@article {518, title = {Vertebrate Hedgehog is secreted on two types of extracellular vesicles with different signaling properties. (Mass spectrometry - Proteomics)}, journal = {Sci Rep}, volume = {4}, year = {2014}, month = {2014 Dec 08}, pages = {7357}, abstract = {

Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3β and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation.

}, keywords = {Animals, Chick Embryo, Exosomes, Extracellular Space, Hedgehog Proteins, HEK293 Cells, Humans, MicroRNAs, Models, Biological, Protein Transport, Signal Transduction, Vertebrates}, issn = {2045-2322}, doi = {10.1038/srep07357}, author = {Vyas, Neha and Walvekar, Ankita and Tate, Dhananjay and Lakshmanan, Vairavan and Bansal, Dhiru and Lo Cicero, Alessandra and Raposo, Graca and Palakodeti, Dasaradhi and Dhawan, Jyotsna} } @article {713, title = {Active remodeling of cortical actin regulates spatiotemporal organization of cell surface molecules.}, journal = {Cell}, volume = {149}, year = {2012}, month = {2012 Jun 08}, pages = {1353-67}, abstract = {

Many lipid-tethered proteins and glycolipids exist as monomers and nanoclusters on the surface of living cells. The spatial distribution and dynamics of formation and breakup of nanoclusters does not reflect thermal and chemical equilibrium and is controlled by active remodeling of the underlying cortical actin. We propose a model for nanoclustering based on active hydrodynamics, wherein cell surface molecules bound to dynamic actin are actively driven to form transient clusters. This consistently explains all of our experimental observations. Using FCS and TIRF microscopy, we provide evidence for the existence of short, dynamic, polymerizing actin filaments at the cortex, a key assumption of the theoretical framework. Our theory predicts that lipid-anchored proteins that interact with dynamic actin must exhibit anomalous concentration fluctuations, and a cell membrane protein capable of binding directly to actin can form nanoclusters. These we confirm experimentally, providing an active mechanism for molecular organization and its spatiotemporal regulation on the plasma membrane.

}, keywords = {Actins, Animals, Cell Line, Tumor, Cell Membrane, CHO Cells, Cricetinae, Cytoskeleton, Humans, Membrane Proteins, Models, Biological, Spectrometry, Fluorescence}, issn = {1097-4172}, doi = {10.1016/j.cell.2012.05.008}, author = {Gowrishankar, Kripa and Ghosh, Subhasri and Saha, Suvrajit and C, Rumamol and Mayor, Satyajit and Rao, Madan} } @article {717, title = {Developmental heterogeneity in DNA packaging patterns influences T-cell activation and transmigration.}, journal = {PLoS One}, volume = {7}, year = {2012}, month = {2012}, pages = {e43718}, abstract = {

Cellular differentiation programs are accompanied by large-scale changes in nuclear organization and gene expression. In this context, accompanying transitions in chromatin assembly that facilitates changes in gene expression and cell behavior in a developmental system are poorly understood. Here, we address this gap and map structural changes in chromatin organization during murine T-cell development, to describe an unusual heterogeneity in chromatin organization and associated functional correlates in T-cell lineage. Confocal imaging of DNA assembly in cells isolated from bone marrow, thymus and spleen reveal the emergence of heterogeneous patterns in DNA organization in mature T-cells following their exit from the thymus. The central DNA pattern dominated in immature precursor cells in the thymus whereas both central and peripheral DNA patterns were observed in na{\"\i}ve and memory cells in circulation. Na{\"\i}ve T-cells with central DNA patterns exhibited higher mechanical pliability in response to compressive loads in vitro and transmigration assays in vivo, and demonstrated accelerated expression of activation-induced marker CD69. T-cell activation was characterized by marked redistribution of DNA assembly to a central DNA pattern and increased nuclear size. Notably, heterogeneity in DNA patterns recovered in cells induced into quiescence in culture, suggesting an internal regulatory mechanism for chromatin reorganization. Taken together, our results uncover an important component of plasticity in nuclear organization, reflected in chromatin assembly, during T-cell development, differentiation and transmigration.

}, keywords = {Animals, Antigens, CD, Antigens, Differentiation, T-Lymphocyte, Bone Marrow Cells, Cell Lineage, Cell Movement, Cell Nucleus, Chromatin, DNA, Hematopoietic Stem Cells, Lectins, C-Type, Lymphocyte Activation, Mice, Microscopy, Confocal, Models, Biological, Models, Statistical, Sequence Analysis, DNA, Spleen, T-Lymphocytes}, issn = {1932-6203}, doi = {10.1371/journal.pone.0043718}, author = {Gupta, Soumya and Marcel, Nimi and Talwar, Shefali and Garg, Megha and R, Indulaxmi and Perumalsamy, Lakshmi R and Sarin, Apurva and Shivashankar, G V} } @article {480, title = {Drosophila protein interaction map (DPiM): a paradigm for metazoan protein complex interactions. [Drosophila facility]}, journal = {Fly (Austin)}, volume = {6}, year = {2012}, month = {2012 Oct-Dec}, pages = {246-53}, abstract = {

Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein-especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex "map" provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map.

}, keywords = {Animals, Cell Line, Computational Biology, Drosophila melanogaster, Drosophila Proteins, Models, Biological, Protein Interaction Mapping, Protein Interaction Maps}, issn = {1933-6942}, doi = {10.4161/fly.22108}, author = {Guruharsha, K G and Obar, Robert A and Mintseris, Julian and Aishwarya, K and Krishnan, R T and VijayRaghavan, K and Artavanis-Tsakonas, Spyros} }