Differential distribution of dynamin isoforms in mammalian cells
Mol Biol Cell 1998 Sep;9(9):2595-609
Dynamins are 100-kD GTPases that are essential for clathrin-coated vesicle formation during receptor-mediated endocytosis. To date, three different dynamin genes have been identified, with each gene expressing at least four different alternatively spliced forms. Currently, it is unclear whether these different dynamin gene products perform distinct or redundant cellular functions. Therefore, the focus of this study was to identify additional spliced variants of dynamin from rat tissues and define the distribution of the dynamin family members in a cultured rat epithelial cell model (Clone 9 cells).
Following long distance RT-PCR of mRNA from different rat tissues, the full-length cDNAs encoding the different dynamin isoforms were sequenced and revealed four additional spliced variants for Dyn 1 and nine for Dyn 3. Thus, in rat tissues there are a total of at least 25 different mRNAs produced from the three dynamin genes. Subsequently, we generated stably transfected Clone 9 cells expressing full-length cDNAs of 6 different spliced forms tagged with green fluorescent protein (GFP).
Confocal or fluorescence microscopy of these transfected cells revealed that many of the dynamin proteins associate with distinct membrane compartments, which include clathrin-coated pits at the plasma membrane and the Golgi apparatus, and several undefined vesicle populations. These results indicate that the dynamin family is more extensive than was originally predicted and suggest that the different dynamin proteins are localized to distinct cytoplasmic or membrane compartments.
More than 25 distinct dynamin mRNAs are expressed in rat tissues. Schematic illustration of the dynamin family of proteins. The three known dynamin gene products are diagrammed, and the corresponding alternatively-spliced sites are indicated.
Amino acid sequences of twelve of the spliced variants were obtained from previously published studies (Cook et al., 1996; Cook et al., 1994; Nakata et al., 1991; Nakata et al., 1993; Robinson et al., 1993; Sontag et al., 1994) and were published previously in two separate reviews (Robinaon et al., 1994; Urrutia et al., 1997) . Sequence information for the additional 13 inserts or substitutions was obtained in this study by long distance RT-PCR of total mRNA from several rat tissues.
We have denoted the clustered sites of alternative splicing within each transcript as ìsplicing regionsî which are colored blue. Dyn 1 and Dyn 2 each have 2 splicing regions, whereas Dyn 3 has 3 splicing regions. Note that Dyn 1 and Dyn 3 each have a spliced insert encoding premature terminations in splicing regions 2 or 1 and 2, respectively. Yellow represents the GTP-binding sites, red and green represent the pleckstrin homology (PH) and proline rich (PR) domains, respectively.
Dynamin 1 localization
Bar, 10 um.
Expression of six distinct dynamin-GFP constructs in stably transfected epithelial cells. To test if transfected Clone 9 cells were expressing dynamin-GFP, total protein from homogenates of the six different dynamin-expressing cell lines was separated by SDS-PAGE and subjected to immunoblot analysis with either the Pan-dynamin antibody (MC65), isoform specific antibodies for Dyn 2 and Dyn 3, or a GFP antibody.
(A) Immunoblots of homogenates from cells expressing 2 spliced variants of either Dyn 1-GFP or Dyn 2-GFP. The Pan-dynamin antibody (MC65) recognized both the endogenous dynamin protein band at 100 kD and the dynamin fusion protein band at 120 kD. As expected, the higher molecular weight band was only seen when blotted with the anti-GFP antibody. For immunoblot analysis of Dyn 3-GFP expressing cells (B) a combination of isoform specific antibodies to Dyn 2 and Dyn 3 was used because the Dyn 3-GFP fusion protein was not recognized by the Pan-MC65 antibody. With these antibodies both the endogenous dynamin and expressed Dyn 3-GFP were seen.
Dynamin 2 localization
Bar, 10 um.
Two variants of Dyn 2-GFP show dramatic differences in affinity for the Golgi apparatus. Double immunofluorescence staining of cells expressing Dyn 2(aa)-GFP with antibodies to clathrin (a-aî) or TGN38 (b-bî) revealed that Dyn 2(aa)-GFP has a strong affinity for membrane tubules of the Golgi apparatus (arrows). Interestingly, Dyn 2(aa) was also localized in the cortical ruffles of transfected cells (a+aî, arrowheads).
In sharp contrast to the Dyn 2(aa) spliced variant which associated with clathrin at both the plasma membrane and the Golgi apparatus, Dyn 2(ab)-GFP, a form different by only a four amino acid deletion in the first splicing region, localized to clathrin-coated pits at the plasma membrane only. No affinity for the Golgi apparatus (arrows) in cells stained with a clathrin antibody (c-cî) or a TGN38 antibody (d-dî) was seen.
Dynamin 3 localization
Bar, 10 um.
Different distributions for two forms of Dyn 1-GFP in Clone 9 cells. Stably transfected Clone 9 cells expressing Dyn 1(ab)-GFP were immunostained with antibodies to clathrin (a-a") or labeled with internalized fluorescent dextran (b-bî) to identify the endosomal/lysosomal compartments. Whereas the Dyn 1(ab)-GFP appeared to co-localize precisely with clathrin-coated pits at the plasma membrane (boxed regions), there was no co-localization with clathrin at the Golgi apparatus.
Large vesiclular structures associated with Dyn 1-GFP patches were seen in these transfected cells and had no colocalization with endocytosed dextran (b-bî) suggesting that these structures do not represent a lysosomal compartment. In contrast to Dyn 1(ab)-GFP, Dyn 1(bb)-GFP exhibited a diffuse cytoplasmic distribution in transfected cells with some localization to the Golgi apparatus (arrows) as confirmed by double immunofluorescence staining for clathrin (c-cî) and the trans-Golgi protein TGN38 (d-dî).
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