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Tracking and Quantifying Developmental Processes in <em>C. elegans</em> Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Goethe University

Nerve growth factor (NGF) is a potent growth factor that plays a key role in neuronal cell differentiation and may also play a role in hematopoietic differentiation. It has been shown that NGF induced synergistic action for the colony formation of CD34 positive hematopoietic progenitor cells treated with macrophage-colony stimulating factor (M-CSF or CSF-1), or stem cell factor (SCF). However, the exact role of NGF in hematopoietic system is unclear. It is also not clear whether NGF mediated signals in hematopoietic cells are identical to those in neuronal cells.

Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis.

The TREX (transcription/export) complex has been conserved throughout evolution from yeast to man and is required for coupled transcription elongation and nuclear export of mRNAs. The TREX complex in mammals and Drosophila is composed of the THO subcomplex (THOC1, THOC2, THOC5, THOC6, and THOC7), THOC3, UAP56, and Aly/THOC4. In human and Drosophila, various studies have shown that THO is required for the export of heat shock mRNAs, but nothing is known about other mRNAs. Our previous study using conditional THOC5 (or FMIP) knockout mice revealed that the presence of THOC5 is critical in hematopoietic cells but not for terminally differentiated cells. In this study, we describe the establishment of a mouse embryo fibroblast cell line (MEF), THOC5 flox/flox. Four days after infection of MEF THOC5 flox/flox with adenovirus carrying Cre-recombinase gene (Ad-GFP-Cre), THOC5 is down-regulated >95% at the protein level, and cell growth is strongly suppressed. Transcriptome analysis using cytoplasmic RNA isolated from cells lacking functional THOC5 reveals that only 2.9% of all genes were down-regulated more than twofold. Although we examined these genes in fibroblasts, one-fifth of all down-regulated genes (including HoxB3 and polycomb CBX2) are known to play a key role in hematopoietic development. We further identified 10 genes that are spliced but not exported to the cytoplasm in the absence of THOC5. These mRNAs were copurified with THOC5. Furthermore, Hsp70 mRNA was exported in the absence of THOC5 at 37°C, but not under heat shock condition (42°C), suggesting that THOC5 may be required for mRNA export under stress and/or upon signaling-induced conditions.

Identification of mRNAs that are spliced but not exported to the cytoplasm in the absence of THOC5 in mouse embryo fibroblasts.

The parkin-associated endothelial-like receptor (PAELR, GPR37) is an orphan G protein-coupled receptor that interacts with and is degraded by parkin-mediated ubiquitination. Mutations in parkin are thought to result in PAELR accumulation and increase neuronal cell death in Parkinson's disease. In this study, we find that the protein interacting with C-kinase (PICK1) interacts with PAELR. Specifically, the Postsynaptic density protein-95/Discs large/ZO-1 (PDZ) domain of PICK1 interacted with the last three residues of the c-terminal (ct) located PDZ motif of PAELR. Pull-down assays indicated that recombinant and native PICK1, obtained from heterologous cells and rat brain tissue, respectively, were retained by a glutathione S-transferase fusion of ct-PAELR. Furthermore, coimmunoprecipitation studies isolated a PAELR-PICK1 complex from transiently transfected cells. PICK1 interacts with parkin and our data showed that PICK1 reduces PAELR expression levels in transiently transfected heterologous cells compared to a PICK1 mutant that does not interact with PAELR. Finally, PICK1 over-expression in HEK293 cells reduced cell death induced by PAEALR over-expression during rotenone treatment and these effects of PICK1 were attenuated during inhibition of the proteasome. These results suggest a role for PICK1 in preventing PAELR-induced cell toxicity.

The protein interacting with C-kinase (PICK1) interacts with and attenuates parkin-associated endothelial-like (PAEL) receptor-mediated cell death.

Entamoeba histolytica is a protist parasite that is the causative agent of amoebiasis, and is a highly motile organism. The motility is essential for its survival and pathogenesis, and a dynamic actin cytoskeleton is required for this process. EhCoactosin, an actin-binding protein of the ADF/cofilin family, participates in actin dynamics, and here we report our studies of this protein using both structural and functional approaches. The X-ray crystal structure of EhCoactosin resembles that of human coactosin-like protein, with major differences in the distribution of surface charges and the orientation of terminal regions. According to in vitro binding assays, full-length EhCoactosin binds both F- and G-actin. Instead of acting to depolymerize or severe F-actin, EhCoactosin directly stabilizes the polymer. When EhCoactosin was visualized in E. histolytica cells using either confocal imaging or total internal reflectance microscopy, it was found to colocalize with F-actin at phagocytic cups. Over-expression of this protein stabilized F-actin and inhibited the phagocytic process. EhCoactosin appears to be an unusual type of coactosin involved in E. histolytica actin dynamics.

EhCoactosin stabilizes actin filaments in the protist parasite Entamoeba histolytica.

The dynamical properties of water at protein-water interfaces are unlike those in the bulk. Here we utilize molecular dynamics simulations to study water dynamics in interstitial regions between two proteins. We consider two natural protein-protein complexes, one in which the Nipah virus G protein binds to cellular ephrin B2 and the other in which the same G protein binds to ephrin B3. While the two complexes are structurally similar, the two ephrins share only a modest sequence identity of ∼50%. X-ray crystallography also suggests that these interfaces are fairly extensive and contain exceptionally large amounts of waters. We find that while the interstitial waters tend to occupy crystallographic sites, almost all waters exhibit residence times of less than hundred picoseconds in the interstitial region. We also find that while the differences in the sequence of the two ephrins result in quantitative differences in the dynamics of interstitial waters, the trends in the shifts with respect to bulk values are similar. Despite the high wetness of the protein-protein interfaces, the dynamics of interstitial waters are considerably slower compared to the bulk-the interstitial waters diffuse an order of magnitude slower and have 2-3 fold longer hydrogen bond lifetimes and 2-1000 fold slower dipole relaxation rates. To understand the role of interstitial waters, we examine how implicit solvent models compare against explicit solvent models in producing ephrin-induced shifts in the G conformational density. Ephrin-induced shifts in the G conformational density are critical to the allosteric activation of another viral protein that mediates fusion. We find that in comparison with the explicit solvent model, the implicit solvent model predicts a more compact G-B2 interface, presumably because of the absence of discrete waters at the G-B2 interface. Simultaneously, we find that the two models yield strikingly different induced changes in the G conformational density, even for those residues whose conformational densities in the apo state are unaffected by the treatment of the bulk solvent. Together, these results show that the explicit treatment of interstitial water molecules is necessary for a proper description of allosteric transitions.

Water Dynamics at Protein-Protein Interfaces: Molecular Dynamics Study of Virus-Host Receptor Complexes.

Formins are highly conserved heterogeneous family of proteins with several isoforms having significant contribution in multiple cellular functions. Formins play crucial role in remodelling of actin cytoskeleton and thus play important role in cell motility. Formins are also involved in many cellular activities like determining cell polarity, cytokinesis and morphogenesis. Formins are multi domain protein with characteristic homodimeric formin homology 2 (FH2) domain. It nucleates the actin filaments and its activity is regulated by the presence of characteristic formin homology 1 (FH1) domain. In higher mammals like human and mouse fifteen different formin isoforms are present. However the function and expression pattern of each and every formin in different adult tissues are not well characterized. Here we have found that multiple formins are expressing in each adult tissue of mouse, irrespective of their origin from the germ layer. Formins are also expressing from early stage of development to the adulthood in brain. The expression of many formins in a single tissue of adult mouse indicates that regulation of actin cytoskeleton dynamics by formins may be crucial for physiological processes like wound healing, tissue repairing, exocytosis, endocytosis, synapse formation and maintenance. Expression of FMNL2 and Fhdc1 are high in adult mouse brain as compare to embryonic stages. Higher expression of FMNL2 and Fhdc1 indicates that FMNL2 and Fhdc1 might be very important for the adult brain functions.

Expression of multiple formins in adult tissues and during developmental stages of mouse brain.

This work reports the development of highly fluorescent materials for the selective and efficient detection of picric acid explosive in the nanomolar range by fluorescence quenching phenomenon. Poly(vinyl alcohol) grafted polyaniline (PPA) and its nanocomposites with 2-mercaptosuccinic acid (MSA)-capped CdTe quantum dots (PPA-Q) and with MSA-capped CdTe/ZnS core/shell quantum dots (PPA-CSQ) are synthesized in a single step free radical polymerization reaction. The thermal stability and photo stability of the polymer increases in the order of PPA < PPA-Q < PPA-CSQ. The polymers show remarkably high selectivity and efficient sensitivity toward picric acid, and the quenching efficiency for PPA-CSQ reaches up to 99%. The detection limits of PPA, PPA-Q, and PPA-CSQ for picric acid are found to be 23, 1.6, and 0.65 nM, respectively, which are remarkably low. The mechanism operating in the quenching phenomenon is proposed to be a combination of a strong inner filter effect and ground state electrostatic interaction between the polymers and picric acid. A portable and cost-effective electronic device for the visual detection of picric acid by the sensory system is successfully fabricated. The device is further employed for quantitative detection of picric acid in real water samples.

Macromolecular Systems with MSA-Capped CdTe and CdTe/ZnS Core/Shell Quantum Dots as Superselective and Ultrasensitive Optical Sensors for Picric Acid Explosive.

Priyanka Dutta

Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II,
School of Medicine,
Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II, School of Medicine

Goethe University

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Priyanka Dutta

Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II,School of Medicine,Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II, School of Medicine

Goethe University

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II,
School of Medicine,
Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II, School of Medicine