RESUMO
Ca(2+) has long been known to play an important role in cellular polarity and guidance. We studied the role of Ca(2+) signaling during random and directed cell migration to better understand whether Ca(2+) directs cell motility from the leading edge and which ion channels are involved in this function by using primary zebrafish keratinocytes. Rapid line-scan and time-lapse imaging of intracellular Ca(2+) (Ca(2+)i) during migration and automated image alignment enabled us to characterize and map the spatiotemporal changes in Ca(2+)i. We show that asymmetric distributions of lamellipodial Ca(2+) sparks are encoded in frequency, not amplitude, and that they correlate with cellular rotation during migration. Directed migration during galvanotaxis increases the frequency of Ca(2+) sparks over the entire lamellipod; however, these events do not give rise to asymmetric Ca(2+)i signals that correlate with turning. We demonstrate that Ca(2+)-permeable channels within these cells are mechanically activated and include several transient receptor potential family members, including TRPV1. Last, we demonstrate that cell motility and Ca(2+)i activity are affected by pharmacological agents that target TRPV1, indicating a novel role for this channel during cell migration.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Queratinócitos/citologia , Queratinócitos/metabolismo , Canais de Cátion TRPV/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Células Epidérmicas , Epiderme/metabolismo , Peixe-ZebraRESUMO
Many cells respond directionally to small DC electrical fields (EFs) by an unknown mechanism, but changes in intracellular Ca(2+) are widely assumed to be involved. We have used zebrafish (Danio rerio) keratocytes in an effort to understand the nature of the EF-cell interaction. We find that the adult zebrafish integument drives substantial currents outward through wounds produced by scale removal, establishing that keratocytes near the wound will experience endogenous EFs. Isolated keratocytes in culture turn toward the cathode in fields as small as 7 mV mm(-1), and the response is independent of cell size. Epidermal sheets are similarly sensitive. The frequency of intracellular Ca(2+) spikes and basal Ca(2+) levels were increased by EFs, but the spikes were not a necessary aspect of migration or EF response. Two-photon imaging failed to detect a pattern of gradients of Ca(2+) across the lamellipodia during normal or EF-induced turning but did detect a sharp, stable Ca(2+) gradient at the junction of the lamellipodium and the cell body. We conclude that gradients of Ca(2+) within the lamellipodium are not required for the EF response. Immunostaining revealed an anode to cathode gradient of integrin beta1 during EF-induced turning, and interference with integrin function attenuated the EF response. Neither electrophoretic redistribution of membrane proteins nor asymmetric perturbations of the membrane potential appear to be involved in the EF response, and we propose a new model in which hydrodynamic forces generated by electro-osmotic water flow mediate EF-cell interactions via effects on focal adhesions.
Assuntos
Cálcio/metabolismo , Movimento Celular/fisiologia , Eletricidade , Células Epidérmicas , Integrina beta1/metabolismo , Animais , Polaridade Celular , Células Cultivadas , Quelantes/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Gadolínio/metabolismo , Humanos , Peixe-ZebraRESUMO
Little is known about the cues that guide migrating neural crest derivatives to their targets. This lack of understanding is especially significant in the case of Schwann cells, which have been transplanted into the central nervous system in an effort to promote axonal myelination after injury or disease. We have investigated the response of Schwann cells, cultured from the peripheral nerves of E7/8 chick embryos, to applied electrical fields. We find that they respond by migrating to the anode, and show a significant anodal bias in directionality at 3 mV mm(-1). This is the smallest electrical field that has been shown to affect cellular movement or growth in culture, and the anodal direction is surprising given the known cathodal responses of neural crest cells. The effective fields are considerably smaller than endogenous electrical fields that have been measured in embryonic tissues.
Assuntos
Movimento Celular/fisiologia , Estimulação Elétrica/métodos , Células de Schwann/citologia , Células de Schwann/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Crista Neural/citologia , Crista Neural/fisiologiaRESUMO
An important basis for the clinical application of small DC electric current to mammalian spinal injury is the responses of neurons in culture to applied electric fields. Our recent finding that zebrafish neurons were unresponsive to applied fields prompted us to critically examine previous results. We conclude that compelling evidence for neuronal guidance and directional stimulation of growth toward either the cathode or anode in an electric field exists only for cultured Xenopus neurons, and not for any mammalian neurons. No basis for the reported success in treating spinal injury exists in the in vitro studies, and considerable research will be required if the conditions of field application in mammalian spinal injury are to be optimized.
Assuntos
Terapia por Estimulação Elétrica/tendências , Campos Eletromagnéticos , Regeneração Nervosa/efeitos da radiação , Traumatismos da Medula Espinal/terapia , Medula Espinal/efeitos da radiação , Animais , Células Cultivadas , Cones de Crescimento/fisiologia , Cones de Crescimento/efeitos da radiação , Humanos , Modelos Animais , Regeneração Nervosa/fisiologia , Especificidade da Espécie , Medula Espinal/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Xenopus laevis/fisiologiaRESUMO
Naturally occurring electric fields (EFs) have been implicated in cell guidance during embryonic development and adult wound healing. Embryonic Xenopus laevis neurons sprout preferentially towards the cathode, turn towards the cathode, and migrate faster towards the cathode in the presence of an external EF in vitro. A recent Phase 1 clinical trial has investigated the effects of oscillating EFs on human spinal cord regeneration. The purpose of this study was to investigate whether embryonic zebrafish neurons respond to an applied EF, and thus extend this research into another vertebrate system. Neural tubes of zebrafish embryos (16-17 somites) were dissected and dissociated neuroblasts were plated onto laminin-coated glass. A 100 mV/mm EF was applied to cell cultures for 4 or 20 h and the responses of neurons to the applied EFs were investigated. After 4h in an EF neurites were significantly shorter than control neurites. No other statistically significant effects were observed. After 20 h, control and EF-exposed neurites were no different in length. No length difference was seen between cathodally- and anodally-sprouted neurites. Application of an EF did not affect the average number of neurons in a chamber. Growth cones did not migrate preferentially towards either pole of the EF and no asymmetry was seen in neurite sprout sites. We conclude that zebrafish neurons do not respond to a 100 mV/mm applied EF in vitro. This suggests that neurons of other vertebrate species may not respond to applied EFs in the same ways as Xenopus laevis neurons.
Assuntos
Eletricidade , Embrião não Mamífero/citologia , Neuritos/efeitos da radiação , Neurônios/efeitos da radiação , Animais , Polaridade Celular/fisiologia , Polaridade Celular/efeitos da radiação , Células Cultivadas , Embrião não Mamífero/efeitos da radiação , Neurônios/citologia , Peixe-Zebra/embriologiaRESUMO
Biased left-right asymmetry is a fascinating and medically important phenomenon. We provide molecular genetic and physiological characterization of a novel, conserved, early, biophysical event that is crucial for correct asymmetry: H+ flux. A pharmacological screen implicated the H+-pump H+-V-ATPase in Xenopus asymmetry, where it acts upstream of early asymmetric markers. Immunohistochemistry revealed an actin-dependent asymmetry of H+-V-ATPase subunits during the first three cleavages. H+-flux across plasma membranes is also asymmetric at the four- and eight-cell stages, and this asymmetry requires H+-V-ATPase activity. Abolishing the asymmetry in H+ flux, using a dominant-negative subunit of the H+-V-ATPase or an ectopic H+ pump, randomized embryonic situs without causing any other defects. To understand the mechanism of action of H+-V-ATPase, we isolated its two physiological functions, cytoplasmic pH and membrane voltage (Vmem) regulation. Varying either pH or Vmem, independently of direct manipulation of H+-V-ATPase, caused disruptions of normal asymmetry, suggesting roles for both functions. V-ATPase inhibition also abolished the normal early localization of serotonin, functionally linking these two early asymmetry pathways. The involvement of H+-V-ATPase in asymmetry is conserved to chick and zebrafish. Inhibition of the H+-V-ATPase induces heterotaxia in both species; in chick, H+-V-ATPase activity is upstream of Shh; in fish, it is upstream of Kupffer's vesicle and Spaw expression. Our data implicate H+-V-ATPase activity in patterning the LR axis of vertebrates and reveal mechanisms upstream and downstream of its activity. We propose a pH- and Vmem-dependent model of the early physiology of LR patterning.
Assuntos
Padronização Corporal , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Prótons , Vertebrados/embriologia , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Embrião de Galinha , Embrião não Mamífero , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Modelos Biológicos , Inibidores da Bomba de Prótons , Fatores de Tempo , Xenopus/embriologia , Peixe-Zebra/embriologiaRESUMO
Microelectrodes, made from a Cl(-)-selective liquid ion exchanger previously used to measure putative Cl- fluxes in Lilium longiflorum pollen tubes, were characterized. The electrodes were poorly selective, possessing only about 10-fold selectivity for Cl- over other anions tested. They had only 2.4-fold selectivity for Cl- over the anionic form of the H+ buffer, MES, indicating that the electrode can indirectly detect H+ gradients. Apparent anion influx was detected along the pollen tube shafts and at the grains while apparent anion efflux was detected near the tip of the tube. During oscillating growth, the peak of the oscillating apparent anion efflux at the tip occurred, on average, 7.9 sec after the peak of the growth oscillations. Consideration of the previously characterized H+ fluxes in lily pollen grains and tubes, as well as the poor anion selectivity of the Cl- electrodes, indicates that the putative Cl- fluxes are in fact changes in the anionic concentration of the buffer resulting from H+ gradients and not changes in Cl- concentration. The claim of a central role for Cl- in lily pollen tube growth is further undermined by the fact that these tubes grow at the same rate if the Cl- content of the growth medium is reduced to trace levels (< or =31 microM), and that the grains have only small reserves of Cl-. These results lead to the conclusion that Cl- fluxes are not a significant component of pollen tube growth and Cl- itself is not required for growth.
Assuntos
Cloretos/metabolismo , Lilium/metabolismo , Pólen/metabolismo , Resinas de Troca Aniônica , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/metabolismo , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Lilium/efeitos dos fármacos , Pólen/efeitos dos fármacos , Fatores de TempoRESUMO
Pollen tube growth requires a Ca2+ gradient, with elevated levels of cytosolic Ca2+ at the growing tip. This gradient's magnitude oscillates with growth oscillation but is always maintained. Ca2+ influx into the growing tip is necessary, and its magnitude also oscillates with growth. It has been widely assumed that stretch-activated Ca2+ channels underlie this influx, but such channels have never been reported in either pollen grains or pollen tubes. We have identified and characterized stretch-activated Ca2+ channels from Lilium longiflorum pollen grain and tube tip protoplasts. The channels were localized to a small region of the grain protoplasts associated with the site of tube germination. In addition, we find a stretch-activated K+ channel as well as a spontaneous K+ channel distributed over the entire grain surface, but neither was present at the germination site or at the tip. Neither stretch-activated channel was detected in the grain protoplasts unless the grains were left in germination medium for at least 1 h before protoplast preparation. The stretch-activated channels were inhibited by a spider venom that is known to block stretch-activated channels in animal cells, but the spontaneous channel was unaffected by the venom. The venom also stopped pollen tube germination and elongation and blocked Ca2+ entry into the growing tip, suggesting that channel function is necessary for growth.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Canais Iônicos/fisiologia , Pólen/fisiologia , Protoplastos/fisiologia , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Canais Iônicos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Pressão , Venenos de Aranha/farmacologiaRESUMO
Previous work has shown that distinct Ca(2+) gradients precede and predict the loci of germination of the zygotes of the brown alga, Silvetia compressa (J. Agardh) E. Serrão, T.O. Cho, S.M. Boo et Brawley, that are polarized by unilateral blue light. We show here that dark-grown S. compressa zygotes also form cytosolic Ca(2+) gradients prior to germination and then germinate from the site of elevated Ca(2+). In no case did germination occur without a prior formation of a Ca(2+) gradient. Using the self-referencing Ca(2+)-selective probe, we measured highly localized influx of Ca(2+) during photopolarization, indicating that extracellular stores supply at least some of the Ca(2+) needed to construct a gradient. Finally, we find that germination was inhibited by a bath-applied inhibitor of calcium/calmodulin-dependent kinase II (CaM kinase II), KN-93 (but not by its inactive analog, KN-92), and by an injected inhibitory peptide for the kinase. KN-93 did not interfere with the photopolarization of the zygotes, consistent with the view that calmodulin is not involved in the initial response to light. The KN-93 results indicate that the requirement for active CaM kinase II for germination ends about 2 h before overt germination. We conclude that Ca(2+) gradients, generated in part by localized calcium entry from the seawater, are an essential part of the process of polarity development and expression in these cells, regardless of the nature of the external cue that directs the orientation of the axis. Calmodulin and CaM kinase II are involved in interpreting (but not in establishing) the calcium gradient, allowing germination to occur at the site of elevated calcium, but CaM kinase II appears not to be involved in the initiation of germination.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Phaeophyceae/crescimento & desenvolvimento , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Polaridade Celular/fisiologia , Luz , Modelos Biológicos , Phaeophyceae/metabolismo , Esporos/crescimento & desenvolvimento , Esporos/metabolismo , Sulfonamidas/farmacologiaRESUMO
A fundamental aspect of biological systems is their spatial organization. In development, regeneration and repair, directional signals are necessary for the proper placement of the components of the organism. Likewise, pathogens that invade other organisms rely on directional signals to target vulnerable areas. It is widely understood that chemical gradients are important directional signals in living systems. Less well recognized are electrical fields, which can also provide directional information. Small, steady electrical fields can directly guide cell movement and growth and can generate chemical gradients of charged macromolecules against the leveling action of diffusion. At the site of a lesion in an ion-transporting epithelium, for example, a substantial electrical field is instantly generated and may extend over many cell diameters. There are numerous other situations in which relatively long-range electrical fields have been shown to exist naturally. Recently, there has been substantial progress in identifying specific processes that are controlled, to some extent, by these endogenous electrical fields. This review highlights these recent data and discusses possible mechanisms by which the fields might affect biological processes.
Assuntos
Eletrofisiologia , Modelos Biológicos , Animais , Transporte Biológico Ativo , Padronização Corporal/fisiologia , Córnea/fisiologia , Feminino , Insetos , Oócitos/metabolismo , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Regeneração/fisiologia , Transdução de SinaisRESUMO
Two mechanisms have been proposed as the primary control of oscillating tip growth in Lilium longiflorum Thunb. pollen tubes: changes in cell wall strength (Holdaway-Clarke et al. 1997) or alternatively, changes in turgor pressure (Messerli et al. 2000). Here we have modified the ionic and osmotic concentrations of the growth medium to test predictions derived from both models. Raising the [Ca2+]o tenfold above normal reduced the amplitude of the [Ca2+]i oscillations and growth oscillations while it raised the basal [Ca2+]i and growth rate such that the average growth rate did not change. Raising the [H+] of the growth medium tenfold reversibly decreased and sometimes eliminated the [Ca2+]i and growth oscillations without changing the average growth rate. Lowering the [H+] tenfold led to irregular frequency and amplitude [Ca2+]i oscillations, reduced the average growth rate of tubes and led to cell bursting in 33% of tubes. Addition of 50 mM H+ buffer, MES, to prevent pH changes in the cell wall increased the period, amplitude and duration of both [Ca2+]i and growth oscillations. Changing the [K+]o did not markedly effect [Ca2+]i oscillations. Reducing the osmolarity of the medium led to transient large-amplitude [Ca2+]i and growth oscillations while reducing large-amplitude oscillations over long periods. In many different conditions under which growth still occurs, lily pollen tubes maintain growth oscillations, albeit with modified frequency, amplitude and duration. We conclude that modifications to both proposed models are necessary to explain oscillating growth in this system.
Assuntos
Flores/crescimento & desenvolvimento , Lilium/crescimento & desenvolvimento , Cálcio/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/fisiologia , Flores/efeitos dos fármacos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Lilium/efeitos dos fármacos , Modelos Biológicos , Concentração Osmolar , Pressão Osmótica/efeitos dos fármacos , Potássio/farmacologiaRESUMO
Reproduction in higher plants requires the directed growth of pollen tubes in order to transmit sperm cells to the ovules at the base of the style. Many signaling processes have been implicated in polarized pollen tube growth. Here, changes in the concentration of calcium, potassium, hydrogen (pH), and chloride are discussed, as they may all contribute to the process of oscillatory growth and guidance observed in pollen tubes.
Assuntos
Íons/metabolismo , Componentes Aéreos da Planta/crescimento & desenvolvimento , Pólen/fisiologia , Flores/metabolismo , Flores/fisiologia , Germinação/fisiologia , Magnoliopsida/metabolismo , Magnoliopsida/fisiologia , Componentes Aéreos da Planta/metabolismo , Componentes Aéreos da Planta/fisiologia , Pólen/metabolismoRESUMO
A pharmacological screen identified the H+ and K+ ATPase transporter as obligatory for normal orientation of the left-right body axis in Xenopus. Maternal H+/K+-ATPase mRNA is symmetrically expressed in the 1-cell Xenopus embryo but becomes localized during the first two cell divisions, demonstrating that asymmetry is generated within two hours postfertilization. Although H+/K+-ATPase subunit mRNAs are symmetrically localized in chick embryos, an endogenous H+/K+-ATPase-dependent difference in membrane voltage potential exists between the left and right sides of the primitive streak. In both species, pharmacologic or genetic perturbation of endogenous H+/K+-ATPase randomized the sided pattern of asymmetrically expressed genes and induced organ heterotaxia. Thus, LR asymmetry determination depends on a very early differential ion flux created by H+/K+-ATPase activity.
Assuntos
Padronização Corporal , Membrana Celular/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Potenciais da Membrana , Omeprazol/análogos & derivados , 2-Piridinilmetilsulfinilbenzimidazóis , Animais , Compostos de Bário/farmacologia , Embrião de Galinha , Cloretos/farmacologia , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Hibridização In Situ , Lansoprazol , Dados de Sequência Molecular , Omeprazol/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Inibidores da Bomba de Prótons , RNA Mensageiro/metabolismo , Fatores de Tempo , XenopusRESUMO
Unidirectional blue light directs the rhizoid-thallus axis in the apolar zygote of the brown alga, Silvetia compressa. This effect is mediated by an increase in the intracellular concentration of guanosine 3', 5'-cyclic monophosphate. In this study we show the identification of a rhodopsin-like protein, by means of antibody reaction, in the plasma membrane of Silvetia eggs. This new result suggests a role for opsins in Silvetia photopolarity.