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 28 March 2006
doi: 10.1242/jcs.02875

Journal of Cell Science 119, 1604-1611 (2006)
Published by The Company of Biologists 2006
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 Related articles in JCS
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 Tothova, J.
Right arrow Articles by Schiaffino, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tothova, J.
Right arrow Articles by Schiaffino, S.
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?

NFATc1 nucleocytoplasmic shuttling is controlled by nerve activity in skeletal muscle

Jana Tothova1,2,*, Bert Blaauw1,2,3,*, Giorgia Pallafacchina1,2,*, Rüdiger Rudolf1,2, Carla Argentini1,2, Carlo Reggiani3,4 and Stefano Schiaffino1,2,4,{ddagger}

1 Department of Biomedical Sciences, University of Padova, Padova, Italy
2 Venetian Institute of Molecular Medicine (VIMM), Padova, Italy
3 Department of Anatomy and Physiology, University of Padova, Padova, Italy
4 CNR Institute of Neurosciences, University of Padova, Padova, Italy


Figure 1
View larger version (103K):

[in a new window]
  		Fig. 1. NFATc1-GFP has a predominantly nuclear localization in most slow muscle fibers and a predominantly cytoplasmic localization in most fast fibers. (A-D) Longitudinal (A,B) and transversal (C,D) sections of mouse soleus (A,C) and tibialis anterior (TA) muscles (B,D) transfected with plasmids coding for NFATc1-GFP fusion protein. Notice the nuclear localization of NFATc1-GFP in soleus (A,C) and the presence of multiple foci of GFP fluorescence when nuclei are examined at higher magnification (A, inset). By contrast, NFATc1-GFP is either homogeneously distributed in the nuclei and cytoplasm of transfected TA muscle fibers (B,D) or some nuclear profiles are GFP-negative in this muscle, as shown by DAPI staining (D, inset). (E-F) Transverse sections of soleus (E) and TA (F) muscles transfected with plasmids coding for a constitutively active flag-tagged mutant of NFATc1 (caNFATc1) and analyzed with anti-flag antibodies (left panels) and nuclear DAPI staining (central panels). Images were merged to demonstrate colocalization (right panels). Bars, 30 µm (A-F); 5 µm (inset in A); 10 µm (inset in D).

 

Figure 2
View larger version (12K):

[in a new window]
  		Fig. 2. Proportion of muscle fibers showing predominantly nuclear localization of NFATc1-GFP in normal mouse soleus and TA muscles, and in muscles subjected to various experimental conditions. Values are expressed as mean ± s.e.m. Muscles were transfected with plasmid coding for NFATc1-GFP and 7 days later were either immediately removed for analysis (control) or subjected to various experimental conditions: anesth, 2 hours anaesthesia; den, 2 hours denervation; overload, soleus muscle from contralateral leg; cain, soleus muscle co-transfected with the calcineurin inhibitor cain; 100 Hz or 20 Hz, TA muscles stimulated for 2 hours at 100 Hz or 20 Hz, respectively; 20 Hz + cain, TA muscles co-transfected with cain and stimulated for 2 hours at 20 Hz.

 

Figure 3
View larger version (54K):

[in a new window]
  		Fig. 3. Nucleus-to-cytoplasm translocation of NFATc1-GFP is rapidly induced by inactivity in slow muscle fibers. Transversal sections of soleus muscles transfected with NFATc1-GFP and examined 2 hours after section of the sciatic nerve (A) or after 2 hours anaesthesia (B). Notice cytoplasmic distribution of NFATc1-GFP (A,B) with no GFP fluorescence in DAPI-stained nuclei (A, inset). Bars, 30 µm (A,B); 10 µm (inset in A).

 

Figure 4
View larger version (15K):

[in a new window]
  		Fig. 4. (A) Force-frequency relation. (B,C) Representative mechanical responses at (B) high stimulation frequency (100 Hz) and (C) low stimulation frequency (20 Hz). Tibialis anterior was stimulated via common peroneal nerve and force was recorded with a force transducer (FT03E, Grass, Warwick, USA) connected to the mouse foot.

 

Figure 5
View larger version (48K):

[in a new window]
  		Fig. 5. Nuclear translocation of NFATc1-GFP is induced in fast muscle fibers by electrostimulation with a tonic low-frequency pattern of impulses but not by a phasic high-frequency pattern. TA muscles transfected with NFATc1-GFP were electrostimulated for 2 hours via the common peroneal nerve with two distinct impulse patterns: (A) a phasic high-frequency (100 Hz) pattern, which resembles the firing pattern of fast motor neurons or (B) a tonic low-frequency (20 Hz) pattern, which resembles the firing pattern of slow motor neurons. Notice that NFATc1-GFP maintains a cytoplasmic localization after stimulation with the `fast' pattern (A), with negative nuclear profiles (see merge of GFP and DAPI staining in A, insets), but shows a nuclear translocation after stimulation with the `slow' pattern (B), with multiple intranuclear foci of fluorescence, similar to those seen in soleus muscle fibers (B, inset, compare with Fig. 1A). Bars, 30 µm (A-B); 10 µm (inset in A); 5 µm (inset in B).

 

Figure 6
View larger version (71K):

[in a new window]
  		Fig. 6. Nucleocytoplasmic shuttling of NFATc1-GFP visualized in muscles of living mice. TA muscle transiently expressing NFATc1-GFP was observed in situ by using two-photon microscopy as described in Materials and Methods, either during application or after suspension of low-frequency stimulation. (A,B) Micrographs depicting maximum-intensity projections of either 15 confocal sections of individual fibers taken at the indicated time points after start (A) or suspension of low-frequency stimulation (B); bar, 25 µm. (C) Graphs show the mean fluorescence intensity of 30 (import, n=3 fibers) or 49 nuclei (export, n=3 fibers). Data (mean ± s.e.m.) were obtained from background-corrected sum plots and were either normalized to the 60-minute values (import, left panel) or 0-minute values (export, right panel). (D) High-resolution confocal micrographs of individual nuclei showing the concentration of NFATc1-GFP in punctuate structures. Bar, 5 µm.

 

Figure 7
View larger version (101K):

[in a new window]
  		Fig. 7. The calcineurin inhibitor cain/cabin1 blocks the nuclear translocation of NFATc1-GFP. (A,B) Soleus muscles co-transfected with plasmids coding for NFATc1-GFP and myc-tagged cain. (C,D) TA muscles co-transfected with plasmids coding for NFATc1-GFP and myc-tagged cain and electrostimulated for 2 hours with a 20 Hz impulse pattern. Serial sections were either stained with anti-myc (A,C,E,G) or examined for GFP fluorescence (B,D,F,H). (E-H) Occasional fibers in electrostimulated TA muscles that do not express cain (asterisks) maintain a nuclear localization of NFATc1, whereas neighboring fibers that do express cain show a cytoplasmic localization of NFATc1-GFP. Bar, 30 µm.

 

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 2006