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. Author manuscript; available in PMC: 2022 Oct 22.
Published in final edited form as: ACS Nano. 2021 Oct 22;15(11):17528–17548. doi: 10.1021/acsnano.1c04435

Figure 3. Structural analysis of the MDA-MB-231 cells during alternative dynein-kinesin balance shift mechanisms.

Figure 3.

(a) - MT ring median-plane cross-section in MDA-MB-231 cells (left) as shown in the schematic (right) in +Dynapyrazole A+Blebbistatin. Note kinesin accumulation at the MT rings (arrows), see also SI3a-d.

(b) - MT rings expand and deform cells into the lenticular discoids (MT ring diameter > cell width). The plots depict the measurements of the cell discoids diameters (diameter of the MT ring) and the cells’ widths (transverse cell dimension). The dashed box depicts the averaged cell discoid diameter and width boundaries (means).

(c) - Top row - Adhesion and spreading of MDA-MB-231 cells on the collagen-1 grids (G’=50 kPa) in control condition (+DMSO) and during kinesin overactivation (+Kinesore), compared to the direct dynein inactivation (+Dynapyrazole A). Kinesin over-activation with +Kinesore does not inhibit the dynein activity, yet induces a significant increase of the biaxial spreading of cells in comparison to the control cells. Alternatively, dynein-kinesin balance shift towards the kinesin activity via the direct dynein inhibition (+Dynapyrazole A) induces a visible reduction of cell spreading and “dendritization” of cells that is similar the loss of cell contractility shape during Blebbistatin-induced myosin II inhibition (Figure 1, +Blebbistatin). The loss of cell contractility in Dynapyrazole A is related to the two main factors 74: the loss of MT-to-actomyosin mechanical crosslinking and force transmission, as well as the loss of the dynein-driven mechanical contractility within MT network (+Dynapyrazole A).

Bottom row - Morphometric analysis of control (+DMSO), and +Kinesore- and +Dynapyrazole A-treated cells indicate a significant increase in cell biaxial spreading, driven by kinesin-mediated MT network expansion.

(d) - Suggested mechanism for the MT-actomyosin interaction in cells on the collagen grids. Dyneins and kinesins control the (1) MT network compaction and (2) MT network expansion respectively. Dyneins also crosslink the MT network to the actomyosin cytoskeleton (stress-fibers). I.e., actomyosin, adhesion-bound matrix, adhesion complex, MTs and MT motor proteins subsystems are united into a single mechanical system. See also SI4a.

(e) - Alternative methods for outbalancing the kinesin activity via +Dynapyrazole A+Blebbistatin and +Kinesor+Blebbistatin induce MT rings (arrows) and cell discoids.

F-actin is labelled with Phalloidin-ATTO 647N. Chromatin is labelled with Hoechst. Pairwise one-way t tests-derived p values are shown on the plots with corresponding n (size of individual cells measurement sets) for each condition, generated in triplicates.