QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

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

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fujii, R. Articles by Takumi, T. Search for Related Content PubMed PubMed Citation Articles by Fujii, R. Articles by Takumi, T.

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.