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First published online 29 November 2005
doi: 10.1242/jcs.02692

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TLS facilitates transport of mRNA encoding an actin-stabilizing protein to dendritic spines

Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan

View larger version (44K): [in a new window]   Fig. 1. TLS colocalized with RNA-containing particles in neuronal dendrites. (A) Mouse hippocampal neurons at culture day 21 (21 DIV) were labeled with SYTO14 (middle) and then immunostained with anti-TLS polyclonal antibody (left). Immunoreactive signals of endogenous TLS overlap with SYTO14-labeled RNA-containing particles (right). Scale bars, 10 µm in top panels, 5 µm in bottom panels. (B) TLS fused to GFP (FL) overlaps with RNA-containing particles stained with ethidium bromide (EtBr). Whereas GFP fused to an N-terminal fragment of TLS (N-TLS) is confined to the nucleus, that fused to the C-terminal fragment of TLS (C-TLS), which contains the RNA-binding domain, is observed in dendrites, where it is colocalized with RNA signals, even in the distal part of dendrites (over 80 µm from the soma). Arrowheads indicate representative TLS/RNA clusters. Bars, 2 µm in top three rows; 5 µm in bottom bottom. (C) Quantitative data obtained from B.

View larger version (26K): [in a new window]   Fig. 2. Reduced RNA translocation in TLS-null neuronal dendrites. (A) EtBr staining of RNA-containing particles in neuronal dendrites of wild-type (+/+) and TLS-null (–/–) neurons. RNA staining of dendrites extending 20 µm from the cell body is shown. Bar, 10 µm. (B) Quantitative data on RNA staining. DHPG treatment increases the RNA content in wild-type neurons (45.1±2.8% increase, P<0.01). In TLS-null neurons, no significant increase is observed after DHPG treatment (12.6±4.1% increase, P=0.299). Without DHPG stimulation, there was no significant difference in the RNA content between wild-type and TLS-null neurons (P=0.59). *P<0.01. (C) TLS overexpression does not change the amount of RNA in either wild-type or TLS-null neurons (P=0.059 and P=0.039, +/+ and –/–, respectively). DHPG treatment significantly upregulates the RNA content in both types of neurons (*P<0.01). Representative RNA staining is shown in A (right panels). Error bars indicate s.e.m.

View larger version (38K): [in a new window]   Fig. 3. Nd1-L mRNA content is decreased in TLS-null hippocampal neurons. (A) Cell in situ hybridization for detection of Nd1-L and ß-actin mRNA. Primary cultures of mouse hippocampal neurons were prepared from wild-type and TLS-null mouse embryos. At 23 DIV, the cells were treated with 50 mM DHPG for 60 minutes or left untreated as a control. The amount of Nd1-L mRNA in dendrites is increased by DHPG treatment. However, it is much lower in TLS-null dendrites and is not increased even after DHPG treatment. Bar, 5 µm. (B) Quantitative representation of the cell in situ hybridization in A. (C) Lower magnification of cell in situ hybridization data. Nd1-L mRNA is unevenly distributed in dendrites (arrowheads) and forms clusters at dendritic branching points (arrows). ß-actin mRNA is evenly distributed in dendrites (right). Bars, left, 20 µm, right, 10 µm. Inset in the middle panel shows the DIC image.

View larger version (49K): [in a new window]   Fig. 4. TLS associates with Nd1-L mRNA in mouse cortex polysome fraction. (A) Schematic representation of IP/RT-PCR. (B) Western blot to show specificity of anti-TLS monoclonal antibody used in this study. IgG: IP with IgG control; TLS: IP with anti-TLS monoclonal antibody; N: brain polysomal extract from TLS-null mouse cortex; P: brain polysomal extract from wild-type mouse cortex. (C) IP/RT-PCR to detect association of TLS with ß-actin mRNA (left) and Nd1-L mRNA (right). IgG: IP with IgG; anti-TLS: IP with anti-TLS antibody; no template: no template for PCR; cDNA: mouse cortex cDNA used as template. 2-log: 2-log DNA ladder; 100 bp: 100 bp DNA ladder.

View larger version (58K): [in a new window]   Fig. 5. Nd1-L mRNA associates with TLS. (A) Gel mobility shift assay using radioactive-labeled Nd1-L 3'-UTR probe. In vitro synthesized 493 nucleotide RNA probe for Nd1-L mRNA was radioactively labeled with 32P. The 32P-labeled probe was incubated with TLS protein prepared from rabbit reticulocytes and the complexes were resolved by native gel electrophoresis. A 50-fold molar excess of cold probe inhibited the binding of the radioactive probe to TLS. From left to right; lane 1, probe only; lane 2, probe with no competitor; lanes 3, 4, and 5, fivefold, 50-fold and 500-fold molar excess, respectively, of cold probe was added to the binding reaction together with the radioactive-labeled probes. (B) 100-fold molar excess of unlabeled irrelevant transcripts (205 nt transcript encoding pcDNA3.1/Myc-His polylinker site; 780 nt transcript encoding ß-actin 3'-UTR containing a zip code sequence) were challenged to in vitro binding of TLS and 32P-labeled Nd1-L probe.

View larger version (42K): [in a new window]   Fig. 6. Overexpression of TLS-GFP in TLS-null neurons increases amount of Nd1-L mRNA in dendrites. Primary cultures of TLS-null hippocampal neurons (21 DIV) were infected with adenovirus containing TLS-GFP to overexpress TLS protein. TLS-GFP-expressing TLS-null neurons were then subjected to cell in situ hybridization using an Nd1-L-specific probe. (A) TLS-null neurons express hardly any Nd1-L mRNA in their dendrites. Bar, 10 µm. (B) A quantitative representation of A. Overexpression of TLS-GFP increases the amount of Nd1-L mRNA by fivefold compared with that observed in TLS-null dendrites (P<0.05, n=50 individual dendrites). (C) Immunostaining with anti-GFP antibody after cell in situ hybridization for Nd1-L mRNA. TLS-GFP was colocalized with Nd1-L transcripts in mature spines of wild-type neurons (top). In TLS-null neurons expressing TLS-GFP, Nd1-L transcripts were also found in spines together with TLS-GFP (bottom, arrows). Bar, 30 µm.

View larger version (35K): [in a new window]   Fig. 7. Abnormal actin organization in TLS-null dendrites. Mouse primary hippocampal neurons prepared from either wild-type or TLS-null mice were fixed at 21 DIV, and then stained with Oregon Green phalloidin to visualize F-actin. (A) Actin organization in wild-type (A1) and TLS-null dendrites (B1). Wild-type dendrites normally have mushroom-shaped spines (arrowheads in A2). By contrast, numbers TLS-null dendrites did not have distinct heads to the spines (B2, arrows) or they produced long, thin, filopodia-like protrusions (B3, arrows). Bar, 20 µm in B1, 5 µm in B3.

View larger version (80K): [in a new window]   Fig. 8. Nd1-L suppresses cytochalasin-induced actin destabilization. Primary culture of mouse hippocampal neurons (21 DIV) were transfected with either RFP plasmids (vector) or plasmids containing RFP fused to Nd1-L (Nd1-L) by nuclear injection. RFP protein was ubiquitously distributed in RFP-expressing neurons. Nd1-L-RFP protein was localized in the cell body and dendrites (third row). When neurons expressing RFP were treated with cytochalasin D (0.2 µM) for 48 hours (vector/CytD), the actin filaments in the dendritic spines were disrupted. However, in Nd1-L-expressing neurons, cytochalasin D did not affect F-actin in the spines (Nd1-L/CytD). A highly magnified view of a dendrite is shown under each larger image. Bar, 30 µm; 5 µm for higher magnification.

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