A complex of catalytically inactive protein phosphatase-1 sandwiched between Sds22 and inhibitor-3

Protein Ser/Thr phosphatase-1 (PP1) associates with a host of proteins to form substrate-specific holoenzymes. Sds22 and Inhibitor-3 (I3) are two independently described ancient interactors of PP1. We show here by various approaches that Sds22 and I3 form a heterotrimeric complex with PP1, both in cell lysates and after purification. The stability of the complex depended on functional PP1 interaction sites in Sds22 and I3, indicating that PP1 is sandwiched between Sds22 and I3. Intriguingly, I3 could not be replaced in this complex by another PP1 interactor with the same PP1 binding motif. In vitro, Sds22 and I3 were potent inhibitors of PP1, but with only some substrates. The inhibition by Sds22 could be reproduced with synthetic Sds22 fragments comprising leucine-rich repeats (LRR) 2 and 5. Sds22 and LRR5 also slowly converted PP1 into a conformation that was inactive with all tested substrates. Cell lysates that were prepared under conditions that prevented the Sds22-induced inactivation of PP1 contained a catalytically inactive complex of Sds22, PP1, and I3, indicating that this complex exists in vivo. Therefore, our studies show that a pool of PP1 is complexly controlled by both Sds22 and I3.     

Arabidopsis GCP2 and GCP3 are part of a soluble gamma-tubulin complex and have nuclear envelope targeting domains

In higher plants, microtubules (MTs) are assembled in distinctive arrays in the absence of a defined organizing center. Three MT nucleation sites have been described: the nuclear surface, the cell cortex and cortical MT branch points. The Arabidopsis thaliana (At) genome contains putative orthologues encoding all the components of characterized mammalian nucleation complexes: gamma-tubulin and gamma-tubulin complex proteins GCP2 to GCP6. We have cloned the cDNA encoding AtGCP2, and show that gamma-tubulin, AtGCP2 and AtGCP3 are part of the same tandem affinity-purified complex and are present in a large membrane-associated complex. In addition, small soluble gamma-tubulin complexes of the size expected for a gamma-tubulin core complex are recruited to isolated nuclei. Using immunogold labelling, AtGCP3 is localized to both the nuclear envelope (NE) and the plasma membrane. To identify domains that could play a role in targeting complexes to these nucleation sites, truncated AtGCP2- and AtGCP3-green fluorescent protein fusion proteins were expressed in BY-2 cells. Several domains from AtGCP2 and AtGCP3 are capable of targeting fusions to the NE. We propose that regulated recruitment of soluble gamma-tubulin-containing complexes is responsible for nucleation at dispersed sites in plant cells and contributes to the formation and organization of the various MT arrays.     

Rab6 and the secretory pathway affect oocyte polarity in Drosophila

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. Here, we describe a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. We found that Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. We also provide evidence that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFalpha homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. Our results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport.     

Species abundance distributions: moving beyond single prediction theories to integration within an ecological framework

Species abundance distributions (SADs) follow one of ecology's oldest and most universal laws – every community shows a hollow curve or hyperbolic shape on a histogram with many rare species and just a few common species. Here, we review theoretical, empirical and statistical developments in the study of SADs. Several key points emerge. (i) Literally dozens of models have been proposed to explain the hollow curve. Unfortunately, very few models are ever rejected, primarily because few theories make any predictions beyond the hollow-curve SAD itself. (ii) Interesting work has been performed both empirically and theoretically, which goes beyond the hollow-curve prediction to provide a rich variety of information about how SADs behave. These include the study of SADs along environmental gradients and theories that integrate SADs with other biodiversity patterns. Central to this body of work is an effort to move beyond treating the SAD in isolation and to integrate the SAD into its ecological context to enable making many predictions. (iii) Moving forward will entail understanding how sampling and scale affect SADs and developing statistical tools for describing and comparing SADs. We are optimistic that SADs can provide significant insights into basic and applied ecological science.     

Conceptual bases for quantifying the role of the environment on gene evolution: the participation of positive selection and neutral evolution

To demonstrate that a given change in the environment has contributed to the emergence of a given genotypic and phenotypic shift during the course of evolution, one should ask to what extent such shifts would have occurred without environmental change. Of course, such tests are rarely practical but phenotypic novelties can still be correlated to genomic shifts in response to environmental changes if enough information is available. We surveyed and re-evaluated the published data in order to estimate the role of environmental changes on the course of species and genomic evolution. Only a few published examples clearly demonstrate a causal link between a given environmental change and the fixation of a genomic variant resulting in functional modification (gain, loss or alteration of function). Many others suggested a link between a given phenotypic shift and a given environmental change but failed to identify the underlying genomic determinant(s) and/or the associated functional consequence(s). The proportion of genotypic and phenotypic variation that is fixed concomitantly with environmental changes is often considered adaptive and hence, the result of positive selection, even though alternative causes, such as genetic drift, are rarely investigated. Therefore, the second aim herein is to review evidence for the mechanisms leading to fixation.     

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

Background  

Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.