Shroom4 (Kiaa1202) is an actin-associated protein implicated in cytoskeletal organization

All animal cells utilize a specialized set of cytoskeletal proteins to determine their overall shape and the organization of their intracellular compartments and organelles. During embryonic development, the dynamic nature of the actin cytoskeleton is critical for virtually all morphogenic events requiring changes in cell shape, migration, adhesion, and division. The behavior of the actin cytoskeleton is modulated by a myriad of accessory proteins. Shroom3 is an actin binding protein that regulates neural tube morphogenesis by eliciting changes in cell shape through a myosin II-dependent pathway. The Shroom-related gene SHROOM4 (formerly called KIAA1202) has also been implicated in neural development, as mutations in this gene are associated with human X-linked mental retardation. To better understand the function of Shrm4 in embryonic development, we have cloned mouse Shroom4 and characterized its protein product in vivo and in vitro. Shroom4 is expressed in a wide range of cell types during mouse development, including vascular endothelium and the polarized epithelium of the neural tube and kidney. In endothelial cells and embryo fibroblasts, endogenous Shroom4 co-distributes with myosin II to a distinct cytoplasmic population of F-actin and ectopic expression of Shroom4 in multiple cell types enhances or induces the formation of this actin-based structure. This localization is mediated, at least in part, by the direct interaction of Shroom4 and F-actin. Our results suggest that Shroom4 is a regulator of cytoskeletal architecture that may play an important role in vertebrate development.     

Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues ¹ demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers ² suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.     

Analysis of a spindle pole body mutant reveals a defect in biorientation and illuminates spindle forces

The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-microm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.     

Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus

Cilia are complex organelles involved in sensory perception and fluid or cell movement. They are constructed through a highly conserved process called intraflagellar transport (IFT). Mutations in IFT genes, such as Tg737, result in severe developmental defects and disease. In the case of the Tg737orpk mutants, these pathological alterations include cystic kidney disease, biliary and pancreatic duct abnormalities, skeletal patterning defects, and hydrocephalus. Here, we explore the connection between cilia dysfunction and the development of hydrocephalus by using the Tg737orpk mutants. Our analysis indicates that cilia on cells of the brain ventricles of Tg737orpk mutant mice are severely malformed. On the ependymal cells, these defects lead to disorganized beating and impaired cerebrospinal fluid (CSF) movement. However, the loss of the cilia beat and CSF flow is not the initiating factor, as the pathology is present prior to the development of motile cilia on these cells and CSF flow is not impaired at early stages of the disease. Rather, our results suggest that loss of cilia leads to altered function of the choroid plexus epithelium, as evidenced by elevated intracellular cAMP levels and increased chloride concentration in the CSF. These data suggest that cilia function is necessary for regulating ion transport and CSF production, as well as for CSF flow through the ventricles.     

Structure of PITPbeta in complex with phosphatidylcholine: comparison of structure and lipid transfer to other PITP isoforms

Phosphatidylinositol transfer protein (PITP) is a ubiquitous eukaryotic protein that preferentially binds either phosphatidylinositol or phosphatidylcholine and catalyzes the exchange of these lipids between membranes. Mammalian cytosolic PITPs include the ubiquitously expressed PITPalpha and PITPbeta isoforms (269-270 residues). The crystal structure of rat PITPbeta complexed to dioleoylphosphatidylcholine was determined to 2.18 A resolution with molecular replacement using rat PITPalpha (77% sequence identify) as the phasing model. A structure comparison of the alpha and beta isoforms reveals minimal differences in protein conformation, differences in acyl conformation in the two isoforms, and remarkable conservation of solvent structure around the bound lipid. A comparison of transfer activity by human and rat PITPs, using small unilamellar vesicles with carefully controlled phospholipid composition, indicates that the beta isoforms have minimal differences in transfer preference between PtdIns and PtdCho when donor vesicles contain predominantly PtdCho. When PtdCho and PtdIns are present in equivalent concentrations in donor vesicles, PtdIns transfer occurs at approximately 3-fold the rate of PtdCho. The rat PITPbeta isoform clearly has the most diminished transfer rate of the four proteins studied. With the two rat isoforms, site-directed mutations of two locations within the lipid binding cavity that possess differing biochemical properties were characterized: I84alpha/F83beta and F225alpha/L224beta. The 225/224 locus is more critical in determining substrate specificity. Following the mutation of this locus to the other amino acid, the PtdCho transfer specific activity became PITPalpha (F225L) approximately PITPbeta and PITPbeta (L224F) approximately PITPalpha. The 225alpha/224beta locus plays a modest role in the specificity of both isoforms toward CerPCho.     

Prolonged maintenance of water balance by adult females of the American spider beetle, Mezium affine Boieldieu, in the absence of food and water resources

Moisture requirements were evaluated for female adults of spider beetles Mezium affine Boieldieu and Gibbium aequinoctiale Boieldieu to determine how they are differentially adapted for life in a dry environment. Features showing extreme desiccation resistance of M. affine were an impermeable cuticle wherein activation energies (43 kJ/mol) were suppressed, daily water losses as little as 0.3%/day with an associated group effect, a low 64% water content and an impressive ability to survive nearly 3 months with no food and water. Behaviorally, the extended period of water stress and fasting was marked by long intervals of physical inactivity (quiescence), as though dead. These characteristics emphasizing water retention rather than gain are shared by G. aequinoctiale and reflect a typical xerophilic water balance profile. Water uptake was restricted to imbibing liquid, as evidenced by uptake of dye-stained droplets of free water and a critical equilibrium activity of 1.00a_v, where the inability to absorb water vapor from the air fails to equilibrate declining water levels (gain≠loss) except at saturation. Four-fold reduction in survival time within dry air and accelerated water loss rates with high activation energies for female adults of the closely related winged Prostephanus truncatus (Say) suggest that the enhanced water conservation of spider beetles is due, in part, to fusion of their elytra supplemented by entering into quiescence.     

Mutational analysis of the Myxococcus xanthus Omega4400 promoter region provides insight into developmental gene regulation by C signaling

Myxococcus xanthus utilizes extracellular signals during development to coordinate cell movement, differentiation, and changes in gene expression. One of these signals, the C signal, regulates the expression of many genes, including Omega4400, a gene identified by an insertion of Tn5 lac into the chromosome. Expression of Tn5 lac Omega4400 is reduced in csgA mutant cells, which fail to perform C signaling, and the promoter region has several sequences similar to sequences found in the regulatory regions of other C-signal-dependent genes. One such gene, Omega4403, depends absolutely on the C signal for expression, and its promoter region has been characterized previously by mutational analysis. To determine if the similar sequences within the Omega4400 and Omega4403 regulatory regions function in the same way, deletion analysis and site-directed mutagenesis of the Omega4400 promoter region were performed. A 7-bp sequence centered at -49 bp, termed a C box, is identical in the Omega4400 and Omega4403 promoter regions, yet mutations in the individual base pairs affected expression from the two promoters very differently. Also, a single-base-pair change within a similar 5-bp element, which is centered at -61 bp in both promoter regions, had very different effects on the activities of the two promoters. Further mutational analysis showed that two regions are important for Omega4400 expression; one region, from -63 to -31 bp, is required for Omega4400 expression, and the other, from -86 to -81 bp, exerts a two- to fourfold effect on expression and is at least partially responsible for the C signal dependence of the Omega4400 promoter. Mutations in sigD and sigE, which are genes that encode sigma factors, abolished and reduced Omega4400 expression, respectively. Expression of Omega4400 in actB or actC mutants correlated well with the altered levels of C signal produced in these mutants. Our results provide the first detailed analysis of an M. xanthus regulatory region that depends partially on C signaling for expression and indicate that similar DNA sequences in the Omega4400 and Omega4403 promoter regions function differently.