RESUMEN

Cellular systems must deal with mechanical forces to satisfy their physiological functions. In this context, proteins with mechanosensitive properties play a crucial role in sensing and responding to environmental changes. The discovery of aquaporins (AQPs) marked a significant breakthrough in the study of water transport. Their transport capacity and regulation features make them key players in cellular processes. To date, few AQPs have been reported to be mechanosensitive. Like mechanosensitive ion channels, AQPs respond to tension changes in the same range. However, unlike ion channels, the aquaporin's transport rate decreases as tension increases, and the molecular features of the mechanism are unknown. Nevertheless, some clues from mechanosensitive ion channels shed light on the AQP-membrane interaction. The GxxxG motif may play a critical role in the water permeation process associated with structural features in AQPs. Consequently, a possible gating mechanism triggered by membrane tension changes would involve a conformational change in the cytoplasmic extreme of the single file region of the water pathway, where glycine and histidine residues from loop B play a key role. In view of their transport capacity and their involvement in relevant processes related to mechanical forces, mechanosensitive AQPs are a fundamental piece of the puzzle for understanding cellular responses.

RESUMEN

In Saccharomyces cerevisiae cAMP regulates different cellular processes through PKA. The specificity of the response of the cAMP-PKA pathway is highly regulated. Here we address the mechanism through which the cAMP-PKA pathway mediates its response to heat shock and thermal adaptation in yeast. PKA holoenzyme is composed of a regulatory subunit dimer (Bcy1) and two catalytic subunits (Tpk1, Tpk2, or Tpk3). PKA subunits are differentially expressed under certain growth conditions. Here we demonstrate the increased abundance and half-life of TPK1 mRNA and the assembly of this mRNA in cytoplasmic foci during heat shock at 37 °C. The resistance of the foci to cycloheximide-induced disassembly along with the polysome profiling analysis suggest that TPK1 mRNA is impaired for entry into translation. TPK1 expression was also evaluated during a recurrent heat shock and thermal adaptation. Tpk1 protein level is significantly increased during the recovery periods. The crosstalk of cAMP-PKA pathway and CWI signalling was also studied. Wsc3 sensor and some components of the CWI pathway are necessary for the TPK1 expression upon heat shock. The assembly in foci upon thermal stress depends on Wsc3. Tpk1 expression is lower in a wsc3∆ mutant than in WT strain during thermal adaptation and thus the PKA levels are also lower. An increase in Tpk1 abundance in the PKA holoenzyme in response to heat shock is presented, suggesting that a recurrent stress enhanced the fitness for the coming favourable conditions. Therefore, the regulation of TPK1 expression by thermal stress contributes to the specificity of cAMP-PKA signalling.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Semivida , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Polirribosomas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Temperatura

RESUMEN

Cells exert traction forces on the extracellular matrix to which they are adhered through the formation of focal adhesions. Spatial-temporal regulation of traction forces is crucial in cell adhesion, migration, cellular division, and remodeling of the extracellular matrix. By cultivating cells on polyacrylamide hydrogels of different stiffness we were able to investigate the effects of substrate stiffness on the generation of cellular traction forces by Traction Force Microscopy (TFM), and characterize the molecular dynamics of the focal adhesion protein zyxin by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). As the rigidity of the substrate increases, we observed an increment of both, cellular traction generation and zyxin residence time at the focal adhesions, while its diffusion would not be altered. Moreover, we found a positive correlation between the traction forces exerted by cells and the residence time of zyxin at the substrate elasticities studied. We found that this correlation persists at the subcellular level, even if there is no variation in substrate stiffness, revealing that focal adhesions that exert greater traction present longer residence time for zyxin, i.e., zyxin protein has less probability to dissociate from the focal adhesion.

Asunto(s)

Estrés Mecánico , Zixina/química , Citoesqueleto de Actina/efectos de los fármacos , Amidas/farmacología , Animales , Bovinos , Adhesión Celular , Citocalasina D/farmacología , Células Endoteliales , Recuperación de Fluorescencia tras Fotoblanqueo , Adhesiones Focales , Proteínas Fluorescentes Verdes , Microscopía Intravital , Cinética , Rayos Láser , Ratones , Ratones Endogámicos BALB C , Piridinas/farmacología , Proteínas Recombinantes de Fusión/química , Vinculina/química , Quinasas Asociadas a rho/antagonistas & inhibidores

RESUMEN

Upon overnutrition, adipocytes activate a homeostatic program to adjust anabolic pressure. An inflammatory response enables adipose tissue (AT) expansion with concomitant enlargement of its capillary network, and reduces energy storage by increasing insulin resistance. Galectin-12 (Gal-12), an endogenous lectin preferentially expressed in AT, plays a key role in adipocyte differentiation, lipolysis, and glucose homeostasis. Here, we reveal biochemical and biophysical determinants of Gal-12 structure, including its preferential recognition of 3-fucosylated structures, a unique feature among members of the galectin family. Furthermore, we identify a previously unanticipated role for this lectin in the regulation of angiogenesis within AT. Gal-12 showed preferential localization within the inner side of lipid droplets, and its expression was upregulated under hypoxic conditions. Through glycosylation-dependent binding to endothelial cells, Gal-12 promoted in vitro angiogenesis. Moreover, analysis of in vivo AT vasculature showed reduced vascular networks in Gal-12-deficient (Lgals12-/-) compared to wild-type mice, supporting a role for this lectin in AT angiogenesis. In conclusion, this study unveils biochemical, topological, and functional features of a hypoxia-regulated galectin in AT, which modulates endothelial cell function through recognition of 3-fucosylated glycans. Thus, glycosylation-dependent programs may control AT homeostasis by modulating endothelial cell biology with critical implications in metabolic disorders and inflammation.

Asunto(s)

Adipocitos/metabolismo , Células Endoteliales/metabolismo , Galectinas/metabolismo , Neovascularización Patológica/metabolismo , Tejido Adiposo/metabolismo , Animales , Fenómenos Fisiológicos Celulares/fisiología , Resistencia a la Insulina/fisiología , Gotas Lipídicas/metabolismo , Lipólisis/fisiología , Ratones Noqueados , Polisacáridos/metabolismo

RESUMEN

Mechanical stimuli play a key role in many cell functions such as proliferation, differentiation and migration. In the mammary gland, mechanical signals such as the distension of mammary epithelial cells due to udder filling are proposed to be directly involved during lactation and involution. However, the evolution of focal adhesions -specialized multiprotein complexes that mechanically connect cells with the extracellular matrix- during the mammary gland development, as well as the influence of the mechanical stimuli involved, remains unclear. Here we present the use of an equibiaxial stretching device for exerting a sustained normal strain to mammary epithelial cells while quantitatively assessing cell responses by fluorescence imaging techniques. Using this approach, we explored changes in focal adhesion dynamics in HC11 mammary cells in response to a mechanical sustained stress, which resembles the physiological stimuli. We studied the relationship between a global stress and focal adhesion assembly/disassembly, observing an enhanced persistency of focal adhesions under strain as well as an increase in their size. At a molecular level, we evaluated the mechanoresponses of vinculin and zyxin, two focal adhesion proteins postulated as mechanosensors, observing an increment in vinculin molecular tension and a slower zyxin dynamics while increasing the applied normal strain.

Asunto(s)

Células Epiteliales/metabolismo , Adhesiones Focales/metabolismo , Imagenología Tridimensional , Glándulas Mamarias Animales/citología , Mecanotransducción Celular , Estrés Mecánico , Animales , Supervivencia Celular , Femenino , Fluorescencia , Recuperación de Fluorescencia tras Fotoblanqueo , Cinética , Ratones Endogámicos BALB C , Fibras de Estrés/metabolismo , Vinculina/metabolismo , Zixina/metabolismo

RESUMEN

KEY MESSAGE: In vitro tomato pollen tubes show a cytoplasmic calcium gradient that oscillates with the same period as growth. Pollen tube growth requires coordination between the tip-focused cytoplasmic calcium concentration ([Ca2+]cyt) gradient and the actin cytoskeleton. This [Ca2+]cyt gradient is necessary for exocytosis of small vesicles, which contributes to the delivery of new membrane and cell wall at the pollen tube tip. The mechanisms that generate and maintain this [Ca2+]cyt gradient are not completely understood. Here, we studied calcium dynamics in tomato (Solanum lycopersicum) pollen tubes using transgenic tomato plants expressing the Yellow Cameleon 3.6 gene under the pollen-specific promoter LAT52. We use tomato as an experimental model because tomato is a Solanaceous plant that is easy to transform, and has an excellent genomic database and genetic stock center, and unlike Arabidopsis, tomato pollen is a good system to do biochemistry. We found that tomato pollen tubes showed an oscillating tip-focused [Ca2+]cyt gradient with the same period as growth. Then, we used a pharmacological approach to disturb the intracellular Ca2+ homeostasis, evaluating how the [Ca2+]cyt gradient, pollen germination and in vitro pollen tube growth were affected. We found that cyclopiazonic acid (CPA), a drug that inhibits plant PIIA-type Ca2+-ATPases, increased [Ca2+]cyt in the subapical zone, leading to the disappearance of the Ca2+ oscillations and inhibition of pollen tube growth. In contrast, 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of Ca2+ released from the endoplasmic reticulum to the cytoplasm in animals cells, completely reduced [Ca2+]cyt at the tip of the tube, blocked the gradient and arrested pollen tube growth. Although both drugs have antagonistic effects on [Ca2+]cyt, both inhibited pollen tube growth triggering the disappearance of the [Ca2+]cyt gradient. When CPA and 2-APB were combined, their individual inhibitory effects on pollen tube growth were partially compensated. Finally, we found that GsMTx-4, a peptide from spider venom that blocks stretch-activated Ca2+ channels, inhibited tomato pollen germination and had a heterogeneous effect on pollen tube growth, suggesting that these channels are also involved in the maintenance of the [Ca2+]cyt gradient. All these results indicate that tomato pollen tube is an excellent model to study calcium dynamics.

Asunto(s)

ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Calcio/metabolismo , Calmodulina/metabolismo , Proteínas Luminiscentes/metabolismo , Tubo Polínico/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Solanum lycopersicum/metabolismo , Compuestos de Boro/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Indoles/farmacología , Péptidos y Proteínas de Señalización Intercelular , Solanum lycopersicum/efectos de los fármacos , Solanum lycopersicum/crecimiento & desarrollo , Péptidos/farmacología , Proteínas de Plantas/antagonistas & inhibidores , Tubo Polínico/efectos de los fármacos , Tubo Polínico/crecimiento & desarrollo , Venenos de Araña/farmacología

RESUMEN

Many cell signaling pathways involve the diffusion of messengers that bind and unbind to and from intracellular components. Quantifying their net transport rate under different conditions then requires having separate estimates of their free diffusion coefficient and binding or unbinding rates. In this paper, we show how performing sets of fluorescence correlation spectroscopy (FCS) experiments under different conditions, it is possible to quantify free diffusion coefficients and on and off rates of reaction-diffusion systems. We develop the theory and present a practical implementation for the case of the universal second messenger, calcium (Ca^{2+}) and single-wavelength dyes that increase their fluorescence upon Ca^{2+} binding. We validate the approach with experiments performed in aqueous solutions containing Ca^{2+} and Fluo4 dextran (both in its high and low affinity versions). Performing FCS experiments with tetramethylrhodamine-dextran in Xenopus laevis oocytes, we infer the corresponding free diffusion coefficients in the cytosol of these cells. Our approach can be extended to other physiologically relevant reaction-diffusion systems to quantify biophysical parameters that determine the dynamics of various variables of interest.

RESUMEN

Ca2+ signals are ubiquitous. One of the key factors for their versatility is the variety of spatio-temporal distributions that the cytosolic Ca2+ can display. In most cell types Ca2+ signals not only depend on Ca2+ entry from the extracellular medium but also on Ca2+ release from internal stores, a process which is in turn regulated by cytosolic Ca2+ itself. The rate at which Ca2+ is transported, the fraction that is trapped by intracellular buffers, and with what kinetics are thus key features that affect the time and spatial range of action of Ca2+ signals. The quantification of Ca2+ diffusion in intact cells is quite challenging because the transport rates that can be inferred using optical techniques are intricately related to the interaction of Ca2+ with the dye that is used for its observation and with the cellular buffers. In this paper, we introduce an approach that uses Fluorescence Correlation Spectroscopy (FCS) experiments performed at different conditions that in principle allows the quantification of Ca2+ diffusion and of its reaction rates with unobservable (non-fluorescent) Ca2+ buffers. To this end, we develop the necessary theory to interpret the experimental results and then apply it to FCS experiments performed in a set of solutions containing Ca2+, a single wavelength Ca2+ dye, and a non-fluorescent Ca2+ buffer. We show that a judicious choice of the experimental conditions and an adequate interpretation of the fitting parameters can be combined to extract information on the free diffusion coefficient of Ca2+ and of some of the properties of the unobservable buffer. We think that this approach can be applied to other situations, particularly to experiments performed in intact cells.

RESUMEN

Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

Asunto(s)

Empalme Alternativo , N-Metiltransferasa de Histona-Lisina/genética , Animales , Azepinas/farmacología , Encéfalo/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/metabolismo , Exones , Transferencia Resonante de Energía de Fluorescencia , Genes Reporteros , Células HeLa , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Metilación/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Neuronas/citología , Neuronas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Quinazolinas/farmacología , Interferencia de ARN , Precursores del ARN/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Tretinoina/farmacología

RESUMEN

Many plasma membrane channels form oligomeric assemblies, and heterooligomerization has been described as a distinctive feature of some protein families. In the particular case of plant plasma membrane aquaporins (PIPs), PIP1 and PIP2 monomers interact to form heterotetramers. However, the biological properties of the different heterotetrameric configurations formed by PIP1 and PIP2 subunits have not been addressed yet. Upon coexpression of tandem PIP2-PIP1 dimers in Xenopus oocytes, we can address, for the first time to our knowledge, the functional properties of single heterotetrameric species having 2:2 stoichiometry. We have also coexpressed PIP2-PIP1 dimers with PIP1 and PIP2 monomers to experimentally investigate the localization and biological activity of each tetrameric assembly. Our results show that PIP2-PIP1 heterotetramers can assemble with 3:1, 1:3, or 2:2 stoichiometry, depending on PIP1 and PIP2 relative expression in the cell. All PIP2-PIP1 heterotetrameric species localize at the plasma membrane and present the same water transport capacity. Furthermore, the contribution of any heterotetrameric assembly to the total water transport through the plasma membrane doubles the contribution of PIP2 homotetramers. Our results also indicate that plasma membrane water transport can be modulated by the coexistence of different tetrameric species and by intracellular pH. Moreover, all the tetrameric species present similar cooperativity behavior for proton sensing. These findings throw light on the functional properties of PIP tetramers, showing that they have flexible stoichiometry dependent on the quantity of PIP1 and PIP2 molecules available. This represents, to our knowledge, a novel regulatory mechanism to adjust water transport across the plasma membrane.

Asunto(s)

Acuaporinas/química , Acuaporinas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Multimerización de Proteína , Agua/metabolismo , Animales , Transporte Biológico , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Concentración de Iones de Hidrógeno , Ósmosis , Protones , Xenopus laevis/metabolismo

RESUMEN

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55(Gag) is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4(+) T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55(Gag) membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55(Gag) with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.

Asunto(s)

Membrana Celular/metabolismo , VIH-1/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ensamble de Virus/fisiología , Replicación Viral/fisiología , Proteínas de Unión al GTP rab/metabolismo , Transporte Biológico Activo/genética , Membrana Celular/genética , Membrana Celular/virología , Endosomas/genética , Endosomas/metabolismo , Endosomas/virología , Humanos , Células Jurkat , Macrófagos/metabolismo , Macrófagos/virología , Proteínas de la Membrana/metabolismo , Antígenos de Histocompatibilidad Menor , Fosfatidilinositol 4,5-Difosfato/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTP

RESUMEN

The gradient of Bicoid (Bcd) is key for the establishment of the anterior-posterior axis in Drosophila embryos. The gradient properties are compatible with the SDD model in which Bcd is synthesized at the anterior pole and then diffuses into the embryo and is degraded with a characteristic time. Within this model, the Bcd diffusion coefficient is critical to set the timescale of gradient formation. This coefficient has been measured using two optical techniques, Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS), obtaining estimates in which the FCS value is an order of magnitude larger than the FRAP one. This discrepancy raises the following questions: which estimate is "correct''; what is the reason for the disparity; and can the SDD model explain Bcd gradient formation within the experimentally observed times? In this paper, we use a simple biophysical model in which Bcd diffuses and interacts with binding sites to show that both the FRAP and the FCS estimates may be correct and compatible with the observed timescale of gradient formation. The discrepancy arises from the fact that FCS and FRAP report on different effective (concentration dependent) diffusion coefficients, one of which describes the spreading rate of the individual Bcd molecules (the messengers) and the other one that of their concentration (the message). The latter is the one that is more relevant for the gradient establishment and is compatible with its formation within the experimentally observed times.

Asunto(s)

Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Proteínas de Homeodominio/metabolismo , Transactivadores/metabolismo , Transporte Activo de Núcleo Celular , Animales , Animales Modificados Genéticamente , Fenómenos Biofísicos , Tipificación del Cuerpo , Biología Computacional , Simulación por Computador , Difusión , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas de Homeodominio/genética , Modelos Biológicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Fluorescencia , Transactivadores/genética

RESUMEN

The plant aquaporin plasma membrane intrinsic proteins (PIP) subfamily represents one of the main gateways for water exchange at the plasma membrane (PM). A fraction of this subfamily, known as PIP1, does not reach the PM unless they are coexpressed with a PIP2 aquaporin. Although ubiquitous and abundantly expressed, the role and properties of PIP1 aquaporins have therefore remained masked. Here, we unravel how FaPIP1;1, a fruit-specific PIP1 aquaporin from Fragaria x ananassa, contributes to the modulation of membrane water permeability (Pf) and pH aquaporin regulation. Our approach was to combine an experimental and mathematical model design to test its activity without affecting its trafficking dynamics. We demonstrate that FaPIP1;1 has a high water channel activity when coexpressed as well as how PIP1-PIP2 affects gating sensitivity in terms of cytosolic acidification. PIP1-PIP2 random heterotetramerization not only allows FaPIP1;1 to arrive at the PM but also produces an enhancement of FaPIP2;1 activity. In this context, we propose that FaPIP1;1 is a key participant in the regulation of water movement across the membranes of cells expressing both aquaporins.

Asunto(s)

Acuaporinas/química , Acuaporinas/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/química , Proteínas de Plantas/genética , Animales , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Fragaria/metabolismo , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos/química , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Modelos Teóricos , Mutagénesis Sitio-Dirigida , Oocitos/metabolismo , Permeabilidad , Multimerización de Proteína , ARN Complementario/metabolismo , Agua/química , Xenopus laevis

RESUMEN

Research done in the last years strongly support the hypothesis that PIP aquaporin can form heterooligomeric assemblies, specially combining PIP2 monomers with PIP1 monomers. Nevertheless, the structural elements involved in the ruling of homo versus heterooligomeric organization are not completely elucidated. In this work we unveil some features of monomer-monomer interaction in Beta vulgaris PIP aquaporins. Our results show that while BvPIP2;2 is able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. The lack of functional interaction between BvPIP2;1 and BvPIP1;1 was further corroborated by dose-response curves of water permeability due to aquaporin activity exposed to different acidic conditions. We also found that BvPIP2;1 is unable to translocate BvPIP1;1-ECFP from an intracellular position to the plasma membrane when co-expressed, as BvPIP2;2 does. Moreover we postulate that the first extracellular loop (loop A) of BvPIP2;1, could be relevant for the functional interaction with BvPIP1;1. Thus, we investigate BvPIP2;1 loop A at an atomic level by Molecular Dynamics Simulation (MDS) and by direct mutagenesis. We found that, within the tetramer, each loop A presents a dissimilar behavior. Besides, BvPIP2;1 loop A mutants restore functional interaction with BvPIP1;1. This work is a contribution to unravel how PIP2 and PIP1 interact to form functional heterooligomeric assemblies. We postulate that BvPIP2;1 loop A is relevant for the lack of functional interaction with BvPIP1;1 and that the monomer composition of PIP assemblies determines their functional properties.

Asunto(s)

Acuaporinas/química , Acuaporinas/metabolismo , Beta vulgaris/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Animales , Permeabilidad de la Membrana Celular , Secuencia Conservada , Concentración de Iones de Hidrógeno , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ósmosis , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Xenopus laevis

RESUMEN

Many natural phenomena display "self-organized criticality" (SOC), (Bak et al., 1987). This refers to spatially extended systems for which patterns of activity characterized by different lengthscales can occur with a probability density that follows a power law with pattern size. Differently from power laws at phase transitions, systems displaying SOC do not need the tuning of an external parameter. Here we analyze intracellular calcium (Ca(2+)) signals, a key component of the signaling toolkit of almost any cell type. Ca(2+) signals can either be spatially restricted (local) or propagate throughout the cell (global). Different models have suggested that the transition from local to global signals is similar to that of directed percolation. Directed percolation has been associated, in turn, to the appearance of SOC. In this paper we discuss these issues within the framework of simple models of Ca(2+) signal propagation. We also analyze the size distribution of local signals ("puffs") observed in immature Xenopus Laevis oocytes. The puff amplitude distribution obtained from observed local signals is not Gaussian with a noticeable fraction of large size events. The experimental distribution of puff areas in the spatio-temporal record of the image has a long tail that is approximately log-normal. The distribution can also be fitted with a power law relationship albeit with a smaller goodness of fit. The power law behavior is encountered within a simple model that includes some coupling among individual signals for a wide range of parameter values. An analysis of the model shows that a global elevation of the Ca(2+) concentration plays a major role in determining whether the puff size distribution is long-tailed or not. This suggests that Ca(2+)-clearing from the cytosol is key to determine whether IP(3)-mediated Ca(2+) signals can display a SOC-like behavior or not.

RESUMEN

The flash photolysis of "caged" compounds is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. These caged compounds are inactive and become active when illuminated with ultraviolet light. This paper describes an inexpensive adaptation of an Olympus confocal microscope that uses as source of ultraviolet light the mercury lamp that comes with the microscope for conventional fluorescence microscopy. The ultraviolet illumination from the lamp (350 - 400 nm) enters through an optical fiber that is coupled to a nonconventional port of the microscope. The modification allows to perform the photolysis of caged compounds over wide areas (∼ 200 µm) and obtain confocal fluorescence images simultaneously. By controlling the ultraviolet illumination exposure time and intensity it is possible to regulate the amount of photolyzed compounds. In the paper we characterize the properties of the system and show its capabilities with experiments done in aqueous solution and in Xenopus Laevis oocytes. The latter demonstrate its applicability for the study of Inositol 1,4,5-trisphosphate-mediated intracellular calcium signals.

Asunto(s)

Señalización del Calcio/fisiología , Inositol 1,4,5-Trifosfato/química , Inositol 1,4,5-Trifosfato/metabolismo , Microscopía Confocal/instrumentación , Fotólisis , Animales , Calcio/química , Calcio/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Diseño de Equipo , Modelos Lineales , Microscopía Confocal/métodos , Oocitos/metabolismo , Rayos Ultravioleta , Xenopus laevis

RESUMEN

In many cell-signaling pathways information is transmitted via the diffusion of messenger molecules. In most cases, messengers react with other substances and diffuse at the same time. Effective diffusion coefficients may be introduced to characterize the net transport rate that results from the combined effect of these two processes. It was shown in [B. Pando, Proc. Natl. Acad. Sci. U.S.A. 103, 5338 (2006)] that even in the simplest scenario in which one bimolecular reaction is involved, two different effective coefficients are relevant. One gives the rate at which small perturbations spread out with time while the other relates the mean square displacement of a single particle to the time elapsed. They coincide in the absence of reactions but may be very different in other cases. Optical techniques provide a relatively noninvasive means by which transport rates can be estimated. In the above mentioned paper it was discussed why, under certain conditions, fluorescence recovery after photobleaching (FRAP), a technique commonly used to estimate diffusion rates in cells, provides information on one of the two effective coefficients. In the present paper we show that, under the same conditions, another commonly used optical technique, fluorescence correlation spectroscopy (FCS), gives information on the other one. This opens up the possibility of combining experiments to obtain information that goes beyond effective transport rates. In the present paper we discuss different ways to do so.

Asunto(s)

Difusión , Fenómenos Ópticos , Recuperación de Fluorescencia tras Fotoblanqueo , Espectrometría de Fluorescencia