The tumour suppressor DiRas3 interacts with C-RAF and downregulates MEK activity to restrict cell migration.

BACKGROUND INFORMATION: The mitogenic pathway, composed of RAF kinases, mitogen-activated protein kinase kinases (MEK) and extracellular signal-regulated kinases (ERK), promotes cell proliferation and migration and is upregulated in many tumours. DiRas3 (ARHI, Noey2), a mainly GTP-bound Ras-like protein with an unusual N-terminal extension, is predominantly lost in ovarian and breast cancers. Its re-expression in these tissues impairs cell proliferation, autophagy, apoptosis and cell migration. Further, loss of DiRas3 correlates with an increase in growth factor-induced ERK phosphorylation. Therefore, DIRAS3 proves to be a curious gene with remarkable tumour suppressing capabilities. However, how DiRas3 interferes with ERK phosphorylation, has remained unknown. RESULTS: We demonstrate that DiRas3 associates in vivo with C-RAF and directly binds in vitro to C-RAF, which is upstream of MEK and ERK. Direct binding of DiRas3 to C-RAF is nucleotide independent, and DiRas3's N-terminal extension alone is not sufficient for binding C-RAF. DiRas3 expression inhibits the activating phosphorylations of MEK and ERK. Serum-induced recruitment of DiRas3 to the plasma membrane depends mainly on its N-terminal extension and less on its C-terminus, bound nucleotide or the presence of Ras-GTP. Correspondingly, removal of the N-terminal extension strongly decreases DiRas3's inhibition of MEK and ERK phosphorylations. Tyrosyl-phosphatases do not contribute significantly to reduction of ERK-phosphorylation byDiRas3. Consistently, downregulation of DiRas3 results in a small but significant and persistent increase in MEK and ERK phosphorylation, but does not increase phosphorylation of P38, AKT and c-Jun NH2-terminal kinase. Finally, downregulation of DiRas3 causes increased cell migration, through a mechanism that is MEK dependent. CONCLUSIONS: These results support a model in which serum signals induce the recruitment of DiRas3 to the plasma membrane, where it is tethered via its N- and C-termini. At the plasma membrane, DiRas3 interacts with C-RAF to specifically suppress the activating phosphorylations on MEK and ERK, thus restricting migration of non-cancer cells. This effect is relatively small, but it is also persistent, suggesting that it contributes to the maintenance of the non-migratory phenotype of non-cancerous tissues, in which DiRas3 is expressed.

Asymmetric division events promote variability in cell cycle duration in animal cells and Escherichia coli.

Asymmetric cell division is a major mechanism generating cell diversity. As cell cycle duration varies among cells in mammalian tissue culture cells, we asked whether their division asymmetry contributes to this variability. We identify among sibling cells an outlier using hierarchical clustering on cell cycle durations of granddaughter cells obtained by lineage tracking of single histone2B-labelled MDCKs. Remarkably, divisions involving outlier cells are not uniformly distributed in lineages, as shown by permutation tests, but appear to emerge from asymmetric divisions taking place at non-stochastic levels: a parent cell influences with 95% confidence and 0.5% error the unequal partitioning of the cell cycle duration in its two progenies. Upon ninein downregulation, this variability propagation is lost, and outlier frequency and variability in cell cycle durations in lineages is reduced. As external influences are not detectable, we propose that a cell-autonomous process, possibly involved in cell specialisation, determines cell cycle duration variability.

Hormetic modulation of differentiation of normal human epidermal keratinocytes undergoing replicative senescence in vitro.

Normal human epidermal keratinocytes (NHEK) show both the Hayflick phenomenon of replicative senescence and differentiation in vitro, depending upon the culture conditions. Using this experimental model system, we have studied age-related changes in the ability of serially passaged NHEK to enter into differentiation in the presence of calcium, as measured by the levels of differentiation markers involucrin, p38 and Hsp27. The results obtained in these studies show that calcium-induced differentiation of NHEK becomes progressively delayed during cellular aging in vitro, which can be modulated by treatments such as mild heat stress, kinetin and curcumin. Whereas all these treatments on their own were able to increase the levels of various differentiation markers to varying extents, their effects were synergistic and rapid in the presence of calcium. Furthermore, all three modulators tested in the present study bring about their effects by inducing stress response pathways in terms of an increase in the levels of stress proteins Hsp90, Hsp70 and heme-oxygenase-1 (HO-1), which is indicative of stress-induced hormesis bringing about the biologically beneficial effects.

Sugar-induced premature aging and altered differentiation in human epidermal keratinocytes.

Normal human epidermal keratinocytes (NHEK) show both the Hayflick phenomenon and differentiation in vitro. The aim of this study was to induce senescence in keratinocytes using two sugars, glucose and glyoxal. Induction of senescence in early-passage NHEK was characterized by monitoring cell morphology, short-term growth characteristics, cell proliferation, and viability assay. In addition, apoptosis, senescence-associated (SA) beta-gal activity, proteasomal activity and glycation, and glycoxidation of total proteins were determined. Our results show that a 3-day treatment with 100 mM glucose or 0.1 mM glyoxal induces in early-passage NHEK various cellular and biochemical characteristics comparable to those observed in serially subcultured late passage NHEK. Furthermore, sugar-treated prematurely aged NHEK showed impaired differentiation, as measured by the quantification of involucrin. There is preliminary evidence that a preexposure of NHEK to mild heat shock (41 degrees C, 1 h, 6 h in advance) can abrogate some of the sugar-induced negative effects, which is an example of mild stress-induced hormesis. This experimental model can be useful to study the effects of potential antiaging interventions.

Kinetin-induced differentiation of normal human keratinocytes undergoing aging in vitro.

Kinetin (N(6)-furfuryladenine) is a cytokinin growth factor having several anti-aging effects reported for human cells and fruit flies. We have observed that short-term culturing of human keratinocytes in the presence of 40 to 200 microM kinetin results in a significant inhibition of cell growth. Studies were undertaken to analyze the process of differentiation as a reason for growth inhibition. Keratinocytes at different passage levels were treated with fetal calf serum (FCS) and calcium as differentiation-inducing positive controls, with different concentrations of kinetin, and with a combination of kinetin and calcium. The induction and progression of differentiation was monitored by morphological observations and by using several differentiation markers, including keratins (K10 and K14), involucrin, epidermal transglutaminase, and some new keratinocyte-specific antibodies isolated by the phage display method. In young keratinocytes, two days of calcium treatment reduced the K14 level by 78%, and increased the levels of K10 and involucrin by 40% and 29%, respectively. In comparison, 40 microM kinetin had no effect on the K14 level, but increased the K10 level by 28% and that of involucrin by four-fold. The combination of calcium and 40 microM kinetin led to a decrease by 23% in the K14 level, to an increase in the level of K10 by 55%, and to a two-fold rise in the involucrin level. These results suggest that the rate, extent, and quality of differentiation depend on the inducing agent, and that kinetin may be useful in promoting the differentiation of human keratinocytes, especially in the presence of calcium.

An analysis toolbox to explore mesenchymal migration heterogeneity reveals adaptive switching between distinct modes.

Mesenchymal (lamellipodial) migration is heterogeneous, although whether this reflects progressive variability or discrete, 'switchable' migration modalities, remains unclear. We present an analytical toolbox, based on quantitative single-cell imaging data, to interrogate this heterogeneity. Integrating supervised behavioral classification with multivariate analyses of cell motion, membrane dynamics, cell-matrix adhesion status and F-actin organization, this toolbox here enables the detection and characterization of two quantitatively distinct mesenchymal migration modes, termed 'Continuous' and 'Discontinuous'. Quantitative mode comparisons reveal differences in cell motion, spatiotemporal coordination of membrane protrusion/retraction, and how cells within each mode reorganize with changed cell speed. These modes thus represent distinctive migratory strategies. Additional analyses illuminate the macromolecular- and cellular-scale effects of molecular targeting (fibronectin, talin, ROCK), including 'adaptive switching' between Continuous (favored at high adhesion/full contraction) and Discontinuous (low adhesion/inhibited contraction) modes. Overall, this analytical toolbox now facilitates the exploration of both spontaneous and adaptive heterogeneity in mesenchymal migration.

A plastic relationship between vinculin-mediated tension and adhesion complex area defines adhesion size and lifetime.

Cell-matrix adhesions are central mediators of mechanotransduction, yet the interplay between force and adhesion regulation remains unclear. Here we use live cell imaging to map time-dependent cross-correlations between vinculin-mediated tension and adhesion complex area, revealing a plastic, context-dependent relationship. Interestingly, while an expected positive cross-correlation dominated in mid-sized adhesions, small and large adhesions display negative cross-correlation. Furthermore, although large changes in adhesion complex area follow vinculin-mediated tension alterations, small increases in area precede vinculin-mediated tension dynamics. Modelling based on this mapping of the vinculin-mediated tension-adhesion complex area relationship confirms its biological validity, and indicates that this relationship explains adhesion size and lifetime limits, keeping adhesions focal and transient. We also identify a subpopulation of steady-state adhesions whose size and vinculin-mediated tension become stabilized, and whose disassembly may be selectively microtubule-mediated. In conclusion, we define a plastic relationship between vinculin-mediated tension and adhesion complex area that controls fundamental cell-matrix adhesion properties.

Tissue-culture light sheet fluorescence microscopy (TC-LSFM) allows long-term imaging of three-dimensional cell cultures under controlled conditions.

Fluorescence long-term imaging of cellular processes in three-dimensional cultures requires the control of media supply, temperature, and pH, as well as minimal photodamage. We describe a system based on a light sheet fluorescence microscope (LSFM), which is optimized for long-term, multi-position imaging of three-dimensional in-gel cell cultures. The system integrates a stable culture condition control system in the optical path of the light-sheet microscope. A further essential element is a biocompatible agarose container suitable for the LSFM, in which any cell type can be cultured in different gel matrices. The TC-LSFM allows studying any in vitro cultured cell type reacting to, dividing in, or migrating through a three-dimensional extracellular matrix (ECM) gel. For this reason we called it "tissue culture-LSFM" (TC-LSFM). The TC-LSFM system allows fast imaging at multiple locations within a millimeter-sized ECM gel. This increases the number of analyzed events and allows testing population effects. As an example, we show the maturation of a cyst of MDCK (canine kidney epithelial) cells over a period of three days. Moreover, we imaged, tracked, and analyzed MDCK cells during the first five days of cell aggregate formation and discovered a remarkable heterogeneity in cell cycle lengths and an interesting cell death pattern. Thus, TC-LSFM allows performing new long-term assays assessing cellular behavior in three-dimensional ECM-gel cultures. For example migration, invasion or differentiation in epithelial cell systems, stem cells, as well as cancer cells can be investigated.

A mathematical method for the 3D analysis of rotating deformable systems applied on lumen-forming MDCK cell aggregates.

Cell motility contributes to the formation of organs and tissues, into which multiple cells self-organize. However such mammalian cellular motilities are not characterized in a quantitative manner and the systemic consequences are thus unknown. A mathematical tool to decipher cell motility, accounting for changes in cell shape, within a three-dimensional (3D) cell system was missing. We report here such a tool, usable on segmented images reporting the outline of clusters (cells) and allowing the time-resolved 3D analysis of circular motility of these as parts of a system (cell aggregate). Our method can analyze circular motility in sub-cellular, cellular, multi-cellular, and also non-cellular systems for which time-resolved segmented cluster outlines are available. To exemplify, we characterized the circular motility of lumen-initiating MDCK cell aggregates, embedded in extracellular matrix. We show that the organization of the major surrounding matrix fibers was not significantly affected during this cohort rotation. Using our developed tool, we discovered two classes of circular motion, rotation and random walk, organized in three behavior patterns during lumen initiation. As rotational movements were more rapid than random walk and as both could continue during lumen initiation, we conclude that neither the class nor the rate of motion regulates lumen initiation. We thus reveal a high degree of plasticity during a developmentally critical cell polarization step, indicating that lumen initiation is a robust process. However, motility rates decreased with increasing cell number, previously shown to correlate with epithelial polarization, suggesting that migratory polarization is converted into epithelial polarization during aggregate development.

ROCK-mediated contractility, tight junctions and channels contribute to the conversion of a preapical patch into apical surface during isochoric lumen initiation.

Epithelial cells assemble into three-dimensional aggregates to generate lumen-containing organ substructures. Cells therein contact the extracellular matrix with their basal surface, neighbouring cells with their contact surface and the lumen with their apical surface. We investigated the development of single MDCK cells into aggregates with lumen using quantitative live-cell imaging to identify morphogenetic rules for lumen formation. In two-cell aggregates, membrane insertion into the contact surface established a preapical patch (PAP) characterized by the presence of the apical marker gp135, microvilli and the absence of E-cadherin. This PAP originated from a compartment that had hallmarks of an apical recycling endosome, and matured through Brefeldin-A-sensitive membrane trafficking and the establishment of tight junctions around itself. As a result of the activity of water and ion channels, an optically resolvable lumen formed. Initially, this lumen enlarged without changes in aggregate volume or cell number but with decreasing cell volumes. Additionally, the ROCK1/2-myosin-II pathway counteracted PAP and lumen formation. Thus, lumen formation results from PAP establishment, PAP maturation, lumen initiation and lumen enlargement. These phases correlate with distinct cell surface and volume patterns, which suggests that such morphometric parameters are regulated by trafficking, ROCK-mediated contractility and hydrostatic pressure or vice versa.