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First published online December 21, 2005
doi: 10.1242/10.1242/jcs.02726

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Myo10 in brain: developmental regulation, identification of a headless isoform and dynamics in neurons

Aurea D. Sousa^*, Jonathan S. Berg^*, Brian W. Robertson, Rick B. Meeker and Richard E. Cheney

Department of Cell and Molecular Physiology, Medical Biomolecular Research Building (MBRB), University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545, USA

View larger version (41K): [in a new window]   Fig. 1. Brain expresses a headless form of Myo10. (A) Schematic map of the human Myo10 gene and bar diagrams of the full-length and headless Myo10 transcripts. The headless transcript begins with an alternate exon (gray) that encodes a 5' UTR specific to headless Myo10. Splicing of this 5' UTR to the exon that begins at M644 of full-length Myo10, results in a headless transcript lacking the first 643 aa of full-length Myo10. Major domains of the Myo10 protein are indicated by colored boxes, vertical lines on the gene map represent exons. The horizontal red lines indicate the locations of the probes used for northern blotting. (B) Multiple-tissue northern blot incubated with three different Myo10 probes. The blot was originally incubated with a probe targeting the 3' UTR common to both transcripts (right) and detected both a 9 kb band (upper arrowhead) in non-brain tissues and a smaller 7 kb band (lower arrowhead) in brain (Berg et al., 2000). The blot was then stripped and reprobed with a sequence from the 5' UTR of the headless transcript (middle), which detected a 7 kb band in brain but not the full-length 9 kb band in other tissues. The blot was then stripped and reprobed a final time with a sequence from the head domain (left). In addition to the 7 kb band in brain, the headless probe appears to react non-specifically with a faint band in tissues such as liver that is slightly larger than the 9 kb band corresponding to full-length Myo10. (C) Sequence alignment of human EST AI878891 with full-length Myo10 cDNA (NM_012334) show the 5' UTR specific to the headless form (capitalized nucleotides) as well as the initial protein-coding exons of the headless form and their perfect match to the full-length Myo10 exon beginning at M644. Exon boundaries are indicated by vertical dotted lines and stop codons in the alternate 5' UTR are highlighted in red. Predicted start methionines at M644 and M747 are shown in green.

View larger version (39K): [in a new window]   Fig. 2. Myo10 expression in brain is developmentally regulated. Western blot of whole cerebri (A) or cerebelli (B) from mice at postnatal days 1 to adult (P>45) were lysed in SDS sample buffer and immunoblotted with antibody no. 3568 against mouse Myo10. Both full-length- (240 kDa) and headless-Myo10 (164 kDa) are expressed in mouse cerebrum where they show clear developmental regulation, with a peak of expression between postnatal days 5-15. Cerebellum from mouse shows a different pattern of Myo10 expression, with prominent and increasing levels of full-length Myo10 expression throughout development. (C) Full-length and headless Myo10 (arrowheads) are detected by immunoblotting in undifferentiated CAD cells as well as in CAD cells differentiated into a neuronal phenotype.

View larger version (130K): [in a new window]   Fig. 3. Immunolocalization of Myo10 in mouse brain. (A-C) Sagittal section from the cerebellum of an adult mouse double-labeled for Myo10 and calbindin, a marker for Purkinje cells. The cell bodies of Purkinje cells (PL) and their dendrites in the molecular layer (ML) are brightly stained by Myo10. Myo10 stains the granule-cell layer (GL) more faintly, and the cerebellar white matter (WM) shows little staining. (D-F) A similar section of the molecular layer at higher magnification shows staining for Myo10 in the dendrite (arrow in E) of a Purkinje cell. (G-I) Sagittal section from the cerebellum of an adult mouse double-labeled for Myo10 and GFAP, a marker for Bergmann glia and other astrocytes. (J-L) A similar section of the molecular layer at higher magnification showing that Myo10 staining is also present in the processes of Bergmann glia (arrow in K). (M-O) Sagittal section from the cerebrum of an adult mouse double-labeled for Myo10 and GFAP showing staining of the ventricular zone (VZ) and subventricular zone (SVZ). Although the parenchyma of the adult mouse cerebrum shows relatively faint Myo10 staining, the ependymal cells lining the ventricle show bright Myo10 staining. The subjacent subventricular astrocytes marked by GFAP staining (red arrow in H) are also Myo10 immunopositive.

View larger version (108K): [in a new window]   Fig. 4. Subcellular localization of endogenous Myo10 in CAD cells. (A) In undifferentiated CAD cells, Myo10 shows a clear localization to the leading edge as well as a more diffuse localization in central cytoplasm. (B) Undifferentiated CAD cells stained with a non-immune control antibody show little or no staining. (C) After 2 days of serum withdrawal to induce CAD-cell differentiation into a neuronal phenotype, Myo10 staining is observed in cell bodies, neurites, the edges of growth cones and the tips of neuronal filopodia. (D) Differentiated CAD cells stained with a non-immune control antibody show little or no staining. Myo10 antibody no. 3568 was used to stain Myo10 (green) and rhodamine-phalloidin was used to label F-actin (red).

View larger version (103K): [in a new window]   Fig. 5. Full-length Myo10, but not headless Myo10, localizes to the tips of filopodia when transfected into CAD cells. (A) In undifferentiated CAD cells, full-length GFP-Myo10 (green) localizes to the tips of the short filopodia along the leading edge. (B) Headless GFP-Myo10 does not localize to filopodial tips and instead shows a more diffuse localization to the cell membrane and the central portion of undifferentiated CAD cells. (C) In CAD cells induced to differentiate into a neuronal phenotype, full-length GFP-Myo10 localizes to neurites as well as the tips of neuronal filopodia. (D) Headless GFP-Myo10 localizes primarily to the soma and neurites of differentiated CAD cells and does not localize to the tips of filopodia. (E) High-magnification images showing localization of full-length GFP-Myo10 to the tips of filopodia in CAD cells at early stages of differentiation. Notice that the filopodia frequently clump together at their tips. Cells were double-labeled with rhodamine-phalloidin to stain F-actin (red).

View larger version (122K): [in a new window]   Fig. 6. Full-length Myo10 localizes to sites of cell-cell contact. (A) In undifferentiated CAD cells, GFP-Myo10 (green) is present at the tips of short filopodia-like structures at sites of cell-cell contact. The panels on the right shows high-magnification views of the inset. (B) GFP-Myo10 is also present at the tips of filopodia at sites of cell-cell contact in differentiated CAD cells. The panels on the right show high-magnification views of the inset and illustrate that Myo10 is present at the tips of filopodia that are contacting neurites. Cells were double-labeled for F-actin by staining with rhodamine-phalloidin (red). Although under the exposure settings used here some filopodia appear only faintly labeled for F-actin relative to the much thicker neurites and cell bodies, longer exposures indicate that all of the filopodia contain F-actin.

View larger version (29K): [in a new window]   Fig. 7. Puncta of Myo10 can move laterally along the leading edge and fuse with one another. (A) Fluorescence image of an undifferentiated CAD transiently transfected with full-length GFP-Myo10 showing 17 relatively large puncta of GFP-Myo10 (labeled a-q) at the tips of filopodial bundles along the leading edge. (B) Outline of the leading-edge region of this cell that was imaged for 12 minutes and then displayed as a kymograph to highlight movements along the lateral edge. (C) Kymograph illustrating the movements of puncta of Myo10 along the leading edge. Although the larger Myo10 puncta maintained relatively stable positions along the leading edge, smaller Myo10 puncta tended to move laterally along the leading edge until they collided and fused with larger puncta, thus generating a kymograph with a dendritic appearance.

View larger version (15K): [in a new window]   Fig. 8. Diagrams of proteins with domain structures similar to the tail of Myo10. Illustrated are the domain structures of human proteins that are structurally similar to headless Myo10. Domain predictions were based on the NCBI/CDD and/or PFAM servers. Regions predicted to form coiled coils by the PAIRCOIL server are indicated by `CC'. Arrows indicate protein identity in %. Although the C-terminal portion of FLJ21019 was not recognized as a FERM domain by CDD or PFAM, database searches with FLJ21019 show that it shares 32% protein identity with the FERM domain of Myo10.

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