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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/jcs.00220

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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Furukawa, R.
Right arrow Articles by Fechheimer, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Furukawa, R.
Right arrow Articles by Fechheimer, 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?

Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton in Dictyostelium

Ruth Furukawa*, Andrew Maselli*, Susanne A. M. Thomson, Rita W. L. Lim, John V. Stokes and Marcus Fechheimer{ddagger}

Department of Cellular Biology, University of Georgia, Athens, Georgia 30602, USA



View larger version (53K):

[in a new window]
  		Fig. 1. Wild-type and calcium-insensitive forms of the 34 kDa protein. (A) Sequence of wild-type 34 kDa and 34 kDa {Delta}EF2 proteins. Nucleotide and amino-acid sequences of the wild-type and modified EF hand 34 kDa proteins are shown. The altered nucleotides and amino acids are shown in bold. (B) Expression and purification of the 34 kDa and 34 kDa {Delta}EF2 proteins. SDS-PAGE gel stained with Coomassie blue. Lanes 1 and 3 were loaded with 100 µg of BL21 (DE3) expressing 34 kDa and 34 kDa {Delta}EF2 proteins, respectively. Lanes 2 and 4 were loaded with 25 µg of purified 34 kDa and 34 kDa {Delta}EF2 proteins, respectively. (C) Western blot showing Dictyostelium AX2 and 34-kDa-null cells expressing the wild-type 34 kDa protein and 34 kDa {Delta}EF2 proteins. Proteins from cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose and probed with a monoclonal antibody B2C reactive to the 34 kDa protein.

 


View larger version (15K):

[in a new window]
  		Fig. 2. Calcium binding to 34 kDa and 34 kDa {Delta}EF2 proteins measured by equilibrium dialysis. (A) Calcium binding. Approximately 10 µM 34 kDa and 34 kDa {Delta}EF2 proteins were dialyzed as a function of [Ca45]CaCl2 concentration in buffer as described in Materials and Methods. The moles of calcium bound per mole of 34 kDa protein are plotted versus the free calcium concentration for each protein. 34 kDa protein ({circ}); 34 kDa {Delta}EF2 protein ([UNK]). (B) Scatchard plot from equilibrium dialysis. The moles of calcium bound per mole of 34 kDa protein divided by the free calcium concentration are plotted versus moles of calcium bound per mole of 34 kDa protein. The straight line indicates the best fit line yielding Kd of 2.4 µM.

 


View larger version (28K):

[in a new window]
  		Fig. 3. Binding of the 34 kDa and 34 kDa {Delta}EF2 proteins to F-actin measured by co-sedimentation. (A) Moles of 34 kDa protein bound per mole of actin subunits in filaments is plotted as a function of the total 34 kDa protein added. 34 kDa protein ({circ}); 34 kDa {Delta}EF2 protein ([UNK]). The 34 kDa {Delta}EF2 protein binds F-actin moderately with a slightly greater affinity than the wild-type protein. (B) The relative amount of 34 kDa protein bound to F-actin in the presence and absence of micromolar concentrations of calcium. The amount of wild-type 34 kDa protein bound to F-actin decreases by 70% in the presence of micromolar calcium whereas the amount of 34 kDa {Delta}EF2 protein bound to F-actin is calcium insensitive.

 


View larger version (123K):

[in a new window]
  		Fig. 4. Bundling of F-actin filaments by the 34 kDa {Delta}EF2 protein is not inhibited by calcium. Mixtures of F-actin and the 34 kDa proteins in the presence of low and high concentrations of calcium were viewed by transmission electron microscopy using negative staining. Top row, wild-type 34 kDa protein and F-actin at low (A) and high (B) concentrations of calcium. Bottom row, 34 kDa {Delta}EF2 protein and F-actin at low (C) and high (D) concentrations of calcium. Mixtures of the wild-type 34 kDa protein and F-actin form bundles in the presence of low but not high concentrations of calcium. By contrast, mixtures of 34 kDa {Delta}EF2 protein and F-actin form bundles that are not calcium regulated. Magnification 14,000x.

 


View larger version (19K):

[in a new window]
  		Fig. 5. Growth curves in 34-kDa-null amoebae expressing the 34 kDa and 34 kDa {Delta}EF2 proteins. (A) 20°C, (B) 15°C, AX2 ({square}), 34-kDa-null ({triangleup}), r34 kDa ({circ}), r34kDa {Delta}EF2 ([UNK]). Cells were grown axenically in shaking cultures, and cell density was measured as a function of time after inoculation. The 34-kDa-null cells grow as well as wild-type (AX-2) at 20°C but more slowly than wild-type at 15°C. Expression of the 34 kDa {Delta}EF2 slows the growth of 34-kDa-null cells at 20°C (A). Expression of the r34 kDa protein restores the normal growth rate to that of 34-kDa-null cells at 15°C. By contrast, 34 kDa {Delta}EF2 fails to rescue the slow growth phenotype (B).

 


View larger version (19K):

[in a new window]
  		Fig. 6. Growth curves in 34 kDa/{alpha}-actinin double null amoebae rescued with 34 kDa and 34 kDa {Delta}EF2 proteins. Cells were grown axenically in shaking cultures, and cell density was measured as a function of time after inoculation. (A) 20°C, (B) 15°C, AX2 ({square}), 34 kDa/{alpha}-actinin double null ({triangleup}), r34 kDa ({circ}), r34 kDa {Delta}EF2 ([UNK]). The 34 kDa/{alpha}-actinin double null cells grow more slowly than wild-type at both 20°C and 15°C. Expression of the wild-type 34 kDa protein restores wild-type growth. By contrast, expression of 34 kDa {Delta}EF2 protein stimulates growth only slightly at 20°C (A) and has no restorative effect on growth of the 34 kDa/{alpha}-actinin double mutant at 15°C (B).

 


View larger version (33K):

[in a new window]
  		Fig. 7. Fluorescence images of amoebae used in ratio mapping. Wild-type AX2 cells were stained with Oregon-green-labeled (A) and TRITC-labeled phalloidin (B) for the F-actin/F-actin ratio map. 34-kDa-null cells were rescued with either wild-type 34 kDa protein (C,D), 34 kDa GFP (E,F) or 34 kDa {Delta}EF2 (G,H). Cells were stained with Oregon-green-labeled phalloidin (C,E,G) and monoclonal antibody B2C against 34 kDa protein followed by a TRITC anti-mouse secondary antibody (D,H) or viewed directly for fluorescence from 34 kDa GFP (F). (C) F-Actin, (D) 34 kDa protein, (E) F-Actin, (F) 34 kDa GFP, (G) F-Actin, (H) 34 kDa {Delta}EF2. Bar, 5 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 2003