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 3 March 2009
doi: 10.1242/jcs.041715

Journal of Cell Science 122, 957-964 (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 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 Murk, K.
Right arrow Articles by Rothkegel, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Murk, K.
Right arrow Articles by Rothkegel, M.
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?

In birds, profilin-2a is ubiquitously expressed and contributes to actin-based motility

Kai Murk1, Sabine Buchmeier1, Brigitte M. Jockusch2 and Martin Rothkegel1,*

1 Cellular Neurobiology, University of Braunschweig, 38092 Braunschweig, Germany
2 Cell Biology, Zoological Institute, University of Braunschweig, 38092 Braunschweig, Germany


Figure 1
View larger version (44K):
[in this window]
[in a new window]

  		Fig. 1. Conservation of murine and chicken profilin amino acid sequences. (A) Sequence comparison of PFN1 and PFN2a from various species, according to the information from protein data bases NCBI (http://www.ncbi.nlm.nih.gov) and Ensembl (http://www.ensembl.org). The numbers are the percentage identity with the respective mouse profilin isoforms. The chicken sequences are highlighted in grey. Note that homology of PFN1 sequences is quite high among mammals, but only moderate among vertebrates, whereas PFN2a is much better conserved among all classes of vertebrates. (B,C) Alignment of chicken and mouse PFN1 and PFN2a amino acid sequences. Mismatches are indicated by asterisks. Mouse and chicken PFN1 sequences (B) differ markedly in the center and in the C-terminal half of the molecule, whereas the sequences for PFN2a (C) show only a single, conserved exchange at position 38. Sequences recognized as epitopes by isoform-specific antibodies are boxed.

 

Figure 2
View larger version (43K):
[in this window]
[in a new window]

  		Fig. 2. Expression pattern of profilin isoforms PFN1 and PFN2a in chicken tissues. (A) Immunoreactivity of a monoclonal antibody against mouse PFN1. (B) Immunoreactivity of a polyclonal rabbit serum raised against mouse PFN2a, after cross-adsorption on recombinant mouse PFN1. The immunoblots show the respective reactions against purified mouse PFN1 and chicken PFN1 and PFN2a, and against total extracts from chicken and mouse fibroblasts and mouse brain. Note that the monoclonal antibody against PFN1 does not react with chPFN2a (A), whereas the polyclonal antibody against PFN2a does not react with either mouse or chicken PFN1 (B). (C,D) Western blot analysis of PFN1 (C) and PFN2a (D) in embryonic (stage E15) and adult tissues. (E,F) Densitometry quantification of PFN1 (E) and PFN2a (F) on immunoblots, with standard calibration curves obtained with recombinant PFN1 and PFN2a. Note that PFN2a is expressed in many embryonic and adult tissues and is not restricted to brain.

 

Figure 3
View larger version (78K):
[in this window]
[in a new window]

  		Fig. 3. Profilin isoforms localize differentially in cultivated embryonic chicken fibroblasts. Double immunostaining of a chicken fibroblast 24 hours after isolation from the embryo (stage E12) with antibodies specific for chPFN1and chPFN2a, respectively, and counterstained for F-actin with phalloidin. The images depict confocal sections through the cell. Note that chPFN1 is primarily concentrated in the nucleus (arrowhead), whereas PFN2a is also associated with an actin filament arc at the base of the leading lamella (arrows) and also with the rim of the leading lamella. Scale bar: 10 µm.

 

Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		Fig. 4. Protein level and localization of PFN1 in chicken fibroblasts depends on culture conditions. (A) Western blot analysis of PFN1 (left panel) and PFN2a (center panel) in extracts of chicken fibroblasts 24 hours after isolation and cultivation for 96 hours in medium supplemented with FCS and chicken embryo extract. Note that the amount of PFN1 approximately doubles during cultivation whereas PFN2a concentration does not significantly change (right panel, PFN content is expressed as percent of total protein). Tubulin, used as a control, does not change. (B,C) Fluorescence images of PFN1 in embryonic chicken fibroblasts in confocal sections after staining with anti-PFN1 and DAPI. (B) Cells were grown for 96 hours in medium supplemented with FCS. (C) Cells from B were starved subsequently for 48 hours in medium without FCS. Scale bars: 10 µm. (C) Statistical analysis of cells as shown in B and C. Note that PFN1 changes from nuclear to a cytosolic over time, but under starvation conditions, PFN1 concentration in the nucleus increases again. (Mean ± s.d.; n=3, at least 250 cells per experiment).

 

Figure 5
View larger version (55K):
[in this window]
[in a new window]

  		Fig. 5. Isoform-specific knockdown of PFNs in cultured chicken fibroblasts does not interfere with vitality. Confocal images of cultured chicken fibroblasts post transfection with RNAi vectors and GFP as a transfection marker. Upper panels, double fluorescence for GFP, and immunostaining for PFN1 and PFN2a of cells 96 hours after transfection with the PFN1-specific vector miRNA1/5 reveals only a very faint fluorescence in GFP-positive, PFN1-knockdown cells, whereas expression of chPFN2a is unaffected. Center panel, double fluorescence for GFP and immunostaining for PFN1 and PFN2a of cells 8 days after transfection with the PFN2a specific vector miRNA 2/3. Staining for PFN2a shows only a faint signal in the GFP-positive transfected cells, whereas PFN1 is still expressed. Lower panels, analogous images obtained with cells 8 days after transfection with the bicistronic miRNA vector 1/5 and 2/3. All GFP-positive cells show only weak fluorescence after immunostaining for both PFN1 and PFN2a. Scale bars: 10 µm.

 

Figure 6
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 6. Lack of PFN2a has a greater effect on cell spreading and adhesion than lack of PFN1. (A) Spreading-adhesion assay. Chicken fibroblasts were allowed to adhere for 30 minutes onto collagen-coated coverslips, subsequently fixed and stained for F-actin. According to morphology and organization of the actin cytoskeleton, cells were categorized into two groups: Well-spread cells (group 1) and compact round cells without substantial spreading (group 2). Scale bar: 10 µm. (B) Statistical analysis of cells subjected to the adhesion test and classified according to the criteria defined in A (Mean ± s.d.; n=3, at least 352 cells per experiment). Red bars, cells transfected 8 days earlier with the PNF1-specific vectors miRNA 1/3 or 1/5. Green bars, cells transfected 8 days earlier with the PFN2a-specific vectors miRNA 2/3 or 2/6. Blue bar, cells transfected with the bicistronic vector miRNA 1/5 and 2/3. Orange bar, cells transfected with the control vector miRNA LacZ. Note that PFN1-deficient cells spread to approximately the same extent as control cells, whereas loss of PFN2a has a statistically significant effect. Double knockdown of both profilin isoforms reduces the number of fast-spreading cells to about 50% that of controls. (C) Morphology of focal contacts of cells after knockdown of either PFN1 (transfected with miRNA1/5), PFN2a (transfected with miRNA2/3) or both (transfected with the bicistronic vector miRNA 1/5 and 2/3), compared with control cells (transfected with miRNA LacZ). Staining with anti-vinculin reveals that loss of either profilin isoform leads to focal contacts (arrows) smaller than those in untransfected cells (arrowheads). (D) Statistical analysis of cells depicted in C. Note that the focal contact area of control cells shows a maximum of around 120 square pixel, whereas the size of focal contacts of cells lacking profilins is shifted towards smaller structures. Again, the effect of loss of PFN2a exceeds that of loss of PFN1, and knocking down both isoforms has the greatest effect. Very large focal contacts (areas measuring more than 300 square pixels) were very rare in cells deficient in PFN2a.

 

Figure 7
View larger version (69K):
[in this window]
[in a new window]

  		Fig. 7. Lack of PFN2a has a greater effect on locomotion than lack of PFN1. (A) Transwell migration assay. Chemotactic movement of chicken fibroblasts in a Transwell-based filter assay. In this example, cells transfected with the PFN1-specific vector comprising miRNA 1/5 and GFP were used to monitor locomotion. Scale bars: 20 µm. (B) Statistical analysis of cells that migrated to the bottom face of the well (at least 220 cells). Note that as in the adhesion-spreading assay, loss of PFN1 (in cells transfected with either miRNA 1/3 or 1/5, red bars) has no statistically significant effect, whereas loss of PFN2a (in cells transfected with miRNA 2/3 or 2/6, green bars) results in reduced migration. Again, in analogy to spreading, loss of both profilin isoforms (blue bar) has the largest effect. Results show mean ± s.d.

 

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