We have characterized the mechanism by which the subcellular distribution of c‐Src is controlled by the phosphorylation of tyrosine 527. Mutation of this tyrosine dramatically redistributes c‐Src from endosomal membranes to focal adhesions. Redistribution to focal adhesions occurs independently of kinase activity and cellular transformation. In cells lacking the regulatory kinase (CSK) that phosphorylates tyrosine 527, c‐Src is also found predominantly in focal adhesions, confirming that phosphorylation of tyrosine 527 affects the location of c‐Src inside the cell. The first 251 amino acids of c‐Src are sufficient to allow association with focal adhesions, indicating that at least one signal for positioning c‐Src in focal adhesions resides in the amino‐terminal half. Point mutations and deletions in the first 251 amino acids of c‐Src reveal that association with focal adhesions requires the myristylation site needed for membrane attachment, as well as the SH3 domain. Expression of the amino‐terminal region alters both the structural and biochemical properties of focal adhesions. Focal adhesions containing this non‐catalytic portion of c‐Src are larger and exhibit increased levels of phosphotyrosine staining. Our results suggest that c‐Src may regulate focal adhesions and cellular adhesion by a kinase‐independent mechanism.

Thefluorescence microscope is routinely used to study cellular structure in many biomedical research laboratories and is increasingly used as a quantitative assay system for cellular dynamics. One of the major causes of image degradation in the fluorescence microscope is blurring. Deconvolution algorithms use a model of the microscope imaging process to either subtract or reassign out-of-focus blur. A variety of algorithms are now commercially available, each with its own characteristic advantages and disadvantages. In this article, we review the imaging process in the fluorescence microscope and then discuss how the various deconvolution methods work. Finally, we provide a summary of practical tips for using deconvolution and discuss imaging artifacts and how to minimize them.

SummaryPHD1 belongs to the family of prolyl-4-hydroxylases (PHDs) that is responsible for posttranslational modification of prolines on specific target proteins. Because PHD activity is sensitive to oxygen levels and certain byproducts of the tricarboxylic acid cycle, PHDs act as sensors of the cell’s metabolic state. Here, we identify PHD1 as a critical molecular link between oxygen sensing and cell-cycle control. We show that PHD1 function is required for centrosome duplication and maturation through modification of the critical centrosome component Cep192. Importantly, PHD1 is also required for primary cilia formation. Cep192 is hydroxylated by PHD1 on proline residue 1717. This hydroxylation is required for binding of the E3 ubiquitin ligase SCFSkp2, which ubiquitinates Cep192, targeting it for proteasomal degradation. By modulating Cep192 levels, PHD1 thereby affects the processes of centriole duplication and centrosome maturation and contributes to the regulation of cell-cycle progression.

Bioimaging data have significant potential for reuse, but unlocking this potential requires systematic archiving of data and metadata in public databases. We propose draft metadata guidelines to begin addressing the needs of diverse communities within light and electron microscopy. We hope this publication and the proposed Recommended Metadata for Biological Images (REMBI) will stimulate discussions about their implementation and future extension.

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