Stopping transformed cancer cell growth by rigidity sensing.

A common feature of cancer cells is the alteration of kinases and biochemical signalling pathways enabling transformed growth on soft matrices, whereas cytoskeletal protein alterations are thought to be a secondary issue. However, we report here that cancer cells from different tissues can be toggled between transformed and rigidity-dependent growth states by the absence or presence of mechanosensory modules, respectively. In various cancer lines from different tissues, cells had over tenfold fewer rigidity-sensing contractions compared with normal cells from the same tissues. Restoring normal levels of cytoskeletal proteins, including tropomyosins, restored rigidity sensing and rigidity-dependent growth. Further depletion of other rigidity sensor proteins, including myosin IIA, restored transformed growth and blocked sensing. In addition, restoration of rigidity sensing to cancer cells inhibited tumour formation and changed expression patterns. Thus, the depletion of rigidity-sensing modules through alterations in cytoskeletal protein levels enables cancer cell growth on soft surfaces, which is an enabling factor for cancer progression.

Proteomic profiling of 24-epibrassinolide-induced chilling tolerance in harvested banana fruit.

The mechanism of 24-epibrassinolide (EBR)-induced chilling tolerance in harvested banana fruit was investigated. Results showed that EBR pretreatment remarkably suppressed the development of chilling injury (CI) in harvested banana fruit during 12 days of cold storage at 8 °C, as indicated by lower CI index in treated fruit. Physiological measurements exhibited that EBR treatment reduced the relative electrolyte leakage and malondialdehyde (MDA) content while increased the chlorophyll fluorescence (Fv/Fm), total soluble solids (TSS) and ratio of TSS and titratable acidity. Furthermore, the differentially accumulated proteins of banana fruit in response to EBR and cold treatment were investigated by employing gel-based proteomic in combination with MALDI-TOF-TOF MS and LC-ESI-MS/MS analyses. There were fifty five protein spots to be successfully identified. Notably, most of up-regulated proteins by EBR treatment were related to energy biosynthesis, stress response and cell wall modification. In contrast, proteins involved in protein degradation and energy consumption were down-regulated by EBR treatment. These results suggest that EBR treatment could enhance the defense ability, promote the synthesis and utilization of energy, as well as maintain the protein function via enhancing protein biosynthesis and inhibiting protein degradation, consequently contributing to improvement of cold tolerance in harvested banana fruit. SIGNIFICANCE: To extend our understanding of chilling injury (CI) of harvested banana fruit, we reported the effect of 24-epibrassinolide (EBR) on CI of banana fruit when stored at 8 °C. It was the first report on the comprehensive proteomic analysis of banana fruit in response to EBR treatment at low temperature. EBR pretreatment significantly reduced CI in harvested banana fruit. Fifty five protein spots were successfully identified. Notably, the most of up-regulated proteins by EBR treatment were related to energy biosynthesis, stress response and cell wall modification. In contrast, proteins involved in protein degradation and energy consumption were down-regulated. These results suggest that exogenous EBR treatment could enhance the defense ability and maintain high energy status. Meanwhile, EBR treatment maintained protein function via enhancing protein biosynthesis and inhibiting protein degradation. These results may help us to understand the molecular mechanism of the chilling tolerance induced by EBR treatment and broaden the current knowledge of the mechanism of CI of harvested banana fruit.

EGFR and HER2 activate rigidity sensing only on rigid matrices.

Epidermal growth factor receptor (EGFR) interacts with integrins during cell spreading and motility, but little is known about the role of EGFR in these mechanosensing processes. Here we show, using two different cell lines, that in serum- and EGF-free conditions, EGFR or HER2 activity increase spreading and rigidity-sensing contractions on rigid, but not soft, substrates. Contractions peak after 15-20 min, but diminish by tenfold after 4 h. Addition of EGF at that point increases spreading and contractions, but this can be blocked by myosin-II inhibition. We further show that EGFR and HER2 are activated through phosphorylation by Src family kinases (SFK). On soft surfaces, neither EGFR inhibition nor EGF stimulation have any effect on cell motility. Thus, EGFR or HER2 can catalyse rigidity sensing after associating with nascent adhesions under rigidity-dependent tension downstream of SFK activity. This has broad implications for the roles of EGFR and HER2 in the absence of EGF both for normal and cancerous growth.

α-Actinin links extracellular matrix rigidity-sensing contractile units with periodic cell-edge retractions.

During spreading and migration, the leading edges of cells undergo periodic protrusion-retraction cycles. The functional purpose of these cycles is unclear. Here, using submicrometer polydimethylsiloxane pillars as substrates for cell spreading, we show that periodic edge retractions coincide with peak forces produced by local contractile units (CUs) that assemble and disassemble along the cell edge to test matrix rigidity. We find that, whereas actin rearward flow produces a relatively constant force inward, the peak of local contractile forces by CUs scales with rigidity. The cytoskeletal protein α-actinin is shared between these two force-producing systems. It initially localizes to the CUs and subsequently moves inward with the actin flow. Knockdown of α-actinin causes aberrant rigidity sensing, loss of CUs, loss of protrusion-retraction cycles, and, surprisingly, enables the cells to proliferate on soft matrices. We present a model based on these results in which local CUs drive rigidity sensing and adhesion formation.

Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices.

Cells test the rigidity of the extracellular matrix by applying forces to it through integrin adhesions. Recent measurements show that these forces are applied by local micrometre-scale contractions, but how contraction force is regulated by rigidity is unknown. Here we performed high temporal- and spatial-resolution tracking of contractile forces by plating cells on sub-micrometre elastomeric pillars. We found that actomyosin-based sarcomere-like contractile units (CUs) simultaneously moved opposing pillars in net steps of ∼2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing.

FHOD1 is needed for directed forces and adhesion maturation during cell spreading and migration.

Matrix adhesions provide critical signals for cell growth or differentiation. They form through a number of distinct steps that follow integrin binding to matrix ligands. In an early step, integrins form clusters that support actin polymerization by an unknown mechanism. This raises the question of how actin polymerization occurs at the integrin clusters. We report here that a major formin in mouse fibroblasts, FHOD1, is recruited to integrin clusters, resulting in actin assembly. Using cell-spreading assays on lipid bilayers, solid substrates, and high-resolution force-sensing pillar arrays, we find that knockdown of FHOD1 impairs spreading, coordinated application of adhesive force, and adhesion maturation. Finally, we show that targeting of FHOD1 to the integrin sites depends on the direct interaction with Src family kinases and is upstream of the activation by Rho kinase. Thus, our findings provide insights into the mechanisms of cell migration with implications for development and disease.

Cells test substrate rigidity by local contractions on submicrometer pillars.

Cell growth and differentiation are critically dependent upon matrix rigidity, yet many aspects of the cellular rigidity-sensing mechanism are not understood. Here, we analyze matrix forces after initial cell-matrix contact, when early rigidity-sensing events occur, using a series of elastomeric pillar arrays with dimensions extending to the submicron scale (2, 1, and 0.5 µm in diameter covering a range of stiffnesses). We observe that the cellular response is fundamentally different on micron-scale and submicron pillars. On 2-µm diameter pillars, adhesions form at the pillar periphery, forces are directed toward the center of the cell, and a constant maximum force is applied independent of stiffness. On 0.5-µm diameter pillars, adhesions form on the pillar tops, and local contractions between neighboring pillars are observed with a maximum displacement of ∼60 nm, independent of stiffness. Because mutants in rigidity sensing show no detectable displacement on 0.5-µm diameter pillars, there is a correlation between local contractions to 60 nm and rigidity sensing. Localization of myosin between submicron pillars demonstrates that submicron scale myosin filaments can cause these local contractions. Finally, submicron pillars can capture many details of cellular force generation that are missed on larger pillars and more closely mimic continuous surfaces.