskeleton. J Cell Biol. 83, 667-673.
7. Howe, C. L., and Mooseker, M. S. (1983) Characterization of the 110-Kdalton Actin-Calmodulin-, and Membrane-Binding Protein from Microvilli of Intestinal Epithelial Cells. J Cell Biol. 97, 974-985.
8. Chen, Z. Y., Hasson, T., Zhang, D. S., Schwender, B. J., Derfler, B. H., Mooseker, M. S., and Corey, D. P. (2001) Myosin-VIIb, a Novel Unconventional Myosin, is a Constituent of Microvilli in Transporting Epithelia. Genomics. 72, 285-296.
9. Heintzelman, M. B., Hasson, T., and Mooseker, M. S. (1994) Multiple Unconventional Myosin Domains of the Intestinal Brush Border Cytoskeleton. J Cell Sci. 107 ( Pt 12), 3535-3543.
10. Tyska, M. J., and Mooseker, M. S. (2002) MYO1A (Brush Border Myosin I) Dynamics in the Brush Border of LLC-PK1-CL4 Cells. Biophys J. 82, 1869-1883.
11. McConnell, R. E., Higginbotham, J. N., Shifrin, D. A.,Jr, Tabb, D. L., Coffey, R. J., and Tyska, M. J. (2009) The Enterocyte Microvillus is a Vesicle-Generating Organelle. J Cell Biol. 185, 1285-1298.
12. Tyska, M. J., Mackey, A. T., Huang, J. D., Copeland, N. G., Jenkins, N. A., and Mooseker, M. S. (2005) Myosin-1a is Critical for Normal Brush Border Structure and Composition. Mol Biol Cell. 16, 2443-2457.
13. Fath, K. R., and Burgess, D. R. (1993) Golgi-Derived Vesicles from Developing Epithelial Cells Bind Actin Filaments and Possess Myosin-I as a Cytoplasmically Oriented Peripheral Membrane Protein. J Cell Biol. 120, 117-127.
|Science Writers||Anne Murray|