Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

Background

TRPV4 and the cellular cytoskeleton have each been reported to influence cellular mechanosensitive processes as well as the development of mechanical hyperalgesia. If and how TRPV4 interacts with the microtubule and actin cytoskeleton at a molecular and functional level is not known.

Methodology and Principal Findings

We investigated the interaction of TRPV4 with cytoskeletal components biochemically, cell biologically by observing morphological changes of DRG-neurons and DRG-neuron-derived F-11 cells, as well as functionally with calcium imaging. We find that TRPV4 physically interacts with tubulin, actin and neurofilament proteins as well as the nociceptive molecules PKCε and CamKII. The C-terminus of TRPV4 is sufficient for the direct interaction with tubulin and actin, both with their soluble and their polymeric forms. Actin and tubulin compete for binding. The interaction with TRPV4 stabilizes microtubules even under depolymerizing conditions in vitro. Accordingly, in cellular systems TRPV4 colocalizes with actin and microtubules enriched structures at submembranous regions. Both expression and activation of TRPV4 induces striking morphological changes affecting lamellipodial, filopodial, growth cone, and neurite structures in non-neuronal cells, in DRG-neuron derived F11 cells, and also in IB4-positive DRG neurons. The functional interaction of TRPV4 and the cytoskeleton is mutual as Taxol, a microtubule stabilizer, reduces the Ca²⁺-influx via TRPV4.

Conclusions and Significance

TRPV4 acts as a regulator for both, the microtubule and the actin. In turn, we describe that microtubule dynamics are an important regulator of TRPV4 activity. TRPV4 forms a supra-molecular complex containing cytoskeletal proteins and regulatory kinases. Thereby it can integrate signaling of various intracellular second messengers and signaling cascades, as well as cytoskeletal dynamics. This study points out the existence of cross-talks between non-selective cation channels and cytoskeleton at multiple levels. These cross talks may help us to understand the molecular basis of the Taxol-induced neuropathic pain development commonly observed in cancer patients.     

Cholesterol in Negatively Charged Lipid Bilayers Modulates the Effect of the Antimicrobial Protein Granulysin

The release of granulysin, a 9-kDa cationic protein, from lysosomal granules of cytotoxic T lymphocytes and natural killer cells plays an important role in host defense against microbial pathogens. Granulysin is endocytosed by the infected target cell via lipid rafts and kills subsequently intracellular bacteria. The mechanism by which granulysin binds to eukaryotic and prokaryotic cells but lyses only the latter is not well understood. We have studied the effect of granulysin on large unilamellar vesicles (LUVs) and supported bilayers with prokaryotic and eukaryotic lipid mixtures or model membranes with various lipid compositions and charges. Binding of granulysin to bilayers with negative charges, as typically found in bacteria and lipid rafts of eukaryotic cells, was shown by immunoblotting. Fluorescence release assays using LUV revealed an increase in permeability of prokaryotic, negatively charged and lipid raft-like bilayers devoid of cholesterol. Changes in permeability of these bilayers could be correlated to defects of various sizes penetrating supported bilayers as shown by atomic force microscopy. Based on these results, we conclude that granulysin causes defects in negatively charged cholesterol-free membranes, a membrane composition typically found in bacteria. In contrast, granulysin is able to bind to lipid rafts in eukaryotic cell membranes, where it is taken up by the endocytotic pathway, leaving the cell intact.     

Ranking differential hubs in gene co-expression networks

Identifying the genes that change their expressions between two conditions (such as normal versus cancer) is a crucial task that can help in understanding the causes of diseases. Differential networking has emerged as a powerful approach to detect the changes in network structures and to identify the differentially connected genes among two networks. However, existing differential network-based methods primarily depend on pairwise comparisons of the genes based on their connectivity. Therefore, these methods cannot capture the essential topological changes in the network structures. In this paper, we propose a novel algorithm, DiffRank, which ranks the genes based on their contribution to the differences between the two networks. To achieve this goal, we define two novel structural scoring measures: a local structure measure (differential connectivity) and a global structure measure (differential betweenness centrality). These measures are optimized by propagating the scores through the network structure and then ranking the genes based on these propagated scores. We demonstrate the effectiveness of DiffRank on synthetic and real datasets. For the synthetic datasets, we developed a simulator for generating synthetic differential scale-free networks, and we compared our method with existing methods. The comparisons show that our algorithm outperforms these existing methods. For the real datasets, we apply the proposed algorithm on several gene expression datasets and demonstrate that the proposed method provides biologically interesting results.     

Development of EST derived SSRs and SNPs as a genomic resource in Indian catfish, <Emphasis Type="Italic">Clarias batrachus</Emphasis>

Clarias batrachus, an Indian catfish species, is endemic to the Indian subcontinent and potential cultivable species. The genomic resources in C. batrachus in the form of ESTs containing microsatellite repeats (EST-SSR) and single nucleotide polymorphisms (SNPs) that are associated with the expressed genes from spleen were mined. From a total of 1,937 ESTs generated, 1,698 unique sequences were obtained, out of which 221 EST-SSRs were identified and 54% could be functionally annotated by similarity searches. A total of 23 contigs containing 3 or more ESTs were found to contain 31 SNP loci, out of which 8 ESTs showed similarity to genes of known function and 1 for hypothetical protein. Nine ESTs with SSRs and/or SNPs identified in this study were reported to be associated with diseases in human and animals. These identified loci can be developed into markers in C. batrachus, which can be useful in linkage mapping, comparative genomics studies and for its genetic improvement programmes.     

Gadolinium-containing phosphatidylserine liposomes for molecular imaging of atherosclerosis

Exteriorized phosphatidylserine (PS) residues in apoptotic cells trigger rapid phagocytosis by macrophage scavenger receptor pathways. Mimicking apoptosis with liposomes containing PS may represent an attractive approach for molecular imaging of atherosclerosis. We investigated the utility of paramagnetic gadolinium liposomes enriched with PS (Gd-PS) in imaging atherosclerotic plaque. Gd-PS-containing Gd-conjugated lipids, fluorescent rhodamine, and PS were prepared and characterized. Cellular uptake in RAW macrophages (fluorescent uptake of rhodamine) was studied on a fluorescence plate reader, while Gd-PS-induced alteration in T1 relaxivity was evaluated using a 1.5 T MRI scanner. RAW cells demonstrate PS-dependent uptake of across a range of concentrations (2, 6, 12, and 20%) in comparison to control liposomes with no PS (0%). In vivo performance of Gd-PS was evaluated in the ApoE^−/− mouse model by collection of serial T1 weighted gradient echo MR images using an 11.7 T MRI system and revealed rapid and significant enhancement of the aortic wall that was seen for at least 4 h after injection. Gd-PS-enriched liposomes enhance atherosclerotic plaque and colocalize with macrophages in experimental atherosclerosis.     

Ladder-like azido-bridged copper(II) complexes: Synthesis, X-ray structure and magnetic study

Two mixed-ligand copper(II) complexes [{Cu(L¹)(μ_1,3-N₃)}{Cu(L)(μ_1,3-N₃)(μ_1,1-N₃)}]_n (1) [HL¹ = 1-(N-ortho-hydroxyacetophenimino)-2,2-dimethyl-aminoethane; L = 2-(dimethylamino)-ethylamine] and [{Cu(L²)(μ_1,3-N₃)}{Cu(L)(μ_1,3-N₃)(μ_1,1-N₃)}]_n (2) [HL² = 1-(N-5-methoxy-ortho-hydroxyacetophenimino)-2,2-dimethyl-aminoethane] have been formed upon addition of aqueous solution of sodium azide to a methanolic solution of copper nitrate trihydrate and corresponding Schiff-base ligands. The ligands, HL¹ and HL² undergo partial hydrolysis of their imine bond during the course of reaction. Both the complexes contain single end-to-end (μ_1,3) azido bridged 1D infinite chains (rail) which propagate parallel to the crystallographic b-axis; neighboring chains are interconnected by pairs through double asymmetric end-on (μ_1,1) azido bridges (rung) to yield a ladder-like structure. In both complexes, rungs (end-on azido bridges) do not connect copper centers of the chains like in a regular ladder; instead they connect only the alternating copper sites of the 1D chain. In a chain the coordination environment around copper(II) ions are not the same: while the {Cu(L¹)(μ_1,3-N₃)} and {Cu(L²)(μ_1,3-N₃)} moieties have a penta-coordinated copper(II) center, the copper(II) ion of the neighboring {Cu(L¹)(μ_1,3-N₃)(μ_1,1-N₃)} or {Cu(L²)(μ_1,3-N₃)(μ_1,1-N₃)} moiety has an octahedral coordination environment. The variable temperature (2-300 K) magnetic susceptibility measurements showed that the magnetic interaction between the metal centers in complexes 1 and 2 is dominantly antiferromagnetic. The results of magnetic model are in good agreement with the experimental data.     

Behavioral estrous signs can predict the time of ovulation in mithun (Bos frontalis)

The objective of this study was to investigate the relationship of different behavioral estrous signs and time of ovulation to identify if behavioral estrous sign(s) can be used as predictor of time of ovulation in mithuns. Data were collected for 54 ovulations from 16 mithuns. The animals were monitored for onset of estrus by observing different behavioral estrous signs at 2 h interval and bull parading thrice a day for 30 min and were further confirmed by plasma progesterone profile. All animals were also observed for any of the estrous signs at every 2 h interval for 30 min and mounting behavior was studied by bull parading at every 2 h for 30 min after onset of estrus. Time of ovulation was detected by rectal palpation at 2 h interval from onset of estrus till ovulation. Behavioral signs of estrus was more intense in primiparous than multiparous mithuns. Ovulation occurred at 26.1+/-1.1 h (ranging between 20 and 31 h) after the onset of estrus. As the method used to determine the onset of estrus is time consuming, labor intensive and no device is yet available to detect onset of estrus automatically, so this cannot be used practically as a predictor of time of ovulation. The mithun cow at estrus to be mounted by bull was recorded in all cases (100%). Ovulation occurred 23.5+/-1.5 h (ranging between 19 and 27 h) after first mounting. Although promising, mounting cannot be assessed automatically, which limits its practical use as a predictor of ovulation. Standing heat was recorded in 98.1% of total estrus studied in mithun cows and ovulation occurred 21.8+/-1.3 h (ranging between 19 and 25 h) after first observed standing heat. Standing heat can be detected automatically using mounting detectors. Hence, standing heat can be used practically as ovulation predictor in mithuns. In conclusion, cow to be mounted by mithun bull is the best predictor of ovulation, but non-availability of devices to detect it automatically restricts its practical application. Standing heat that recorded 98.1% estrus cases in mithun cows, can also be detected automatically using mounting detector, therefore be used widely as an ovulation predictor in field condition for mithun cows.