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Process formation results from the imbalance between motor-mediated forces

Lotfi Ferhat*, Guillaume Rami, Igor Medina, Yehezkel Ben-Ari and Alfonso Represa

INMED/INSERM U29, 163 rue de Luminy, BP 13, 13273 Marseille Cedex 09, France



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  		Fig. 1. Transfection of cultured HEK 293 cells. (A) A cell transfected with RFP. Diffuse RFP protein is observed in the cytoplasm and the nucleus but the staining in the latter is stronger. (B,C) Two examples of cells transfected with ac.CaP-RFP. These ac.CaP-RFP cells display striking morphological changes compared with RFP-transfected cells. (D) A cell transfected with ac.CaP{Delta}-GFP that does not have the actin-binding domains. The staining observed is diffuse in the cytoplasm and the nucleus. The ac.CaP{Delta}-GFP-transfected cells did not display any morphological change compared with RFP-transfected cells. Bar, 15 µm.

 


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  		Fig. 2. Microfilament organisation in HEK 293 cells transfected with acidic calponin. Panels A and B illustrate untransfected control cells and ac.CaP-GFP-transfected cells stained with Texas Red-X phalloidin, respectively. The organisation of microfilaments is dramatically altered in ac.CaP-GFP cells (C). Indeed, microfilaments are thicker and display a twisted organisation compared with control cells. Panel D corresponds to the overlay of B and C. The yellow color indicates that ac.CaP-GFP mainly binds to F-actin. Note the accumulation of calponin and microfilaments within the tip of the process (see arrows). Bar, 15 µm.

 


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  		Fig. 3. Microtubule organisation in HEK 293 cells transfected with acidic calponin. Panels A and B illustrate the same control cells double stained with Texas Red-X phalloidin and ß-tubulin antibodies, respectively. Panel C corresponds to the overlay of A and B. It shows that microtubules radiated through the cytoplasm as individual filaments until stopped by the cortical actin filaments. Panel D shows an ac.CaP-RFP-transfected cell. Panel E illustrates the same cell counterstained with ß-tubulin antibodies. Panel F corresponds to the overlay of D and E. It shows that ac.CaP-RFP-transfected cells displayed a clear-cut reorganisation of microtubules compared with control cells. Thick bundles of microtubules were observed within the cell bodies and extensions. Moreover, the ac.CaP-RFP (in red) was not co-distributed with microtubules (in green) but rather adjacent. Panel G shows a process of an ac.CaP-RFP cell. Panel H illustrate the same process counterstained with ß-tubulin antibodies. Panel I corresponds to the overlay of G and H. It shows that ac.CaP filaments/microfilaments (in red) and microtubules (in green) clearly form ‘curves’ within this process (see arrows). Bar, 15 µm.

 


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  		Fig. 4. Inhibition of dynein activity blocks process formation induced by acidic calponin. Panel A shows a cell that overexpresses ac.CaP-RFP and extends several processes. Panel B shows that a cell overexpressing dynamitin-GFP does not display any morphological change. The Red color corresponds to cells expressing ac.CaP-RFP, the green color indicates cells overexpressing dynamitin-GFP. Panel C corresponds to the overlay of panels A and B. Panels D-F illustrate the same cells double transfected with ac.CaP-RFP (in red) and dynamitin-GFP (in green). Panel F corresponds to the overlay of panels D and E. It shows that all cells overexpressing both ac.CaP-RFP and dynamitin-GFP do not develop processes. Note also that cells double transfected with ac.CaP-RFP and dynamitin-GFP are smaller than cells expressing either ac.CaP-RFP (A) or dynamitin-GFP (B). Bar, 30 µm.

 

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