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 7 October 2008
doi: 10.1242/jcs.036152

Journal of Cell Science 121, 3581-3588 (2008)
Published by The Company of Biologists 2008
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 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 Google Scholar
Google Scholar
Right arrow Articles by Undyala, V. V.
Right arrow Articles by Beningo, K. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Undyala, V. V.
Right arrow Articles by Beningo, K. A.
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?

The calpain small subunit regulates cell-substrate mechanical interactions during fibroblast migration

Vishnu V. Undyala1, Micah Dembo2, Katherine Cembrola3, Benjamin J. Perrin4, Anna Huttenlocher4, John S. Elce5, Peter A. Greer5,6, Yu-li Wang3 and Karen A. Beningo1,*

1 Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA
2 Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA
3 Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01605, USA
4 Department of Pediatrics and Pharmacology, University of Wisconsin Medical School, Madison, WI 53706, USA
5 Department of Biochemistry, Queen's University, Kingston, Ontario, K7L 3N6 Canada
6 Department of Pathology and Molecular Medicine, Cancer Research Institute, Queen's University, Kingston, Ontario, K7L 3N6 Canada


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 1. Inhibition of traction forces in Capn4–/– fibroblasts. (A,B) Vector plots of traction stress indicate magnitude and direction of traction stresses exerted by cells on the substrate, for Capn4–/– cells stably re-expressing the small subunit (A) and Capn4–/– cells (B). (C) Bar graph shows average integrated traction forces magnitude from each type of the cells. Capn4–/– cells show a significant reduction in traction forces compared with wild-type or rescued cells (Student's t-test P=0.0003).

 

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

  		Fig. 2. Defects in the dynamics of traction forces and energy output for Capn4–/– fibroblasts. (A) Color rendering of the magnitude of traction stress at 6, 12, 20 and 28 minutes in rescued Capn4–/– fibroblasts and in Capn4–/– cells shows weak, scattered traction forces for Capn4–/– fibroblasts, whereas control cells exert strong forces at the leading and trailing edges. Color scale represents a minimum magnitude of 1x103 dynes/cm2 and a maximum of 2.9x105 dynes/cm2. (B) Plots of average integrated traction forces against time show a much higher degree of dynamics for rescued Capn4–/– cells than Capn4–/– fibroblasts. (C) Dynamics of traction forces measured by taking a ratio between average integrated traction forces and the s.d. measured over a period of 45 minutes. Rescued Capn4–/– fibroblasts have a much smaller value than Capn4–/– cells (four cells, each recorded over 45 minutes), indicating a larger fluctuation in forces. The difference in the energy stored in the substrate is even more striking (Student's t-test P=0.00001), with a much lower output for Capn4–/– fibroblasts than rescued cells (n=13 and n=23, respectively).

 

Figure 3
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 3. Generation of normal traction forces by cells defective in calpain 1 or calpain 2. (A) Average integrated traction forces generated by MEF cells treated with siRNA for either Capn4, Capn1 or Capn2 or cells overexpressing calpastatin. Neither silencing of calpain 1 or calpain 2, nor overexpression of calpastatin, causes a detectable reduction in traction forces. Data are compiled from measurements of a minimum of 15 cells for each cell type. Although wild-type MEF cells transfected with siRNA against Capn4 show a significant reduction in the magnitude of traction stress (Student's t-test P=0.0001) (n=19). (B,C) Immunofluorescence of the calpain small subunit in MEFs transfected with control RNA (B), or with siRNA against Capn4 (C) shows a striking reduction in the amount of small subunit upon siRNA-mediated gene silencing. (D) RT-PCR demonstrates an 88% reduction in the amount of Capn4 mRNAs. (E) Level of calpastatin protein expressed in wild-type MEF cells transfected with GFP plasmid alone and cells in which calpastatin-GFP has been overexpressed.

 

Figure 4
View larger version (79K):
[in this window]
[in a new window]

  		Fig. 4. Involvement of calpain 1, calpain 2 and the calpain small subunit in cellular responses to mechanical forces. Images of Capn4–/– cells or rescued cells are recorded at 0 minutes, prior to micromanipulation, and at 20, 40, and 60 minutes after pushing on the substrate with a blunted microneedle against the direction of cell migration. Thin arrows indicate the direction of cell migration and thick arrowheads show the direction of pushing. Scale bar: 10 µm. The chart indicates a response (+) or a failure to respond (–) to stimulation under each of the cellular conditions.

 

Figure 5
View larger version (43K):
[in this window]
[in a new window]

  		Fig. 5. Failure of cells defective in calpain to respond to the engagement of dorsal integrins. Capn4–/– (B,D) or rescued (A,C) fibroblasts were cultured on the surface (A,B), or within two layers (C,D), of fibronectin-coated polyacrylamide substrates. Only rescued cells respond to the engagement of dorsal integrins by adopting an elongated shape. Scale bar: 10 µm. The response is quantified by taking the ratio of the length to the width of cells (E). In addition, similar results are obtained with siRNA-induced gene silencing using NIH3T3 cells. Each bar represents mean ± s.e.m. of 25 cells from three experiments.

 

Figure 6
View larger version (54K):
[in this window]
[in a new window]

  		Fig. 6. Capn4–/– fibroblasts are defective in adhesiveness and adhesion: stress fiber linkage. (A) Rescued Capn4–/– fibroblasts, Capn1- and Capn2-knockdown and calpastatin-overexpressing MEF cells, but not Capn4–/– cells, adhere strongly to the substrate in a centrifugation assay. Each bar represents mean ± s.e.m. results from three separate experiments, expressed as a percentage of control as defined by wild-type fibroblasts. Actin and vinculin immunofluorescence of representative control fibroblasts (B,C) and Capn4–/– fibroblasts (D,E) show the abnormal organization of these structures in the knockout cells. Scale bar: 10 µm. (F) Colocalization analysis of actin and vinculin indicates a decreased association of prominent actin fibers with vinculin-containing adhesions in the Capn4–/– fibroblasts compared with the control and Capn1- and Capn2-silenced fibroblasts, as well as cells overexpressing calpastatin. The analysis involves counting the total number of vinculin-containing adhesions and those associated with actin stress fibers in the anterior region (n=15 cells).

 

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 2008