Differential distribution of dynamin isoforms in mammalian cells

Abstract

Dynamins are 100-kDa 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 to define the distribution of the dynamin family members in a cultured rat epithelial cell model (Clone 9 cells). After long-distance reverse transcription (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 dynamin I and nine for dynamin III. 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 six different spliced forms tagged with green fluorescent protein. 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.

Figure 1

Amplification of three different dynamin isoforms (Dyn 1–3) from rat brain by long-distance RT-PCR. cDNA was synthesized from 10 μg of total RNA from rat brain using an oligo(dT)n primer. Amplification of the genes encoding full-length dynamin isoforms was performed using long-distance RT-PCR. Specific primers (A) were designed by MacVector and using the dynamin cDNA sequences from GenBank. The 5′ PCR primers were complementary to the initiation sequence. The 3′ PCR primers included the corresponding stop codon for each dynamin-coding region, thus discriminating the two alternative C termini of Dyn 1 and Dyn 3. After long-distance PCR, the reaction products were analyzed by agarose gel electrophoresis (B). Approximately 2.6-kb bands represent each dynamin isoform.

Figure 2

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 12 of the spliced variants were obtained from previously published studies (Nakata et al., 1991, 1993; Robinson et al., 1993; Cook et al., 1994, 1996; Sontag et al., 1994) and were published previously in two separate reviews (Robinson 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 “splicing regions,” which are blue. Dyn 1 and Dyn 2 each have two splicing regions, whereas Dyn 3 has three 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.

Figure 3

RT-PCR detects only Dyn 1 and Dyn 3 transcripts in purified populations of rat sensory neurons (DRGs). (A) Phase microscopic images of isolated neuronal cell populations used to test for the expression of all three dynamin genes. Cell types examined: a mixed culture of rat sensory neurons (DRGs) that includes glial cells and fibroblasts (arrows), a highly enriched culture of rat DRGs in which the supportive cells have been selectively eliminated, and a widely used, rat neoplastic adrenal cell line (PC-12). (B) RT-PCR of total mRNA isolated from each population of cells depicted in A. All three dynamin gene products were detected in the mixed population of DRGs. In contrast, removal of the supportive, nonneuronal cells reduced the amount of Dyn 2 to nearly undetectable levels, but the levels of Dyn 1 and Dyn 3 remained unchanged. PC-12 cells expressed nearly equal amounts of all three dynamin transcripts.

Figure 4

Expression of six distinct dynamin-GFP constructs in stably transfected epithelial cells. To test whether 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 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 two 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 kDa and the dynamin fusion protein band at 120 kDa. As expected, the higher-molecular-mass 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.

Figure 5

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–a") or TGN38 (b–b") 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 and a", arrowheads). In sharp contrast to the Dyn 2(aa) spliced variant that 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 second 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–c") or a TGN38 antibody (d–d") was seen. Bar, 10 μm.

Figure 6

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–b") to identify the endosomal and lysosomal compartments. Whereas the Dyn 1(ab)-GFP appeared to colocalize precisely with clathrin-coated pits at the plasma membrane (boxed regions), there was no colocalization 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–b") 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–c") and the trans-Golgi protein TGN38 (d–d"). Bar, 10 μm.

Figure 7

Dyn 3-GFP forms expressed in Clone 9 cells distribute diffusely to the cytoplasm or to unidentified vesicular structures. Fluorescence images of Dyn 3(baa)-GFP-transfected cells reveal a prominent population of brightly labeled vesicles, which did not colocalize with clathrin (a–a") at either the plasma membrane (boxes) or the Golgi apparatus (arrows). These dynamin-associated vesicles did not colocalize with endocytosed dextran (boxed regions), suggesting they are not late endocytic compartments (b–b"). In contrast, cells expressing Dyn 3(aaa)-GFP [a form differing from Dyn 3(baa) by only a 10-amino-acid insertion at the first splicing region] showed only a diffuse cytoplasmic fluorescence with a slight association with the Golgi apparatus, as demonstrated by costaining with an antibody to clathrin (c–c"). Bar, 10 μm.