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Mammalian cells redirect their movement in response to changes in the physical properties of their extracellular matrix (ECM) adhesive scaffolds, including changes in available substrate area, shape, or flexibility. Yet, little is known about the cell's ability to discriminate between different types of spatial signals. Here we utilize a soft-lithography-based, microcontact printing technology in combination with automated computerized image analysis to explore the relationship between ECM geometry and directional motility. When fibroblast cells were cultured on fibronectin-coated adhesive islands with the same area (900 micrometers2) but different geometric forms (square, triangle, pentagon, hexagon, trapezoid, various parallelograms) and aspect ratios, cells preferentially extended new lamellipodia from their corners. In addition, by imposing these simple geometric constraints through ECM, cells were directed to deposit new fibronectin fibrils in these same corner regions. These data indicate that mammalian cells can sense edges within ECM patterns that exhibit a wide range of angularity and that they use these spatial cues to guide where they will deposit ECM and extend new motile processes during the process of directional migration.

This paper reports a simple and versatile technique for generating structures on the surfaces of poly(dimethylsiloxane) (PDMS), approximately sinusoidal waves with periods between 0.1 and 10 μm, and the use of these structures to study cell contact guidance. The features are generated by stretching PDMS slabs mechanically, oxidizing them in an oxygen plasma, and allowing them to relax. These surface features are similar to photolithographically fabricated grooves that have traditionally been used to investigate cell contact guidance, although their edges are rounded rather than angular. Bovine capillary endothelial cells align and elongate on these features. The morphology and cytoskeletal structure of the aligned cells are similar to those of cells described in previous studies of contact guidance on surfaces with other types of topography. These observations and comparisons indicate that sharp edges in the features defining the grooves are not essential in eliciting contact guidance. This technique provides a method for fabricating microfeatures for the studies of the interactions between cells and their environment that does not require a cleanroom or access to photolithographic tools.

There are many experiments in which it would be useful to treat a part of the surface or interior of a cell with a biochemical reagent. It is difficult, however, to achieve subcellular specificity, because small molecules diffuse distances equal to the extent of the cell in seconds. This paper demonstrates experimentally, and analyzes theoretically, the use of multiple laminar fluid streams in microfluidic channels to deliver reagents to, and remove them from, cells with subcellular spatial selectivity. The technique made it possible to label different subpopulations of mitochondria fluorescently, to disrupt selected regions of the cytoskeleton chemically, to dislodge limited areas of cell-substrate adhesions enzymatically, and to observe microcompartmental endocytosis within individual cells. This technique does not require microinjection or immobilization of reagents onto nondiffusive objects; it opens a new window into cell biology.

Syncytia arising from the fusion of cells expressing the HIV‐1‐encoded Env gene with cells expressing the CD4/CXCR4 complex undergo apoptosis following the nuclear translocation of mammalian target of rapamycin (mTOR), mTOR‐mediated phosphorylation of p53 on Ser15 (p53S15), p53‐dependent upregulation of Bax and activation of the mitochondrial death pathway. p53S15 phosphorylation is only detected in syncytia in which nuclear fusion (karyogamy) has occurred. Karyogamy is secondary to a transient upregulation of cyclin B and a mitotic prophase‐like dismantling of the nuclear envelope. Inhibition of cyclin‐dependent kinase‐1 (Cdk1) prevents karyogamy, mTOR activation, p53S15 phosphorylation and apoptosis. Neutralization of p53 fails to prevent karyogamy, yet suppresses apoptosis. Peripheral blood mononuclear cells from HIV‐1‐infected patients exhibit an increase in cyclin B and mTOR expression, correlating with p53S15 phosphorylation and viral load. Cdk1 inhibition prevents the death of syncytia elicited by HIV‐1 infection of primary CD4 lymphoblasts. Thus, HIV‐1 elicits a pro‐apoptotic signal transduction pathway relying on the sequential action of cyclin B–Cdk1, mTOR and p53.

“Micro‐landscapes” suitable for investigating the behavior of living cells under different stimuli are cast from poly(dimethylsiloxane) by simultaneously etching a slab and a relief (see Figure) with a Bu4NF solution and flushing out dissolved material with N‐methylpyrrolidone. It is crucial that all liquid streams during the etching process are laminar. Various accessible topographies for the relief and examples of applications are given.

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