Local 3D matrix confinement determines division axis through cell shape.

How the division axis is determined in mammalian cells embedded in three-dimensional (3D) matrices remains elusive, despite that many types of cells divide in 3D environments. Cells on two-dimensional (2D) substrates typically round up completely to divide. Here, we show that in 3D collagen matrices, mammalian cells such as HT1080 human fibrosarcoma and MDA-MB-231 breast cancer cells exhibit division modes distinct from their Counterparts on 2D substrates, with a markedly higher fraction of cells remaining highly elongated through mitosis in 3D matrices. The long axis of elongated mitotic cells accurately predicts the division axis, independently of matrix density and cell-matrix interactions. This 3D-specific elongated division mode is determined by the local confinement produced by the matrix and the ability of cells to protrude and locally remodel the matrix via ß1 integrin. Elongated division is readily recapitulated using collagen-coated microfabricated channels. Cells depleted of ß1 integrin still divide in the elongated mode in microchannels, suggesting that 3D confinement is sufficient to induce the elongated cell-division phenotype.

Non-allosteric enzyme switches possess larger effector-induced changes in thermodynamic stability than their non-switch analogs.

The ability to regulate cellular protein activity offers a broad range of biotechnological and biomedical applications. Such protein regulation can be achieved by modulating the specific protein activity or through processes that regulate the amount of protein in the cell. We have previously demonstrated that the nonhomologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 ß-lactamase (BLA) can result in genes that confer maltose-dependent resistance to ß-lactam antibiotics even though the encoded proteins are not allosteric enzymes. We showed that these phenotypic switches-named based on their conferral of a switching phenotype to cells-resulted from a specific interaction with maltose in the cell that increased the switches cellular accumulation. Since phenotypic switches represent an important class of engineered proteins for basic science and biotechnological applications in vivo, we sought to elucidate the phenomena behind the increased accumulation and switching properties. Here, we demonstrate the key role for the linker region between the two proteins. Experimental evidence supports the hypothesis that in the absence of their effector, some phenotypic switches possess an increased rate of unfolding, decreased conformational stability, and increased protease susceptibility. These factors alone or in combination serve to decrease cellular accumulation. The effector functions to increase cellular accumulation by alleviating one or more of these defects. This perspective on the mechanism for phenotypic switching will aid the development of design rules for switch construction for applications and inform the study of the regulatory mechanisms of natural cellular proteins.