Analysis of senescence-responsive stress fiber proteome reveals reorganization of stress fibers mediated by elongation factor eEF2 in HFF-1 cells

Abstract

Stress fibers (SFs), which are actomyosin structures, reorganize in response to various cues to maintain cellular homeostasis. Currently, the protein components of SFs are only partially identified, limiting our understanding of their responses. Here we isolate SFs from human fibroblasts HFF-1 to determine with proteomic analysis the whole protein components and how they change with replicative senescence (RS), a state where cells decline in ability to replicate after repeated divisions. We found that at least 135 proteins are associated with SFs, and 63 of them are upregulated with RS, by which SFs become larger in size. Among them, we focused on eEF2 (eukaryotic translation elongation factor 2) as it exhibited upon RS the most significant increase in abundance. We show that eEF2 is critical to the reorganization and stabilization of SFs in senescent fibroblasts. Our findings provide a novel molecular basis for SFs to be reinforced to resist cellular senescence.

Doxycycline Attenuated Ethanol-Induced Inflammaging in Endothelial Cells: Implications in Alcohol-Mediated Vascular Diseases

“…Doxycycline reduced the relative mRNA expression of MCP-1 (Control = 1.00 ± 0.08, ethanol = 2.90 ± 0.10, ethanol + doxycycline = 2.57 ± 0.11, n = 3, *** p < 0.001, * p < 0.05, Figure 6 C), E-selectin (Control = 0.95 ± 0.05, ethanol = 7.54 ± 0.36, ethanol + doxycycline = 6.00 ± 0.71, n = 3, *** p < 0.001, * p < 0.05, Figure 6 D), ICAM-1 (Control = 1.00 ± 0.13, ethanol = 8.52 ± 0.21, ethanol + doxycycline = 7.21 ± 0.38, n = 3, *** p < 0.001, ** p < 0.01, Figure 6 E) and VCAM-1 (Control = 1.00 ± 0.08, ethanol = 1.95 ± 0.19, ethanol + doxycycline = 1.41 ± 0.15, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05 Figure 6 F) in HUVECs treated with ethanol. The senescent cells are known to show impaired migration [ 24 , 25 ]; therefore, we investigated the effects of ethanol and ethanol combined with doxycycline on endothelial cell migration. Ethanol retarded the migration of HUVECs in wound assay, and doxycycline recovered the migration of HUVECs exposed to ethanol, percentage area covered by migrating endothelial cells at different time points after making a scratch (at 0 h: Control = 0.00 ± 0.01%, ethanol = 0.00 ± 0.00%, ethanol + doxycycline = 0.00 ± 0.00%, after 6 h: Control = 36.27 ± 7.03%, ethanol = 24.20 ± 5.36%, ethanol + doxycycline = 33.00 ± 25.87%, after 12 h: Control = 78.18 ± 7.57%, ethanol = 50.83 ± 9.41%, ethanol + doxycycline = 79.65 ± 4.35%, after 24 h: Control = 99.77 ± 0.23%, ethanol = 82.30 ± 13.40%, ethanol + doxycycline = 96.56 ± 5.64%, n = 3, ** p < 0.01, Figure 6 G,H).…”

“…The migration of endothelial cells is an important process for angiogenesis and repair/healing of damaged tissue. Previous studies have reported that the senescent cells show retarded migration and are not efficient in the repair/healing of damaged tissue [ 24 , 25 ]. Ethanol treatment reduced the migration of HUVECs, and doxycycline significantly recovered the migration of ethanol-treated HUVECs ( Figure 6 G,H).…”

A statistical mechanics model for determining the length distribution of actin filaments under cellular tensional homeostasis

“…Our prediction is in agreement with experimental observations made on cells 41 . Exploring the involvement of other actin-regulating proteins 44 will be the subject of future challenging but exciting investigations, for which our approach would provide a basis. To this end, as we discussed just after the introduction of the overall entropy of Eq.…”

A model for determining the length distribution of actin filaments in cells

“…Our prediction is in agreement with experimental observations made on cells (40). Exploring the involvement of other actin-regulating proteins (43) will be the subject of future challenging but exciting investigations, for which our approach would provide a basis. To this end, as we discussed just after the introduction of the overall entropy of Eq.…”