A Brownian Ratchet Model Explains the Biased Sidestepping of Single-Headed Kinesin-3 KIF1A.

The kinesin-3 motor KIF1A is involved in long-ranged axonal transport in neurons. To ensure vesicular delivery, motors need to navigate the microtubule lattice and overcome possible roadblocks along the way. The single-headed form of KIF1A is a highly diffusive motor that has been shown to be a prototype of a Brownian motor by virtue of a weakly bound diffusive state to the microtubule. Recently, groups of single-headed KIF1A motors were found to be able to sidestep along the microtubule lattice, creating left-handed helical membrane tubes when pulling on giant unilamellar vesicles in vitro. A possible hypothesis is that the diffusive state enables the motor to explore the microtubule lattice and switch protofilaments, leading to a left-handed helical motion. Here, we study the longitudinal rotation of microtubules driven by single-headed KIF1A motors using fluorescence-interference contrast microscopy. We find an average rotational pitch of ≃1.5µm, which is remarkably robust to changes in the gliding velocity, ATP concentration, microtubule length, and motor density. Our experimental results are compared to stochastic simulations of Brownian motors moving on a two-dimensional continuum ratchet potential, which quantitatively agree with the fluorescence-interference contrast experiments. We find that single-headed KIF1A sidestepping can be explained as a consequence of the intrinsic handedness and polarity of the microtubule lattice in combination with the diffusive mechanochemical cycle of the motor.

Aryl Hydrocarbon Receptor Promotes Liver Polyploidization and Inhibits PI3K, ERK, and Wnt/ß-Catenin Signaling.

Aryl hydrocarbon receptor (AhR) deficiency alters tissue homeostasis. However, how AhR regulates organ maturation and differentiation remains mostly unknown. Liver differentiation entails a polyploidization process fundamental for cell growth, metabolism, and stress responses. Here, we report that AhR regulates polyploidization during the preweaning-to-adult mouse liver maturation. Preweaning AhR-null (AhR-/-) livers had smaller hepatocytes, hypercellularity, altered cell cycle regulation, and enhanced proliferation. Those phenotypes persisted in adult AhR-/- mice and correlated with compromised polyploidy, predominance of diploid hepatocytes, and enlarged centrosomes. Phosphatidylinositol-3-phosphate kinase (PI3K), extracellular signal-regulated kinase (ERK), and Wnt/ß-catenin signaling remained upregulated from preweaning to adult AhR-null liver, likely increasing mammalian target of rapamycin (mTOR) activation. Metabolomics revealed the deregulation of mitochondrial oxidative phosphorylation intermediates succinate and fumarate in AhR-/- liver. Consistently, PI3K, ERK, and Wnt/ß-catenin inhibition partially rescued polyploidy in AhR-/- mice. Thus, AhR may integrate survival, proliferation, and metabolism for liver polyploidization. Since tumor cells tend to be polyploid, AhR modulation could have therapeutic value in the liver.

A Tunable Magnetic Domain Wall Conduit Regulating Nanoparticle Diffusion.

We demonstrate a general and robust method to confine on a plane strongly diffusing nanoparticles in water by using size tunable magnetic channels. These virtual conduits are realized with pairs of movable Bloch walls located within an epitaxially grown ferrite garnet film. We show that once inside the magnetic conduit the particles experience an effective local parabolic potential in the transverse direction, while freely diffusing along the conduit. The stiffness of the magnetic potential is determined as a function of field amplitude that varies the width of the magnetic channel. Precise control of the degree of confinement is demonstrated by tuning the applied field. The magnetic conduit is then used to realize single files of nonpassing particles and to induce periodic condensation of an ensemble of particles into parallel stripes in a completely controllable and reversible manner.

Pattern selection by dynamical biochemical signals.

The development of multicellular organisms involves cells to decide their fate upon the action of biochemical signals. This decision is often spatiotemporally coordinated such that a spatial pattern arises. The dynamics that drive pattern formation usually involve genetic nonlinear interactions and positive feedback loops. These complex dynamics may enable multiple stable patterns for the same conditions. Under these circumstances, pattern formation in a developing tissue involves a selection process: why is a certain pattern formed and not another stable one? Herein we computationally address this issue in the context of the Notch signaling pathway. We characterize a dynamical mechanism for developmental selection of a specific pattern through spatiotemporal changes of the control parameters of the dynamics, in contrast to commonly studied situations in which initial conditions and noise determine which pattern is selected among multiple stable ones. This mechanism can be understood as a path along the parameter space driven by a sequence of biochemical signals. We characterize the selection process for three different scenarios of this dynamical mechanism that can take place during development: the signal either 1) acts in all the cells at the same time, 2) acts only within a cluster of cells, or 3) propagates along the tissue. We found that key elements for pattern selection are the destabilization of the initial pattern, the subsequent exploration of other patterns determined by the spatiotemporal symmetry of the parameter changes, and the speeds of the path compared to the timescales of the pattern formation process itself. Each scenario enables the selection of different types of patterns and creates these elements in distinct ways, resulting in different features. Our approach extends the concept of selection involved in cellular decision-making, usually applied to cell-autonomous decisions, to systems that collectively make decisions through cell-to-cell interactions.

Single-molecule diffusion in a periodic potential at a solid-liquid interface.

We used single-molecule tracking experiments to observe the motion of small hydrophobic fluorescent molecules at the interface between water and a solid surface that exhibited periodic chemical patterns. The dynamics were characterized by non-ergodic, continuous time random walk statistics. The step-size distributions displayed enhanced probability of steps to periodic distances, consistent with theoretical predictions for diffusion in an atomic/molecular scale periodic potential. Surprisingly, this general behavior was observed here for surfaces exhibiting characteristic length scales three orders of magnitude larger than atomic/molecular dimensions, and may provide a new way to understand and control solid-liquid interfacial diffusion for molecular targeting applications.

Localización de nódulos pulmonares con arpón guiado por tomografía axial computarizada previa a la resección videotoracoscópica. Experiencia en 52 casos / Locating pulmonary nodules with a computed axial tomography-guided harpoon prior to videothoracoscopic resection. Experience with 52 cases

Introducción La resección videotoracoscópica (VTC) de los nódulos pulmonares (NP) periféricos requiere en ocasiones la práctica de una minitoracotomía para su localización mediante palpación. El objetivo de este estudio es evaluar la eficacia como método de localización preoperatoria de los NP de la colocación de un arpón guiado por TAC. Material y métodos Desde noviembre de 2004 hasta enero de 2011, 52 pacientes fueron programados para localización preoperatoria de 55 NP mediante la colocación de un arpón guiado por TAC. Resultados Un total de 52 pacientes (31 hombres y 21 mujeres) con edades entre 28 y 84 años (media: 62,2 años) con NP < 20mm (media: 9,57mm). De ellos, 35 tenían historia oncológica. Se colocaron 55 arpones (a 3 pacientes, 2 arpones simultáneos). En la VTC, 52 arpones fueron hallados correctamente anclados al NP. No se observaron complicaciones. En el grupo de 35 pacientes con antecedentes oncológicos, los nódulos resultaron ser malignos en 26 (74,3%). En los 17 no oncológicos fueron malignos el 70,6%. La estancia hospitalaria osciló entre 4 y 72 h, con 19 pacientes incluidos en un programa de cirugía ambulatoria (36,5%).Conclusiones La identificación preoperatoria de los NP permite su resección VTC directa. La colocación de un arpón guiado por TAC en los NP constituye un procedimiento seguro y efectivo que puede llevarse a cabo en un programa de cirugía ambulatoria (AU)

Objective Videothoracoscopic (VTC) resection of peripheral pulmonary nodules (PN) occasionally requires performing a mini-thoracotomy to locate them using palpation. The aim of this study is to evaluate the usefulness of inserting a CT-guided harpoon as a method for locating PN prior to surgery. Material and methods A study was conducted on a total of 52 patients who were scheduled for locating 55 PN prior to surgery by inserting a CT-guided harpoon, from November 2004 to January 2011.ResultsOf the 52 patients, of whom 35 had a history of cancer, 31 were male and 21 were female, with ages between 28 and 84 years (mean: 62.2 years) with a PN <20mm (mean: 9.57mm). A total of 55 harpoons were inserted (3 patients had 2 simultaneous harpoons). Using the VTC it was observed that 52 harpoons were correctly anchored to the PN. There were no complications. In the group of 35 patients with an oncology history, the nodules were malignant in 26 cases (74.3%), and there were 17 (70.6%) with malignant PN in those with no oncology history. The hospital stay varied between 4 and 72h, with 19 patients (36.5%) included in a one-day surgery program. Conclusions The preoperative identification of peripheral pulmonary nodules enables them to be removed directly with VTC. The insertion of a CT-guided harpoon in the PN is a safe and effective procedure that can be performed in a one-day surgery program (AU)

Functional consequences of Wnt-induced dishevelled 2 phosphorylation in canonical and noncanonical Wnt signaling.

Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594-609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/ß-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ε as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser(594), Thr(595), and Ser(597) of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates ß-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.

[Locating pulmonary nodules with a computed axial tomography-guided harpoon prior to videothoracoscopic resection. Experience with 52 cases]. / Localización de nódulos pulmonares con arpón guiado por tomografía axial computarizada previa a la resección videotoracoscópica. Experiencia en 52 casos.

OBJECTIVE: Videothoracoscopic (VTC) resection of peripheral pulmonary nodules (PN) occasionally requires performing a mini-thoracotomy to locate them using palpation. The aim of this study is to evaluate the usefulness of inserting a CT-guided harpoon as a method for locating PN prior to surgery. MATERIAL AND METHODS: A study was conducted on a total of 52 patients who were scheduled for locating 55 PN prior to surgery by inserting a CT-guided harpoon, from November 2004 to January 2011. RESULTS: Of the 52 patients, of whom 35 had a history of cancer, 31 were male and 21 were female, with ages between 28 and 84 years (mean: 62.2 years) with a PN <20mm (mean: 9.57mm). A total of 55 harpoons were inserted (3 patients had 2 simultaneous harpoons). Using the VTC it was observed that 52 harpoons were correctly anchored to the PN. There were no complications. In the group of 35 patients with an oncology history, the nodules were malignant in 26 cases (74.3%), and there were 17 (70.6%) with malignant PN in those with no oncology history. The hospital stay varied between 4 and 72h, with 19 patients (36.5%) included in a one-day surgery program. CONCLUSIONS: The preoperative identification of peripheral pulmonary nodules enables them to be removed directly with VTC. The insertion of a CT-guided harpoon in the PN is a safe and effective procedure that can be performed in a one-day surgery program.

Asymmetric stochastic switching driven by intrinsic molecular noise.

Low-copy-number molecules are involved in many functions in cells. The intrinsic fluctuations of these numbers can enable stochastic switching between multiple steady states, inducing phenotypic variability. Herein we present a theoretical and computational study based on Master Equations and Fokker-Planck and Langevin descriptions of stochastic switching for a genetic circuit of autoactivation. We show that in this circuit the intrinsic fluctuations arising from low-copy numbers, which are inherently state-dependent, drive asymmetric switching. These theoretical results are consistent with experimental data that have been reported for the bistable system of the gallactose signaling network in yeast. Our study unravels that intrinsic fluctuations, while not required to describe bistability, are fundamental to understand stochastic switching and the dynamical relative stability of multiple states.

Differential regulation of TP73 isoforms by 1α,25-dihydroxyvitamin D3 and survivin in human colon and breast carcinomas.

We evaluate whether 1,25(OH)(2)D(3) downregulates TP73 variants in colon and breast carcinomas, the role of survivin in this context, and the significance of this network in the clinic. Tumor cells were treated/untreated with 1,25(OH)(2)D(3) and transiently transfected with survivin. Levels of survivin and TP73 variants were evaluated by quantitative RT-PCR and Western blotting. In 75 colon and 60 breast cancer patients, the expressions of survivin and TP73 isoforms were determined. Tumor characteristics were examined in each patient. Survivin protein levels were also evaluated in a subgroup of patients and cell lines. Decrease in survivin and TAp73 transcripts and protein and ΔNp73 mRNA was detected after 1,25(OH)(2)D(3) treatment. Ectopic survivin expression led to an increase in the TAp73, ΔNp73, ΔEx2p73, and ΔEx2-3p73 transcripts. In cancer patients, direct correlations were observed between TP73 variants and survivin levels. 1,25(OH)(2)D(3) negatively regulate survivin and TP73 variants in colon and breast cancer cells. Positive regulation of TP73 isoforms by survivin may exist, which reinforces the possibility that the downregulation of TP73 forms by 1,25(OH)(2)D(3) is survivin-dependent.

The EWS/FLI1 oncogenic protein inhibits expression of the Wnt inhibitor DICKKOPF-1 gene and antagonizes beta-catenin/TCF-mediated transcription.

Tumours of the Ewing family, which comprise Ewing's sarcoma and peripheral primitive neuroectodermal tumours, are highly aggressive and mostly affect children and adolescents. They are characterized by chromosomal translocations leading to the generation of fusion proteins between EWS (or very rarely FUS) and members of the E-twenty-six (ETS) family of transcription factors that are capable of transforming cells. EWS/FLI1, the most frequent fusion, is thought to cause transformation through activation or repression of specific target genes. We present evidence demonstrating that the Wnt inhibitor and beta-catenin/T-cell factor (TCF)-responsive gene DICKKOPF-1 (DKK-1) is a transcriptional target of EWS/FLI1, which can inhibit both basal and beta-catenin-induced transactivation of the DKK-1 promoter. Moreover, our data indicate that EWS/FLI1 has a more general effect on beta-catenin/TCF-mediated transcription since it can block transactivation of a consensus beta-catenin/TCF reporter construct. Consistently, Ewing tumour cells expressing different EWS/ETS translocations cannot engage beta-catenin/TCF-dependent transcription, whereas silencing of EWS/FLI1 restores beta-catenin responsiveness in A673 and RD-ES Ewing tumour cells. Accordingly, gene set enrichment analysis shows that beta-catenin/TCF target genes are significantly enriched among genes downregulated by EWS/FLI1 in the Ewing cell line A673. Mechanistically, the inhibitory effect of EWS/FLI1 can be overcome by a constitutively active TCF4 protein (TCF4-VP16). Moreover, EWS/FLI1 binds lymphoid enhancer factor 1, a TCF family member, and interferes with its binding to beta-catenin, which could explain its negative effect on beta-catenin/TCF-mediated transcription. Our results show that EWS/FLI1 inhibits both DKK-1 expression as well as beta-catenin/TCF-dependent transcription, which could contribute to progression of tumours of the Ewing family.

Wnt proteins induce dishevelled phosphorylation via an LRP5/6- independent mechanism, irrespective of their ability to stabilize beta-catenin.

Wnt glycoproteins play essential roles in the development of metazoan organisms. Many Wnt proteins, such as Wnt1, activate the well-conserved canonical Wnt signaling pathway, which results in accumulation of beta-catenin in the cytosol and nucleus. Other Wnts, such as Wnt5a, activate signaling mechanisms which do not involve beta-catenin and are less well characterized. Dishevelled (Dvl) is a key component of Wnt/beta-catenin signaling and becomes phosphorylated upon activation of this pathway. In addition to Wnt1, we show that several Wnt proteins, including Wnt5a, trigger phosphorylation of mammalian Dvl proteins and that this occurs within 20 to 30 min. Unlike the effects of Wnt1, phosphorylation of Dvl in response to Wnt5a is not concomitant with beta-catenin stabilization, indicating that Dvl phosphorylation is not sufficient to activate canonical Wnt/beta-catenin signaling. Moreover, neither Dickkopf1, which inhibits Wnt/beta-catenin signaling by binding the Wnt coreceptors LRP5 and -6, nor dominant-negative LRP5/6 constructs could block Wnt-mediated Dvl phosphorylation. We conclude that Wnt-induced phosphorylation of Dvl is independent of LRP5/6 receptors and that canonical Wnts can elicit both LRP-dependent (to beta-catenin) and LRP-independent (to Dvl) signals. Our data also present Dvl phosphorylation as a general biochemical assay for Wnt protein function, including those Wnts that do not activate the Wnt/beta-catenin pathway.

Truncated mutants of the putative Wnt receptor LRP6/Arrow can stabilize beta-catenin independently of Frizzled proteins.

Secreted signaling proteins of the Wnt family are known to regulate a diverse range of developmental processes, and their signaling pathway through beta-catenin is frequently activated in cancer. The identification of both Frizzled and LRP5/6 (LRP: low-density lipoprotein receptor-related protein) proteins as components of cell-surface receptors for Wnt proteins has raised questions about their individual functions. We have investigated this issue through a structure-function analysis of Frizzled and LRP proteins that have been implicated in Wnt1 signaling. Consistent with other reports, we find that LRP6/Arrow proteins deleted for their extracellular domain are able to activate the Wnt/beta-catenin signaling pathway. Importantly, our results demonstrate that this signaling from LRP6/Arrow derivatives can occur in a Frizzled- and ligand-independent manner. Furthermore, we show that the PPSP motifs within the intracellular domain of LRP6 are required for signaling. In contrast to results with LRP6, overexpression of Frizzled proteins did not activate the pathway. Based on evidence of ligand binding to both Frizzled and LRP6, current models suggest that both proteins are components of a Wnt receptor complex that signals to beta-catenin. In light of these models, our data imply that LRP5/6/Arrow proteins constitute the distal signal-initiating component of these receptors. The results also support the notion that LRP5/6 are candidate oncogenes.

Thyroid hormone receptors/THR genes in human cancer.

Thyroid hormone (triiodothyronine, T3) is a pleiotropic regulator of growth, differentiation and tissue homeostasis in higher organisms that acts through the control of target gene expression. Most, if not all, major T3 actions are mediated by specific high affinity nuclear receptors (TR) which are encoded by two genes, THRA and THRB. Several TRalpha and TRbeta receptor isoforms are expressed. Abundant and contradictory literature exists on the relationship between circulating thyroid hormone levels, thyroid diseases and human cancer. In 1986, a connection between TR and cancer became evident when the chicken TRalpha1 was characterized as the c-erbA proto-oncogene, the cellular counterpart of the retroviral v-erbA oncogene. V-erbA causes erythroleukemias and sarcomas in birds, and hepatocellular carcinomas in transgenic mice. In recent years, many studies have analyzed the presence of quantitative (abnormal levels) or qualitative (mutations) alterations in the expression of THR genes in different types of human neoplasias. While their role in tumor generation or progression is currently unclear, both gross chromosomal and minor mutations (deletions, aberrant splicing, point mutations) and changes in the level of expression of THRA and THRB genes have been found. Together with other in vitro data indicating connections between TR and p53, Rb, cyclin D and other cell cycle regulators and oncogenes, these results suggest that THRA and THRB may be involved in human cancer.

Expression of thyroid hormone receptor/erbA genes is altered in human breast cancer.

The relation between thyroid status and diseases and cancer is unclear. No detailed analysis of thyroid hormone receptor (TR) expression in human breast cancer has been reported. We have analysed the expression and mutational status of the TRalpha1, encoded by the c-erbA proto-oncogene, TRbeta1 and TRbeta2 isoforms in 70 sporadic breast cancers. Alterations in the RNA level of TRbeta1, TRalpha1, or both were found in a number of patients. No expression of TRbeta2 RNA was detected. Western blotting analysis confirmed the differences in expression at the protein level in those cases where sufficient tumor sample was available. Additionally, tumor-specific truncated TRbeta1 RNA was found in six patients. Strikingly, three transcripts shared the same breakpoint. Only one tumor carried the corresponding deletion at the genomic DNA level, suggesting that the remaining abnormal TRbeta1 transcripts are aberrant splicing products. Though no significant correlation was found between TRbeta1 alteration and any clinical parameter, it showed a tendency to associate with early age of onset (<50 years). Our results reveal specific alterations in the expression of TRbeta and TRalpha genes in a subset of breast cancer patients, suggesting that deregulation of thyroid hormone target genes may be involved in the generation of this neoplasia.