Improvements to the Cell Migration Knowledgebase

We have improved the Cell Migration Knowledgebase in order to make all available information on genes, proteins and their orthologs quicker and easier to find. Information about all members of an ortholog set is now displayed on gene and protein pages. For example, when looking at the details of a human gene or protein, you will find the equivalent phosphorylation sites in other organisms. In addition, the ortholog set and family pages now show all major names by which the genes are known, and we provide links to other important databases. We also show how many consortium products are available for each gene. Go to the following link for an example: Paxillin family.

Paxillin is a scaffold protein that localizes to the intracellular surface of cell adhesion sites. It recruits regulatory and structural proteins that control the dynamic changes in cell adhesion, cytoskeletal reorganization and gene expression important for cell migration. Recruitment occurs through the interactions of its multiple protein-binding modules, which are often regulated by phosphorylation. Paxillin coordinates the spatial and temporal action of the Rho family of small GTPases that regulate the actin cytoskeleton by recruiting GTPase activators, suppressors and effector proteins.

Causal mapping (CMAP): Generating hypotheses about how molecular and cellular systems are regulated.

Considerable efforts have been made to develop models aimed at increasing our understanding of biological processes at a systems-level. Gabriel Weinreb and colleagues previously introduced a systems biology tool called causal mapping (CMAP) for studying complex cellular and molecular mechanisms. CMAP is a coarse-grained graphical modeling approach in which the system being studied is described as a map of its functional elements. A CMAP network therefore looks very similar to how signaling pathways are commonly represented by molecular cell biologists; a mapped model of known and hypothetical interactions — such as inhibitory or activating functions — between the elements of the system. In PLoS ONE, Gabriel Weinreb and colleagues now report the use of the CMAP methodology as a tool for generating hypotheses about the mechanisms that regulate molecular and cellular systems. The authors apply this method to the complex phenomenon of cortical oscillation, which is observed in spreading cells following microtubule depolymerization and known to involve intracellular calcium and the activation of the Rho pathway. They also show that CMAP enables different hypotheses to be ranked according to a fitness index and suggest experimental approaches to distinguish between these competing hypotheses. By generating a variety of hypotheses to explain complex biological systems, the ultimate aims of these tools are to understand the underlying mechanisms of biological processes and to guide further experimental design.

• Weinreb GE, Kapustina MT, Jacobson K, Elston TC. In silico generation of alternative hypotheses using causal mapping (CMAP). PLoS ONE 2009;4(4):e5378. Epub 2009 Apr 29. PubMed

Methods: Screening for bright chemosensors

Regulation of protein activity is often achieved by phosphorylation in both eukaryotes and prokaryotes. Thus, protein kinases play a major role in controlling numerous cellular processes by catalysing the transfer of the γ-phosphoryl ATP group to the side chains of Ser, Thr and/or Tyr residues. Developing tools for monitoring kinase activity is important for both the basic understanding of signaling networks and for the pharmaceutical industry. Barbara Imperiali’s group has recently developed sulfonamido-oxine (Sox)-based fluorescent peptides for assaying Ser/Thr and Tyr kinases. Upon phosphorylation, the chromophore of Sox-containing substrates binds Mg2+ and undergoes chelation-enhanced fluorescence (CHEF). However, substrate specificity remains the main challenge in studying kinase activity using this and other techniques. In Bioorganic and Medicinal Chemistry Letters, Barbara Imperiali and colleagues now report the development of a new method for identifying Sox-based probes with improved specificity for Ser/Thr kinases. In this method, a combinatorial peptide library is first exposed to the desired kinase. After chemical modification of the phosphopeptides in the library, the products are identified using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). This method is proposed to be both specific and versatile, and its reliability has been confirmed by identifying the best known sequence for Protein Kinase A.

• González-Vera JA, Luković E, Imperiali B. A rapid method for generation of selective Sox-based chemosensors of Ser/Thr kinases using combinatorial peptide libraries. Bioorg Med Chem Lett. 2009 Feb 15;19(4):1258-60. PubMed

Podosomes, ruffled border and osteoclast behavior: Down to the bone

Bone maintains its functionality by undergoing continuous remodeling as a result of the coordinated activity of osteoblasts and osteoclasts. Whereas osteoblasts form new bone, osteoclasts resorb it. Bone resorption requires a specialized apparatus that consists of a sealing zone through which osteoclasts attach to the bone surface. The specialized ‘ruffled border’ organelle acidifies and secretes proteolytic enzymes into the cavity between the cell membrane and the bone surface. This induces the local dissolution of the mineral, which is followed by digestion of the remaining collagen. Podosomes - small, actin-associated adhesion sites - play a key role in assembling this system as they first assemble into clusters that develop into the sealing zone. The bone resorption activity of osteoclasts is regulated at many levels and appears to be dependent on the chemical and mechanical properties of the underlying bone. However, how these bone properties are sensed remains unclear. To address this question, Lia Addadi and colleagues compared the behavior of osteoclasts cultured on three distinct surfaces - glass, calcite crystals and bone. Using light and electron microscopy, the authors show that the structure and organization of podosomes is independent from the substrate matrix, suggesting that their assembly is intrinsically regulated. In contrast, the integrity and stability of podosomes, as well as the structure of the ruffled border, appeared to be surface-induced. Further studies will identify the local characteristics of the bone surface underlying the differences in osteoclast behavior.

• Geblinger D, Geiger B, Addadi L. Surface-induced regulation of podosome organization and dynamics in cultured osteoclasts. Chembiochem. 2009;10(1):158-65. PubMed

Cofilin activity regulation: It’s all about cycling

Cell migration requires the assembly of actin filaments at the leading edge to push the cell edge forward. Actin filaments are polarized and their polymerization occurs mainly at one end — the barbed end. The rate of actin assembly is controlled by the number of available free barbed ends, which is, in turn, regulated by numerous actin-binding proteins. Among these, cofilin is known to increase the number of barbed ends because of its capacity to sever existing filaments and its actin-nucleating activity. However, how cofilin is activated remains controversial. In an ‘Opinion article’ published in the Journal of Cell Science, Jacco van Rheenen and colleagues now show that cofilin is a key regulator of cell migration in inflammatory cells as well as regulating cancer cell metastasis. Furthermore, the authors propose that cofilin activity undergoes a single common cycle in both cell types. The initial activation of cofilin in tumour cells depends on phospholipase Cγ (PLCγ), whereas in inflammatory cells cofilin also requires dephosphorylation through Rac2. These are thought to be different starting points for the same cycle, in which cofilin activation/inactivation is accompanied by cycling between the cytosol, actin filaments and the plasma membrane.

• van Rheenen J, Condeelis J, Glogauer M. A common cofilin activity cycle in invasive tumor cells and inflammatory cells. J Cell Sci.2009 Feb 1;122(Pt3):305-11. PubMed

Chemosensors: Specificity for kinases

Intracellular phosphorylation plays a major role in the control of numerous cellular processes and its deregulation can lead to serious illness. Developing sensitive and selective tools for monitoring kinase activities is important both for understanding signaling cascades and for drug development. Recently, fluorescence-based assays suitable for high-throughput kinase screening in complex mixtures such as unfractionated cell lysates have been developed. However, as the number of kinases is very high, the major challenge in the field of kinase analysis remains the specificity of the sensor. In Journal of the American Chemical Society, Barbara Imperiali and colleagues now report the development of a general fluorescence-based sensor design with high capacity to discriminate between multiple kinases. The sensors are based on the previously reported β-turn focused (BTF) design that exploits the chelation-enhanced fluorescence (CHEF) of the sulfonamido-oxine amino acid Sox. In the BTF design, part of the kinase recognition motif is placed N- or C-terminal to the phosphorylation site and a constrained β-turn motif. In the absence of phosphorylation, the affinity for Mg2+ is low, but this increases dramatically upon phosphorylation because of a chelate effect. The drawback of the original design is that it could not implement an extended peptide recognition sequence because of the requirement for the constrained β-turn motif. The authors have now circumvented this limitation through alkylation of a cysteine residue within the sequence with a Sox-based chromophore. The resulting chemosensor peptides still show robust fluorescence changes on phosphorylation but they are sufficiently flexible to coordinate Mg2+ without the need for a β-turn, thus allowing the incorporation of extended kinase recognition motifs. The new design strategy greatly increases specificity as exemplified with the development of highly optimized chemosensors for Akt, MK2, PKC and Src.

• Lucovic E, Gonzalez-Vera J,A, Imperiali B. Recognition-Domain Focused Chemosensors: Versatile and Efficient Reporters of Protein Kinase Activity. J Am Chem Soc 2008;130(38):12821-7. PubMed