Spermatogenesis and sertoli cell activity in mice lacking Sertoli cell receptors for follicle stimulating hormone and androgen

Spermatogenesis in the adult male depends on the action of FSH and androgen. Ablation of either hormone has deleterious effects on Sertoli cell function and the progression of germ cells through spermatogenesis. In this study we generated mice lacking both FSH receptors (FSHRKO) and androgen receptors on the Sertoli cell (SCARKO) to examine how FSH and androgen combine to regulate Sertoli cell function and spermatogenesis. Sertoli cell number in FSHRKO-SCARKO mice was reduced by about 50% but was not significantly different from FSHRKO mice. In contrast, total germ cell number in FSHRKO-SCARKO mice was reduced to 2% of control mice (and 20% of SCARKO mice) due to a failure to progress beyond early meiosis. Measurement of Sertoli cell-specific transcript levels showed that about a third were independent of hormonal action on the Sertoli cell, whereas others were predominantly androgen dependent or showed redundant control by FSH and androgen. Results show that FSH and androgen act through redundant, additive, and synergistic regulation of spermatogenesis and Sertoli cell activity. In addition, the Sertoli cell retains a significant capacity for activity, which is independent of direct hormonal regulation

A Biobrick Library for Cloning Custom Eukaryotic Plasmids

Researchers often require customised variations of plasmids that are not commercially available. Here we demonstrate the applicability and versatility of standard synthetic biological parts (biobricks) to build custom plasmids. For this purpose we have built a collection of 52 parts that include multiple cloning sites (MCS) and common protein tags, protein reporters and selection markers, amongst others. Importantly, most of the parts are designed in a format to allow fusions that maintain the reading frame. We illustrate the collection by building several model contructs, including concatemers of protein binding-site motifs, and a variety of plasmids for eukaryotic stable cloning and chromosomal insertion. For example, in 3 biobrick iterations, we make a cerulean-reporter plasmid for cloning fluorescent protein fusions. Furthermore, we use the collection to implement a recombinase-mediated DNA insertion (RMDI), allowing chromosomal site-directed exchange of genes. By making one recipient stable cell line, many standardised cell lines can subsequently be generated, by fluorescent fusion-gene exchange. We propose that this biobrick collection may be distributed peer-to-peer as a stand-alone library, in addition to its distribution through the Registry of Standard Biological Parts (http://partsregistry.org/)

Insight into the Assembly Properties and Functional Organisation of the Magnetotactic Bacterial Actin-like Homolog, MamK

Magnetotactic bacteria (MTB) synthesize magnetosomes, which are intracellular vesicles comprising a magnetic particle. A series of magnetosomes arrange themselves in chains to form a magnetic dipole that enables the cell to orient itself along the Earth’s magnetic field. MamK, an actin-like homolog of MreB has been identified as a central component in this organisation. Gene deletion, fluorescence microscopy and in vitro studies have yielded mechanistic differences in the filament assembly of MamK with other bacterial cytoskeletal proteins within the cell. With little or no information on the structural and behavioural characteristics of MamK outside the cell, the mamK gene from Magnetospirillium gryphiswaldense was cloned and expressed to better understand the differences in the cytoskeletal properties with its bacterial homologues MreB and acitin. Despite the low sequence identity shared between MamK and MreB (22%) and actin (18%), the behaviour of MamK monitored by light scattering broadly mirrored that of its bacterial cousin MreB primarily in terms of its pH, salt, divalent metal-ion and temperature dependency. The broad size variability of MamK filaments revealed by light scattering studies was supported by transmission electron microscopy (TEM) imaging. Filament morphology however, indicated that MamK conformed to linearly orientated filaments that appeared to be distinctly dissimilar compared to MreB suggesting functional differences between these homologues. The presence of a nucleotide binding domain common to actin-like proteins was demonstrated by its ability to function both as an ATPase and GTPase. Circular dichroism and structural homology modelling showed that MamK adopts a protein fold that is consistent with the ‘classical’ actin family architecture but with notable structural differences within the smaller domains, the active site region and the overall surface electrostatic potential

Ena/VASP proteins have an anti-capping independent function in filopodia formation

Author Posting. © American Society for Cell Biology, 2007. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 18 (2007): 2579-2591, doi:10.1091/mbc.E06-11-0990.Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at filopodial tips. Mutation of the G-actin–binding motif (GAB) partially compromised stabilization of Ena/VASP at filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.This work was supported in part by National Institutes of Health Grants GM7542201 to D.A.A., GM58801 to F.B.G., and GM62431 to G.G.B. and by Cell Migration Consortium Grants GM64346 to D.A.A and G.G.B

Viable systems for planning - a case study on needs and planning for supported housing

SUPPORTING PEOPLE HAS GIVEN a new urgency to discussions of needs analysis, strategic planning and the user voice. This article explores a two‐year research project developed by Manchester City Council, Manchester Methodists Housing Association and the Housing Corporation, which aimed to design a model for identifying supported housing needs, based on a collaborative, interdepartmental approach to needs assessment.</jats:p