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Human myosin-Vc is a novel class V myosin expressed in epithelial cells

Department of Cell and Molecular Physiology, Medical Science Research Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

View larger version (40K): [in a new window]   Fig. 3. The relative mRNA distribution of the class V myosins in northern and dot blots shows that Myo5a is most abundantly expressed in brain, whereas Myo5b and Myo5c have a broad distribution. Myo5c is particularly abundant in secretory or epithelial tissues. Human mRNA blots (Clontech) were hybridized to probes for Myo5a, Myo5c and Myo5c. Subsequent incubations with control probes (ubiquitin or ß-actin) revealed that the mRNA loadings were in fact well normalized (not shown).

View larger version (21K): [in a new window]   Fig. 1. Discovery and cloning of a novel class V myosin. (A) cDNA clones comprising the full coding sequence of human MYO5C. Combining the sequence of clones 2, 5 and 7 and EST 178419 results in 6839 bp of cDNA including 19 bp of 5'UTR, 5229 bp of coding sequence and 1591 bp of 3'UTR containing an Alu repeat and ending in a poly-A tail. Black shading indicates the sequenced portion of the clones. The locations of the mRNA probe, the His-tagged tail construct used to generate antibodies 199 and 200, and the GFP-tail construct are also depicted. Arrows indicate PCR primers used for amplification. (B) The three class V myosins in vertebrates. Note that the Myo5c tail lacks a PEST region but contains a small region similar to the alternatively spliced exon D mapped in Myo5a (Seperack et al., 1995). The human Myo5a sequence used here consists of a GenBank sequence (Y07759) into which we have inserted the 25 aa sequence of exon F. This Myo5a sequence thus includes all three alternatively spliced exons and will differ somewhat from actual splice forms in size and numbering. The diagrams of Myo5b and Myo5c are based on GenBank sequences U60416 and AF272390, respectively. (C) Predicted structure of the three class V myosins. Interestingly, the length of the initial segment of coiled-coil is predicted to be 30 nm for all three myosins.

View larger version (92K): [in a new window]   Fig. 2. Alignment of the three class V myosins in vertebrates. Bolded letters denote identical residues in all three sequences. The head, ATP binding site and globular tail are marked. Solid boxes mark each of six IQ motifs in the neck domain. Regions predicted (Lupas et al., 1991) to form a coiled-coil with greater than 90% probability are denoted by dashed underlines. Carets indicate the boundaries of Myo5a exons A through F that have been mapped by PCR (Lambert et al., 1998; Seperack et al., 1995), and shading indicates the three exons known to undergo tissue-specific alternative splicing. Note that the human Myo5a sequence reported to bind the dynein light chain (dotted bracket) does not contain exons D and F (Naisbitt et al., 2000). The sequence corresponding to the mouse Myo5a construct reported to bind kinesin (Huang et al., 1999) is also indicated (solid bracket). The position of a conserved serine in the globular tail (`serine 1650' for mouse Myo5a), whose phosphorylation regulates binding of Myo5a to melanosomes, is marked by a diamond (Karcher et al., 2001). Rab11a-binding by rabbit Myo5b is reported to require the sequence corresponding to aa 1397-1418 and 1797-1811 of rat Myo5b (Lapierre et al., 2001). The sequence and location of a putative vacuole-binding site in the globular tail of Myo2p is also shown; asterisks denote functionally crucial residues (Catlett et al., 2000). The GenBank accession number for Myo5c is AF272390.

View larger version (40K): [in a new window]   Fig. 4. Myo5c protein is abundant in epithelial and secretory tissue. (A) Immunoblot showing the protein distribution of class V myosins in mouse tissues. Myo5a is abundant in brain, whereas Myo5b and Myo5c are more widely distributed in non-neuronal tissues. Gels were run in triplicate, samples were loaded equally by wet tissue weight and blots were probed with antibodies to Myo5a, Myo5b or Myo5c. It is unclear if the lower band detected by the Myo5b antibodies in brain represents a breakdown product. (B) Immunoblot showing that Myo5c is enriched in the mucosa of mouse colon, but not the underlying muscle wall. The Ponceau stain of this blot shows that myosin-II and actin are found primarily with the muscle wall. The mucosa was dissected from 0.4 g of colon by scraping. The mucosal layer and the muscle wall were then separately homogenized in 4 ml of buffer as for the other mouse tissue homogenates, and equal volumes were loaded. (C) Immunblots of untransfected and transfected Hela cells. Blotting of HeLa cell lysates reveals that these cells endogenously express both Myo5a and Myo5c (left panels, 50 µg load). HeLa cells transfected with the GFP-Myo5c tail express a protein of the expected size, 120 kDa (right panels, 20 µg load).

View larger version (95K): [in a new window]   Fig. 5. Myo5c is expressed in epithelial cells of the colon and pancreas. In human colon sections double-labeled for Myo5c (A) and F-actin (B), Myo5c localizes to the single layer of epithelial cells constituting the intestinal crypts, as seen in longitudinal section (arrow) and cross section (arrowhead). Myo5c is particularly enriched in the apical portion of the epithelial cells, where it colocalizes with F-actin. The basolateral portions of these cells, which extend 30 µm, exhibit some Myo5c staining but relatively little F-actin staining. The surrounding interstitial cells are faintly stained by F-actin but exhibit little or no Myo5c staining and appear black. (C) Non-immune serum IgG exhibits little or no staining of the adjacent section. (D) Higher magnification view of a single crypt showing Myo5c staining of individual intestinal epithelial cells. These cells, which surround the intestinal lumen, exhibit bright apical staining and fainter staining of cytoplasm and lateral cell margins. (E) In mouse pancreas sections, Myo5c localizes to the exocrine pancreas. (F,G) Higher magnification of the same section, triple-labeled for Myo5c (green), F-actin (red) and DAPI to label nuclei (blue), shows that Myo5c colocalizes with F-actin in the apical portions (arrows, F,G) of epithelial cells. Note that Myo5c does not colocalize with all F-actin-containing structures (arrowheads, F,G).

View larger version (104K): [in a new window]   Fig. 6. Overexpressing the GFP-Myo5c tail leads to the formation of puncta and tubulovesicular structures. In transfected cells, the GFP-Myo5c tail forms puncta and tubules (A,B,D,G), suggestive of a membranous compartment, whereas GFP alone has a diffuse distribution (C). (D-F) Antibodies to Myo5c stain the puncta. (G-I) The puncta formed by overexpression of the GFP-Myo5c tail do not colocalize with endogenous Myo5a, which shows a striking localization to small puncta in the periphery of the cell.

View larger version (108K): [in a new window]   Fig. 7. The GFP-Myo5c tail does not colocalize with or perturb the Golgi compartment, late endosomes or lysosomes. Arrows denote cells expressing the GFP-Myo5c tail, whereas arrowheads mark untransfected cells. (A-C) The GFP-Myo5c tail does not colocalize with golgin97, a membrane marker for the Golgi compartment. (D-F) Although the mannose-6-phosphate receptor cycles between the TGN, late endosomes, and lysosomes, at steady state the majority of the receptor is found on late endosomes, where it does not colocalize with the GFP-Myo5c tail. (G-I) The GFP-Myo5c tail also fails to colocalize with LAMP1, a membrane marker for lysosomes.

View larger version (111K): [in a new window]   Fig. 8. The overexpressed GFP-Myo5c tail colocalizes with and perturbs a compartment containing the transferrin receptor and rab8, but not rab11a. In all panels, arrows denote transfected cells and arrowheads denote untransfected cells. (A-C) The overexpressed GFP-Myo5c tail colocalizes with the transferrin receptor, a marker for the endosomal pathway. Furthermore, the distribution of the transferrin receptor is perturbed in cells expressing the GFP-Myo5c tail (arrows) as compared to untransfected cells (arrowheads). (D-I) Transfected cells were also stained for rab proteins to define specific membrane compartments. The GFP-Myo5c tail puncta colocalize with rab8 (D-F), but not rab11a (G-I). Similar to the results with the transferrin receptor, at least some of the rab8 distribution appears perturbed in transfected cells (arrow, F), whereas the distribution of rab11a does not appear to be significantly altered in transfected cells (arrows, I) versus untransfected cells (arrowhead, I).

View larger version (103K): [in a new window]   Fig. 9. In cells expressing the GFP-Myo5c tail, transferrin is taken up normally and colocalizes with the Myo5c tail after a short delay, but transferrin trafficking is inhibited. Cells were incubated in TRITC-transferrin for 10 minutes, followed by incubation in unlabeled transferrin for various `chase' times of 0, 5, 20 or 60 minutes. (A-C) Cells expressing the GFP-Myo5c tail (arrows) appear to internalize transferrin similar to untransfected cells (arrowhead). After 10 minutes of internalization, transferrin does not significantly colocalize with the GFP-Myo5c tail puncta, but by 20-60 minutes (D-I), transferrin shows obvious colocalization with the GFP-Myo5c tail. Furthermore, transferrin remains in cells expressing the GFP-Myo5c tail (arrows) for much longer than in untransfected cells (arrowheads), indicating that the GFP-Myo5c tail impedes transferrin trafficking. (J-K) Close examination of cells after 5 minutes of chase, when transferrin first begins to colocalize with the GFP-Myo5c tail, reveals that transferrin appears to associate with the margins of the GFP-Myo5c tail puncta (arrows).

View larger version (28K): [in a new window]   Fig. 10. A working model of class V myosins in membrane trafficking in HeLa cells. Myo5a exhibits a striking localization to small puncta of an unknown identity in the periphery. In HeLa cells Myo5b appears to associate with a major recycling compartment containing rab11a, as a dominant negative construct consisting of the Myo5b tail inhibits transferrin recycling and colocalizes with the transferrin receptor and rab11a (Lapierre et al., 2001). By contrast, the Myo5c tail associates with an apparently distinct transferrin-accessible compartment containing rab8 but not rab11a. Although the precise nature of the pathway associated with Myo5c in HeLa cells is unclear, this pathway is likely to be of particular importance in the epithelial cells where this myosin is most abundantly expressed.