This paper reports on a model surface that is inert in biological fluids and that is important for studies in biointerfacial science. A self-assembled monolayer (SAM) terminated in the mannitol group was found to prevent the adsorption of proteins and the attachment of cells. Surface plasmon resonance spectroscopy showed that the mannitol-terminated SAM prevented the adsorption of several proteins and was indistinguishable from a SAM presenting tri(ethylene glycol)groups. In a second set of experiments, monolayers were patterned into circular regions of hexadecanethiolate with the surrounding area terminated in the mannitol group in order to evaluate the inert surface for patterning cell attachment. 3T3 fibroblasts, attached to the circular regions, proliferated to occupy these regions completely and remained patterned on these regions for 25 days. The use of oligo(ethylene glycol)-terminated SAMs, by contrast, showed a loss in fidelity of the pattern after one week in culture. The mannitol-terminated monolayers significantly extend the time course for maintaining patterned cells and will have immediate utility in fundamental and applied biology.

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffinembedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

The adhesion of mammalian cells is mediated by the binding of cell-surface integrin receptors to peptide ligands from the extracellular matrix and the clustering of these receptors into focal adhesion complexes. This paper examines the effect of one mechanistic variable, ligand affinity, on the assembly of focal adhesions (FAs) in order to gain mechanistic insight into this process. This study uses self-assembled monolayers of alkanethiolates on gold as a substrate to present either a linear or cyclic Arg-Gly-Asp peptide at identical densities. Inhibition assays showed that the immobilized cyclic RGD is a higher affinity ligand than linear RGD. 3T3 Swiss fibroblasts attached to substrates presenting the cyclic peptide at twice the rate they attached to substrates presenting the linear peptide. Quantitation of focal adhesions revealed that cells on cyclic RGD had twice the number of FAs as did cells on linear RGD and that these focal adhesions were on average smaller. These findings show that affinity affects the assembly of integrins into focal adhesions and support a model based on competing rates of nucleation and growth of FAs to explain the change in distribution of FAs with ligand affinity. This study is important because it provides a model system that is well-suited for biophysical studies of integrin-mediated cell adhesion and reveals insight into one mechanism utilized by cells to perceive environmental changes.

In both metazoan development and metastatic cancer, migrating cells must carry out a detailed, complex program of sensing cues, binding substrates, and moving their cytoskeletons. The linker cell in Caenorhabditis elegans males undergoes a stereotyped migration that guides gonad organogenesis, occurs with precise timing, and requires the nuclear hormone receptor NHR-67. To better understand how this occurs, we performed RNA-seq of individually staged and dissected linker cells, comparing transcriptomes from linker cells of third-stage (L3) larvae, fourth-stage (L4) larvae, and nhr-67-RNAi-treated L4 larvae. We observed expression of 8,000-10,000 genes in the linker cell, 22-25% of which were up-or downregulated 20-fold during development by NHR-67. Of genes that we tested by RNAi, 22% (45 of 204) were required for normal shape and migration, suggesting that many NHR-67-dependent, linker cell-enriched genes play roles in this migration. One unexpected class of genes up-regulated by NHR-67 was tandem pore potassium channels, which are required for normal linker-cell migration. We also found phenotypes for genes with human orthologs but no previously described migratory function. Our results provide an extensive catalog of genes that act in a migrating cell, identify unique molecular functions involved in nematode cell migration, and suggest similar functions in humans.major sperm protein | nematode development | transcriptional profiling | twin pore potassium channels | conserved uncharacterized proteins C ell migration is pervasive in animal development and is also an unwanted part of human cancer. Several different varieties of cell migration, ranging from social protozoa to the human immune system, have been extensively studied (1), but there remains a need for detailed analysis of the different components of migration in a simple cell type that can be exhaustively probed through anatomy, genetics, and genomics. We have undertaken to develop the linker cell (LC) of Caenorhabditis elegans as such a model. The LC is a single cell, attached to the front of a proliferating male gonad, which carries out a stereotypic, long-range migration to generate the shape of the mature gonad (Fig. 1A). During 25 h of the second to fourth larval stages (L2 through L4), the LC moves anteriorly along the ventral body wall, turns dorsally, proceeds posteriorly, switches sides once more to the ventral bodywall, continues posteriorward, reaches the cloaca, and eventually dies (2-4). Throughout this process, the proliferating and lengthening male gonad follows the LC's path to the cloaca. Stage-specific changes occur during the middle to late stages of migration (L3 and L4 larval stages): the LC changes shape from a spheroid to a pointed ellipsoid; it increases its speed of migration; and the netrin receptor gene unc-5 is down-regulated within the LC, which causes it to switch body walls ventrally ( Fig. 1A; ref. 3). These changes between the L3 and L4 stages require the orphan nuclear receptor NHR-67, whose orthologs TAILLESS and TLX...