Abstract
SUN proteins participate in diverse cellular activities, many of which are connected to the nuclear envelope. Recently, the family member SUN1 has been linked to novel biological activities. These include the regulation of nucleoli, intranuclear compartments that assemble ribosomal subunits. We show that SUN1 associates with nucleoli in several mammalian epithelial cell lines. This nucleolar localization is not shared by all cell types, as SUN1 concentrates at the nuclear envelope in ganglionic neurons and non-neuronal satellite cells. Database analyses and Western blotting emphasize the complexity of SUN1 protein profiles in different mammalian cells. We constructed a STRING network which identifies SUN1-related proteins as part of a larger network that includes several nucleolar proteins. Taken together, the current data highlight the diversity of SUN1 proteins and emphasize the possible links between SUN1 and nucleoli.
Keywords: Nucleus, Nucleolus, Nuclear envelope, SUN1
Specifications Table
Subject area | Biology |
More specific subject area | Cell biology |
Type of data | Fluorescence microscopy, 3D reconstruction, Western blot, sequence alignment, STRING network |
How data was acquired | Confocal microscopy, Western blotting, database search and analysis |
Data format | Analyzed |
Experimental factors | Oxidative stress |
Data source location | McGill University, Montreal, Canada and NCBI |
Data accessibility | https://www.ncbi.nlm.nih.gov/protein/ |
Value of the data
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SUN1-related proteins can localize to nucleoli.
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SUN1 nucleolar association is maintained during oxidative stress.
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SUN1 nucleolar localization is cell type specific.
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SUN1 is part of a larger network with links to the nucleolus.
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Data provide the foundation to define the mechanisms through which SUN1 controls nucleolar functions.
1. Data
SUN (Sad1-UNC84 homology) proteins connect the nuclear lamina to the cytoskeleton [1], [2], [3]. Most SUN proteins studied to date concentrate in the inner nuclear membrane, where they interact with other membrane components and the nuclear lamina. In the perinuclear space, SUN domains bind KASH (Klarsicht, ANC-1 and Syne homology) proteins that are embedded in the outer nuclear membrane. In this scenario, SUN proteins contain domains in the nucleoplasm, the inner nuclear membrane and the perinuclear space.
Members of the SUN protein family contribute to a wide variety of biological activities, including mechanotransduction to the nucleus [4], formation of bipolar spindles and progression through mitosis [5], DNA double strand break repair [6] and HIV replication [7]. Moreover, SUN1 and SUN2 exhibit cell-type specific functions that are critical to nucleokinesis in the developing cerebellum [8]. While there is some functional overlap between SUN1 and SUN2, both proteins make also unique contributions to cell physiology.
Our data focus on SUN1, a protein with established links to human health. For example, SUN1 promotes proper myonuclear positioning [9], and SUN1 is a disease modifier gene for Emery–Dreyfus muscular dystrophy [9]. In addition, SUN1 can regulate adhesion to the extracellular matrix and thus affects the formation of invadopodia in cancer cells [10]. Recently, novel SUN1 activities have been described that go beyond the interaction with nuclear membranes or the lamina, suggesting that SUN1 controls nucleolar function [11], mRNA export [12] and sperm development [13]. Multiple SUN1 isoforms exist [13], [14], [15] that can differ in subcellular localization, association with binding partners and cellular function. These diverse properties of SUN1 proteins are not fully understood. Several of these properties are addressed in Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5 and Table 1.
Table 1.
Cells |
Ab-1 |
Ab-2 |
||
---|---|---|---|---|
IF | WB | IF | WB | |
LLC-PK1 | Nucleoli | >180 kD; | Nucleoli | Multiple bands |
~95 kD | ||||
HK-2 | Cytoplasm, nucleus, nucleoli, (nuclear envelope) | >180 kD; | Cytoplasm, nucleus | ~140 kD (major); additional bands of larger and smaller molecular mass |
~95 kD | ||||
HeLa | Cytoplasm, nucleus, nucleoli, (NE) | >180 kD; | Nucleus, cytoplasm | ~140 kD (major); additional bands of larger and smaller molecular mass |
~95 kD | ||||
Neurons | Cytoplasm, (nucleus) | >180 kD; | NE | ~100 kD (major) |
smaller bands | ||||
Satellite cells | Cytoplasm, nucleus | >180 kD; | NE | ~100 kD (major) |
smaller bands |
Supplemental File 1. SUN1 interactors identified by STRING are listed. The properties of individual nodes are described. The presence of SUN1 interactors in nucleoli is based on data published for spatial proteomics that investigated the proteome of different subcellular compartments [17].
2. Experimental design, materials and methods
2.1. Antibodies
The following antibodies were used for immunostaining at the dilutions indicated: Sun1 (BethylLab A303-438A, 1:125; ab-1) or Sun1-specific antibodies (1:50; ab-2), kindly provided by M. Alsheimer (University of Würzburg, Germany), RPA194 (Santa Cruz, sc-48385; 1:500), lamin A/C (Santa Cruz, sc-6215; 1:500). For Western blotting ab-1 and ab-2 were diluted 1:1,000. Fig. 1A shows the regions recognized by ab-1 and ab-2.
2.2. Cell culture
Conditions for growth of LLC-PK1 cells (renal proximal tubule, porcine) and superior cervical ganglion (SCG, mouse) neurons have been published [[18] and references therein]. HeLa (cervix adenocarcinoma, human) and HK2 (renal proximal tubule, human) cells were grown according to standard protocols. For immunostaining, mouse SCG neurons and mouse ganglionic non-neuronal cells were co-cultured and analyzed with the same methods.
2.3. Stress exposure
Oxidative stress was induced with 0.5 mM arsenite added in growth medium for 30 min; controls were treated with the vehicle water in growth medium.
2.4. Immunofluorescence
Two different methods were employed to detect Sun1 by immunostaining. Cells were either fixed with formaldehyde (ab-1) or incubated with cold methanol (ab-2), essentially as described [18]. All secondary antibodies (Jackson ImmunoResearch) were affinity-purified and pre-adsorbed to mammalian proteins to minimize non-specific binding.
In brief, for ab-1, cells were washed once in PBS, fixed in 3.7% formaldehyde/PBS for 20 min at room temperature and rinsed with PBS. Membranes were permeabilized with 0.1% Triton X-100/2 mg/ml BSA/0.1% NaN3 for 5 min at room temperature. Non-specific binding sites were blocked with 0.05% Tween 20, 5% FBS, 1 mM NaN3 in PBS for 1 hour at room temperature.
For staining with ab-2, cultured cells were washed twice with PBS. Cells were fixed and permeabilized with 100% cold methanol for 10 min at −20 °C. After rinsing with PBS, non-specific binding sites were blocked with 5% serum in PBS for 1 h at room temperature. Primary antibodies were diluted in 5% serum/PBS and cleared by 5 min centrifugation at 13,000 rpm (microcentrifuge). Samples were incubated with the supernatant overnight at 4 °C. Following two washes in PBS (15 min/wash step), affinity-purified secondary antibodies were added for 1 h at room temperature. Samples were washed three times with PBS (5 min/wash step), and nuclei were stained with DAPI.
2.5. Microscopy and 3D reconstruction
Image acquisition and protocols for 3D reconstruction followed standard protocols [18], [19], [20]. In brief, confocal images were acquired with a Zeiss LSM510 microscope in the multi-track mode. Filter settings were chosen to minimize cross-talk between the channels. Images were processed in Photoshop. 3D reconstructions were carried out with Imaris software.
2.6. Western blotting
Crude extracts were prepared, separated by SDS-PAGE and analyzed by Western blotting essentially as described [19].
2.7. SUN1 interaction network
SUN1 interactions were analyzed with STRING [21]. The analyses were performed at the highest confidence setting of 0.9. Proteomics data for HeLa cells have been published [17].
Acknowledgements
We thank Manfred Alsheimer, University of Würzburg, for his generous gift of antibodies against SUN1. This work was supported by grants from NSERC (Natural Sciences and Engineering Research Council of Canada) to US and JFP (RGPIN-04137-15, RGPIN-262240-11).
Footnotes
Transparency data associated with this article can be found in the online version at doi:10.1016/j.dib.2017.05.028.
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.dib.2017.05.028.
Transparency document. Supplementary material
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Appendix A. Supplementary material
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