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Immobile survival of motoneuron (SMN) protein stored in Cajal bodies can be mobilized by protein interactions

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Abstract

Reduced levels of survival of motoneuron (SMN) protein lead to spinal muscular atrophy, but it is still unknown how SMN protects motoneurons in the spinal cord against degeneration. In the nucleus, SMN is associated with two types of nuclear bodies denoted as gems and Cajal bodies (CBs). The 23 kDa isoform of fibroblast growth factor-2 (FGF-223) is a nuclear protein that binds to SMN and destabilizes the SMN-Gemin2 complex. In the present study, we show that FGF-223 depletes SMN from CBs without affecting their general structure. FRAP analysis of SMN-EGFP in CBs demonstrated that the majority of SMN in CBs remained mobile and allowed quantification of fast, slow and immobile nuclear SMN populations. The potential for SMN release was confirmed by in vivo photoconversion of SMN-Dendra2, indicating that CBs concentrate immobile SMN that could have a specialized function in CBs. FGF-223 accelerated SMN release from CBs, accompanied by a conversion of immobile SMN into a mobile population. Furthermore, FGF-223 caused snRNP accumulation in CBs. We propose a model in which Cajal bodies store immobile SMN that can be mobilized by its nuclear interaction partner FGF-223, leading to U4 snRNP accumulation in CBs, indicating a role for immobile SMN in tri-snRNP assembly.

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Abbreviations

CB:

Cajal body

FRAP:

Fluorescence recovery after photobleaching

SMN:

Survival of motoneuron protein

SMA:

Spinal muscular atrophy

FGF:

Fibroblast growth factor

SnRNP:

Small nuclear ribonucleoprotein particle

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Acknowledgments

The authors thank Kerstin Kuhlemann and Hildegard Streich for expert technical help. We are grateful to Dr. R. Bauerfeind and W. Posselt (Central Laser Microscopy Facility, Hannover Medical School) for expert technical advice on laser confocal microscopy. M.K.S. was supported by grants NYSTEM, contracts C026415 and C026714.

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Authors and Affiliations

  1. Institute of Neuroanatomy, Hannover Medical School, OE 4140, Carl-Neuberg-Str.1, 30625, Hannover, Germany

    Benjamin Förthmann, Hella Brinkmann, Andreas Ratzka, Claudia Grothe & Peter Claus

  2. Center for Systems Neuroscience, 30625, Hannover, Germany

    Benjamin Förthmann, Claudia Grothe & Peter Claus

  3. Department of Pathology and Anatomical Sciences, State University of New York, Buffalo, NY, 14214, USA

    Michal K. Stachowiak

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  1. Benjamin Förthmann
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  2. Hella Brinkmann
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Correspondence to Peter Claus.

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18_2012_1242_MOESM1_ESM.jpg

Supplemental Fig. 1 The velocities of CBs after overexpression of FGF-2 are not altered For live cell imaging, single CBs of pSMN-EGFP and pFGF-223-DsRed2 or (control) pFGF-218-DsRed2 transfected HEK293T cells were measured every minute up to 20 minutes unless the focused nuclear body left the volume as defined by z-sections. The total distance per minute for each CB was measured. All distances per minute were plotted as a velocity distribution (FGF-223-DsRed2 n = 149 single distances, FGF-218-DsRed2 n = 163 single distances). The overexpression of FGF-223 showed no influence on the distribution of CB velocities compared to FGF-218 overexpressing cells (χ2 -test, n.s., non-significant) (JPEG 529 kb)

18_2012_1242_MOESM2_ESM.jpg

Supplemental Fig. 2 Accumulation of U4 snRNPs at CBs after reduction of immobile SMN by FGF-223 HEK293T cells were transfected with pECFP (A, D, G, H, I) or pFGF-218-ECFP (B, E, K, L, M) or pFGF-223-ECFP (C, F, N, O, P) 24 hours before fixation. Cells were examined after fluorescence in situ hybridization (FISH, A3-P3) with probes for U4, U5 and U6 snRNAs and immunostainings with anti-coilin (A2-P2). Arrows point to the CBs in the merged images (A4-P4) that were enlarged in the inserted PDM images. Here, Intensity Correlation Analyses are demonstrated (LUT; orange, positive PDM; blue, negative PDM). Colocalized pixels (both channels vary synchronically from the mean pixel intensity) are represented by positive PDM (yellow). Scale bar, 2 μm (A1-L4), 0.2 μm (insets). For FGF-223 transfected cells and also controls probed against U4 snRNA, three individual cells have been shown each. Supplemental Fig. 2 (JPEG 7747 kb)

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Förthmann, B., Brinkmann, H., Ratzka, A. et al. Immobile survival of motoneuron (SMN) protein stored in Cajal bodies can be mobilized by protein interactions. Cell. Mol. Life Sci. 70, 2555–2568 (2013). https://doi.org/10.1007/s00018-012-1242-8

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