Probing DNA conformational changes with high temporal resolution by tethered particle motion

The tethered particle motion (TPM) technique informs about conformational changes of DNA molecules, e.g. upon looping or interaction with proteins, by tracking the Brownian motion of a particle probe tethered to a surface by a single DNA molecule and detecting changes of its amplitude of movement. We discuss in this context the time resolution of TPM, which strongly depends on the particle-DNA complex relaxation time, i.e. the characteristic time it takes to explore its configuration space by diffusion. By comparing theory, simulations and experiments, we propose a calibration of TPM at the dynamical level: we analyze how the relaxation time grows with both DNA contour length (from 401 to 2080 base pairs) and particle radius (from 20 to 150 nm). Notably we demonstrate that, for a particle of radius 20 nm or less, the hydrodynamic friction induced by the particle and the surface does not significantly slow down the DNA. This enables us to determine the optimal time resolution of TPM in distinct experimental contexts which can be as short as 20 ms.     

The nebulette repeat domain is necessary for proper maintenance of tropomyosin with the cardiac sarcomere

Nebulette is a cardiac-specific isoform of the giant actin-binding protein nebulin. Nebulette, having a mass of ∼ 100 kDa, is only predicted to extend 150 nm from the edge of the Z-lines. Overexpression of the nebulette C-terminal linker and/or SH3 domains in chicken cardiomyocytes results in a loss of endogenous nebulette with a concomitant loss of tropomyosin (TPM) and troponin, as well as a shortening of the thin filaments. These data suggest that nebulette's position in the sarcomere is important for the maintenance of TPM, troponin and thin filament length. To evaluate this hypothesis, N-terminal nested truncations tagged with GFP were expressed in chicken cardiomyocytes and the cells were analyzed for the distribution of myofilament proteins. Minimal effects on the myofilaments were observed with N-terminal deletions of up to 10 modules; however, deletion of 15 modules replicated the phenotype observed with expression of the C-terminal fragments. Expression of internal deletions of nebulette verifies that a site between module 10 and 15 is important for TPM maintenance within the sarcomeric lattice. We have additionally isolated TPM cDNAs from a yeast two hybrid (Y2H) analysis. These data indicate the importance of the nebulette-TPM interactions in the maintenance and stability of the thin filaments.     

Identification of novel tropomyosin 1 genes of pufferfish (Fugu rubripes) on genomic sequences and tissue distribution of their transcripts

Fugu genome database enabled us to identify two novel tropomyosin 1 (TPM1) genes through in silico data mining and isolation of their corresponding cDNAs in vivo. The duplicate TPM1 genes in Japanese pufferfish Fugu rubripes suggest that additional an ancient segmental duplication or whole genome duplication occurred in fish lineage, which, like many other reported Fugu genes, showed reduction in genomic size in comparison with their human homologue. Computer analysis predicted that the coiled-coil probabilities, that were thought to be the most major function of TPM, were the same between the two TPM1 isoforms. We confirmed that the tissue expression profiles of the two TPM1 genes differed from each other, which implied that changes in expression pattern could fix duplicated TPM1 genes although the two TPM1 isoforms appear to have similar function.     

Maximizing exosome colloidal stability following electroporation

Development of exosome-based semisynthetic nanovesicles for diagnostic and therapeutic purposes requires novel approaches to load exosomes with cargo. Electroporation has previously been used to load exosomes with RNA. However, investigations into exosome colloidal stability following electroporation have not been considered. Herein, we report the development of a unique trehalose pulse media (TPM) that minimizes exosome aggregation following electroporation. Dynamic light scattering (DLS) and RNA absorbance were employed to determine the extent of exosome aggregation and electroextraction post electroporation in TPM compared to common PBS pulse media or sucrose pulse media (SPM). Use of TPM to disaggregate melanoma exosomes post electroporation was dependent on both exosome concentration and electric field strength. TPM maximized exosome dispersal post electroporation for both homogenous B16 melanoma and heterogeneous human serum-derived populations of exosomes. Moreover, TPM enabled heavy cargo loading of melanoma exosomes with 5 nm superparamagnetic iron oxide nanoparticles (SPION5) while maintaining original exosome size and minimizing exosome aggregation as evidenced by transmission electron microscopy. Loading exosomes with SPION5 increased exosome density on sucrose gradients. This provides a simple, label-free means of enriching exogenously modified exosomes and introduces the potential for MRI-driven theranostic exosome investigations in vivo.     

Topiramate modulation of kainate-induced calcium currents is inversely related to channel phosphorylation level

Topiramate (TPM) is a structurally novel broad-spectrum anticonvulsant known to modulate the activity of several ligand- and voltage-gated ion channels in neurons. These include an inhibitory effect on the AMPA and kainate subtypes of glutamate receptors, mixed modulatory effects (usually positive) on some types of GABAA receptors, negative modulatory effects on some types of voltage-gated Na+ and Ca2+ channels, and a positive modulatory effect on at least one type of K+ channel. The nature of these effects at the molecular level has not been established, but two previous studies have implicated the phosphorylation state of these receptor/channel complexes as an influencing factor in the activity of TPM. Here, we report that the ability of TPM to inhibit a kainate-induced accumulation of free Ca2+ in cultured neurons from rat cerebral cortex is inversely related to the level of cAMP-dependent protein kinase (cAPK) mediated phosphorylation of kainate-activated receptors/channels. Specifically, when cell cultures were pre-treated with forskolin or dibutyryl cAMP, indirect activators of cAPK, the activity of TPM was abolished, whereas when the cells were pre-treated with H89, an inhibitor of cAPK, the relative activity of TPM was enhanced. The results of this study support the hypothesis that TPM binds to phosphorylation sites on AMPA and kainate receptors, but only in the dephosphorylated state and thereby exerts an allosteric modulatory effect on channel conductance.     

Modulation of myosin function by isoform-specific properties of Saccharomyces cerevisiae and muscle tropomyosins.

Tropomyosin is an extended coiled-coil protein that influences actin function by binding longitudinally along thin filaments. The present work compares cardiac tropomyosin and the two tropomyosins from Saccharomyces cerevisiae, TPM1 and TPM2, that are much shorter than vertebrate tropomyosins. Unlike cardiac tropomyosin, the phase of the coiled-coil-forming heptad repeat of TPM2 is discontinuous; it is interrupted by a 4-residue deletion. TPM1 has two such deletions, which flank the 38-residue partial gene duplication that causes TPM1 to span five actins instead of the four of TPM2. Each of the three tropomyosin isoforms modulates actin-myosin interactions, with isoform-specific effects on cooperativity and strength of myosin binding. These different properties can be explained by a model that combines opposite effects, steric hindrance between myosin and tropomyosin when the latter is bound to a subset of its sites on actin, and also indirect, favorable interactions between tropomyosin and myosin, mediated by mutually promoted changes in actin. Both of these effects are influenced by which tropomyosin isoform is present. Finally, the tropomyosins have isoform-specific effects on in vitro sliding speed and on the myosin concentration dependence of this movement, suggesting that non-muscle tropomyosin isoforms exist, at least in part, to modulate myosin function.     

Impact of the zebra mussel Dreissena polymorpha invasion on the budget of suspended material in a shallow lagoon ecosystem

The role of the zebra mussel Dreissena polymorpha in redistribution of total particulate material (TPM) between the water column and bottom sediment was estimated using the TPM budget for a mussel bed in the Curonian lagoon, the Baltic Sea. Seasonal clearance rates were derived from the TPM budget assuming two resuspension scenarios: no resuspension and full resuspension of biodeposits. Estimated clearance rates for both scenarios were compared with the rates calculated from the population clearance rate model. Seasonal clearance rates estimated using the population model (1.1 and 11.8 l g⁻¹ SFDW day⁻¹) fitted well into the interval of seasonal clearance rates calculated from TPM budgets assuming no resuspension of biodeposits (3.2 and 21.4 l g SFDW⁻¹ day⁻¹). In the scenario with biodeposits resuspension clearance rates were much higher (57.4 and 148.9 g SFDW⁻¹ day⁻¹). The ratio of clearance to residence time was highly dependent on the fate of biodeposits. Therefore its use in interpretation of the species impact on TPM was limited. An alternative measure based on the ratio of the amount of TPM biodeposited to TPM transported into the bed was used. It was found that zebra mussels are able to deposit between 10 and 30% of the incoming TPM, and the amount of biodeposited material was correlated with water residence time. Results indicate that the impact of zebra mussels on TPM in the lagoon is small relative to the high transport rates of TPM over the bed. However, annual biosedimentation rate (~590 g m⁻²) in the mussel bed was higher than physical deposition rate (~380 g m⁻²) in accumulation areas devoid of large suspension feeders. We suggest that a local impact due to enhanced availability of organic material to other trophic groups of associated benthic organisms may be more significant than effects on TPM pathways at an ecosystem scale.     

Molecular evolution in bacteria: Surfaces, cathodes and anodes

Molecular evolution is examined in bacteria with an emphasis on mineral surfaces, membranes, cathodes and anodes. In early molecular evolution, cathode-anode system may have been naturally occurring on a nm to µm scale. Secondly, the cathode-anode system could have been separated by a primitive, permeable lipid or microsphere on a mineral surface, that was a precursor of a more advanced membrane with a charge differential on either side of the membrane. These aspects will be considered from a theoretical evolutionary perspective.     

Single-Cell Force Spectroscopy of Interaction of Lipopolysaccharides from <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis> with J774 Macrophage Membrane Using Optical Tweezers

In order to investigate quantitatively the role of lipopolysaccharides (LPS) from outer bacterial membrane at the initial state of bacterium adhesion to a host cell membrane, a model system for single cell force spectroscopy was developed and used. The system comprised of an LPS-coated microsphere placed into optical trap and a J774 macrophage being approached the microsphere to initiate their binding and then moved back to rupture the bond. An “object shadow” phenomenon was discovered, manifested as large-scale variations of the signal of photodetector registering the trapped microsphere displacement, such variations emerging long before the actual interaction between the macrophage and microsphere. The theory and the measurements technique were developed for registration of the force required for detachment of bounded microsphere from the object investigated by means of optical tweezers under the “object shadow” conditions. Characteristic spectra of binding force between J774 macrophage and microspheres functionalized with various LPS, as well as LPS plus complementary antibodies preparations were obtained at the rate of detachment force application of 3–6 pN/s. Force spectrum characteristic of Yersinia pseudotuberculosis LPS possessing O-antigen had a maximum at ~14 pN with half-width of ~23 pN. The treatment of O-antigen with complementary antibodies resulted in transformation of this spectrum into a spectrum with maximum at ~10 pN and half-width of ~14 pN, being almost identical to the spectrum of Y. pestis LPS devoid of O-antigen, with a maximum at ~9 pN and half-width of ~13 pN. A possible mechanism of force spectra formation has been proposed under assumptions of nonspecific binding of O-antigen and probable receptor-type binding of LPS core region to the macrophage surface. The elastic modulus of macrophage envelope, as estimated using analysis of displacement of the contacting microsphere as an indenter, was ≈0.17 pN/nm.     

A probabilistic model for ligand-cytoskeleton transmembrane adhesion: predicting the behavior of microspheres on the surface of migrating cells

A theoretical model describing the attachment and cytoskeletal coupling of microspheres to the dorsal surface of motile cells was developed. Integral membrane receptors beneath a ligand-coated microsphere are allowed to be either free, attached to the microsphere, bound to the rearward moving actin network, or linked to both the bead and the cytoskeleton, and to switch between these four states. The binding transitions being modeled as chemical reactions governed by rate constants taken from literature, the chance for a receptor to be in each binding state over time is obtained by solving mass-balance equations for the probability functions. The population of n such receptors beneath the microsphere is accounted for by a binomial distribution for each state. Adhesion and transmembrane coupling (resulting in microsphere transport) being defined by a minimal number of ligand-receptor and receptor-cytoskeleton bonds, respectively, the probabilities of attachment and transport of the microsphere over time are expressed in terms of state probability distributions. It is found that increasing the ligand density raises the attachment and transport probabilities, in good quantitative agreement with recent experiments using optical tweezers and accurate position tracking. Increasing the bead size does not affect attachment, but raises the transport probability with a marked transition for bead diameter around 100 nm, as for experimental data. Increasing the restraining force decreases the transport probability, probably by inducing a rupture of receptor-cytoskeleton bonds. This study thus provides a framework that helps understand the process of cortical flow associated with cell locomotion.     

Expression of a novel cardiac-specific tropomyosin isoform in humans

Tropomyosins are a family of actin binding proteins encoded by a group of highly conserved genes. Humans have four tropomyosin-encoding genes: TPM1, TPM2, TPM3, and TPM4, each of which is known to generate multiple isoforms by alternative splicing, promoters, and 3' end processing. TPM1 is the most versatile and encodes a variety of tissue specific isoforms. The TPM1 isoform specific to striated muscle, designated TPM1alpha, consists of 10 exons: 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b. In this study, using RT-PCR with adult and fetal human RNAs, we present evidence for the expression of a novel isoform of the TPM1 gene that is specifically expressed in cardiac tissues. The new isoform is designated TPM1kappa and contains exon 2a instead of 2b. Ectopic expression of human GFP.TPM1kappa fusion protein can promote myofibrillogenesis in cardiac mutant axolotl hearts that are lacking in tropomyosin.     

Topiramate reduces AMPA-induced Ca(2+) transients and inhibits GluR1 subunit phosphorylation in astrocytes from primary cultures

Topiramate (TPM) is a structurally novel broad spectrum anticonvulsant known to have a negative modulatory effect on the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate subtypes of glutamate receptors and some types of voltage-gated Na(+) and Ca(2+) channels, and a positive modulatory effect on some types of gamma-aminobutyric acid(A) (GABA(A)) receptors and at least one type of K(+) channels in neurons. In an earlier work, we showed that the negative modulatory effect of TPM (100 mum) on AMPA/kainate receptors in neurons is dependent on TPM modulation of the phosphorylation state of these receptors. In this work, we investigated the effect of TPM on AMPA-induced intracellular calcium ([Ca(2+)](i)) responses in cultured rat cortical astrocytes, with special interest in intracellular mechanisms. Here, we report that the ability of TPM (1-100 mum) to inhibit AMPA-induced accumulation of Ca(2+) in astrocytes is inversely related to the level of protein kinase A (PKA) -mediated phosphorylation of channels activated by AMPA. The level of receptor phosphorylation was further determined with western blot using phosphorylation specific antibodies that recognize the glutamate receptor 1 (GluR1) subunit phosphorylated on Ser845. These results demonstrated that, even in cultured cortical astrocytes, TPM significantly reduced the phophorylation level of GluR1 subunits. Furthermore, it was shown that TPM binds to AMPA receptors in the dephosphorylated state and thereby exerts an allosteric modulatory effect on the ion channel.     

Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport

Disruption of the yeast tropomyosin gene TPM1 results in the apparent loss of actin cables from the cytoskeleton (Liu, H., and A. Bretscher. 1989. Cell. 57:233-242). Here we show that TPM1 disrupted cells grow slowly, show heterogeneity in cell size, have delocalized deposition of chitin, and mate poorly because of defects in both shmooing and cell fusion. The transit time of alpha-factor induced a-agglutinin secretion to the cell surface is longer than in isogenic wild-type strains, and some of the protein is mislocalized. Many of the TPM1-deleted cells contain abundant vesicles, similar in morphology to late secretory vesicles, but without an abnormal accumulation of intermediates in the delivery of either carboxypeptidase Y to the vacuole or invertase to the cell surface. Combinations of the TPM1 disruption with sec13 or sec18 mutations, which affect early steps in the secretory pathway, block vesicle accumulation, while combinations with sec1, sec4 or sec6 mutations, which affect a late step in the secretory pathway, have no effect on the vesicle accumulation. The phenotype of the TPM1 disrupted cells is very similar to that of a conditional mutation in the MYO2 gene, which encodes a myosin-like protein (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). The myo2-66 conditional mutation shows synthetic lethality with the TPM1 disruption, indicating that the MYO2 and TPM1 gene products may be involved in the same, or parallel function. We conclude that tropomyosin, and by inference actin cables, may facilitate directed vesicular transport of components to the correct location on the cell surface.     

The effects of ventilation and the dilution of smoke upon the clastogenic and aneugenic activity of tobacco particulate matter in cultured mammalian cells

We demonstrate here that tobacco particulate matter (TPM) produced from both non-ventilated and ventilated cigarettes of varying tar contents induced structural and numerical aberrations in cultured Chinese hamster cells. Our data indicate that TPM from ventilated cigarettes is of lower potency in inducing both clastogenic and aneugenic effects compared with TPM from non-ventilated cigarettes. These observations provide support for the concept that the genotoxic activity (to cultured Chinese hamster cells) of cigarette smoke is reduced by increased ventilation to a greater extent than a 1:1 ratio between yield reduction and smoke dilution.     

Expression of a novel tropomyosin isoform in axolotl heart and skeletal muscle

TPM1κ is an alternatively spliced isoform of the TPM1 gene whose specific role in cardiac development and disease is yet to be elucidated. Although mRNA studies have shown TPM1κ expression in axolotl heart and skeletal muscle, it has not been quantified. Also the presence of TPM1κ protein in axolotl heart and skeletal muscle has not been demonstrated. In this study, we quantified TPM1κ mRNA expression in axolotl heart and skeletal muscle. Using a newly developed TPM1κ specific antibody, we demonstrated the expression and incorporation of TPM1κ protein in myofibrils of axolotl heart and skeletal muscle. The results support the potential role of TPM1κ in myofibrillogenesis and sarcomeric function. J. Cell. Biochem. 110: 875–881, 2010. © 2010 Wiley‐Liss, Inc.     

Accumulation of systematic TPM1 mediates inflammation and neuronal remodeling by phosphorylating PKA and regulating the FABP5/NF‐κB signaling pathway in the retina of aged mice

The molecular mechanisms underlying functional decline during normal brain aging are poorly understood. Here, we identified the actin‐associated protein tropomyosin 1 (TPM1) as a new systemic pro‐aging factor associated with function deficits in normal aging retinas. Heterochronic parabiosis and blood plasma treatment confirmed that systemic factors regulated age‐related inflammatory responses and the ectopic dendritic sprouting of rod bipolar (RBC) and horizontal (HC) cells in the aging retina. Proteomic analysis revealed that TPM1 was a potential systemic molecule underlying structural and functional deficits in the aging retina. Recombinant TPM1 protein administration accelerated the activation of glial cells, the dendritic sprouting of RBCs and HCs and functional decline in the retina of young mice, whereas anti‐TPM1 neutralizing antibody treatment ameliorated age‐related structural and function changes in the retina of aged mice. Old mouse plasma (OMP) induced glial cell activation and the dendritic outgrowth of RBCs and HCs in young mice, and yet TMP1‐depleted OMP failed to reproduce the similar effect in young mice. These results confirmed that TPM1 was a systemic pro‐aging factor. Moreover, we demonstrated that systematic TPM1 was an immune‐related molecule, which elicited endogenous TPM1 expression and inflammation by phosphorylating PKA and regulating FABP5/NF‐κB signaling pathway in normal aging retinas. Interestingly, we observed TPM1 upregulation and the ectopic dendritic sprouting of RBCs and HCs in young mouse models of Alzheimer''s disease, indicating a potential role of TPM1 in age‐related neurodegenerative diseases. Our data indicate that TPM1 could be targeted for combating the aging process.