Mitochondria can recognize and assemble fragments of a beta-barrel structure

β-barrel proteins are found in the outer membranes of eukaryotic organelles of endosymbiotic origin as well as in the outer membrane of Gram-negative bacteria. Precursors of mitochondrial β-barrel proteins are synthesized in the cytosol and have to be targeted to the organelle. Currently, the signal that assures their specific targeting to mitochondria is poorly defined. To characterize the structural features needed for specific mitochondrial targeting and to test whether a full β-barrel structure is required, we expressed in yeast cells the β-barrel domain of the trimeric autotransporter Yersinia adhesin A (YadA). Trimeric autotransporters are found only in prokaryotes, where they are anchored to the outer membrane by a single 12-stranded β-barrel structure to which each monomer is contributing four β-strands. Importantly, we found that YadA is solely localized to the mitochondrial outer membrane, where it exists in a native trimeric conformation. These findings demonstrate that, rather than a linear sequence or a complete β-barrel structure, four β-strands are sufficient for the mitochondria to recognize and assemble a β-barrel protein. Remarkably, the evolutionary origin of mitochondria from bacteria enables them to import and assemble even proteins belonging to a class that is absent in eukaryotes.     

High refractive index silicone gels for simultaneous total internal reflection fluorescence and traction force microscopy of adherent cells

Substrate rigidity profoundly impacts cellular behaviors such as migration, gene expression, and cell fate. Total Internal Reflection Fluorescence (TIRF) microscopy enables selective visualization of the dynamics of substrate adhesions, vesicle trafficking, and biochemical signaling at the cell-substrate interface. Here we apply high-refractive-index silicone gels to perform TIRF microscopy on substrates with a wide range of physiological elastic moduli and simultaneously measure traction forces exerted by cells on the substrate.     

Integrin alpha4beta1 promotes focal adhesion kinase-independent cell motility via alpha4 cytoplasmic domain-specific activation of c-Src

The fibronectin binding integrins alpha5beta1 and alpha4beta1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For alpha5beta1, beta1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for alpha5beta1-stimulated cell motility and that exogenous expression of human alpha4 in FAK-null fibroblasts forms a functional alpha4beta1 receptor that promotes robust cell motility equal to the alpha5beta1 stimulation of wild-type and FAK-reconstituted fibroblasts. alpha4beta1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the alpha4 cytoplasmic domain, independent of direct paxillin binding to alpha4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. alpha4 cytoplasmic domain-initiated signaling led to a approximately 4-fold activation of c-Src which did not require paxillin binding to alpha4. Notably, alpha4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase alpha overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. alpha4beta1-stimulated cell motility of triple-null Src(-/-), c-Yes(-/-), and Fyn(-/-) fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for alpha5beta1-stimulated FAK activation, our results support the existence of a novel alpha4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway.     

Integrin regulation

Integrin signaling is bidirectional. 'Inside-out' signals regulate integrin affinity for adhesive ligands, and ligand-dependent 'outside-in' signals regulate cellular responses to adhesion. Integrin extracellular domains are yielding to high-resolution structural analyses, and intracellular proteins involved in integrin signaling are being identified. However, a key unresolved question is how integrins propagate signals across the plasma membrane.     

Impact of Admission and Cache Replacement Policies on Response Times of Jobs on Data Grids

Caching techniques have been used widely to improve the performance gaps of storage hierarchies in computing systems. Little is known about the impact of policies on the response times of jobs that access and process very large files in data grids, particularly when data and computations on the data have to be co-located on the same host. In data intensive applications that access large data files over wide area network environment, such as data-grids, the combination of policies for job servicing (or scheduling), caching and cache replacement can significantly impact the performance of grid jobs. We present preliminary results of a simulation study that combines an admission policy with a cache replacement policy when servicing jobs submitted to a storage resource manager.The results show that, in comparison to a first come first serve policy, the response times of jobs are significantly improved, for practical limits of disk cache sizes, when the jobs that are back-logged to access the same files are taken into consideration in scheduling the next file to be retrieved into the disk cache. Not only are the response times of jobs improved, but also the metric measures for caching policies, such as the hit ratio and the average cost per retrieval, are improved irrespective of the cache replacement policy used. Ekow Otoo is research staff scientist with the scientific data management group at Lawrence Berkeley National Laboratory, University of California, Berkeley. He received his B.Sc. degree in Electrical Engineering from the University of Science and Technology, Kumasi, Ghana and a post graduate diploma in Computer Science from the University of Ghana, Legon. In 1977, he received his M.Sc. degree in Computer Science from the University of Newcastle Upon Tyne in Britain and his Ph.D. degree in Computer Science from McGill University, Montreal, Canada in 1983. He joined the faculty of the School of Computer Science, Carleton University, in 1983 and from 1987 to 1999, he was a tenured faculty member of the School of Computer Science, Carleton University, Ottawa, Canada. He has served as research consultant to Bell Northern Research, Ottawa, Canada, and as a research project consultant to the GIS Division, Geomatics Canada, Natural Resources Canada, from 1990 to 1998. Ekow Otoo is a member of the ACM and IEEE. His research interests include database management systems, data structures and algorithms, parallel I/O for high performance computing, parallel and distributed computing. Doron Rotem is currently a senior staff scientist and a member of the Data Management group at the Lawrence Berkeley National Lab. His research interests include Grid Computing, Workflow, Scientific Data Management and Paralled and Distributed Computing and Algorithms. He has published over 80 papers in international journals and conferences in these areas. Prior to that, Dr Rotem co-founded and served as a CTO of a startup company, called CommerceRoute, that made software products in the area of workflow and data integration and before that, he was an Associate Professor in the Department of Computer Science, University of Waterloo, Canada. Dr. Rotem holds a B.Sc degree in Mathematics and Statistics from the Hebrew University, Jerusalem, Israel and a Ph.D. in Computer Science from the University of the Witwatersrand, Johannesburg, South Africa. Arie Shoshani is a senior staff scientist at Lawrence Berkeley National Laboratory. He joined LBNL in 1976. He heads the Scientific Data Management Group. He received his Ph.D. from Princeton University in 1969. From 1969 to 1976, he was a researcher at System Development Corporation, where he worked on the Network Control Program for the ARPAnet, distributed databases, database conversion, and natural language interfaces to data management systems. His current areas of work include data models, query languages, temporal data, statistical and scientific database management, storage management on tertiary storage, and grid storage middleware. Arie is also the director of a Scientific Data Management (SDM) Integrated Software Infrastructure Center (ISIC), one of seven centers selected by the SciDAC program at DOE in 2001. In this capacity, he is coordinating the work of collaborators from 4 DOE laboratories and 4 universities (see: http://sdmcenter.lbl.gov). Dr. Shoshani has published over 65 technical papers in refereed journals and conferences, chaired several workshops, conferences, and panels in database management; and served on numerous program committees for various database conferences. He also served as an associate editor for the ACM Transactions on Database Systems. He was elected a member of the VLDB Endowment Board, served as the Publication Board Chairperson for the VLDB Journal, and as the Vice-President of the VLDB Endowment. His home page is http://www.lbl.gov/arie.     

Strong and ubiquitous expression of transgenes targeted into the beta-actin locus by Cre/lox cassette replacement

Conventional approaches to produce transgenic mice recurrently yield unpredictable patterns and levels of transgene expression, a situation calling for the development of new techniques to overcome these drawbacks in the context of overexpression studies. Here we present an efficient method for rapid and reproducible transgenesis using the recombinase mediated cassette exchange (RMCE) (Bouhassira et al.: Blood 90:3332-3344, 1997) procedure. A lox511-EGFP-TK/neo-loxP cassette was placed under the control of the endogenous mouse beta-actin promoter. Heterozygous mice revealed strong and ubiquitous EGFP expression throughout embryogenesis and adulthood. Reproducibly, the same expression pattern was obtained with RMCE when it was used to replace the EGFP-harboring cassette by ECFP or placental alkaline phosphatase (PLAP) reporter genes (DePrimo et al.: Transgenic Res 5:459-466, 1996). Furthermore, the RMCE procedure proved efficient as well in embryonic stem (ES) cells as directly in zygotes. Our results demonstrate ubiquitous expression of floxed transgenes in the endogenous beta-actin locus and they support the general use of the beta-actin locus for targeted transgenesis.     

Fine-needle aspiration of the thyroid: an overview

Thyroid nodules (TN) are a common clinical problem. Fine needle aspiration (FNA) of the thyroid now is practiced worldwide and proves to be the most economical and reliable diagnostic procedure to identify TNs that need surgical excision and TNs that can be managed conservatively. The key for the success of thyroid FNA consists of an adequate or representative cell sample and the expertise in thyroid cytology. The FNA cytologic manifestations of TNs may be classified into seven working cytodiagnostic groups consisting of a few heterogenous lesions each to facilitate the differential diagnosis. Recent application of diagnostic molecular techniques to aspirated thyroid cells proved to be useful in separating benign from malignant TNs in several cases of indeterminate lesions.     

Structural basis for phosphatidylinositol phosphate kinase type Igamma binding to talin at focal adhesions

The cytoskeletal protein talin binds to a short C-terminal sequence in phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), activating the enzyme and promoting the local production of phosphatidylinositol 4,5 bisphosphate, which regulates focal adhesion dynamics as well as clathrin-mediated endocytosis in neuronal cells. Here we show by crystallographic, NMR, and calorimetric analysis that the phosphotyrosine binding (PTB)-like domain of talin engages the PIPKIgamma C terminus in a mode very similar to that of integrin binding. However, PIPKIgamma binds in the canonical PTB-peptide mode with an SPLH motif replacing the classic NPXY motif. The tighter packing of the SPLH motif against the hydrophobic core of talin may explain the stronger binding of PIPKIgamma. Two tyrosine residues flanking the SPLH motif (Tyr-644 and Tyr-649) have been implicated in the regulation of talin binding. We show that phosphorylation at Tyr-644, a Src phosphorylation site in vivo, has little effect on the binding mode or strength, which is consistent with modeling studies in which the phosphotyrosine makes surface-exposed salt bridges, and we suggest that its strong activating effect arises from the release of autoinhibitory restraints in the full-length PIPKIgamma. Modeling studies suggest that phosphorylation of Tyr-649 will likewise have little effect on talin binding, whereas phosphorylation of the SPLH serine is predicted to be strongly disruptive. Our data are consistent with the proposal that Src activity promotes a switch from integrin binding to PIPKIgamma binding that regulates focal adhesion turnover.