FAK displacement from focal adhesions: a promising strategy to target processes implicated in cancer progression and metastasis.

Background: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed or activated in several advanced-stage solid cancers. It is known to play both kinase-dependent and -independent roles in promoting tumor progression and metastasis. Numerous inhibitors, targeting either the enzymatic or scaffolding activities of FAK have been generated, with varying degree of success.

Methods of Calpain Inhibition to Determine the Role of Calpains in Embryo Development in Amphibians.

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that the role of calpains in cell migration and other processes has been extensively studied in vitro, the same does not apply to cell migration and morphogenetic events during embryogenesis, in vivo. Herein, we describe the use of three different methods to selectively block calpain activity in vivo in order to investigate the impact on Xenopus gastrulation and neurulation, namely, a calpain inhibitor, a dominant negative, and a morpholino antisense oligonucleotide (MO).

Determining Temporal and Spatial Expression of Calpains in Amphibians.

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that Calpains have been identified in the Xenopus genome, their expression patterns and role have not been characterized. Therefore, herein, we describe two methods to determine temporal and spatial expression of Calpain 2 during Xenopus development, namely, RT-PCR and whole-mount in situ hybridization (WISH).

Addressing the Functional Determinants of FAK during Ciliogenesis in Multiciliated Cells.

We previously identified focal adhesion kinase (FAK) as an important regulator of ciliogenesis in multiciliated cells. FAK and other focal adhesion (FA) proteins associate with the basal bodies and their striated rootlets and form complexes named ciliary adhesions (CAs). CAs display similarities with FAs but are established in an integrin independent fashion and are responsible for anchoring basal bodies to the actin cytoskeleton during ciliogenesis as well as in mature multiciliated cells.

Making the connection: ciliary adhesion complexes anchor basal bodies to the actin cytoskeleton.

Cilia have been associated with diverse developmental and physiological processes, and defects in cilia underlie a number of genetic conditions. Several lines of evidence support a critical role of the actin cytoskeleton in ciliogenesis and ciliary function. Here, we show that well-characterized focal adhesion (FA) proteins, including FAK, Paxillin, and Vinculin, associate with the basal bodies of multiciliated cells and form complexes (CAs) that interact with the actin cytoskeleton.

In vivo, site-specific, covalent conjugation of quantum dots to proteins via split-intein splicing.

The ability to target proteins with nanostructures and/or nanodevices in vivo is important for understanding and controlling their biological function. Quantum dots (QDs) serve as an ideal model nanostructure due to their superior optical properties that permit visual confirmation of in vivo targeting and localization and due to their potential as a bio-imaging tool. Here, we describe the site-specific covalent conjugation of quantum dots to target proteins in vivo using an intein-based method.

Split-inteins for simultaneous, site-specific conjugation of quantum dots to multiple protein targets in vivo.

Background: Proteins labelled with Quantum Dots (QDs) can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within live cells or in vivo. However one of the major problems regarding the use of QDs for biological imaging is the difficulty of targeting QDs onto proteins. We have recently developed a DnaE split intein-based method to conjugate Quantum Dots (QDs) to the C-terminus of target proteins in vivo.