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Dynamic movements of Ro52 cytoplasmic bodies along microtubules

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Abstract

The RING-finger protein Ro52/TRIM21 is known as an autoantigen and is recognized by anti-Ro/SSA antibodies, which are commonly found in patients with Sjögren’s syndrome and systemic lupus erythematosus. Recently, Ro52 has been shown to localize to distinct structures called cytoplasmic bodies and function as an E3 ubiquitin ligase. However, the Ro52 cytoplasmic bodies have not been well characterized. In this study, we investigated the Ro52 cytoplasmic bodies using fluorescence microscopy. This analysis revealed that the Ro52 cytoplasmic bodies are diffusely located in the cytoplasm and exist independently of TRIM5α cytoplasmic bodies. Our results further showed that the Ro52 cytoplasmic bodies are not stained with MitoTracker dye and are not colocalized with the proteasome subunit Rpt5, the caveolae component caveolin-1, the endosome markers (EEA1, Rab5, and Rab7), and the lysosome marker LAMP2. These results indicate that the Ro52 cytoplasmic bodies are not mitochondria, proteasome-enriched structures, caveolae, endosomes, or lysosomes. Importantly, the Ro52 cytoplasmic bodies are highly motile and are located along the microtubule network. These results suggest that the Ro52 cytoplasmic bodies are unidentified structures that are transported along the microtubule network.

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Abbreviations

HTLV-1:

Human T-cell leukemia virus type 1

TRIM:

Tripartite motif

HEK:

Human embryonic kidney

HRP:

Horseradish peroxidase

EGFP:

Enhanced green fluorescent protein

PCR:

Polymerase chain reaction

DAPI:

4′,6-Diamidine-2′-phenylindole dihydrochloride

WGA:

Wheat germ agglutinin

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Acknowledgments

We thank Dr. Roger Tsien for providing mRFP cDNA. This work was supported in part by National Institutes of Health Grant R01AG024497 (to T.K.).

Author information

Correspondence to Tetsu Kamitani.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplemental Movie 1. High motility of Ro52 cytoplasmic bodies. To observe live images of Ro52 cytoplasmic bodies, live cell fluorescence microscopy was performed. HT1080 cells expressing Ro52-EGFP were taken as images in a 60-frame sequence captured at 2-second intervals using live cell fluorescence microscopy with a 100× objective lens. These images were used to generate Supplemental Movie 1 (MOV 1168 kb)

Supplemental Movie 2. Ro52 cytoplasmic bodies showing long tracks. To clearly visualize the tracks, we identified the cytoplasmic bodies showing long tracks in the original 60 frames (2 s-intervals). The individual cytoplasmic bodies were then labeled with 5 different colors (purple, yellow, red, green, and blue) to generate Supplemental Movie 2 (MOV 1138 kb)

Supplemental Movie 1. High motility of Ro52 cytoplasmic bodies. To observe live images of Ro52 cytoplasmic bodies, live cell fluorescence microscopy was performed. HT1080 cells expressing Ro52-EGFP were taken as images in a 60-frame sequence captured at 2-second intervals using live cell fluorescence microscopy with a 100× objective lens. These images were used to generate Supplemental Movie 1 (MOV 1168 kb)

Supplemental Movie 2. Ro52 cytoplasmic bodies showing long tracks. To clearly visualize the tracks, we identified the cytoplasmic bodies showing long tracks in the original 60 frames (2 s-intervals). The individual cytoplasmic bodies were then labeled with 5 different colors (purple, yellow, red, green, and blue) to generate Supplemental Movie 2 (MOV 1138 kb)

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Tanaka, M., Tanji, K., Niida, M. et al. Dynamic movements of Ro52 cytoplasmic bodies along microtubules. Histochem Cell Biol 133, 273–284 (2010) doi:10.1007/s00418-009-0669-y

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Keywords

  • Ro52
  • TRIM21
  • Ubiquitin ligase
  • Cytoplasmic bodies
  • Microtubule