Note: Tormenta: An open source Python-powered control software for camera based optical microscopy

Until recently, PC control and synchronization of scientific instruments was only possible through closed-source expensive frameworks like National Instruments' LabVIEW. Nowadays, efficient cost-free alternatives are available in the context of a continuously growing community of open-source software developers. Here, we report on Tormenta, a modular open-source software for the control of camera-based optical microscopes. Tormenta is built on Python, works on multiple operating systems, and includes some key features for fluorescence nanoscopy based on single molecule localization.Fil: Barabas, Federico Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Masullo, Luciano Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentin

Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models

ERBB2 is a member of the ERBB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ERBB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ERBB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ERBB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ERBB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ERBB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ERBB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ERBB2: a major population located in large clusters and a minor population outside these structures. Upon ERBB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ERBB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ERBB2 and beta1 integrin activity in breast cancer cells.Instituto de Investigaciones Bioquímicas de La Plat

Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models

ERBB2 is a member of the ERBB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ERBB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ERBB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ERBB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ERBB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ERBB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ERBB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ERBB2: a major population located in large clusters and a minor population outside these structures. Upon ERBB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ERBB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ERBB2 and beta1 integrin activity in breast cancer cells.Instituto de Investigaciones Bioquímicas de La Plat

Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.Fil: Unsain, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Bordenave, Martín Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Martinez, Gaby F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Jalil, Sami. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Von Bilderling, Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Barabas, Federico Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Masullo, Luciano Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Johnstone, Aaron D.. McGill University; CanadáFil: Barker, Philip A.. University of British Columbia; CanadáFil: Bisbal, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Caceres, Alfredo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentin

A fluorescence nanoscopy marker for corticotropin-releasing hormone type 1 receptor: Computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and: In situ affinity constant in cells

Class B G protein-coupled receptors (GPCRs) are involved in a variety of human pathophysiological states. These groups of membrane receptors are less studied than class A GPCRs due to the lack of structural information, delayed small molecule drug discovery, and scarce fluorescence detection tools available. The class B corticotropin-releasing hormone type 1 receptor (CRHR1) is a key player in the stress response whose dysregulation is critically involved in stress-related disorders: psychiatric conditions (i.e. depression, anxiety, and addictions), neuroendocrinological alterations, and neurodegenerative diseases. Here, we present a strategy to label GPCRs with a small fluorescent antagonist that permits the observation of the receptor in live cells through stochastic optical reconstruction microscopy (STORM) with 23 nm resolution. The marker, an aza-BODIPY derivative, was designed based on computational docking studies, then synthesized, and finally tested in biological cells. Experiments on hippocampal neurons demonstrate antagonist effects in similar concentrations as the well-established antagonist CP-376395. A quantitative analysis of two color STORM images enabled the determination of the binding affinity of the new marker in the cellular environment.Fil: Szalai, Alan Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Armando, Natalia Giannina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Barabas, Federico Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Giordano, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Bari, Sara Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Cavasotto, Claudio Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Silberstein Cuña, Susana Iris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Aramendia, Pedro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentin

Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models

<div><p>ErbB2 is a member of the ErbB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ErbB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ErbB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ErbB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ErbB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ErbB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ErbB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ErbB2: a major population located in large clusters and a minor population outside these structures. Upon ErbB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ErbB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ErbB2 and beta1 integrin activity in breast cancer cells.</p></div

ErbB2 and beta1 integrin coexist in membrane clusters of cells with overexpressed receptors.

<p>(A) HC11 cells transfected with beta1 integrin-ECFP-shRNA and ErbB2-mYFP plasmids plated on FN-coated coverslips. Similar to A4-Hela, it is possible to observe membrane structures where both proteins colocalize in a very reproducible fashion. (B) Quantification of Pearson’s correlation, Manders’ overlap, M1 and M2 coefficients for MCF7 (red bars), SKBR3 (green bars), A4-HeLa (yellow bars) and HC11 cotransfected with beta1 integrin-ECFP-shRNA and ErbB2-mYFP plasmids (blue bars). Scale bar: 10um. ** p<0.01 vs. MCF7 and SKBR3 (ANOVA followed by Tukey's test), n = 23 for MCF7, SKBR3 and A4-HeLa, n = 15 for HC11. Data set available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174230#pone.0174230.s001" target="_blank">S1 Fig</a>.</p

Model proposed for ErbB2 and beta1 integrin interaction dynamics in the cell membrane.

<p>(A) When cells are not attached to the substrate, ErbB2 and beta1 integrin coexist in membrane clusters described as lipid rafts by Mocanu and collaborators. (B) When integrins are activated by a ligand, such as FN, they leave the ErbB2 clusters and recruit other protein forming the FAs. Although there is no colocalization between ErbB2 clusters and FAs, ErbB2 molecules outside these structures could interact with FAs proteins such as FAK or p130CAS as described in the literature. (C) When ErbB2 and beta1 integrin are overexpressed the system is saturated and the equilibrium between free, FAs and ErbB clusters-linked beta1 integrin is disrupted. In this condition, overexpression of ErbB2 has been found to induce autophosphorylation and ligand-independent activation of signaling pathways such as Akt or MAPK. In this context, self-activation of ErbB2 could lead to FA protein phosphorylation as they could be physically close to each other due to beta1 integrin location in ErbB2 clusters.</p

Immunolabeling of MCF7 cells in suspension expressing the fusion protein ErbB2-TagRFP.

<p>MCF7 cells expressing the fusion protein ErbB2-TagRFP in suspension were immunolabeled for beta1 integrin. 3D-reconstruction allows us to determine that both proteins coexist in conspicuous domains in the surface of the cell membrane. This is a representative image of 20 acquired cells. All cells showed clusters with positive colocalization (r > 0.5). Scale bar: 5um.</p

STORM images of MCF7 expressing the ErbB2-TagRFP fusion protein and SKBR3 cells.

<p>Cells were labeled with a specific antibody for ErbB2 (OP15, Millipore) and a secondary antibody coupled with the Alexa Fluor 647 fluorophore. It was possible to determine the presence of two different receptor populations: one inside the clusters and another outside these structures, seen as small spots, representing monomers or low stoichiometry aggregates of ErbB2.</p