Distinct in vivo dynamics of vertebrate SUMO paralogues

Abstract

There are three mammalian SUMO paralogues: SUMO-1 is approximately 45% identical to SUMO-2 and SUMO-3, which are 96% identical to each other. It is currently unclear whether SUMO-1, -2, and -3 function in ways that are unique, redundant, or antagonistic. To address this question, we examined the dynamics of individual SUMO paralogues by using cell lines that stably express each of the mammalian SUMO proteins fused to the yellow fluorescent protein (YFP). Whereas SUMO-2 and -3 showed very similar distributions throughout the nucleoplasm, SUMO-1 was uniquely distributed to the nuclear envelope and to the nucleolus. Photobleaching experiments revealed that SUMO-1 dynamics was much slower than SUMO-2 and -3 dynamics. Additionally, the mobility of SUMO paralogues differed between subnuclear structures. Finally, the timing and distributions were dissimilar between paralogues as cells exited from mitosis. SUMO-1 was recruited to nuclear membrane as nuclear envelopes reformed in late anaphase, and accumulated rapidly into the nucleus. SUMO-2 and SUMO-3 localized to chromosome earlier and accumulated gradually during telophase. Together, these findings demonstrate that mammalian SUMO-1 shows patterns of utilization that are clearly discrete from the patterns of SUMO-2 and -3 throughout the cell cycle, arguing that it is functionally distinct and specifically regulated in vivo.

Figure 3.

YFP-SUMO response to stress. (A) YFP-SUMO-2 and YFP-SUMO-3 show increased conjugation with heat stress, but YFP-SUMO-1 does not. Western analysis of heat stressed (43°C; 10 min) or control (37°C; 10 min) YFP-SUMO–expressing (full-length) cell lines, indicating preferential up-regulation of YFP-SUMO-2 and YFP-SUMO-3 conjugation. Blot was probed with GFP antibodies (top) and actin antibodies as a loading/blotting control (bottom). (B) As₂O₃ treatment and increase of YFP-SUMO-1 signal at the PML bodies of live cells. YFP-SUMO-1 was imaged in cells before (0 h) and after 3.5 h of As₂O₃ treatment. Note the number and intensity increase of PML nuclear bodies inside the nuclei after treatment. Images were presented as glow-scale intensity coding palette shown on the right. Bar, 10 μm.

Figure 8.

Time-lapse imaging of YFP-SUMO chimeras during mitosis. Mitotic divisions of transgenic cells were recorded every 3 min after beginning of cell membrane constriction at late anaphase (designated as 0 min). Single metaphase pictures were taken not to induce artifacts or bleaching of signal while laser scanning. Upper right corner of metaphase pictures shows the time of capture before the start of cytokinesis. Insets at A and C show fixed metaphase cells, also stained with the DNA dye DAPI. YFP-SUMO-1 (A) differed from YFP-SUMO-2 (C) and SUMO-3 (E) with enhanced spindle signal and a distinct perinuclear enrichment. Nonconjugatable YFP-tagged SUMO1 (G) showed only a diffused cytoplasmic signal excluded from the chromosomal area at metaphase and anaphase. B, D, F, and H show corresponding DIC pictures of mitotic cells. Bars, 10 μm.

Figure 1.

Expression of YFP-SUMO fusion proteins in HeLa cells. (A) Expression levels of YFP-SUMOs. Western analysis of cells expressing YFP conjugates of both full length and unconjugatable (single glycine) forms of SUMO-1, SUMO-2, and SUMO-3. Same blot was probed with GFP antibodies (left), antibodies against human SUMO-1 (middle), or SUMO-2/3 (right). Asterisks denote YFP-SUMO-1–conjugated Ran-GAP1; arrow indicates the positions of full-length and unconjugatable forms of YFP-SUMO-1, YFP-SUMO-2, and YFP-SUMO-3 (left). Lanes are as indicated at the bottom of the panels. The letter “G” stands for single glycine (unconjugatable) versions of respective YFP-conjugated SUMO cell lines. Last lane is the untransfected HeLa parent line. Molecular weight markers (top to bottom) are 210, 134, 82, 41, 32, and 18 kDa. (B) YFP-SUMO expression does not alter mitotic index. Mitotic index frequencies of control HeLa cells and cells expressing YFP-SUMO conjugates. More than 500 cells were scored for each cell line in triplicate experiments by using phase contrast microscopy analyses of live cells.

Figure 2.

Distribution of YFP-SUMO chimeras in interphase cells. (A) Distribution of YFP-SUMOs in live cells. Stable transgenic cell lines were cultured on coverslip-bottom chambers and observed live for interphase localization patterns: (a) YFP-SUMO-1, (b) YFP-SUMO-2, (c) YFP-SUMO-3, (d) YFP-SUMO-1G, (e) YFP-SUMO-2G, (f) YFP-SUMO-3G, and (g) YFP. Arrows in d–f indicate PML body-like signal at the nucleoplasm of nonconjugatable SUMO transgenic cells. Fluorescence pictures (left) were paired with corresponding DIC images (right), indicating position of nuclei and nucleoli. (B) All SUMO paralogues localize to PML bodies. Colocalization of formaldehyde fixed YFP fusion proteins (green) with immunolocalized endogenous PML protein (red). S1, S2, and S3 stands for YFP-conjugated SUMO-1, SUMO-2, and SUMO-3, respectively. DNA is stained with DAPI (blue) and merged images are shown at the last column. (C) Distribution of YFP-SUMO-1 and RanGAP1 in fixed cells. Colocalization of formaldehyde-fixed YFP-SUMO-1 (green) with immunolocalized endogenous RanGAP1 (red). DNA is stained with DAPI (blue) and merged image is shown at the last column. Bars, 10 μm.

Figure 4.

FRAP analysis of YFP-SUMO chimeras. A spot of 2-μm-radius circle (dotted) was bleached within the nuclei or nucleoli of transgenic cell line, as indicated, and recovery of fluorescence was recorded in the bleached area (see Materials and Methods). Recovery of YFP-SUMO-1 (A) was slower than that of YFP-SUMO-2 (B) and YFP-SUMO-3 (C). Nucleolar FRAP analysis of YFP-SUMO-1 is shown in E. Bright field picture of the area where the nucleolus is located (dotted square) is shown as an inset of E. D shows Histone2B-YFP FRAP as a control experiment. Bar, 10 μm.

Figure 5.

FRAP analysis of YFP-SUMO chimeras. Average recovery curves of five individual cells from each cell line and standard deviations are shown in A to D. Half recovery durations and standard deviations are plotted in E.

Figure 6.

FLIP analysis of YFP-SUMO chimeras. A spot of 2-μm-radius circle (black line) was bleached within the nuclei or nucleoli of transgenic cells expressing YFP-SUMO-1 (A), YFP-SUMO-2 (B), or YFP-SUMO-3 (C) and loss of fluorescence at 10 μm away from the bleaching foci (white circle) was recorded and plotted (see Materials and Methods). Similar experiments were performed with nonconjugatable YFP-SUMO-1G– (D), YFP-SUMO-2G– (E), or YFP-SUMO-3G (F)–expressing cells, as well as with cells expressing YFP alone (G) or Histone2B-YFP (H). Nucleolar FLIP for YFP-SUMO-1 is shown in I. Bright field picture of the area where the nucleolus is located (dotted square) is shown as an inset in I. Bar, 10 μm.

Figure 7.

FLIP analysis of YFP-SUMO chimeras. Average depletion curves of five individual cells from each line and their standard deviations are shown as indicated in A and B. Half depletion durations are plotted in C, with standard deviations.