spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Duchaîne, T. F.
Right arrow Articles by DesGroseillers, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Duchaîne, T. F.
Right arrow Articles by DesGroseillers, L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Staufen2 isoforms localize to the somatodendritic domain of neurons and interact with different organelles

Thomas F. Duchaîne1,*, Indradeo Hemraj2,*, Luc Furic1, Anke Deitinghoff2, Michael A. Kiebler2 and Luc DesGroseillers1,3,{ddagger}

1 Department of Biochemistry, University of Montreal, Montreal, H3C 3J7, Canada
3 Centre de Recherches en Sciences Neurologiques, University of Montreal, Montreal, H3C 3J7, Canada
2 Max-Planck-Institute for Developmental Biology, Tübingen, Germany
* These authors contributed equally to this work



View larger version (101K):

[in a new window]
  		Fig. 6. Stau2 does not colocalize with Stau1 in distal dendrites. (A) Confocal images from fixed, mature hippocampal neurons in culture. Neurons were labeled with mouse polyclonal anti-Stau2 (green) and rabbit anti-Stau1 (red) antibodies. The third panel represents the superposition of both green and red signals. (B) Higher magnification of images taken with conventional fluorescence microscopy. Neurons were labeled with mouse polyclonal anti-Stau2 (green) and rabbit anti-Stau1 (red) antibodies. The middle and right panels represent the superposition of both green and red signals at different magnification. The average diameter of the cell body of a typical CA1 pyramidal neuron is between 8 and 12 µm.

 


View larger version (62K):

[in a new window]
  		Fig. 1. Molecular analysis of Stau2 isoforms. (A) Alternative splicing in the 5' end of the Stau2 gene. Alternative splicing generates two transcripts, T1 and T2, that differ by the insertion of 131 nucleotides in T1 compared with T2. Arrows indicate the first in frame ATG initiation codon for translation of Stau262 and Stau259/52, respectively. Note that the two ATG are not in frame in the T1 transcript and therefore translation initiation of Stau262 starts at the second ATG. * indicates the presence of stop codons in the three open reading frames. (B) Amino-acid sequence alignment of Stau2 dsRBDs and the consensus dsRBD sequence. (C) Alignment of the amino-acid sequence of mouse Stau262 (62), Stau259 (59), Stau252 (52), mouse Stau155 (St1) and Drosophila Staufen (Dro). The sequence of Stau252 is from Buchner et al. (Buchner et al., 1999Go). The position of dsRBDs (dsRBD1 to dsRBD5) is indicated above the sequence. Dots indicate amino-acid residues identical to those of Stau2. All identical amino-acid residues are boxed. Dashes represent gaps in the amino-acid sequences. These sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession numbers AF459099 and AF459100.

 


View larger version (26K):

[in a new window]
  		Fig. 2. Tissue distribution of Stau2 proteins and transcripts. (A) Western blot analysis of Stau2 in neurons. Brain extracts were separated by SDS-PAGE and stained with anti-Stau2 antibodies. Lane 1, mouse monoclonal; lane 2, rabbit polyclonal; lane 3, mouse polyclonal. (B) Northern blot analysis of Stau2 expression in mouse tissues. mRNAs isolated from multiple tissues were transferred to a nylon membrane and hybridized with either a mouse Stau2 cDNA probe (top panel), a mouse Stau1 cDNA probe (middle panel) or an actin cDNA probe (lower panel). Lane 1, brain; lane 2, heart; lane 3, spleen; lane 4, kidney; lane 5, testis; lane 6, ovary. Blots were exposed for 16 hours. (C) RT-PCR analysis of the Stau2 transcripts in brain. Total RNA was isolated from mouse brain and RT-PCR amplified with primers flanking the alternatively spliced exon. PCR amplification was performed with different amounts of starting RNA and cycle numbers. Control RNA (1 µg, no reverse transcription step) was amplified for 32 cycles.

 


View larger version (21K):

[in a new window]
  		Fig. 3. RNA-binding activity of Stau2 dsRBDs. Full-length mouse Stau2 fused to MBP (FL) and different dsRBDs fused to GST (1 to 5) were expressed in bacteria. Crude protein extracts were resolved by SDS PAGE and transferred on nitrocellulose membranes. Filters were incubated with either [32P]bicoid 3'UTR RNA (A) or anti-GST antibodies (B). MBP, overexpressed MBP protein; BSA, 5 µg BSA. (C) A schematic representation of the fusion proteins with a summary of their RNA-binding capacity. Black and grey boxes represent the major and minor RNA-binding domains, respectively, whereas white boxes represent regions with RNA-binding consensus sequence but lacking RNA-binding activity in vitro. The hatched box indicates the position of the region similar to the MAP1B microtubule-binding domain. Boxes with dotted lines represent the rearranged dsRBD5 domain.

 


View larger version (93K):

[in a new window]
  		Fig. 4. Stau2 is found in the somatodendritic compartment of neurons and colocalizes with tubulin in dendrites. (A) Hippocampal neurons in culture were fixed and labeled with rabbit polyclonal anti-Stau2. Inset: high magnification of Stau2 in dendrites. (B) Hippocampal neurons in culture were fixed and labeled with rabbit anti-Stau2 (red) or mouse monoclonal anti-tubulin (green) antibodies. The lower panel represents the superposition of both red and green signals. The average diameter of the cell body of a typical CA1 pyramidal neuron is between 8 and 12 µm.

 


View larger version (77K):

[in a new window]
  		Fig. 5. Stau2 is found in dendrites but not in axons. (A) Hippocampal neurons in culture were fixed and labeled with rabbit anti-Stau2 (green) or mouse monoclonal anti-MAP2 (red) antibodies. Arrows indicate axons (negative in MAP2) with little or no staining for Stau2. The arrowhead indicates a very thin dendrite (MAP2 and Stau2 positive) comparable in size to the marked axon. (B) Hippocampal neurons in culture were fixed and labeled with rabbit anti-Stau2 (green) or mouse monoclonal anti-tau1 (red) antibodies. Arrows indicate axons (negative for Stau2). In (A,B), the upper left panels show phase contrast microscopy. The lower right panels show superposition of both red and green signals. The average diameter of the cell body of a typical CA1 pyramidal neuron is between 8 and 12 µm.

 


View larger version (31K):

[in a new window]
  		Fig. 7. Stau2 splice isoforms are found in high-density particles. (A) Rat cortical neurons (6 DIV) were lysed, homogenized and cytoplasmic and nuclear extracts were prepared by low-speed centrifugation. The supernatant (cytosolic fraction, C) and pellet (nuclear fraction, N) were recovered and analyzed by western blotting with monoclonal anti-Stau2 antibodies. The purity of each fraction was tested with antibodies directed against a nuclear (histone H1) and a cytosolic (ribosomal protein P) protein. (B) Western blot analysis of the S100 and P100 fractions following different treatments. Cytoplasmic extracts were either left untreated (-) or were treated for 30 minutes prior to centrifugation with 0.5 M KCl (KCl), 0.5% Nonidet P40 (NP40), 25 mM EDTA (EDTA), 300 U/ml micrococcal nuclease (RNase) and 0.5% Nonidet P40 and 25 mM EDTA (NP40+EDTA). S100/P100 fractions were analyzed with monoclonal anti-Stau2 (Stau2), anti-ribosomal protein L7a (L7a), anti-{alpha}-tubulin (Tub), and anti-calnexin (CNX) antibodies. The same results were obtained in two independent experiments.

 


View larger version (53K):

[in a new window]
  		Fig. 8. Stau259 and Stau252 isoforms are associated with ribosomes. (A) Cytoplasmic extracts from rat cortical neurons in culture were prepared, placed on a 20-60% discontinuous sucrose gradient and centrifuged at 175,000 g for 3 hours. Fractions were recovered and analyzed by western blotting with monoclonal anti-Stau2 (Stau2), anti-L7a (L7a) and anti-calnexin (CNX) antibodies. The same results were obtained from three independent experiments. R, ribosomes. (B) Cytoplasmic extracts from neurons in culture were treated with 25 mM EDTA to dissociate ribosomal subunits and centrifuged at 250,000 g for 4 hours on a 10-40% continuous sucrose gradient. Fractions were recovered and analyzed by western blotting with monoclonal anti-Stau2 antibodies. Similar results were obtained with rabbit polyclonal anti-Stau2 antibodies (data not shown). The position of the 40S and 60S ribosomal subunits was determined with a spectrophotometer set at 254 nm.

 


View larger version (23K):

[in a new window]
  		Fig. 9. Co-immunoprecipitation of Stau259 with ribosomes. Cytoplasmic extracts from rat cortical neurons in culture were prepared and immunoprecipitated with (A) normal human serum (NHS) and anti-ribosomal protein P (P) or (B) pre-immune serum (PI) and rabbit polyclonal anti-Stau2 (Stau2) antibodies. Immunoprecipitates were analyzed by western blotting with anti-L7a, monoclonal anti-Stau2 and anti-P as indicated. Results similar to those in (A) were obtained when the immunoprecipitation was carried out with the anti-L7a antibodies (data not shown). (C) The Stau2 immunoprecipitates (as in B) were also analyzed by northern blotting for the presence of 18S rRNA (small ribosomal subunit). These experiments were performed three times with reproducible results.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2002