Daniel Sánchez

Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system.

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.

Noncontact Measurement of the Local Mechanical Properties of Living Cells Using Pressure Applied via a Pipette

Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1-150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells.

The use of scanning ion conductance microscopy to image A6 cells

BACKGROUND Continuous high spatial resolution observations of living A6 cells would greatly aid the elucidation of the relationship between structure and function and facilitate the study of major physiological processes such as the mechanism of action of aldosterone. Unfortunately, observing the micro-structural and functional changes in the membrane of living cells is still a formidable challenge for a microscopist. METHOD Scanning ion conductance microscopy (SICM), which uses a glass nanopipette as a sensitive probe, has been shown to be suitable for imaging non-conducting surfaces bathed in electrolytes. A specialized version of this microscopy has been developed by our group and has been applied to image live cells at high-resolution for the first time. This method can also be used in conjunction with patch clamping to study both anatomy and function and identify ion channels in single cells. RESULTS This new microscopy provides high-resolution images of living renal cells which are comparable with those obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Continuous 24h observations under normal physiological conditions showed how A6 kidney epithelial cells changed their height, volume, and reshaped their borders. The changes in cell area correlated with the density of microvilli on the surface. Surface microvilli density ranged from 0.5 microm(-2) for extended cells to 2.5 microm(2) for shrunk cells. Patch clamping of individual cells enabled anatomy and function to be correlated. CONCLUSIONS Scanning ion conductance microscopy provides unique information about living cells that helps to understand cellular function. It has the potential to become a powerful tool for research on living renal cells.

Localized and non-contact mechanical stimulation of dorsal root ganglion sensory neurons using scanning ion conductance microscopy.

Mechanosensitive ion channels convert external mechanical force into electrical and chemical signals in cells, but their physiological function in different tissues is not clearly understood. One reason for this is that there is as yet no satisfactory physiological method to stimulate these channels in living cells. Using the nanopipette-probe of the Scanning Ion Conductance Microscope (SICM), we have developed a new technique to apply local mechanical stimulus to living cells to an area of about 0.385 microm2, determined by the pipette diameter. Our method prevents any physical contact and damage to the cell membrane by use of a pressure jet applied via the nanopipette. The study used whole-cell patch-clamp recordings and measurements of intracellular Ca2+ concentration to validate the application of the mechanical stimulation protocols in human and rat dorsal root ganglia (DRG) sensory neurons. We were able, for the first time, to produce a non-contact, controlled mechanical stimulation on living neurites of human DRG neurons. Our methods will enable the identification and characterisation of compounds being developed for the treatment of clinical mechanical hypersensitivity states.

Sea urchin sperm cation-selective channels directly modulated by cAMP

Components of the sea urchin outer egg jelly layer such as speract drastically change second messenger levels and membrane permeability in sperm. Ion channels are deeply involved in the sperm–egg dialogue in sea urchin and other species. Yet, due to the small size of sperm, studies of ion channels and their modulation by second messengers in sperm are scarce. In this report we offer the first direct evidence that cation‐selective channels upwardly regulated by cAMP operate in sea urchin sperm. Due to their poor selectivity among monovalent cations, channel activation in seawater could contribute to sperm membrane repolarization during the speract response.

Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural‐crest‐related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest‐like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage‐gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.—Sviderskaya, E. V., Easty, D. J., Lawrence, M. A., Sánchez, D. P., Negulyaev, Y. A., Patel, R. H., Anand, P., Korchev, Y. E., Bennett, D. C. Functional neurons and melanocytes induced from immortal lines of postnatal neural crest‐like stem cells. FASEB J. 23, 3179–3192 (2009). www.fasebj.org

Phylogenetic Relationships in Echinocereus (Cactaceae, Cactoideae)

Abstract Echinocereus is the third most species-rich genus in the Cactaceae. It is distributed in North America from Mexico to the central U. S. A. Previous molecular phylogenetic studies have indicated that the genus is polyphyletic, but incomplete taxon sampling and unclear resolution have hindered the formal re-evaluation of generic and infrageneric circumscriptions. To address this problem, we analyzed six plastid regions (matK, rbcL, psbA-trnH, trnQ-rps16, rpl16, and trnL-F) using maximum parsimony and Bayesian inference criteria for 59 species, including all previously proposed infrageneric entities and representing the full range of morphological variation known in the genus. Our results support the monophyly of Echinocereus if E. pensilis is excluded and reestablished as the monotypic genus Morangaya. Two additional morphological characters, erumpent flower buds and green stigma lobes, further support the circumscription of Echinocereus sensu stricto. Phylogenetic analyses recovered nine main clades in Echinocereus s. s., one of which corresponds to the Triglochidiati section; the remaining clades did not correspond to any other recognized sections. We suggest a re-evaluation of previously proposed infrageneric entities.

Measuring Ion Fluxes in Sperm

Publisher Summary The chapter discusses different strategies to study ion fluxes in sperm. Ion channels and transporters in the sperm plasma membrane participate crucially in fertilization. Ion-permeability changes are deeply involved in how sperm sense environmental cues and signals from the outer envelope of the egg to achieve fertilization. Combining the in vivo measurements of intracellular ions and membrane potential in sperm populations and single cells with electrophysiological approaches, such as the smart patch-clamp, and reconstitution strategies in planar bilayers reveal how sperm ion channels participate in sperm motility and the acrosome reaction. This chapter describes ion transport protocols for sea urchin sperm. Egg ligands immediately induce sperm responses. It is important to measure the rapid kinetics of their responses to understand the underlying signaling mechanisms. To perform a successful time-resolved measurement, it is essential that sperm be exposed to egg ligands as rapidly as possible. There are two different methods to achieve this: (1) rapid mixing (stopped-flow fluorometry) and (2) photolysis of caged (photoactivatable) ligands. The chapter presents techniques used in the study of ion fluxes in single sea urchin sperm.

Análisis morfométrico de las especies de Echinocereus sección Triglochidiati (Cactaceae) en México

ResumenEchinocereus sección Triglochidiati ha sido aceptada en los más recientes tratamientos taxonómicos del género, sin embargo el número de especies que incluye ha variado entre una y 14. El presente trabajo tiene como objetivo delimitar el número de especies de la sección Triglochidiati para México a través de análisis multivariados, así como identificar los caracteres cuantitativos que permiten reconocerlas. Se utilizaron el análisis discriminante canónico y el discriminante clasificatorio para 11 supuestos taxones nativos de México y 20 caracteres morfométricos. El número de costillas del tallo, número de espinas centrales, diámetro del tallo, longitud de la espina radial (posición 09), longitud de la flor, razón longitud del tubo receptacular / perianto y longitud de los tricomas del tubo receptacular de la flor son caracteres que permitieron diferenciar seis taxones: E. acifer, E. arizonicus, E. coccineus, E. koehresianus, E. polyacanthus y E. scheeri. Se incluye una clave de identificación de las especies.AbstractEchinocereus section Triglochidiati has been accepted in the most recent taxonomic treatments of the genus. However, the number of included species has varied from one to 14. This study aims to define the species number of section Triglochidiati in Mexico through multivariate analysis, and to identify quantitative characters that can be used to recognize them. We used canonical discriminant analysis and classification discriminant analysis for 11 putative taxa native to Mexico and 20 morphometric characters. The number of stem ribs, number of central spines, stem diameter, length of radial spines (position 09), length of flower, receptacular tube length / perianth length ratio, and the receptacular tube trichome length permitted the differentiation of six taxa: E. acifer, E. arizonicus, E. coccineus, E. koehresianus, E. polyacanthus and E. scheeri. A key to the species is included.

7 – Regulation of Sperm Ion Currents

Publisher Summary This chapter focuses on the regulation of sperm ion currents—that is, the participation of the sperm ion channels in the information exchange between gametes and between gametes and their environment. In all species whose sperm cells possess an acrosome, successful fertilization requires the acrosome reaction. This reaction allows spermatozoa to penetrate through the outer vestments of the egg and to recognize and fuse with the egg plasma membrane. Induction of this fundamental process involves short-range interactions of spermatozoa with components from the eggs outer layers, and also with other components of the female reproductive tract for internal fertilizers. Spermatozoa experience important alterations in their ionic milieu that influence their functional state. This chapter ends with concluding remark that cell signaling is fundamental in determining the behavior of organisms. The propagation of life in many species depends on the dialogue between gametes, ion channels being elementary tools of cell communication. Currently, there is background information about some of the ion channels present in spermatozoa. Future study will determine the molecular mechanisms that regulate these channels in the cell. Combining molecular biological strategies and electrophysiology in spermatogenic cells, and the transfer of ion channels directly from spermatozoa to planar bilayers, opens new avenues to explore the participation of channels in spermatogenesis, and their regulation in mature spermatozoa cells.