EB1 is a member of a conserved protein family that localizes to growing microtubule plus ends. EB1 proteins also recruit cell polarity and signaling molecules to microtubule tips. However, the mechanism by which EB1 recognizes cargo is unknown. Here, we have defined a repeat sequence in adenomatous polyposis coli (APC) that binds to EB1's COOH-terminal domain and identified a similar sequence in members of the microtubule actin cross-linking factor (MACF) family of spectraplakins. We show that MACFs directly bind EB1 and exhibit EB1-dependent plus end tracking in vivo. To understand how EB1 recognizes APC and MACFs, we solved the crystal structure of the EB1 COOH-terminal domain. The structure reveals a novel homodimeric fold comprised of a coiled coil and four-helix bundle motif. Mutational analysis reveals that the cargo binding site for MACFs maps to a cluster of conserved residues at the junction between the coiled coil and four-helix bundle. These results provide a structural understanding of how EB1 binds two regulators of microtubule-based cell polarity.
In yeast, microtubules are organized by the spindle pole body (SPB). The SPB is a disk-like multilayered structure that is embedded in the nuclear envelope via its central plaque, whereas the outer and inner plaques are exposed to the cytoplasm and nucleoplasm, respectively. How the SPB assembles is poorly understood. We show that the inner/central plaque is composed of a stable SPB subcomplex, containing the ␥-tubulin complex-binding protein Spc110p, calmodulin, Spc42p, and Spc29p. Spc29p acts as a linker between the central plaque component Spc42p and the inner plaque protein Spc110p. Evidence is provided that the calmodulin-binding site of Spc110p inf luences the binding of Spc29p to Spc110p. Spc42p also was identified as a component of a cytoplasmic SPB subcomplex containing Spc94p͞Nud1p, Cnm67p, and Spc42p. Spc29p and Spc42p may be part of a critical interface of nucleoplasmic and cytoplasmic assembled SPB subcomplexes that form during SPB duplication. In agreement with this, overexpressed Spc29p was found to be a nuclear protein, whereas Spc42p is cytoplasmic. In addition, an essential function of SPC29 during SPB assembly is indicated by the SPB duplication defect of conditional lethal spc29(ts) cells and by the genetic interaction of SPC29 with CDC31 and KAR1, two genes that are involved in SPB duplication.In the yeast Saccharomyces cerevisiae, microtubule-organizing functions are provided by the spindle pole body (SPB) (for review, see ref. 1). The SPB is a multilayered structure that duplicates once per cell cycle and is embedded in the nuclear envelope through the central plaque structure (ref. 2; see Fig. 6). The outer plaque is located on the cytoplasmic side of the SPB, whereas the inner plaque extends into the nucleoplasm. An additional SPB substructure is the half bridge, a one-sided extension of the central plaque.Some insight into the SPB duplication process came from electron microscopic studies and from the phenotype of conditional-lethal mutants. In early G 1 phase of the cell cycle, the half bridge elongates and develops an appendix toward the cytoplasmic side, which was named the satellite. The satellite is probably a precursor of the newly formed SPB. After start of the cell cycle, the SPB fully duplicates, resulting in two SPBs side by side (3). CDC31 (4, 5) and KAR1 (6, 7) code for components of the SPB half bridge required early in SPB duplication. In mps2-1 and ndc1-1 cells, a fully functional SPB is associated with a partially duplicated SPB containing an outer and a central plaque, sitting on top of the cytoplasmic side of the nuclear envelope (8, 9). The mps2/ndc1 phenotype suggests that the outer plaque and some part of the central plaque are built from cytoplasmic components, whereas the inner plaque is probably assembled in the nucleus. This view is further supported by the fact that SPB components of the inner plaque such as Spc110p (10) are imported into the nucleus, whereas the outer plaque component Spc72p is not (11). Finally, the cytoplasmic and nuclear assembled ...
TLR7-10 localization is not limited to endometrial epithelium but is also present in the stroma of the endometrial tissue. Endometrial TLR2-6, 9 and 10 genes are cyclically expressed during the menstrual cycle.
Purpose: del(17p), del(11q), and associated p53 dysfunction predict for short survival and chemoresistance in B-cell chronic lymphocytic leukemia (CLL). DNA-dependent protein kinase (DNA-PK) is activated by DNA damage and mediates DNA double-strand break repair. We hypothesized that inhibiting DNA-PK would sensitize CLL cells to drug-induced DNA damage and that this approach could increase the therapeutic index of agents used to treat CLL. Experimental Design: Fifty-four CLL cases were characterized for poor prognosis markers [del(17p), del(11q), CD38, and ZAP-70]. In selected cases, DNA-PK catalytic subunit (DNAPKcs) expression and activity and p53 function were also measured. Ex vivo viability assays established sensitivity to fludarabine and chlorambucil and also tested the ability of a novel DNA-PK inhibitor (NU7441) to sensitize CLL cells to these drugs. The effects of NU7441on fludarabine-induced DNA damage repair were also assessed (Comet assays and detection of gH2AX). Results: DNA-PKcs levels correlated with DNA-PK activity and varied 50-fold between cases but were consistently higher in del(17p) (P = 0.01) and del(11q) cases. NU7441 sensitized CLL cells to chlorambucil and fludarabine, including cases with del(17p), del(11q), p53 dysfunction, or high levels of DNA-PKcs. NU7441 increased fludarabine-induced double-strand breaks and abrogated drug-induced autophosphorylation of DNA-PKcs at Ser 2056. High DNA-PK levels predicted for reduced treatment-free interval. Conclusions: These data validate the concept of targeting DNA-PKcs in poor risk CLL, and demonstrate a mechanistic rationale for use of a DNA-PK inhibitor. The novel observation that DNA-PKcs is overexpressed in del(17p) and del(11q) cases indicates that DNA-PK may contribute to disease progression in CLL.
Centrioles are evolutionarily conserved microtubule-based structures at the core of the animal centrosome that are essential for nucleating the axoneme of cilia. We hypothesized that centriole proteins have been under-represented in proteomic studies of the centrosome, because of the larger amount of pericentriolar material making up the centrosome. In this study, we have overcome this problem by determining the centriolar proteome of mammalian sperm cells, which have a pair of centrioles but little pericentriolar material. Mass spectrometry of sperm centrioles identifies known components of centrioles and many previously uncharacterized candidate centriole proteins. Assessment of localization of a subset of these candidates in cultured cells identified CCDC113, CCDC96, C4orf47, CCDC38, C7orf31, CCDC146, CCDC81 and CCDC116 as centrosome-associated proteins. We examined the highly conserved protein CCDC113 further and found that it is a component of centriolar satellites, is in a complex with the satellite proteins HAP1 and PCM1, and functions in primary cilium formation.
The treatment of cartilage with mediators initiates the breakdown of proteoglycan followed by collagen. This is accompanied by the modulation of different proteinases and inhibitors that include members of the MMP family and TIMPs. We have evidence that a chondrocyte membrane-associated metalloproteinase cleaves aggrecan. This activity is rapidly induced after stimulation with IL-1 and OSM and is not inhibited by TIMPs-1 and -2 but is inhibited by synthetic MMP inhibitors. This same combination of cytokines also upregulates the collagenases with the subsequent release of collagen fragments, and there is a close correlation between the amount of collagen released and collagenase activity produced. Collagen release can be prevented after treatment with specific inhibitors of MAP kinases, inhibitors of MMP transcription, synthetic metalloproteinase inhibitors, TIMPs and treatment of cartilage with agents that upregulate TIMPs. The results from bovine cartilage culture models show that collagen release occurs when TIMP levels are low, collagenases are upregulated and then subsequently activated.
Adapting to blurred or sharpened images alters perceived blur of a focused image (M. A. Webster, M. A. Georgeson, & S. M. Webster, 2002). We asked whether blur adaptation results in (a) renormalization of perceived focus or (b) a repulsion aftereffect. Images were checkerboards or 2-D Gaussian noise, whose amplitude spectra had (log–log) slopes from −2 (strongly blurred) to 0 (strongly sharpened). Observers adjusted the spectral slope of a comparison image to match different test slopes after adaptation to blurred or sharpened images. Results did not show repulsion effects but were consistent with some renormalization. Test blur levels at and near a blurred or sharpened adaptation level were matched by more focused slopes (closer to 1/f) but with little or no change in appearance after adaptation to focused (1/f) images. A model of contrast adaptation and blur coding by multiple-scale spatial filters predicts these blur aftereffects and those of Webster et al. (2002). A key proposal is that observers are pre-adapted to natural spectra, and blurred or sharpened spectra induce changes in the state of adaptation. The model illustrates how norms might be encoded and recalibrated in the visual system even when they are represented only implicitly by the distribution of responses across multiple channels.
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