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The bright-field (BF) optical microscope is a traditional bioimaging tool that has been recently tested for depth discrimination during evaluation of specimen morphology; however, existing approaches require dedicated instrumentation or extensive computer modeling. We report a direct method for three-dimensional (3D) imaging in BF microscopy, applicable to label-free samples, where we use Köhler illumination in the coherent regime and conventional digital image processing filters to achieve optical sectioning. By visualizing fungal, animal tissue, and plant samples and comparing with light-sheet fluorescence microscopy imaging, we demonstrate the accuracy and applicability of the method, showing how the standard microscope is an effective 3D imaging device.

Scattering of light from randomly textured materials is ubiquitous and of great interest in biology and for diverse applications—including filtering, light trapping in solar cells, and speckle photography. One attractive means to build these materials is by harnessing the complexity present in structures of biological origin. Here, we report on the development of a random phase diffuser based on intertwined filamentous cells (hyphae) of the fungus Trichoderma atroviride. A fungal colony (mycelium) is grown on the surface of a gel medium, and then removed, fixated, and dehydrated, resulting in a free-standing, two-dimensional random mesh (1 cm × 1 cm × 5 μm) composed of rigid hyphae separated by air gaps. A laser beam incident on the bioplate results in speckle patterns of nearly equal intensity in transmission and reflection. By modeling the bioplate as composed of optical phase elements and computing Fraunhofer diffraction, we recover the overall shape of the observed diffuse light spot. As the hyphal density composing the sample is increased, all optical power is in the speckle pattern, and approximate Lambertian transmissivity is reached. Altogether, our observations suggest that a planar fungal colony can scatter light efficiently by imparting a random phase. These results underscore the potential of a biological structure to develop optical elements and to use light scattering to evaluate morphology in complex structures—such as filamentous mycelia.

Numerous essential physiological processes depend on the TMEM16A-mediated Ca2+-activated chloride fluxes. Extensive structure–function studies have helped to elucidate the Ca2+ gating mechanism of TMEM16A, revealing a Ca2+-sensing element close to the anion pore that alters conduction. However, substrate selection and the substrate–gating relationship in TMEM16A remain less explored. Here, we study the gating–permeant anion relationship on mouse TMEM16A expressed in HEK 293 cells using electrophysiological recordings coupled with site-directed mutagenesis. We show that the apparent Ca2+ sensitivity of TMEM16A increased with highly permeant anions and SCN− mole fractions, likely by stabilizing bound Ca2+. Conversely, mutations at crucial gating elements, including the Ca2+-binding site 1, the transmembrane helix 6 (TM6), and the hydrophobic gate, impaired the anion permeability and selectivity of TMEM16A. Finally, we found that, unlike anion-selective wild-type channels, the voltage dependence of unselective TMEM16A mutant channels was less sensitive to SCN−. Therefore, our work identifies structural determinants of selectivity at the Ca2+ site, TM6, and hydrophobic gate and reveals a reciprocal regulation of gating and selectivity. We suggest that this regulation is essential to set ionic selectivity and the Ca2+ and voltage sensitivities in TMEM16A.

Diatoms are unicellular microalgae with a rigid cell wall, able to glide on surfaces by releasing nanopolymeric fibers through central slits known as raphes. Here we consider the model Nitszchia communis to perform quantitative studies on two complementary aspects involved in diatom gliding. Using video microscopy and automated image analysis, we measure the motion of test beads as they are pulled by extracellular polymeric substances (EPS) fibers at the diatom raphe (particle streaming). A multimodal distribution of particle speed is found, evidencing the appearance of short-time events of high speed and acceleration (known as jerky motion) and suggesting that different mechanisms contribute to set diatom velocity during gliding. Furthermore, we use optical tweezers to obtain force-extension records for extracellular diatom nanofibers; records are well described by the worm-like chain model of polymer elasticity. In contrast to previous studies based on application of denaturing force (in the nN regime), application of low force (up to 6 pN) and using enable us to obtain the persistence length of intact fibers. From these measurements, mechanical parameters of EPS fibers such as radius and elastic constant are estimated. Furthermore, by modeling particle streaming as a spring in parallel with a dashpot, we show that the time involved in the release of mechanical energy after fiber detachment from beads (elastic snapping) agrees with our observations of jerky motion. We conclude that the smooth and jerky motions displayed by gliding diatoms correspond to molecular motors and elastic snapping, respectively, thus providing quantitative elements that incorporate to current models of the mechanics behind diatom locomotion.

The evaluation of morphology is fundamental to comprehend how fungi grow, develop, and interact with the environment. Although fungal growth has been extensively studied associated to two-dimensional geometries, lack of appropriate experimental tools has limited exploration of the complex three-dimensional (3D) structures exhibited by mycelia in more general contexts. In this paper, we report the construction of a light-sheet fluorescence microscope (LSFM) capable of performing time-lapse visualization of 3D biological structures (4D microscopy), and the use of this instrument to follow the dynamics of fungal growth. LSFM uses scanning of selective plane illumination and digital reconstruction to provide 3D images of the specimen. We describe the optical, electronic, and computational means to implement two-color LSFM, and provide detailed procedures for aligning and testing the setup. We successfully demonstrate use of both autofluorescence and specific tagging to image Trichoderma atroviride and Neurospora crassa strains growing in liquid media, over extended times (~12 h) and volumes (~400 × 1500 × 800 μm3) at single-hypha resolution. The excellent image contrast provided by LSFM enables us to visualize the dynamics of mycelial architecture, interactions among hyphae, and measure rates of 3D apical extension. Altogether, our work shows a powerful imaging tool to perform 3D morphological analysis of fungi, from hyphae to mycelium.

The aggregation of proteins is of importance in fields ranging from protein homeostasis to disease. The light-sensing protein Vivid (VVD) regulates responses to blue-light illumination in the filamentous fungus Neurospora crassa. Consisting of a single light‑oxygen-voltage domain, VVD is characterized by cycling between dark and lit states that correspond to formation and disruption of a photoadduct between the flavin cofactor and the apoprotein. Recently, in vitro assays have shown that VVD undergoes self-oxidative damage and aggregation resulting from excessive blue-light illumination. To explore the aggregation process of VVD, here we study the kinetics of aggregation and how it is influenced by environmental factors such as initial protein concentration, temperature, and light. We found that the aggregation kinetics of VVD is consistent with a second-order reaction model involving kinetic control, where thermal decay from lit-VVD to dark-VVD is necessary for aggregation to proceed. The height of the energy barrier separating the lit and dark VVD states is measured as (80 ± 2) kJ mol−1. Application of the kinetic model to the observed dependence of aggregation vs. temperature allowed us to further estimate the energy involved in the nucleation of dark-VVD, (257 ± 24) kJ mol−1. Finally, we show that VVD aggregation levels increase as the time of blue light exposure is augmented, suggesting possible mechanisms for protein damage. These results demonstrate how aggregation of a photoreceptor depends not only on environmental factors but on the intrinsic response of the protein to illumination.

The growth of filamentous fungi is a complex process that involves hyphal elongation and branching. Microscopic observations provide a wealth of information on fungal growth, although often requiring laborious manual intervention to record and analyze images. Here, we introduce a novel tool for automated tracking of growth in fungal hyphae that affords quantitative analysis of growth rate and morphology. We supplied a student-grade bright field microscope with stepper motors to enable computer-control of the microscope stage. In addition, we developed an image-processing routine that detects in real-time the tip of a hypha and tracks it as the hypha elongates. To achieve continuous observation of hyphal growth, our system automatically maintains the observed sample within field-of-view and performs periodic autofocus correction in the microscope. We demonstrate automated, continuous tracking of hyphal growth in Trichoderma atroviride with sampling rates of seconds and observation times of up to 14 h. Tracking records allowed us to determine that T. atroviride hyphae grow with characteristic elongation rates of ∼70 nm/s. Surprisingly, we found that prior to the occurrence of an apical branching event the parental hypha stopped growing during a few minutes. These arrest events presented occasionally for subapical branching as well. Finally, from tracking data we found that the persistence length (a measure of filament extension before presenting a change in direction) associated to T. atroviride hyphae is 362 µm. Altogether, these results show how integration of image analysis and computer control enable quantitative microscopic observations of fungal hyphae dynamics.

El azar juega un papel determinante para los seres vivos. En el caso de los microorganismos sujetos a lo inesperado, es posible emplear modelos matemáticos para describir y predecir el comportamiento promedio; es decir, a nivel de una colonia o al observar un individuo durante mucho tiempo. En este texto nos enfocamos en describir las caminatas que organismos tales como bacterias y diatomeas llevan a cabo en la microescala, un mundo en donde el azar se encuentra a la vuelta de cualquier esquina.

The microscopic development of a mycelium is of importance in all aspects of fungal biology and biotechnology. However, the mechanics of three-dimensional (3D) hyphal growth has been not explored. Using light-sheet fluorescence microscopy, we follow the 3D growth of Trichoderma atroviride in liquid medium and observe two direct collision events among hyphae. In both cases, a hypha undergoing tip extension collides with the side of another hypha, causing mechanical deformation that remains after the collision. From these data we estimate that the force developed by hyphae during tip elongation is at least 260 pN.

The in vitro transport of cargo by motor proteins constitutes a model system to understand mechanisms of vesicle trafficking inside cells. Here we apply the classic bead assay with a short, stiff kinesin protein to test the effect of multiple motors on essential transport parameters: distance, force and microtubule binding rate. Measurements of unloaded run length show that the transition from single- to multiple-motor behavior can be characterized by the appearance of extended runs, in accordance with a recently proposed model that quantifies the probability of multiple-motor engagement. In this transition, application of mechanical load using optical tweezers allows us to register maximum force values above single kinesin levels (8 pN). Yet, averages of run length and maximum force undergo little change as the probability of multiple-motor participation increases. In contrast, the measured rate of bead binding to microtubules scales linearly with the average number of motors per bead. These observations suggest that multiple motors bound randomly to the same cargo mainly increase the probability of attachment of these cargoes to the cytoskeletal filament network.

Bright field (BF) optical microscopy is regarded as a poor method to observe unstained biological samples due to intrinsic low image contrast. We introduce quantitative image restoration in bright field (QRBF), a digital image processing method that restores out-of-focus BF images of unstained cells. Our procedure is based on deconvolution, using a point spread function modeled from theory. By comparing with reference images of bacteria observed in fluorescence, we show that QRBF faithfully recovers shape and enables quantify size of individual cells, even from a single input image. We applied QRBF in a high-throughput image cytometer to assess shape changes in Escherichia coli during hyperosmotic shock, finding size heterogeneity. We demonstrate that QRBF is also applicable to eukaryotic cells (yeast). Altogether, digital restoration emerges as a straightforward alternative to methods designed to generate contrast in BF imaging for quantitative analysis.

En este trabajo se muestran los resultados de los experimentos realizados en el verano del 2014 en el IPICYT, para corroborar la existencia del efecto Talbot el cual consiste en la reaparición de una imagen nítida de un objeto periódico iluminado por una fuente de luz cada cierta distancia después del objeto. Esta distancia se le conoce como distancia de Talbot y da la escala espacial de este fenómeno. Se utilizaron rejillas unidimensionales periódicas, aperiódicas y bidimensionales con estructura hexagonal, todas fueron creadas previamente con el programa grafico Adobe Illustrator con las frecuencias espaciales que se calcularon que darían una distancia de Talbot que permite la observación del efecto en el laboratorio. Las rejillas se iluminaron con un láser de He-Ne de longitud de onda de 633 nm (rojo) y posteriormente se localizó a que distancia se volvía a formar la auto-imagen de la rejilla.

Summary Long DNA molecules remain difficult to image by atomic force microscopy (AFM) because of their tendency to entanglement and spontaneous formation of networks. We present a comparison of two different DNA deposition methods operating at room temperature and humidity conditions, aimed at reproducible imaging of isolated and relaxed λ DNA conformations by AFM in air. We first demonstrate that a standard deposition procedure, consisting in adsorption of DNA in the presence of divalent cations followed by washing and air-drying steps, yields a coexistence of different types of λ DNA networks with a only a few isolated DNA chains. In contrast, deposition using a spin-coating-based technique results in reproducible coverage of a significant fraction of the substrate area by isolated and relaxed λ DNA molecules, with the added benefit of a reduction in the effect of a residual layer that normally embeds DNA strands and leads to an apparent DNA height closer to the expected value. Furthermore, we show that deposition by spin-coating is also well-suited to visualize DNA–protein complexes. These results indicate that spin-coating is a simple, powerful alternative for reproducible sample preparation for AFM imaging. SCANNING 36:561–569, 2014. © 2014 Wiley Periodicals, Inc.

Fungal blue-light photoreceptors have been proposed as integrators of light and oxidative stress. However, additional elements participating in the integrative pathway remain to be identified. In Trichoderma atroviride , the blue-light regulator (BLR) proteins BLR-1 and -2 are known to regulate gene transcription, mycelial growth, and asexual development upon illumination, and recent global transcriptional analysis revealed that the histone deacetylase-encoding gene hda-2 is induced by light. Here, by assessing responses to stimuli in wild-type and Δ hda-2 backgrounds, we evaluate the role of HDA-2 in the regulation of genes responsive to light and oxidative stress. Δ hda-2 strains present reduced growth, misregulation of the con-1 gene, and absence of conidia in response to light and mechanical injury. We found that the expression of hda-2 is BLR-1 dependent and HDA-2 in turn is essential for the transcription of early and late light-responsive genes that include blr-1 , indicating a regulatory feedback loop. When subjected to reactive oxygen species (ROS), Δ hda-2 mutants display high sensitivity whereas Δ blr strains exhibit the opposite phenotype. Consistently, in the presence of ROS, ROS-related genes show high transcription levels in wild-type and Δ blr strains but misregulation in Δ hda-2 mutants. Finally, chromatin immunoprecipitations of histone H3 acetylated at Lys9/Lys14 on cat-3 and gst-1 promoters display low accumulation of H3K9K14ac in Δ blr and Δ hda-2 strains, suggesting indirect regulation of ROS-related genes by HDA-2. Our results point to a mutual dependence between HDA-2 and BLR proteins and reveal the role of these proteins in an intricate gene regulation landscape in response to blue light and ROS. IMPORTANCE Trichoderma atroviride is a free-living fungus commonly found in soil or colonizing plant roots and is widely used as an agent in biocontrol as it parasitizes other fungi, stimulates plant growth, and induces the plant defense system. To survive in various environments, fungi constantly sense and respond to potentially threatening external factors, such as light. In particular, UV light can damage biomolecules by producing free-radical reactions, in most cases involving reactive oxygen species (ROS). In T. atroviride , conidiation is essential for its survival, which is induced by light and mechanical injury. Notably, conidia are typically used as the inoculum in the field during biocontrol. Therefore, understanding the linkages between responses to light and exposure to ROS in T. atroviride is of major basic and practical relevance. Here, the histone deacetylase-encoding gene hda-2 is induced by light and ROS, and its product regulates growth, conidiation, blue light perception, and oxidative stress responses.

Kinesin is a homodimeric motor with two catalytic heads joined to a stalk via short neck linkers (NLs). We measured the torsional properties of single recombinant molecules by tracking the thermal angular motions of fluorescently labeled beads bound to the C terminus of the stalk. When kinesin heads were immobilized on microtubules (MTs) under varied nucleotide conditions, we observed bounded or unbounded angular diffusion, depending on whether one or both heads were attached to the MT. Free rotation implies that NLs act as swivels. From data on constrained diffusion, we conclude that the coiled-coil stalk domains are ≈30-fold stiffer than its flexible “hinge” regions. Surprisingly, while tracking processive kinesin motion at low ATP concentrations, we observed occasional abrupt reversals in the directional orientations of the stalk. Our results impose constraints on kinesin walking models and suggest a role for rotational freedom in cargo transport.

How cells migrate has been investigated primarily for the case of trajectories composed by joined straight segments. In contrast, little is known when cellular motion follows intrinsically curved paths. Here, we use time-lapse optical microscopy and automated trajectory tracking to investigate how individual cells of the diatom Nitzschia communis glide across surfaces under isotropic environmental conditions. We find a distinct kind of random motion, where trajectories are formed by circular arcs traveled at constant speed, alternated with random stoppages, direction reversals and changes in the orientation of the arcs. Analysis of experimental and computer-simulated trajectories show that the circular random motion of diatom gliding is not optimized for long-distance travel but rather for recurrent coverage of limited surface area. These results suggest that one main biological role for this type of diatom motility is to efficiently build the foundation of algal biofilms.

In transmitted optical microscopy, absorption structure and phase structure of the specimen determine the three-dimensional intensity distribution of the image. The elementary impulse responses of the bright field microscope therefore consist of separate absorptive and phase components, precluding general application of linear, conventional deconvolution processing methods to improve image contrast and resolution. However, conventional deconvolution can be applied in the case of pure phase (or pure absorptive) objects if the corresponding phase (or absorptive) impulse responses of the microscope are known. In this work, we present direct measurements of the phase point- and line-spread functions of a high-aperture microscope operating in transmitted bright field. Polystyrene nanoparticles and microtubules (biological polymer filaments) serve as the pure phase point and line objects, respectively, that are imaged with high contrast and low noise using standard microscopy plus digital image processing. Our experimental results agree with a proposed model for the response functions, and confirm previous theoretical predictions. Finally, we use the measured phase point-spread function to apply conventional deconvolution on the bright field images of living, unstained bacteria, resulting in improved definition of cell boundaries and sub-cellular features. These developments demonstrate practical application of standard restoration methods to improve imaging of phase objects such as cells in transmitted light microscopy.

We have constructed and tested a simple and versatile optical tweezers apparatus made from individual optical components, capable of visualizing individual microtubules (∼25 nm in diameter) and studying biomolecules at the single-molecule level, using a minimal set of parts. Our design is based on a conventional, inverted microscope operating under plain bright field illumination. The illuminating system of the microscope is composed of a current-stabilized LED source, a collector lens system, a field diaphragm, a condenser iris diaphragm, a relay lens (LR) that images the condenser iris diaphragm onto the condenser back focal plane (BFP), and the condenser. All of these elements are adjusted for Koehler illumination. On the other hand, a single IR laser beam, coupled into the microscope, enables both standard optical trapping and BFP detection for the measurement of molecular displacements and forces. A convenient feature of our optical setup is that the relay lens LR effectively decouples the illuminating system from the optical tweezers detection stage. In practice, we can close the condenser iris diaphragm almost entirely (∼90%) to maximize contrast for sample visualization, while simultaneously using the full numerical aperture of the condenser for BFP detection. We are able to further optimize imaging of the microscope by using computer-based digital image processing, effectively removing the background, reducing noise, and enhancing contrast. Altogether, our system yields excellent sample visualization in real-time, without the use of any specialized optics and without affecting the optical tweezers optical path. We have tested the optical trapping instrument by measuring the persistence length of double-stranded DNA at high ionic strength (150 mM of Na+), and by following the stepping of the motor protein kinesin on clearly imaged microtubules. The approach presented here provides a straightforward alternative for the study of biomaterials and individual biomolecules.

We present details of the design, construction, and testing of a single-beam optical tweezers apparatus capable of measuring and exerting torque, as well as force, on microfabricated, optically anisotropic particles (an "optical torque wrench"). The control of angular orientation is achieved by rotating the linear polarization of a trapping laser with an electro-optic modulator (EOM), which affords improved performance over previous designs. The torque imparted to the trapped particle is assessed by measuring the difference between left- and right-circular components of the transmitted light, and constant torque is maintained by feeding this difference signal back into a custom-designed electronic servo loop. The limited angular range of the EOM (+/-180 degrees ) is extended by rapidly reversing the polarization once a threshold angle is reached, enabling the torque clamp to function over unlimited, continuous rotations at high bandwidth. In addition, we developed particles suitable for rotation in this apparatus using microfabrication techniques. Altogether, the system allows for the simultaneous application of forces (approximately 0.1-100 pN) and torques (approximately 1-10,000 pN nm) in the study of biomolecules. As a proof of principle, we demonstrate how our instrument can be used to study the supercoiling of single DNA molecules.

Microtubules are filament-shaped, polymeric proteins (~25 nm in diameter) involved in cellular structure and organization. We demonstrate the imaging of individual microtubules using a conventional bright-field microscope, without any additional phase or polarization optics. Light scattered by microtubules is discriminated through extensive use of digital image-processing, thus removing background, reducing noise and enhancing contrast. The setup builds on a commercial microscope, with the inclusion of a minimal and inexpensive set of components, suitable for implementation in the student laboratory. We show how this technique can be applied to a demonstrative biophysical assay, by tracking the motions of microtubules driven by the motor protein kinesin.

We designed, constructed, and tested a single-beam optical trapping instrument employing twin electro-optic deflectors (EODs) to steer the trap in the specimen plane. Compared with traditional instruments based on acousto-optic deflectors (AODs), EOD-based traps offer a significant improvement in light throughput and a reduction in deflection-angle (pointing) errors. These attributes impart improved force and position resolution, making EOD-based traps a promising alternative for high-precision nanomechanical measurements of biomaterials.

We present an efficient alternative to remove lateral chromatic aberration (LCA) in bright field light microscopy images. Our procedure is based on error calibration using time-sequential acquisition at different wavelengths, and error correction through digital image warping. Measurement of the displacements of fiducial marks in the red and green images relative to blue provide calibration factors that are subsequently used in test images to realign color channels digitally. We demonstrate quantitative improvement in the position and boundaries of objects in target slides and in the color content and morphology of specimens in stained biological samples. Our results show a reduction of LCA content below the 0.1% level.

We constructed an optical tweezers instrument capable of exerting torque and measuring the angular motions of small trapped particles, based on the rotation of linear polarization of the trapping laser beam. To change polarization, we employed an electro-optic modulator (EOM), which allows for a much simpler setup than a previous design (La Porta, A. and Wang, M.D. 2004. Optical torque wrench: angular trapping, rotation, and torque detection of quartz microparticles. Phys. Rev. Lett. 19:190801). Torque is monitored by measuring the difference between circularly left-handed and right-handed components of the transmitted beam: constant torque is implemented by feeding this angular signal back into a custom-designed electronic servo loop. The limited dynamic range of the EOM (±180°) is extended by monitoring the drive signal with a microcontroller, which triggers a switch to flip the output polarization by ±180° once a pre-set threshold is reached (within 10 μs). These features enable us to maintain constant torque over unlimited rotations at high bandwidth (∼100 kHz). In addition, we developed optically birefringent, non-spherical particles suitable for this instrument using nanofabrication techniques. The polarization-sensitive method employed by the apparatus precludes the use of Wollaston prisms to perform differential interference contrast (DIC) imaging. However, by exploiting conventional video-enhancement techniques (including background subtraction, contrast enhancement, and frame averaging), we report that individual microtubules (∼25 nm in diameter) can be visualized without DIC optics at ∼5 frames per second. Altogether, our instrument allows for the simultaneous application of force and torque to the study of macromolecules of interest. We are presently extending our previous studies (Gutierrez-Medina, B., et al. 2339-Pos. Torsional properties of kinesin. Biophys. J. 2008. 94:2339-Pos) on the torsional properties of the molecular motor kinesin to investigate the effect of torque on its stepwise motion.

Kinesin is a dimeric motor with twin catalytic heads joined to a common stalk. Kinesin molecules move processively along microtubules in a hand-over-hand walk, with the two heads advancing alternately. Recombinant kinesin constructs with short stalks have been found to “limp”, i.e., exhibit alternation in the dwell times of successive steps. Limping behavior implies that the molecular rearrangements underlying even- and odd-numbered steps must differ, but the mechanism by which such rearrangements lead to limping remains unsolved. Here, we used an optical force clamp to measure individual, recombinant dimers and test candidate explanations for limping. Introducing a covalent cross-link into the stalk region near the heads had no effect on limping, ruling out possible stalk misregistration during coiled-coil formation as a cause. Limping was equally unaffected by mutations that produced 50-fold changes in stalk stiffness, ruling out models where limping arises from an asymmetry in torsional strain. However, limping was enhanced by perturbations that increased the vertical component of load on the motor, including increases in bead size or net load, and decreases in the stalk length. These results suggest that kinesin heads take different vertical trajectories during alternate steps, and that the rates for these motions are differentially sensitive to load.

"TMEM16A es una proteína canal de cloruro (Cl-) activado por incrementos en el calcio (Ca2+) intracelular que se encuentra en las membranas biológicas. La salida del ion Cl- a través de TMEM16A regula la secreción de fluidos epiteliales, contracción del músculo liso y sensibilidad al dolor por calor, entre otros procesos biológicos. El canal es un homodímero. Cada monómero tiene un poro para el paso de los iones. El canal cuenta con un sitio de unión a Ca2+ adyacente al poro. El sitio de unión está formado por las cadenas laterales de E654, E702, E705, E734 y D738. En cada sitio de unión se unen dos iones Ca2+. A pesar de que una de las propiedades fundamentales de los canales iónicos es su capacidad para seleccionar al ion que atraviesa por el poro, TMEM16A presenta una selectividad iónica baja. Estudios variados han identificado la siguiente secuencia de selectividad: tiocianato (SCN-) > nitrato (NO3-) > ioduro (I-) > bromuro (Br-) > Cl- > fluoruro. Sin embargo, se desconocen los elementos de la proteína involucrados en la selección del ion Cl-. El objetivo de mi trabajo de tesis es determinar si la naturaleza química del sitio de unión a Ca2+ participa en el proceso de selectividad aniónica en el canal TMEM16A. Usualmente, la selectividad iónica se establece usando la permeabilidad del ion en el poro. El ordenamiento de mayor a menor de la permeabilidad define la secuencia de selectividad. Para determinar si el sitio de unión a Ca2+ participa en la selección del anión permeante, registré la corriente eléctrica que fluye a través del canal TMEM16A silvestre y de los canales con el sitio de unión a Ca2+ mutado (mutantes E702Q, E705Q y E702Q/E705Q) usando la técnica de fijación del voltaje mediante Patch-clamp en configuración de célula completa. Realicé los experimentos con diferentes soluciones extracelulares conteniendo uno de los siguientes aniones principales: SCN-, NO3-, I-, Br-, Cl- o Glutamato. A partir de las medidas del potencial de inversión determiné la permeabilidad para estos aniones relativa a la permeabilidad del ion Cl-. Mis datos muestran que la selectividad aniónica del canal TMEM16A silvestre es SCN- > NO3- > I- > Br- > Cl- > Glutamato, mientras que la selectividad de las mutantes E702Q y E705Q es I- = SCN- > NO3- = Br- > Cl- > Glutamato y I- = NO3- = Br- = SCN- > Cl- > Glutamato, respectivamente." "The calcium-activated chloride (Cl-) channel TMEM16A has been associated with epithelial fluid secretion, smooth muscle contraction, and heat pain sensibility. TMEM16A is a homodimer with two pores, one in each monomer. Each pore function serves as permeation pathway for ions. TMEM16A has two identical Ca2+ binding sites, one in each monomer, located adjacent to the permeation pathway and near the intracellular side. The Ca2+ binding site is formed by the side chains of E654, E702, E705, E734, and D738. Two Ca2+ ions are bound to each site in a voltage- dependent manner. A fundamental property of ionic channels is their ability to select among different ions, a property named ionic selectivity. Typically, ionic selectivity is reported relative to the physiological permeant ion. Hence, relative to chloride, TMEM16A has low ionic selectivity for other anions. Various studies agree about the selectivity sequence of TMEM16A: thiocyanate (SCN-) > nitrate (NO3-) > iodide (I-) > bromide (Br-) > Cl- > fluoride. However, it is unknown the element or domains involved in anion selection. The aim of my thesis is to determine whether the chemical nature of the Ca2+ binding site plays a role in anion selectivity by the TMEM16A channel. Experimentally, the ionic selectivity is quantitated by the permeability ratio (Permeability of anion X-/ Permeability of Cl-) at the pore calculated using the reversal potentials and the Goldman-Hodgkin-Katz equation. The arrangement of permeability ratios from high to low values defines the selectivity sequence. To determine if de Ca2+ binding site participates in the ionic selectivity I registered the current flowing through the wild type TMEM16A channel and through channels with the Ca2+ binding site muted (E702Q, E705Q, and E702Q / E705Q mutants). I used the “patch-clamp” technique in whole-cell configuration to record the macroscopic currents. I recorded the channel activity exposed to different extracellular solutions containing different anions SCN- or NO3-, or I-, or Br-, or Cl-, or Glutamate. First, I recorded the current in the present of Cl- in the bath and then replaced 100% of Cl- with a given test anion X- and recorded again the currents from the same cell. With the resulting currents, I constructed current versus voltage curves to determine the reversal potential in the presence of Cl- and in the presence of anion X-."

"The vegetative growth of filamentous fungi comprises cell elongation and branching. This complex process involves Intracellular transport of molecules, vesicles and organelles within the cell interior, assisted by motor proteins, which, under the study of Nanosciences, are considered the smallest motors. Here, the growth process of Trichoderma atroviride was studied. First, a novel tool for automated tracking of growth in fungal cells (called hyphae) is introduced. This tool allows performing quantitative analysis of growth rate and morphology. An image-processing routine that detects in real-time the tip of a hypha and tracks it as the hypha elongates was developed. Tracking records allowed to determine that T. atroviride hyphae grow with characteristic elongation rates of ~ 0.07 μm/s. Prior to the occurrence of an apical branching event the parental hypha stopped growing during a few minutes. From tracking data, it was found that the persistence length (a measure of filament extension before presenting a change in direction) associated to T. atroviride hyphae is 362 μm. Second, the absence of Kinesin-1, one of the main nano-motors transporters of essential growth vesicles (reported for other filamentous fungi), and how it affects hyphal elongation, branching and morphology of T. atroviride was studied. By developing a Kinesin-1 deletion mutant strain it was shown that T. atroviride elongation rate drops up to ~ 60%, while branching highly increases. Several experiments following T. atroviride growth stages were carried out (germination, vegetative growth and conidiation) and determined that: The absence of Kinesin-1 modifies T. atroviride germination resulting in delayed conidia germination times. Also, about 50% of conidia presented bipolar germination. Vegetative growth was highly reduced with characteristic elongation rates of ~ 0.03 μm/s. Conidiation was also reduced in ΔKinesin-1 producing ~30x108 conidia vs ~8x108 WT. Altogether, these preliminary results show how integration of image analysis and computer control enable quantitative microscopic observations of fungal hyphae dynamics and how the absence of the nano-motor protein Kinesin-1 results in severe modifications of hyphal elongation, branching, conidiation and morphology." "El crecimiento vegetativo de los hongos filamentosos se define por el alargamiento y la ramificación celular. Este crecimiento es un proceso complejo que involucra el transporte intracelular de moléculas, vesículas y organelos, mediado principalmente por nano-motores. En este proyecto, se estudió el proceso de crecimiento de Trichoderma atroviride en dos aspectos principales: Primero, se presenta una herramienta para el seguimiento automático del crecimiento de células de hongos filamentosos (llamadas hifas) para realizar un análisis cuantitativo de la tasa de crecimiento y aspectos de su morfología. Se desarrolló una rutina de procesamiento de imágenes que detecta en tiempo real la punta de una hifa y la rastrea a medida que la hifa se alarga. Los registros de seguimiento permitieron determinar que las hifas de T. atroviride crecen con tasas de elongación características de ~ 0.07 μm / s. Se encontró que antes de que ocurriera un evento de ramificación apical, la hifa parental detiene su crecimiento durante unos minutos. Finalmente, se determinó que la longitud de persistencia (una medida de la extensión del filamento antes de presentar un cambio en la dirección) asociada a las hifas de T. atroviride es de 362 μm. Segundo, se estudió que la ausencia de la Cinesina-1, uno de los principales nano-motores transportadores de vesículas de crecimiento esenciales (reportados para otros hongos filamentosos), afecta la elongación de las hifas, la ramificación y la morfología de T. atroviride. Al desarrollar en el laboratorio una cepa mutante ΔCinesina-1, se observó que la tasa de elongación de T. atroviride disminuye hasta en un 60%, mientras que la ramificación aumenta considerablemente. Se llevaron a cabo varios experimentos morfológicos siguiendo las etapas de crecimiento de T. atroviride (germinación, crecimiento vegetativo y conidiación) y se encontró que: la ausencia de la Cinesina-1 afectó la germinación de T. atroviride dando como resultado que el ~ 50% de los conidios presentan germinación bipolar. El crecimiento vegetativo se vió altamente disminuido con tasas de elongación características de ~ 0.03 μm/s. El proceso de conidiación también se vió afectado en la cepa ΔCinesina-1 produciendo ~ 30x108 conidias vs ~ 8x108 en la cepa silvestre. En conjunto, estos resultados preliminares muestran cómo la integración del análisis de imágenes y el control por computadora permiten realizar observaciones microscópicas cuantitativas de la dinámica de las hifas de los hongos y cómo la ausencia de la proteína nano-motora Cinesina-1 produce modificaciones en la elongación de hifas, la ramificación, la conidiación y la morfología."