Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of cancer cells critical in cancer progression. However, the underlying mechanisms of how TRCs maintain their self-renewing capability remain elusive. Here we show that relatively undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening, histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability. In contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is unresponsive to matrix stiffness or applied forces. Silencing H3K9 methyltransferase G9a or SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner. Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both in vitro and in vivo. Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC self-renewal.

Protein functions are largely affected by their conformations. This is exemplified in proteins containing modular domains. However, the evolutionary dynamics that define and adapt the conformation of such modular proteins remain elusive. Here we show that cis-interactions between the C-terminal phosphotyrosines and SH2 domain within the protein tyrosine phosphatase Shp2 can be tuned by an adaptor protein, Grb2. The competitiveness of two phosphotyrosines, namely pY542 and pY580, for cis-interaction with the same SH2 domain is governed by an antagonistic combination of contextual amino acid sequence and position of the phosphotyrosines. Specifically, pY580 with the combination of a favorable position and an adverse sequence has an overall advantage over pY542. Swapping the sequences of pY542 and pY580 results in one dominant form of cis-interaction and subsequently inhibits the trans-regulation by Grb2. Thus, the antagonistic combination of sequence and position may serve as a basic design principle for proteins with tunable conformations.

Reactive oxygen species (ROS) have been shown to play crucial roles in regulating various cellular functions, e.g. focal adhesion (FA) dynamics and cell migration upon growth factor stimulation. However, it is not clear how ROS are regulated at subcellular FA sites to impact cell migration. We have developed a biosensor capable of monitoring ROS production at FA sites in live cells with high sensitivity and specificity, utilizing fluorescence resonance energy transfer (FRET). The results revealed that platelet derived growth factor (PDGF) can induce ROS production at FA sites, which is mediated by Rac1 activation. In contrast, integrins, specifically integrin αvβ3, inhibits this local ROS production. The RhoA activity can mediate this inhibitory role of integrins in regulating ROS production. Therefore, PDGF and integrin αvβ3 coordinate to have an antagonistic effect in the ROS production at FA sites to regulate cell adhesion and migration.

A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescent labeling with high efficiency, specificity, and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ~100% efficiency while maintaining the biological function. We demonstrate that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in singlemolecule pull-down analysis. We further show the unique power of our method in a series of single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

DNA helicases are multi-functional motor proteins which translocate along DNA using the energy released from ATP hydrolysis. Translocation is coupled to diverse enzymatic activities, such as duplex unwinding, strand degradation and protein displacement. Resolving such complex processes requires our ability to detect rare, or short-lived intermediates and observe their dynamics. To this end, we have developed a generic fluorescence-based assay, which enables monitoring of DNA unwinding by single helicases in vitro. Biotinylated double-stranded DNA fragments are specifically immobilised on poly-ethylene glycol-coated surfaces through biotin-neutravidin interaction. Total internal reflection fluorescence (TIRF) microscopy is used to achieve high signal-to-noise ratio. To probe DNA unwinding, we use a fluorescently labelled mutant of the E.Coli single-stranded DNA-binding protein (SSB), which specifically binds single-stranded DNA. DNA unwinding events mediated by single helicases are observed as fluorescent spots of increasing intensity, as increasing numbers of SSB molecules bind to the ssDNA product of the helicase. Using an objective-based TIRF microscope, these events are recorded at video rate. The increase in fluorescence intensity directly correlates to the rate at which single helicases unwind DNA. Here, we show that the assay is a powerful tool for probing DNA unwinding by various helicases as a function of DNA sequence, ATP and salt concentration. In comparison with bulk measurements, our single molecule data demonstrate that DNA unwinding involves multiple phases which are masked during ensemble averaging. Hence, this assay adds to the single molecule toolbox available for studying DNA processing enzymes.