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. 2018 Jan 2;217(1):231–249. doi: 10.1083/jcb.201703044

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© 2018 Gagliardi et al.

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Figure 9. — Actin polymerization at the outermost bundle terminals balances centripetal forces generated by the iEAAR. (A) A cell coexpressing mCherry-MRCKα 1–478 and GFP-actin shows a long phase where shape, iEAAR, and associated bundles are maintained with minimal morphological variation followed by a shorter phase of sudden cell collapse and synchronous shortening of bundles. (B) Quantification of the length and shortening rate of one bundle from time-lapse experiment in A. Two distinct phases are identifiable: (1) a tug-of-war phase characterized by slow bundle shortening and (2) a collapse phase where the bundle is subjected to a rapid shortening until its complete adsorption within the iEAAR. Inset, enlargement of the shortening rate in the tug-of-war phase showing a slow and noisy reduction of the length of the bundle. (C) A cell cotransfected with GFP-actin, GST-MRCKα 1–478, and vinculin-RFP showing an iEAAR ring with shorter, downward bundles originating from cell–matrix adhesions and long bundles originating from cell edges, likely the site of cell–cell adhesions. (D) A cell cotransfected with GFP-actin and mCherry-MRCKα 1–478 was bleached at the bundles’ terminals and monitored by time-lapse confocal microscopy. (E) Kymograph showing actin polymerization of a photobleached bundle terminal. (F) Subconfluent MCF10A cells were cotransfected with GFP-actin and mCherry-MRCKα 1–478 then treated with 1 µM latrunculin A, 3 µM cytochalasin D, or cotreated with 100 μM blebbistatin and 3 μM cytochalasin D. Blockage of actin polymerization caused by latrunculin A and cytochalasin D causes rapid cell collapse, which is prevented by blebbistatin treatment. (G) Shortening rate of bundles in GFP-actin and mCherry-MRCKα 1–478 expressing cells treated with 1 µM latrunculin A, 3 µM cytochalasin D, or cotreated with 100 μM blebbistatin and 3 μM cytochalasin D. Each color encodes for a different cell, whereas curves of the same color represent different bundles in the same cell. Inset, enlargement of the shortening rate in cells cotreated with latrunculin A and cytochalasin D.