The impairment of the assembly of vimentin filaments leads to suppression of the formation and maturation of focal contacts and an alteration of the type of cellular protrusions

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Abstract

Cell migration is largely determined by the type of protrusions formed by the cell. Mesenchymal migration is accomplished by the formation of lamellipodia and/or filopodia, and membrane blebs lay at the basis of amoeboid migration. Changing the conditions of migration can lead to an alteration of the character of cell movement, for example, inhibition of Arp2/3-dependent actin polymerization by the CK-666 inhibitor leads to transition from mesenchymal to amoeboid motility type. The ability of cells to switch from one type of motility to another is called migration plasticity. The cellular mechanisms regulating migratory plasticity are poorly understood. One of the factors determining the possibility of migratory plasticity may be the presence and/or organization of vimentin intermediate filaments (VIFs). To investigate whether the organization of the VIF network affects the ability of fibroblasts to form membrane blebs, we used rat embryonic fibroblasts REF52 with normal VIF organization, with VIF knockout (REF–/–) and with a mutation inhibiting the assembly of full-length VIFs (REF117). Blebs formation was induced by treatment of cells with CK-666. Vimentin knockout did not lead to statistically significant increase of number of cells with blebs. In fibroblasts with short fragments of vimentin the number of cells forming blebs both spontaneously and in presence of CK-666 increased significantly. Disruption of the VIF organization did not lead to the significant changes in microtubules network or myosin light chain phosphorylation, but caused a significant reduction of the focal contact system. The most pronounced and statistically significant decrease in both the size and number of focal adhesions were observed in REF117. We believe that the regulation of membrane blebbing by VIFs is mediated by their effect on the focal adhesion system. Analysis of migration of fibroblasts with different organization of VIFs in a three-dimensional collagen gel showed that the organization of VIFs determines the type of cell protrusions, which in turn determines the character of cell movement. A novel role of VIF as a regulator of membrane blebbing, essential for the manifestation of migratory plasticity, is shown.

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About the authors

A. O. Zholudeva

Blokhin National Medical Research Center of Oncology, Ministry of Health of the Russian Federation

Email: tonya_alex@yahoo.com
Russian Federation, 115478 Moscow

N. S. Potapov

Lomonosov Moscow State University

Email: tonya_alex@yahoo.com

Faculty of Biology

Russian Federation, 119234 Moscow

E. A. Kozlova

Lomonosov Moscow State University

Email: tonya_alex@yahoo.com

Faculty of Biology

Russian Federation, 119234 Moscow

M. E. Lomakina

Blokhin National Medical Research Center of Oncology, Ministry of Health of the Russian Federation

Email: tonya_alex@yahoo.com
Russian Federation, 115478 Moscow

A. Y. Alexandrova

Blokhin National Medical Research Center of Oncology, Ministry of Health of the Russian Federation

Author for correspondence.
Email: tonya_alex@yahoo.com
Russian Federation, 115478 Moscow

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Supplementary files

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2. Fig. 1. Cytoskeleton of fibroblasts with different organization of the IMF. Immunofluorescence staining. Upper row – actin (red) and vimentin (green), lower row – microtubules. Scale bar – 10 μm

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3. Fig. 2. Differences in the ability to membrane blebbing in fibroblasts with different organization of the IMF. a – Morphology of control cells (upper row) and cells treated with 200 μM CK-666 for 1 h (lower row). DIC microscopy, scale bar 20 μm. Cells with membrane blebbing are indicated by white arrows. The insets show a high magnification of cells with blebs, highlighted in the figure by a dotted square. b – Scatter plot illustrating the area of ​​REF52, REF–/–, REF117 cells spread on the substrate; mean values ​​with standard deviations are presented. The results of 4 experiments are presented, statistical processing was performed using the GraphPad Prism program, the Kruskall-Wallis criterion (with Dunn's post hoc test): *** p < 0.001, **** p < 0.0001, ns – statistically insignificant changes, N = 60 cells for each group. c – The proportion of cells forming blebs in cultures before and after treatment with CK-666. For statistical analysis, ANOVA was used: *** p < 0.001, **** p < 0.0001, ns – statistically insignificant changes, N = 30 visual fields for each group

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4. Fig. 3. Expression of MLC; pMLC and differences in the FC system in fibroblasts with different vimentin organization. a – Representative Western blot recording changes in the expression of vimentin, myosin light chain (MLC), phosphorylated myosin light chain (pMLC). Gamma-tubulin was used to control protein loading. b – Morphology of FC in cells of the studied cultures, immunofluorescent staining with antibodies to vinculin. Scale bar is 20 μm. c – Western blot demonstrating vinculin expression in three cultures, actin was used to control protein loading, densitometry was performed based on the analysis of four experiments. d – Diagrams illustrating changes in the sizes (left) and numbers (right) of FC in the studied cultures. The diagram shows the 90% interval of area values ​​and the number of individual contacts per cell. Statistical processing was performed using the GraphPad Prism program. To test the statistical significance of the differences in the area of ​​the FC in the three REF cultures, the Kruskal–Wallis test (with Dunn's post hoc test) was used: *** p < 0.001, **** p < 0.0001, ns – statistically insignificant changes, N ≥ 1004 contacts

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5. Fig. 4. Diversity of protrusions formed by cells with different organization of the HMF in a three-dimensional collagen gel. a – Morphology of the formed protrusions, time from the start of shooting for each selected frame is marked, scale segment is 40 μm; b – tracks of movement of cells with different organization of the HMF in a three-dimensional collagen gel

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