Department of Biology Works

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An integrated proteomics analysis of bone tissues in response to mechanical stimulation
(2010-07) Li, Jillian; Zhang, Fan; Chen, Jake Yue
Bone cells can sense physical forces and convert mechanical stimulation conditions into biochemical signals that lead to expression of mechanically sensitive genes and proteins. However, it is still poorly understood how genes and proteins in bone cells are orchestrated to respond to mechanical stimulations. In this research, we applied integrated proteomics, statistical, and network biology techniques to study proteome-level changes to bone tissue cells in response to two different conditions, normal loading and fatigue loading. We harvested ulna midshafts and isolated proteins from the control, loaded, and fatigue loaded Rats. Using a label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) experimental proteomics technique, we derived a comprehensive list of 1,058 proteins that are differentially expressed among normal loading, fatigue loading, and controls. By carefully developing protein selection filters and statistical models, we were able to identify 42 proteins representing 21 Rat genes that were significantly associated with bone cells' response to quantitative changes between normal loading and fatigue loading conditions. We further applied network biology techniques by building a fatigue loading activated protein-protein interaction subnetwork involving 9 of the human-homolog counterpart of the 21 rat genes in a large connected network component. Our study shows that the combination of decreased anti-apoptotic factor, Raf1, and increased pro-apoptotic factor, PDCD8, results in significant increase in the number of apoptotic osteocytes following fatigue loading. We believe controlling osteoblast differentiation/proliferation and osteocyte apoptosis could be promising directions for developing future therapeutic solutions for related bone diseases.
p21-activated kinase regulates mast cell degranulation via effects on calcium mobilization and cytoskeletal dynamics
(2009-03) Allen, Jayme D; Jaffer, Zahara M; Park, Su-Jung; Burgin, Sarah; Hofmann, Clemens; Sells, Mary A; Chen, Shi; Derr-Yellin, Ethel; Michels, Elizabeth G; McDaniel, Andrew; Bessler, Waylan K; Ingram, David A; Atkinson, Simon J; Travers, Jeffrey B; Chemoff, Jonathan; Clapp, D Wade
Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcϵRI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, Wsh/Wsh mast cell–deficient mice locally reconstituted with Pak1−/− bone marrow–derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1−/− BMMCs exhibited a reduction in FcϵRI-induced degranulation. Further, Pak1−/− BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcϵRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases.
Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation
(1991-08) Atkinson, Simon J; Stewart, Murray
We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.
Molecular basis of myosin assembly: coiled-coil interactions and the role of charge periodicities
(1991-01) Atkinson, Simon J; Stewart, Murray
Complementation of alternating zones of positive and negative charge in the myosin rod enables molecules to interact in a number of ways. This accounts for the complexity of the molecular organisation of thick filaments. However, directed mutagenesis of expressed LMM cDNA indicated that charge zone complementation is not a major driving force in myosin polymerisation. Instead, it probably serves to prevent unfavourable interaction geometries.
Mechanism of Actin Polymerization in Cellular ATP Depletion
(2003-11) Atkinson, Simon J; Hosford, Melanie A; Molitoris, Bruce A
Cellular ATP depletion in diverse cell types results in the net conversion of monomeric G-actin to polymeric F-actin and is an important aspect of cellular injury in tissue ischemia. We propose that this conversion results from altering the ratio of ATP-G-actin and ADP-G-actin, causing a net decrease in the concentration of thymosinactin complexes as a consequence of the differential affinity of thymosin β4 for ATP- and ADP-G-actin. To test this hypothesis we examined the effect of ATP depletion induced by antimycin A and substrate depletion on actin polymerization, the nucleotide state of the monomer pool, and the association of actin monomers with thymosin and profilin in the kidney epithelial cell line LLC-PK1. ATP depletion for 30 min increased F-actin content to 145% of the levels under physiological conditions, accompanied by a corresponding decrease in G-actin content. Cytochalasin D treatment did not reduce F-actin formation during ATP depletion, indicating that it was predominantly not because of barbed end monomer addition. ATP-G-actin levels decreased rapidly during depletion, but there was no change in the concentration of ADP-G-actin monomers. The decrease in ATP-G-actin levels could be accounted for by dissociation of the thymosin-G-actin binary complex, resulting in a rise in the concentration of free thymosin β4 from 4 to 11 μM. Increased detection of profilin-actin complexes during depletion indicated that profilin may participate in catalyzing nucleotide exchange during depletion. This mechanism provides a biochemical basis for the accumulation of F-actin aggregates in ischemic cells.
Cytokine-induced F-actin reorganization in endothelial cells involves RhoA activation
(2009-03) Campos, Silvia B; Ashworth, Sharon L; Wean, Sarah; Hosford, Melanie; Sandoval, Ruben M; Hallett, Mark A; Atkinson, Simon J; Molitoris, Bruce A
Acute ischemic kidney injury results in marked increases in local and systemic cytokine levels. IL-1α, IL-6, and TNF-α orchestrate various inflammatory reactions influencing endothelial permeability by altering cell-to-cell and cell-to-extracellular matrix attachments. To explore the role of actin and the regulatory proteins RhoA and cofilin in this process, microvascular endothelial cells (MS1) were exposed to individual cytokines or a cytokine cocktail. Within minutes, a marked, time-dependent redistribution of the actin cytoskeleton occurred with the formation of long, dense F-actin basal stress fibers. The concentration of F-actin, normalized to nuclear staining, significantly increased compared with untreated cells (up 20%, P ≤ 0.05). Western blot analysis of MS1 lysates incubated with the cytokine cocktail for 4 h showed an increase in phosphorylated/inactive cofilin (up 25 ± 15%, P ≤ 0.05) and RhoA activation (up to 227 ± 26% increase, P ≤ 0.05) compared with untreated cells. Decreasing RhoA levels using small interfering RNA blocked the effect of cytokines on stress fiber organization. Treatment with Y-27632, an inhibitor of the RhoA effector p160-ROCK, decreased levels of phosphorylated cofilin and reduced stress fiber fluorescence by 22%. In cells treated with Y-27632 followed by treatment with the cytokine cocktail, stress fiber levels were similar to control cells and cofilin phosphorylation was 55% of control levels. Taken together, these studies demonstrate cytokine stimulation of RhoA, which in turn leads to cofilin phosphorylation and formation of numerous basal actin stress fibers. These results suggest cytokines signal through the Rho-ROCK pathway, but also through another pathway to affect actin dynamics.
RhoA/Rho kinase (ROCK) alters fetuin-A uptake and regulates calcification in bovine vascular smooth muscle cells (BVSMC)
(2010-09) Chen, Neal X; Chen, Xianming; O'Neill, Kalisha D; Atkinson, Simon J; Moe, Sharon M
RhoA/Rho kinases (ROCK) play a critical role in vascular smooth muscle cell (VSMC) actin cytoskeleton organization, differentiation, and function and are implicated in the pathogenesis of cardiovascular disease. We have previously determined that an important step in the regulation of calcification is fetuin-A endocytosis, a process that is dependent on changes in the cytoskeleton, which, in turn, is known to be affected by the RhoA/ROCK signaling pathway. In the present study, bovine VSMC (BVSMC) were treated with the ROCK inhibitor Y-27632 or transfected with ROCK small interfering (si) RNA to knock down ROCK expression. Both conditions resulted in reduced actin stress fibers and increased Cy5-labeled fetuin-A uptake. Inhibition of ROCK by Y-27632 or siRNA also significantly increased BVSMC alkaline phosphatase (ALP) activity and calcification of BVSMC and rat aorta organ cultures. Cells were then incubated in calcification media in the presence or absence of Y-27632 and matrix vesicles (MV) isolated by collagenase digestion. These MV, isolated from BVSMC incubated with Y-27632, had increased ALP activity and increased ability of MV to subsequently calcify collagen by 66%. In contrast, activation of RhoA, which is upstream of ROCK, by transfecting plasmids encoding the dominant active Rho GTPase mutant (Rho-L63) led to decreased fetuin-A uptake and reduced calcification in BVSMC. These results demonstrate that the RhoA/ROCK signaling pathway is an important negative regulator of vascular calcification.
Myosin II Light Chain Phosphorylation Regulates Membrane Localization and Apoptotic Signaling of Tumor Necrosis Factor Receptor-1
(2001-05) Jin, Yijun; Atkinson, Simon J; Marrs, James A; Gallagher, Patricia J
Activation of myosin II by myosin light chain kinase (MLCK) produces the force for many cellular processes including muscle contraction, mitosis, migration, and other cellular shape changes. The results of this study show that inhibition or potentiation of myosin II activation via over-expression of a dominant negative or wild type MLCK can delay or accelerate tumor necrosis factor-α (TNF)-induced apoptotic cell death in cells. Changes in the activation of caspase-8 that parallel changes in regulatory light chain phosphorylation levels reveal that myosin II motor activities regulate TNF receptor-1 (TNFR-1) signaling at an early step in the TNF death signaling pathway. Treatment of cells with either ionomycin or endotoxin (lipopolysaccharide) leads to activation of myosin II and increased translocation of TNFR-1 to the plasma membrane independent of TNF signaling. The results of these studies establish a new role for myosin II motor activity in regulating TNFR-1-mediated apoptosis through the translocation of TNFR-1 to or within the plasma membrane.
Sequences, Structural Models, and Cellular Localization of the Actin-related Proteins Arp2 and Arp3 from Acanthamoeba
(1995-10) Kelleher, Joseph F; Atkinson, Simon J; Pollard, Thomas D
We cloned and sequenced the two actin-related proteins (Arps) present in the profilin-binding complex of Acanthamoeba (Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994, J. Cell Biol. 127:107-115). The sequence of Arp2 is more similar to other Arp2s than to actin, while the sequence of Arp3 is more similar to other Arp3s than to actin. Phylogenetic analysis of all known Arps demonstrates that most group into three major families, which are likely to be shared across all eukaryotic phyla. Together with conventional actins, the Arps form a larger family distinct from structurally related ATPases such as Hsp70's and sugar kinases. Atomic models of the Arps based on their sequences and the structure of actin provide some clues about function. Both Arps have atoms appropriately placed to bind ATP and divalent cation. Arp2, but not Arp3, has a conserved profilin-binding site. Neither Arp has the residues required to copolymerize with actin, but an Arp heterodimer present in the profilin-binding complex might serve as a pointed end nucleus for actin polymerization. Both Acanthamoeba Arps are soluble in cell homogenates, and both are concentrated in the cortex of Acanthamoeba. The cellular concentrations are 1.9 microM Arp2 and 5.1 microM Arp3, substoichiometric to actin (200 microM) but comparable to many actin-binding proteins.
A Fluorescently Tagged C-Terminal Fragment of p47phox Detects NADPH Oxidase Dynamics during Phagocytosis
(2009-03) Li, Xing Jun; Tian, Wei; Stull, Natalie D; Grinstein, Sergio; Atkinson, Simon J; Dinauer, Mary C
The assembly of cytosolic p47phox and p67phox with flavocytochrome b558 at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47phox and p67phox are linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67phox translocation in neutrophils, but is not required for oxidase activity in model systems. Here we examined phagocytosis-induced NADPH oxidase assembly, showing the sequential recruitment of YFP-tagged p67phox to the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47phox PRR. This fragment was recruited in a flavocytochrome b558-dependent, p67phox-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67phox SH3b domain and suggest that the p47phox/p67phox tail-to-tail interaction is disrupted after oxidase assembly such that the p67phox-SH3b domain becomes accessible. Superoxide generation was sustained within phagosomes, indicating that this change does not correlate with loss of enzyme activity. This study defines a sequence of events during phagocytosis-induced NADPH oxidase assembly and provides experimental evidence that intermolecular interactions within this complex are dynamic and modulated after assembly on phagosomes.

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