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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 21 April 2009
doi: 10.1242/jcs.044339

Journal of Cell Science 122, 1499-1506 (2009)
Published by The Company of Biologists 2009
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
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 Google Scholar
Google Scholar
Right arrow Articles by Lalli, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lalli, G.
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?

RalA and the exocyst complex influence neuronal polarity through PAR-3 and aPKC

Giovanna Lalli

The Wolfson Centre for Age-Related Diseases, King's College London, London SE1 1UL, UK


Figure 1
View larger version (46K):
[in this window]
[in a new window]

  		Fig. 1. RalA is required for neuronal polarity. (A) Cortical neurons stained for RalA and neuron-specific β-III tubulin 48 hours after nucleofection of control (top) or RalA siRNA (centre). Control cells positive for RalA display a polarized morphology, whereas a considerable portion of RalA-depleted cells lacks a major process. These RalA-depleted cells (visualized by co-transfection of GFP with RalA siRNA) are not polarized, as shown by lack of tau-1 staining (bottom, arrowhead). The arrow indicates a tau-1-positive axon of an untransfected cell. Scale bars: 40 µm. (B) (Left) Quantification of polarization in cortical neurons 48 hours after nucleofection of control or RalA siRNA (mean ± s.e.m.: control siRNA, 74.53±0.87; RalA siRNA, 41.77±3.53; ***P<0.001). (Centre) Quantification of minor neurite length in control and RalA siRNA (mean ± s.e.m.: control siRNA, 26.80±0.87; RalA siRNA, 58.70±1.70; ***P<0.001). (Right) Active RalA levels increase at early stages of polarization. Neurons were lysed at the indicated time points after plating and assayed for active RalA levels by a Ral-GTP pull down. A representative blot is shown (top), together with data quantification (bottom; mean ± s.e.m.: 10 hours, 1.88±0.36; 24 hours, 1.66±0.29; *P<0.05). (C) At early stages of neuronal polarization (10 hours), RalA concentrates in one emerging neurite tip before PAR-3 (top row, arrowheads). Partial colocalization of RalA with PAR-3 at axon tips shown by a confocal section of a polarized neuron at later times (24 hours) (bottom row, arrowheads). Insets show a magnification of the axon growth cone. Scale bars: 20 µm. (D) Cortical neurons nucleofected with GFP, myc-tagged dominant-negative (RalA28N) or constitutively active (RalA72L) RalA, or an HA-tagged constitutively active RalGEF (Rlf-CAAX) stained with anti-myc or anti-HA and anti-tau-1 (right) antibodies 24 hours after plating. GFP-expressing neurons have a tau-1-positive axon (top row), whereas many cells transfected with Ral mutants or Rlf-CAAX lack tau-1 staining (arrowheads). Arrows indicate tau-1-positive axons of untransfected cells. Scale bar: 40 µm. (E) Quantification of the neuronal polarity defect (mean ± s.e.m.: GFP, 57.47±2.22; RalA28N, 34.23±0.67; RalA72L, 19.6±2.46; Rlf-CAAX, 24.53±5.42; **P<0.01). (F) Cortical neurons nucleofected with GFP or the indicated myc-tagged RalA constructs stained with anti-myc antibody 24 hours after nucleofection. Cells expressing constitutively active RalA (RalA72L) show an unpolarized phenotype also observed in cells expressing the active RalA mutants unable to interact with RalBP1 (RalA72LD49N) or with PLD (RalA72L{Delta}N11). By contrast, neurons expressing the active RalA mutant unable to interact with the exocyst complex (RalA72LD49E) display a polarized morphology. Scale bar: 20 µm. (G) (Top) Quantification of the polarity defect in neurons expressing the indicated constructs (mean ± s.e.m.: GFP, 67.12±2.36; RalA72L, 24.63±2.57; RalA72LD49N, 30.83±3.43; RalA72LD49E, 65.6±2.20; RalA72L{Delta}N11, 30.55±3.89; ***P<0.001). (Bottom) Western blot analysis shows similar expression levels of the myc-tagged RalA constructs.

 

Figure 2
View larger version (52K):
[in this window]
[in a new window]

  		Fig. 2. Cortical neurons lacking exocyst subunits are unpolarized. (A) Cortical neurons nucleofected with the indicated siRNA and co-stained for the indicated exocyst subunits and β-III tubulin 48 hours after plating. Cells depleted of Sec6, Sec8 or Exo84 lack a major process (arrowheads). Cells retaining exocyst subunits in the same field are shown as a staining control. (B) Neurons nucleofected with the indicated siRNA plus GFP stained for tau-1 48 hours after plating. Control cells show a tau-1-positive axon (asterisk), whereas cells lacking exocyst subunits display multiple tau-1-negative neurites (arrowheads). Arrows indicate tau-1-positive axons of untransfected cells. Scale bars: 40 µm. (C) Quantification of the neuronal polarity defect in cells lacking the indicated exocyst subunits (mean ± s.e.m.: control siRNA, 73.5±1.01; Sec6 siRNA, 24.62±3.79; Sec8 siRNA, 23.17±0.37; Exo84 siRNA, 30.18±2.68; ***P<0.001). (D) Depletion of exocyst subunits increases the length of the tau-1-negative minor neurites (mean ± s.e.m.: control siRNA, 26.80±0.87; Sec6 siRNA, 55.32±1.78; Sec8 siRNA, 53.77±1.87; Exo84 siRNA, 56.15±2.49; ***P<0.001). (E) The number of minor neurites emerging from the cell body increases only in Exo84-depleted neurons (mean ± s.e.m.: control siRNA, 3.82±0.13; RalA siRNA, 4.09±0.13; Sec6 siRNA, 3.90±0.11; Sec8 siRNA, 4.12±0.11; Exo84 siRNA, 5.68±0.30; ***P<0.001). (F) Depletion of exocyst subunits decreases total branch density (mean ± s.e.m.: control siRNA, 1.60±0.06; Sec6 siRNA, 0.89±0.06; Sec8 siRNA, 0.96±0.05; Exo84 siRNA, 0.61±0.06; ***P<0.001).

 

Figure 3
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 3. The exocyst complex interacts with PAR-3 and aPKC during neuronal polarization. (A) Confocal sections of neurons showing colocalization of Sec6, Sec8 and Exo84 with PAR-3 at the tips of emerging axons (arrowheads). Insets show a magnification of the axon growth cones. Panels on the right are projections of the confocal sections and show total neuronal morphology. Scale bars: 20 µm. (B) Gradual assembly of the exocyst complex assessed by co-IP of endogenous Sec8 and Exo84 using an anti-Sec6 antibody at the indicated times after plating. Endogenous Sec8 is immunoprecipitated in similar amounts throughout the analyzed time interval, whereas Exo84 and PAR-3 are precipitated in increasing amounts with time (top panel and graph). Quantification of fold increase in the interaction of Sec6 with the indicated proteins at 12 hours was calculated relative to 3 hours and normalized to the amount of correspondent protein in lysates (mean ± s.e.m.: Exo84, 1.96c0.32; Sec8, 1.11±0.09; PAR-3, 1.97±0.10). (C) Sec6, Sec8 and Exo84 co-immunoprecipitate with PAR-3 during neuronal polarization. The PAR-3-exocyst subunit interaction increases from 3 hours to 12 hours (bottom graph, mean ± s.e.m.: Sec6, 2.50±0.45; Sec8, 2.40±0.41; Exo84, 2.94±0.63). (D) Sec6, Sec8 and Exo84 co-immunoprecipitate with aPKC during neuronal polarization (top panel). The aPKC-exocyst subunit interaction increases from 3 hours to 12 hours (bottom graph, mean ± s.e.m.: Sec6, 2.48±0.48; Sec8, 2.48±0.54; Exo84, 2.16±0.41). ns lanes, negative control IP with a non-specific antibody.

 

Figure 4
View larger version (57K):
[in this window]
[in a new window]

  		Fig. 4. RalA regulates the interaction of PAR-3 with the exocyst. (A) Cortical neurons nucleofected with the indicated siRNA, fixed 48 hours after plating and stained for PAR-3, the protein of interest (RalA, Sec6 or Sec8, data not shown) or co-transfected GFP in the case of Exo84 siRNA and F-actin to visualize neuronal morphology. In control cells, PAR-3 accumulates at the axonal tip (top left). Unpolarized neurons lacking RalA (top right), Sec6 (bottom left) or Exo84 (bottom right) show a faint diffuse PAR-3 staining. Insets show a magnification of the numbered growth cones. Scale bars: 20 µm. (B) RalA co-immunoprecipitates with Sec6 at different time points during neuronal polarization. Asterisk indicates the RalA band visible under the light chain of the anti-Sec6 antibody used for the IP. The RalA band is not present in a control IP with a non-specific antibody (ns lane). (C) Cortical neurons were nucleofected with GFP or myc-tagged RalA28N. IP with an anti-Sec6 antibody was carried out 24 hours after plating. Co-IP of PAR-3 is significantly decreased in neurons transfected with RalA28N. ns lane, negative control IP with a non-specific antibody. (D) Quantification of fold decrease in Sec6-PAR-3 interaction in RalA28N or RalA72L-nucleofected neurons compared with GFP-transfected cells (mean ± s.e.m.: RalA28N, 0.37±0.07; RalA72L, 0.40±0.10).

 

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 2009