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The bacteriophage Mu transposase protein can form high-affinity protein-DNA complexes with the ends of transposable elements of the Tn 3 family 总被引:1,自引:0,他引:1
The 37 kb transposable bacteriophage Mu genome encodes a transposase protein which can recognize and bind to a consensus sequence repeated three times at each extremity of its genome. A subset of this consensus sequence (5'-PuCGAAA(A)-3') is found in the ends of many class II prokaryotic transposable elements. These elements, like phage Mu, cause 5 bp duplications at the site of element insertion, and transpose by a cointegrate mechanism. Using the band retardation assay, we have found that crude protein extracts containing overexpressed Mu transposase can form high-affinity protein-DNA complexes with Mu att R and the ends of the class II elements Tn 3 (right) and IS101. No significant protein-DNA complex formation was observed with DNA fragments containing the right end of the element IS102, or a non-specific pBR322 fragment of similar size. These results suggest that the Mu transposase protein can specifically recognize the ends of other class II transposable elements and that these elements may be evolutionarily related. 相似文献
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Shalaka Mulherkar Feng Liu Qin Chen Anjana Narayanan Anthony D. Couvillon H. David Shine Kimberley F. Tolias 《PloS one》2013,8(6)
The assembly of neuronal circuits during development requires the precise navigation of axons, which is controlled by attractive and repulsive guidance cues. In the developing spinal cord, ephrinB3 functions as a short-range repulsive cue that prevents EphA4 receptor-expressing corticospinal tract and spinal interneuron axons from crossing the midline, ensuring proper formation of locomotor circuits. Here we report that the small GTPase RhoA, a key regulator of cytoskeletal dynamics, is also required for ephrinB3/EphA4-dependent locomotor circuit formation. Deletion of RhoA from neural progenitor cells results in mice that exhibit a rabbit-like hopping gait, which phenocopies mice lacking ephrinB3 or EphA4. Consistent with this locomotor defect, we found that corticospinal tract axons and spinal interneuron projections from RhoA-deficient mice aberrantly cross the spinal cord midline. Furthermore, we determined that loss of RhoA blocks ephrinB3-induced growth cone collapse of cortical axons and disrupts ephrinB3 expression at the spinal cord midline. Collectively, our results demonstrate that RhoA is essential for the ephrinB3/EphA4-dependent assembly of cortical and spinal motor circuits that control normal locomotor behavior. 相似文献
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Tolias KF Hartwig JH Ishihara H Shibasaki Y Cantley LC Carpenter CL 《Current biology : CB》2000,10(3):153-156
Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly. 相似文献
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Characterization of a Rac1- and RhoGDI-Associated Lipid Kinase Signaling Complex 总被引:10,自引:0,他引:10
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Kimberley F. Tolias Anthony D. Couvillon Lewis C. Cantley Christopher L. Carpenter 《Molecular and cellular biology》1998,18(2):762-770
Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways. 相似文献
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Sarah E. Ross Alan R. Mardinly Alejandra E. McCord Jonathan Zurawski Sonia Cohen Cynthia Jung Linda Hu Stephanie I. Mok Anar Shah Erin M. Savner Christos Tolias Roman Corfas Suzhen Chen Perrine Inquimbert Yi Xu Roderick R. McInnes Frank L. Rice Gabriel Corfas Qiufu Ma Clifford J. Woolf Michael E. Greenberg 《Neuron》2010,65(6):886-898
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Yan-Fung Wong Qing Sheng Janet WL Chung Jacky KF Chan King L Chow 《BMC developmental biology》2010,10(1):82
Background
C. elegans TGF-β-like Sma/Mab signaling pathway regulates both body size and sensory ray patterning. Most of the components in this pathway were initially identified by genetic screens based on the small body phenotype, and many of these mutants display sensory ray patterning defect. At the cellular level, little is known about how and where these components work although ray structural cell has been implicated as one of the targets. Based on the specific ray patterning abnormality, we aim to identify by RNAi approach additional components that function specifically in the ray lineage to elucidate the regulatory role of TGF-β signaling in ray differentiation. 相似文献9.
Dimitri Yatsenko Kre?imir Josi? Alexander S. Ecker Emmanouil Froudarakis R. James Cotton Andreas S. Tolias 《PLoS computational biology》2015,11(3)
Ambitious projects aim to record the activity of ever larger and denser neuronal populations in vivo. Correlations in neural activity measured in such recordings can reveal important aspects of neural circuit organization. However, estimating and interpreting large correlation matrices is statistically challenging. Estimation can be improved by regularization, i.e. by imposing a structure on the estimate. The amount of improvement depends on how closely the assumed structure represents dependencies in the data. Therefore, the selection of the most efficient correlation matrix estimator for a given neural circuit must be determined empirically. Importantly, the identity and structure of the most efficient estimator informs about the types of dominant dependencies governing the system. We sought statistically efficient estimators of neural correlation matrices in recordings from large, dense groups of cortical neurons. Using fast 3D random-access laser scanning microscopy of calcium signals, we recorded the activity of nearly every neuron in volumes 200 μm wide and 100 μm deep (150–350 cells) in mouse visual cortex. We hypothesized that in these densely sampled recordings, the correlation matrix should be best modeled as the combination of a sparse graph of pairwise partial correlations representing local interactions and a low-rank component representing common fluctuations and external inputs. Indeed, in cross-validation tests, the covariance matrix estimator with this structure consistently outperformed other regularized estimators. The sparse component of the estimate defined a graph of interactions. These interactions reflected the physical distances and orientation tuning properties of cells: The density of positive ‘excitatory’ interactions decreased rapidly with geometric distances and with differences in orientation preference whereas negative ‘inhibitory’ interactions were less selective. Because of its superior performance, this ‘sparse+latent’ estimator likely provides a more physiologically relevant representation of the functional connectivity in densely sampled recordings than the sample correlation matrix. 相似文献
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Rac homologues and compartmentalized phosphatidylinositol 4, 5-bisphosphate act in a common pathway to regulate polar pollen tube growth 总被引:28,自引:0,他引:28
Kost B Lemichez E Spielhofer P Hong Y Tolias K Carpenter C Chua NH 《The Journal of cell biology》1999,145(2):317-330
Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-delta1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process. 相似文献