Hormone-sensing cells require Wip1 for paracrine stimulation in normal and premalignant mammary epithelium

10.1186/bcr3381Breast Cancer Research15

MAPK-Activated Protein Kinase 2 Is Required for Mouse Meiotic Spindle Assembly and Kinetochore-Microtubule Attachment

MAPK-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple physiological functions in mitosis. Here, we show for the first time the unique distribution pattern of MK2 in meiosis. Phospho-MK2 was localized on bipolar spindle minus ends and along the interstitial axes of homologous chromosomes extending over centromere regions and arm regions at metaphase of first meiosis (MI stage) in mouse oocytes. At metaphase of second meiosis (MII stage), p-MK2 was localized on the bipolar spindle minus ends and at the inner centromere region of sister chromatids as dots. Knockdown or inhibition of MK2 resulted in spindle defects. Spindles were surrounded by irregular nondisjunction chromosomes, which were arranged in an amphitelic or syntelic/monotelic manner, or chromosomes detached from the spindles. Kinetochore–microtubule attachments were impaired in MK2-deficient oocytes because spindle microtubules became unstable in response to cold treatment. In addition, homologous chromosome segregation and meiosis progression were inhibited in these oocytes. Our data suggest that MK2 may be essential for functional meiotic bipolar spindle formation, chromosome segregation and proper kinetochore–microtubule attachments

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Effective dispersion of fullerene with methacrylate copolymer in organic solvent and poly(methyl methacrylate)

Dispersion of fullerene, C 60, by addition of polymethacrylate dispersant in methyl methacrylate (MMA) and incorporation of C 60 into poly(methyl methacrylate) (PMMA) were investigated. Copolymers synthesized by radical copolymerization of MMA and 2-naphthyl methacrylate (NMA), poly(MMA-co-NMA), effectively dispersed C 60 in MMA to form clusters of 20 nm. In these cases, addition of minimal 110 naphthyl groups per unit C 60 molecule afforded to give clusters with minimum of 20 nm sizes. Furthermore, block copolymers, poly(MMA-b-NMA) with MMA/NMA mole ratio from 12:1 to 20:1, also efficiently dispersed C 60 to give formation of clusters of 20 nm size by addition of minimal 40 naphthyl groups per unit C 60 molecule, which was corresponding to approximate nine layers of naphthyl group in block copolymer adsorbed on the surface of the cluster. Hybrid films of C 60/PMMA, prepared by casting of C 60-dispersed solution containing PMMA, exhibited absorbance at 400 nm linearly increased with C 60 content

Kinase in Motion : Insights into the Dynamic Nature of p38α by High-Pressure NMR Spectroscopic Studies

Protein kinases are highly dynamic and complex molecules. Here we present high-pressure and relaxation studies of the activated p38α mitogen-activated protein kinase (MAPK). p38α plays a central role in inflammatory diseases such as rheumatoid arthritis and is therefore a highly attractive pharmaceutical target. The combination of high pressure and NMR spectroscopy allowed for a detailed per-residue based assessment of the structural plasticity of p38α and the accessibility of low-lying excited-energy conformations throughout the kinase structure. Such information is uniquely accessible through the combination of liquid-state NMR and high pressure and is of considerable value for the drug discovery process. The interactions of p38α and DFG-in and DFG-out ligands were studied under the application of high pressure, and we demonstrate how we can alter kinase dynamics by pressure in a similar way to what has previously only been observed by ligand binding. Pressure is shown to be a mild and efficient tool for manipulation of intermediate-timescale dynamics