Directional control of neurite outgrowth: emerging technologies for Parkinson's disease using magnetic nanoparticles and magnetic field gradients.

A challenge in current stem cell therapies for Parkinson's disease (PD) is controlling neuronal outgrowth from the substantia nigra towards the targeted area where connectivity is required in the striatum. Here we present progress towards controlling directional neurite extensions through the application of iron-oxide magnetic nanoparticles (MNPs) labelled neuronal cells combined with a magnetic array generating large spatially variant field gradients (greater than 20 T m-1). We investigated the viability of this approach in both two-dimensional and organotypic brain slice models and validated the observed changes in neurite directionality using mathematical models. Results showed that MNP-labelled cells exhibited a shift in directional neurite outgrowth when cultured in a magnetic field gradient, which broadly agreed with mathematical modelling of the magnetic force gradients and predicted MNP force direction. We translated our approach to an ex vivo rat brain slice where we observed directional neurite outgrowth of transplanted MNP-labelled cells from the substantia nigra towards the striatum. The improved directionality highlights the viability of this approach as a remote-control methodology for the control and manipulation of cellular growth for regenerative medicine applications. This study presents a new tool to overcome challenges faced in the development of new therapies for PD.

Stick-slip dynamics of cell adhesion triggers spontaneous symmetry breaking and directional migration of mesenchymal cells on one-dimensional lines.

Directional cell motility relies on the ability of single cells to establish a front-rear polarity and can occur in the absence of external cues. The initiation of migration has often been attributed to the spontaneous polarization of cytoskeleton components, while the spatiotemporal evolution of cell-substrate interaction forces has yet to be resolved. Here, we establish a one-dimensional microfabricated migration assay that mimics the complex in vivo fibrillar environment while being compatible with high-resolution force measurements, quantitative microscopy, and optogenetics. Quantification of morphometric and mechanical parameters of NIH-3T3 fibroblasts and RPE1 epithelial cells reveals a generic stick-slip behavior initiated by contractility-dependent stochastic detachment of adhesive contacts at one side of the cell, which is sufficient to trigger cell motility in 1D in the absence of pre-established polarity. A theoretical model validates the crucial role of adhesion dynamics, proposing that front-rear polarity can emerge independently of a complex self-polarizing system.

Optogenetic dissection of Rac1 and Cdc42 gradient shaping.

During cell migration, Rho GTPases spontaneously form spatial gradients that define the front and back of cells. At the front, active Cdc42 forms a steep gradient whereas active Rac1 forms a more extended pattern peaking a few microns away. What are the mechanisms shaping these gradients, and what is the functional role of the shape of these gradients? Here we report, using a combination of optogenetics and micropatterning, that Cdc42 and Rac1 gradients are set by spatial patterns of activators and deactivators and not directly by transport mechanisms. Cdc42 simply follows the distribution of Guanine nucleotide Exchange Factors, whereas Rac1 shaping requires the activity of a GTPase-Activating Protein, ß2-chimaerin, which is sharply localized at the tip of the cell through feedbacks from Cdc42 and Rac1. Functionally, the spatial extent of Rho GTPases gradients governs cell migration, a sharp Cdc42 gradient maximizes directionality while an extended Rac1 gradient controls the speed.

Subcellular control of Rac-GTPase signalling by magnetogenetic manipulation inside living cells.

Many cell functions rely on the coordinated activity of signalling pathways at a subcellular scale. However, there are few tools capable of probing and perturbing signalling networks with a spatial resolution matching the intracellular dimensions of their activity patterns. Here we present a generic magnetogenetic approach based on the self-assembly of signalling complexes on the surface of functionalized magnetic nanoparticles inside living cells. The nanoparticles act as nanoscopic hot spots that can be displaced by magnetic forces and trigger signal transduction pathways that bring about a cell response. We applied this strategy to Rho-GTPases, a set of molecular switches known to regulate cell morphology via complex spatiotemporal patterns of activity. We demonstrate that the nanoparticle-mediated activation of signalling pathways leads to local remodelling of the actin cytoskeleton and to morphological changes.

Kinetics of diffusion-limited catalytically activated reactions: an extension of the Wilemski-Fixman approach.

We study the kinetics of diffusion-limited catalytically activated A+B-->B reactions taking place in three-dimensional systems, in which an annihilation of diffusive A particles by diffusive traps B may happen only if the encounter of an A with any of the Bs happens within a special catalytic subvolumen: these subvolumens being immobile and uniformly distributed within the reaction bath. Suitably extending the classical approach of Wilemski and Fixman [J. Chem. Phys. 58, 4009 (1973)] to such three-molecular diffusion-limited reactions, we calculate analytically an effective reaction constant and show that it comprises several terms associated with the residence and joint residence times of Brownian paths in finite domains. The effective reaction constant exhibits a nontrivial dependence on the reaction radii, the mean density of catalytic subvolumens, and particles' diffusion coefficients. Finally, we discuss the fluctuation-induced kinetic behavior in such systems.

Optimal search strategies for hidden targets.

What is the fastest way of finding a randomly hidden target? Experimental observations reveal that the search behavior of foraging animals is generally intermittent: active search phases randomly alternate with phases of fast ballistic motion. Here, we study the efficiency of this two state search strategy by calculating analytically the mean first passage time at the target. We model the perception mechanism involved in the active search phase by a diffusive process. We show that the search strategy is optimal when the average duration of "motion phases" varies like the power either 3/5 or 2/3 of the average duration of "search phases" depending on the regime. This scaling accounts for experimental data over a wide range of species, which suggests that the kinetics of search trajectories is a determining factor optimized by foragers and that the perception activity is adequately described by a diffusion process.

Kinetics of target site localization of a protein on DNA: a stochastic approach.

It is widely recognized that the cleaving rate of a restriction enzyme on target DNA sequences is several orders-of-magnitude faster than the maximal one calculated from the diffusion-limited theory. It was therefore commonly assumed that the target site interaction of a restriction enzyme with DNA has to occur via two steps: one-dimensional diffusion along a DNA segment, and long-range jumps coming from association-dissociation events. We propose here a stochastic model for this reaction which comprises a series of one-dimensional diffusions of a restriction enzyme on nonspecific DNA sequences interrupted by three-dimensional excursions in the solution until the target sequence is reached. This model provides an optimal finding strategy which explains the fast association rate. Modeling the excursions by uncorrelated random jumps, we recover the expression of the mean time required for target site association to occur given by Berg et al. in 1981, and we explicitly give several physical quantities describing the stochastic pathway of the enzyme. For competitive target sites we calculate two quantities: processivity and preference. By comparing these theoretical expressions to recent experimental data obtained for EcoRV-DNA interaction, we quantify: 1), the mean residence time per binding event of EcoRV on DNA for a representative one-dimensional diffusion coefficient; 2), the average lengths of DNA scanned during the one-dimensional diffusion (during one binding event and during the overall process); and 3), the mean time and the mean number of visits needed to go from one target site to the other. Further, we evaluate the dynamics of DNA cleavage with regard to the probability for the restriction enzyme to perform another one-dimensional diffusion on the same DNA substrate following a three-dimensional excursion.

Lattice theory of trapping reactions with mobile species.

We present a stochastic lattice theory describing the kinetic behavior of trapping reactions A+B-->B, in which both the A and B particles perform an independent stochastic motion on a regular hypercubic lattice. Upon an encounter of an A particle with any of the B particles, A is annihilated with a finite probability; finite reaction rate is taken into account by introducing a set of two-state random variables--"gates," imposed on each B particle, such that an open (closed) gate corresponds to a reactive (passive) state. We evaluate here a formal expression describing the time evolution of the A particle survival probability, which generalizes our previous results. We prove that for quite a general class of random motion of the species involved in the reaction process, for infinite or finite number of traps, and for any time t, the A particle survival probability is always larger in the case when A stays immobile, than in situations when it moves.

Catalytic reactions with bulk-mediated excursions: mixing fails to restore chemical equilibrium.

In this paper we analyze the effect of the bulk-mediated excursions (BME) of reactive species on the long-time behavior of the catalytic Langmuir-Hinshelwood-like A+B-->0 reactions in systems in which a catalytic plane (CP) is in contact with a liquid phase, containing concentrations of reactive particles. Such BME result from repeated particles desorption from the CP, subsequent diffusion in the liquid phase, and eventual readsorption on the CP away from the initial detachment point. This process leads to an effective superdiffusive transport along the CP. We consider both "batch" reactions, in which all particles of reactive species were initially adsorbed onto the CP, and reactions followed by a steady inflow of particles onto the CP. We show that for batch reactions the BME provide an effective mixing channel and here the mean-field-type behavior emerges. On the contrary, for reaction followed by a steady inflow of particles, we observe essential departures from the mean-field behavior and find that the mixing effect of the BME is insufficient to restore chemical equilibrium. We show that a steady state is established as t--> infinity, in which the limiting value of the mean coverage of the CP depends on the particles' diffusion coefficient in the bulk liquid phase, and that the spatial distributions of adsorbed particles are strongly correlated. Moreover, we show that the relaxation to such a steady state is a power-law function of time, in contrast to the exponential time dependence describing the approach to equilibrium in perfectly stirred systems.

Pascal principle for diffusion-controlled trapping reactions.

In this paper, we analyze the long-time behavior of the survival probability P(A)(t) of an A particle, that performs lattice random walk in the presence of randomly moving traps B. We show that for both perfect and imperfect trapping reactions, for arbitrary spatial dimension d and for a rather general class of random walks, P(A)(t) is less than or equal to the survival probability of an immobile target A in the presence of randomly moving traps.

Trapping reactions with randomly moving traps: exact asymptotic results for compact exploration.

In a recent paper, Bray and Blythe have shown that the survival probability P(A)(t) of an A particle diffusing with a diffusion coefficient D(A) in a one-dimensional system with diffusive traps B is independent of D(A) in the asymptotic limit t--> infinity and coincides with the survival probability of an immobile target in the presence of diffusive traps. Here, we show that this remarkable behavior has a more general range of validity and holds for systems of an arbitrary dimension d, integer or fractal, provided that the traps are "compactly exploring" the space, i.e., the "fractal" dimension d(w) of traps' trajectories is greater than d. For the marginal case when d(w)=d, as exemplified here by conventional diffusion in two-dimensional systems, the decay form is determined up to a numerical factor in the characteristic decay time.

Close but distinct regions of human herpesvirus 8 latency-associated nuclear antigen 1 are responsible for nuclear targeting and binding to human mitotic chromosomes.

Human herpesvirus 8 is associated with all forms of Kaposi's sarcoma, AIDS-associated body cavity-based lymphomas, and some forms of multicentric Castleman's disease. Herpesvirus 8, like other gammaherpesviruses, can establish a latent infection in which viral genomes are stably maintained as multiple episomes. The latent nuclear antigen (LANA or LNAI) may play an essential role in the stable maintenance of latent episomes, notably by interacting concomitantly with the viral genomes and the metaphase chromosomes, thus ensuring an efficient transmission of the neoduplicated episomes to the daughter cells. To identify the regions responsible for its nuclear and subnuclear localization in interphase and mitotic cells, LNAI and various truncated forms were fused to a variant of green fluorescent protein. This enabled their localization and chromosome binding activity to be studied by low-light-level fluorescence microscopy in living HeLa cells. The results demonstrate that nuclear localization of LNAI is due to a unique signal, which maps between amino acids 24 and 30. Interestingly, this nuclear localization signal closely resembles those identified in EBNA1 from Epstein-Barr virus and herpesvirus papio. A region encompassing amino acids 5 to 22 was further proved to mediate the specific interaction of LNA1 with chromatin during interphase and the chromosomes during mitosis. The presence of putative phosphorylation sites in the chromosome binding sites of LNA1 and EBNA1 suggests that their activity may be regulated by specific cellular kinases.

Production of HIV-1 by human B cells infected in vitro: characterization of an EBV genome-negative B cell line chronically synthetizing a low level of HIV-1 after infection.

Human immunodeficiency virus type 1 (HIV-1) has tropism for helper T lymphocytes and cells of the monocyte/ macrophage lineages. HIV-1 can also infect other cell types, including B cells. We show here that 10% of fresh circulating B cells from HIV-1-seronegative donors (i) express the CD4 receptor and CCR5 and CXCR4, two recently described coreceptors for HIV-1 and (ii) are permissive to HIV-1 with de novo proviral DNA integration following ex vivo infection by either SI (syncytium-inducing) or NSI (non-syncytium-inducing) isolates. To get further information on the interaction between HIV and B cells, the susceptibility of several EBV-positive or -negative B cell lines to infection by SI and NSI isolates was checked. Following infection of an EBV- CD4+ CXCR4+ CCR5- B cell line (DG75) by an SI HIV-1 isolate, we obtained a cell line which chronically produced low-level infectious HIV-1 for 2 years (HIV-DG75). Immunocytochemical data, combined with in situ PCR data, established that HIV-DG75 cells consist of at least three populations uninfected cells, infected virus-producing cells, and infected but nonproducing cells. Moreover, HIV-DG75 cells which express p24 antigen do not go into apoptosis, contrary to T lymphocytes. We infer from these results that B cells could constitute a reservoir of infectious virus in infected patients.

Complementary advantages of fluorescence and SIMS microscopies in the study of cellular localization of two new antitumor drugs.

Low light level fluorescence microscopy studies have been carried out on MCF7-P human mammary tumor cells to localize the intracellular distribution of two new anticancer drugs, Pazelliptine and Intoplicine, which are currently under clinical evaluation. These two molecules are thought to act at the nuclear level, through DNA topoisomerase interactions. Because fluorescence of these compounds appears strongly quenched by intercalation in double strand DNA, secondary ion mass spectrometry (SIMS) imaging was used to check the presence of the drugs in the nuclear compartment. In spite of chemical structure similitudes, pazelliptine and intoplicine appear to be distributed in quite different ways within the cells. Incubation for 1 and 24 hours also allowed us to bring to light strong differences in the distribution kinetics. Pazelliptine quickly enters into the nucleoli but is no longer present in the nucleus after 24 hours incubation. Intoplicine was not detected by fluorescence in the nucleus, however SIMS microscopy allowed us to show its accumulation within this cellular compartment as a function of time of exposure. This study shows the complementarity of fluorescence and SIMS microscopies.

Inhibition and enhancement of eukaryotic gene expression by potential non-B DNA sequences.

In a transient or constitutive expression assay we have examined the effect of non-B DNA sequences d(CA)40 and d(CAAAAATGCC)n on gene expression in eukaryotic cells. These sequences were cloned adjacent to the weak eukaryotic promoter (CGTATTTATTTG) and located upstream from the coding sequence of galactokinase enzyme. Recombinants were micro-injected in cultured cells (Chinese hamster fibroblasts R1610, mutant gal-K-) and expression levels have been determined. The alternating purine-pyrimidine tract found in d(CA)40 able to assume the Z-DNA conformation shows an inhibitory effect on gene expression. In addition, our results suggest a new potential role of Z-DNA motifs in vivo to stimulate recombination. The sequences d(CAAAAATGCC)n able to adopt another non-B structure, corresponding to curved (or bended) helix conformation, strongly enhance gene expression and this enhancement depends on sequence redundancy.

Inhibition and enhancement of eukaryotic gene expression by potential non-B DNA sequences.

In a transient or constitutive expression assay we have examined the effect of non-B DNA sequences d(CA)40 and d(CAAAAATGCC)n on gene expression in eukaryotic cells. These sequences were cloned adjacent to the weak eukaryotic promoter (CGTATTTATTTG) and located upstream from the coding sequence of galactokinase enzyme. Recombinants were micro-injected in cultured cells (Chinese hamster fibroblasts R1610, mutant gal-K-) and expression levels have been determined. The alternating purine-pyrimidine tract found in d(CA)40 able to assume the Z-DNA conformation shows an inhibitory effect on gene expression. In addition, our results suggest a new potential role of Z-DNA motifs in vivo to stimulate recombination. The sequences d(CAAAAATGCC)n able to adopt another non-B structure, corresponding to curved or bended helix conformation, strongly enhance gene expression and this enhancement depends on sequence redundancy.

Receptor binding of NBD-labeled fluorescent estrogens and progestins in whole cells and cell-free preparations.

We have studied the interactions of four fluorescent steroid conjugates with either the estrogen or progesterone receptor, both in whole cells and cell-free receptor preparations. The fluorophore, nitrobenzoxadiazole (NBD), was conjugated with a synthetic progestin, with a steroidal estrogen, a non-steroidal estrogen, and with an antiestrogen. With all compounds, receptor-specific binding could be detected by fluorescence measurements following extraction from the protein into an organic solvent. In the native state, however, the NBD-ligand-receptor complex is essentially non-emissive, although these ligands fluoresce strongly when associated with non-specific binders such as albumin. The binding site concentrations and relative affinities determined by fluorescence (after extraction) correspond well with those determined by [3H]estradiol or [3H]R5020 binding to their respective receptors. In T47D breast cancer cells, the NBD-progestin showed receptor-mediated uptake and nuclear localization. These compounds have provided valuable information about the interactions of low and medium affinity ligands with their receptors; however, the successful use of fluorescent ligands for detecting steroid receptors under native-bound conditions, by "imaging" modalities (fluorescence microscopy and flow cytometry) will require the development of fluorophores that are emissive while receptor bound or assay protocols that enable the environment of ligands associated with the receptor to be controlled.

Occurrence of two cholecystokinin binding sites in guinea-pig brain cortex.

Saturation experiments of the highly potent cholecystokinin analogue [3H]Boc(diNle28,31)CCK27-33 ([3H]BNDL-CCK7, 100 Ci/mmol) with guinea pig brain cortex in a large concentration range (0.05 nM to 30 nM) show the presence of two different binding sites (A site: KD = 0.13 nM, Bmax = 35 fmol/mg; B site: KD = 6.4 nM, Bmax = 92 fmol/mg). Both sites exhibit different sensitivity to sodium ions and therefore can be selectively investigated at [3H]BDNL-CCK7 concentration lower than 1 nM for the A site in Tris buffer and in Krebs buffer for the B site. The selectivity factors KIB/KIA of various CCK related peptides vary from 58 for CCK4 to 26 for CCK8 and 4 for the antagonist (Nle28,31) CCK27-32-NH2. The occurrence of two different CCK binding sites in the brain could explain biphasic pharmacological effects of CCK8.

Resonance Raman spectroscopic evidence that carp deoxyhemoglobin remains in a T-like quaternary structure at high pH: implications for cooperativity.

Resonance Raman spectroscopy shows the Fe-proximal imidazole stretching band to shift from 215 to 219 cm-1 between human deoxyhemoglobin (deoxy-Hb) and a Hb sample which is 75% oxygenated, demonstrating that the T-R quaternary structure switch can be monitored by resonance Raman spectroscopy in native Hb at equilibrium. For deoxy-Hb from carp, the band is at 215 cm-1 at pH 9 as well as pH 6, contrary to previous reports of an elevated frequency at high pH. The invariance of this frequency over a large affinity difference is in contrast to a recent report of continuously varying vFe-ImH frequencies for human mutant deoxy-Hb's. The band shifts to 219 cm-1 for carp Hb at pH 9 when O2 is bound to only 20% of the hemes. The spectra are consistent with a T-R switch upon binding approximately 0.5 O2 per Hb, on the average, although the number may be higher if the binding affinity is higher for alpha- than for beta-chains. The 0.5 value, in conjunction with the weak cooperativity observed for carp Hb at pH 9, is incompatible with a value of the allosteric constant, L = (T0)/(R0), large enough to prevent the vFe-ImH band from shifting detectably at pH 9 in the absence of O2. The possibility of functionally important intermediate structures is discussed.