TRP channels in Drosophila photoreceptors: the lipid connection

The light-sensitive current in Drosophila photoreceptors is mediated by transient receptor potential (TRP) channels, at least two members of which (TRP and TRPL) are activated downstream of phospholipase C (PLC) in response to light. Recent evidence is reviewed suggesting that Drosophila TRP channels are activated by one or more lipid products of PLC activity: namely diacylglycerol (DAG), its metabolites (polyunsaturated fatty acids) or the reduction in phosphatidylinositol 4,5-bisphosphate (PIP(2)). The most compelling evidence for this view comes from analysis of rdgA mutants which are unable to effectively metabolise DAG due to a defect in DAG kinase. The rdgA mutation leads to constitutive activation of both TRP and TRPL channels and dramatically increases sensitivity to light in hypomorphic mutations of PLC and G protein.     

Light-regulated interaction of Dmoesin with TRP and TRPL channels is required for maintenance of photoreceptors

Recent studies in Drosophila melanogaster retina indicate that absorption of light causes the translocation of signaling molecules and actin from the photoreceptor's signaling membrane to the cytosol, but the underlying mechanisms are not fully understood. As ezrin-radixin-moesin (ERM) proteins are known to regulate actin-membrane interactions in a signal-dependent manner, we analyzed the role of Dmoesin, the unique D. melanogaster ERM, in response to light. We report that the illumination of dark-raised flies triggers the dissociation of Dmoesin from the light-sensitive transient receptor potential (TRP) and TRP-like channels, followed by the migration of Dmoesin from the membrane to the cytoplasm. Furthermore, we show that light-activated migration of Dmoesin results from the dephosphorylation of a conserved threonine in Dmoesin. The expression of a Dmoesin mutant form that impairs this phosphorylation inhibits Dmoesin movement and leads to light-induced retinal degeneration. Thus, our data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells.     

Drosophila stathmin is required to maintain tubulin pools

Stathmin, or Oncoprotein 18 (Op18), is the founding member of a phosphoprotein family that can regulate the microtubule cytoskeleton by sequestering tubulin and promoting microtubule catastrophe. Stathmin is subject to spatially and temporally controlled regulatory phosphorylation, which inhibits its interaction with tubulin. Drosophila Stathmin has similar properties to the mammalian proteins. We find that Drosophila Stathmin is required for specific microtubule-dependent processes: maintenance of oocyte identity within a germline cyst and localization of polarity determinants. Unexpectedly, microtubules are less abundant in stathmin mutant cells compared to normal cells, showing that a key function of Stathmin in vivo is the long-term maintenance of the microtubule cytoskeleton. The microtubule network re-forms more slowly after coldshock in stathmin mutant follicle cells. Surprisingly, stathmin mutant animals and tissues show a marked decrease in total tubulin-protein levels, and this might explain the effect on the microtubule cytoskeleton. Stathmin overexpression also increases tubulin protein. Free alpha- and beta-tubulin have been shown to negatively autoregulate their own synthesis. We suggest that Stathmin serves to maintain a noninhibitory, soluble, and releasable tubulin pool.     

DAG lipase activity is necessary for TRP channel regulation in Drosophila photoreceptors

In Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses.     

Light-dependent modulation of Shab channels via phosphoinositide depletion in Drosophila photoreceptors

The Drosophila phototransduction cascade transforms light into depolarizations that are further shaped by activation of voltage-dependent K+ (Kv) channels. In whole-cell recordings of isolated photoreceptors, we show that light selectively modulated the delayed rectifier (Shab) current. Shab currents were increased by light with similar kinetics to the light-induced current itself (latency approximately 20 ms), recovering to control values with a t(1/2) of approximately 60 s in darkness. Genetic disruption of PLCbeta4, responsible for light-induced PIP(2) hydrolysis, abolished this light-dependent modulation. In mutants of CDP-diaclyglycerol synthase (cds(1)), required for PIP(2) resynthesis, the modulation became irreversible, but exogenously applied PIP(2) restored reversibility. The modulation was accurately and reversibly mimicked by application of PIP(2) to heterologously expressed Shab channels in excised inside-out patches. The results indicate a functionally implemented mechanism of Kv channel modulation by PIP(2) in photoreceptors, which enables light-dependent regulation of signal processing by direct coupling to the phototransduction cascade.     

Dynactin is required to maintain nuclear position within postmitotic Drosophila photoreceptor neurons

How a nucleus is positioned within a highly polarized postmitotic animal cell is not well understood. In this work, we demonstrate that the Dynactin complex (a regulator of the microtubule motor protein Dynein) is required to maintain the position of the nucleus within post-mitotic Drosophila melanogaster photoreceptor neurons. We show that multiple independent disruptions of Dynactin function cause a relocation of the photoreceptor nucleus toward the brain, and that inhibiting Dynactin causes the photoreceptor to acquire a bipolar appearance with long leading and trailing processes. We find that while the minus-end directed motor Dynein cooperates with Dynactin in positioning the photoreceptor nucleus, the plus-end directed microtubule motor Kinesin acts antagonistically to Dynactin. These data suggest that the maintenance of photoreceptor nuclear position depends on a balance of plus-end and minus-end directed microtubule motor function.     

Genetic dissection of light-induced Ca2+ influx into Drosophila photoreceptors

Invertebrate photoreceptors use the inositol-lipid signaling cascade for phototransduction. A useful approach to dissect this pathway and its regulation has been provided by the isolation of Drosophila visual mutants. We measured extracellular changes of Ca2+ [delta Ca2+]o in Drosophila retina using Ca(2+)-selective microelectrodes in both the transient receptor potential (trp) mutant, in which the calcium permeability of the light-sensitive channels is greatly diminished and in the inactivation-but-no-afterpotential C (inaC) mutant which lacks photoreceptor-specific protein kinase C (PKC). Illumination induced a decrease in extracellular [Ca2+] with kinetics and magnitude that changed with light intensity. Compared to wild-type, the light-induced decrease in [Ca2+]o (the Ca2+ signal) was diminished in trp but significantly enhanced in inaC. The enhanced Ca2+ signal was diminished in the double mutant inaC;trp indicating that the effect of the trp mutation overrides the enhancement observed in the absence of eye-PKC. We suggest that the decrease in [Ca2+]o reflects light-induced Ca2+ influx into the photoreceptors and that the trp mutation blocks a large fraction of this Ca2+ influx, while the absence of eye specific PKC leads to enhancement of light-induced Ca2+ influx. This suggestion was supported by Ca2+ measurements in isolated ommatidia loaded with the fluorescent Ca2+ indicator, Ca Green-5N, which indicated an approximately threefold larger light-induced increase in cellular Ca2+ in inaC relative to WT. Our observations are consistent with the hypothesis that TRP is a light activated Ca2+ channel and that the increased Ca2+ influx observed in the absence of PKC is mediated mainly via the TRP channel.     

TRP channels in Drosophila photoreceptors: the lipid connection

The light-sensitive current in Drosophila photoreceptors is mediated by transient receptor potential (TRP) channels, at least two members of which (TRP and TRPL) are activated downstream of phospholipase C (PLC) in response to light. Recent evidence is reviewed suggesting that Drosophila TRP channels are activated by one or more lipid products of PLC activity: namely diacylglycerol (DAG), its metabolites (polyunsaturated fatty acids) or the reduction in phosphatidylinositol 4,5-bisphosphate (PIP₂). The most compelling evidence for this view comes from analysis of rdgA mutants which are unable to effectively metabolise DAG due to a defect in DAG kinase. The rdgA mutation leads to constitutive activation of both TRP and TRPL channels and dramatically increases sensitivity to light in hypomorphic mutations of PLC and G protein.     

Calcium influx through L-type channels is required for selective activation of extracellular signal-regulated kinase by gonadotropin-releasing hormone.

The hypothalamic decapeptide gonadotropin-releasing hormone stimulates mobilization of two discrete pools of calcium in clonal (alphaT3-1) and primary pituitary gonadotropes. A multidisciplinary approach was implemented to investigate the effects of discrete calcium fluctuations on the signaling pathways linking the gonadotropin-releasing hormone receptor to activation of mitogen-activated protein kinases and immediate early genes. Blockade of calcium influx through nifedipine-sensitive voltage-gated calcium channels reduced buserelin-induced activation of extracellular signal-regulated kinase (ERK) and c-Fos while activation of c-Jun N-terminal kinase and c-Jun was unaffected. Inhibition of buserelin-stimulated ERK activity by nifedipine was also observed in rat pituitary cells in primary culture. Direct activation of alphaT3-1 cell L-type calcium channels with the agonist Bay-K 8644 resulted in phosphorylation of ERK and induction of c-Fos. However, simple voltage-induced channel activation did not produce a sufficient calcium signal, since depolarization with 35 mM KCl failed to induce activation of ERK. Depletion of intracellular calcium stores with thapsigargin did not affect buserelin-induced ERK activation. An inhibitor of protein kinase C decreased calcium influx through nifedipine-sensitive calcium channels and phosphorylation of ERK induced by buserelin. Pharmacological inhibition of protein kinase C did not block Bay-K 8644-induced ERK activation. These observations suggest that calcium influx through L-type channels is required for GnRH-induced activation of ERK and c-Fos and that the influence of calcium lies downstream of protein kinase C.     

Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes

To estimate calpain proteolysis, we measured the hydrolysis rate of a fluorogenic calpain substrate in individual resting normal and dystrophic mdx mouse myotubes in culture. Hydrolysis rates were high during myoblast and myotube alignment and fusion. After alignment and fusion ceased, hydrolysis rates declined. For normal myotubes, hydrolysis remained low after the development of contractile activity. In contrast, after the development of contractile activity, dystrophic mdx myotubes had abnormally high levels of hydrolysis that were dependent on external calcium and that could be abolished by calpeptin, an inhibitor of calpain. We eliminated the direct effects of contraction during measurements of hydrolysis by the addition of tetrodotoxin. Substrate hydrolysis by lysosomes or proteosomes was controlled for using NH(4)Cl and clasto-lactacystin beta-lactone, respectively. Increased activity of the calcium-activated protease in mature mdx myotubes was linked to the abnormal activity of calcium-specific leak channels because an antagonist of these channels reduced the higher levels of hydrolysis in dystrophic myotubes to nearly normal levels. The abnormal activity of these channels is linked to an increased frequency of transient sarcolemmal disruptions in the more fragile mdx myotubes (, ). Treatment of mdx myotubes with a pro-drug of methylprednisolone also reduced calpain substrate hydrolysis to nearly normal levels. However, this inhibition only required 2.5 h of pretreatment, which was not long enough to act by the known effects of prednisolone on calcium homeostasis.     

Calcium-dependent inactivation of light-sensitive channels in Drosophila photoreceptors

Whole-cell voltage clamp recordings were made from photoreceptors of dissociated Drosophila ommatidia under conditions when the light- sensitive channels activate spontaneously, generating a "rundown current" (RDC). The Ca2+ and voltage dependence of the RDC was investigated by applying voltage steps (+80 to -100 mV) at a variety of extracellular Ca2+ concentrations (0-10 mM). In Ca(2+)-free Ringer large currents are maintained tonically throughout 50-ms-long voltage steps. In the presence of external Ca2+, hyperpolarizing steps elicit transient currents which inactivate increasingly rapidly as Ca2+ is raised. On depolarization inactivation is removed with a time constant of approximately 10 ms at +80 mV. The Ca(2+)-dependent inactivation is suppressed by 10 mM internal BAPTA, suggesting it requires Ca2+ influx. The inactivation is absent in the trp mutant, which lacks one class of Ca(2+)-selective, light-sensitive channel, but appears unaffected by the inaC mutant which lacks an eye-specific protein kinase C. Hyperpolarizing voltage steps applied during light responses in wild- type (WT) flies before rundown induce a rapid transient facilitation followed by slower inhibition. Both processes accelerate as Ca2+ is raised, but the time constant of inhibition (12 ms with 1.5 mM external Ca2+ at -60 mV) is approximately 10 times slower than that of the RDC inactivation. The Ca(2+)-mediated inhibition of the light response recovers in approximately 50-100 ms on depolarization, recovery being accelerated with higher external Ca2+. The Ca2+ and voltage dependence of the light-induced current is virtually eliminated in the trp mutant. In inaC, hyperpolarizing voltage steps induced transient currents which appeared similar to those in WT during early phases of the light response. However, 200 ms after the onset of light, the currents induced by voltage steps inactivated more rapidly with time constants similar to those of the RDC. It is suggested that the Ca(2+)-dependent inactivation of the light-sensitive channels first occurs at some concentration of Ca2+ not normally reached during the moderate illumination regimes used, but that the defect in inaC allows this level to be reached.     

Spontaneous activation of light-sensitive channels in Drosophila photoreceptors

In Drosophila photoreceptors light induces phosphoinositide hydrolysis and activation of Ca(2+)-permeable plasma membrane channels, one class of which is believed to be encoded by the trp gene. We have investigated the properties of the light-sensitive channels under conditions where they are activated independently of the transduction cascade. Whole-cell voltage clamp recordings were made from photoreceptors in a preparation of dissociated Drosophila ommatidia. Within a few minutes of establishing the whole-cell configuration, there is a massive spontaneous activation of cation-permeable channels. When clamped near resting potential, this "rundown current" (RDC) accelerates over several seconds, peaks, and then relaxes to a steady- state which lasts indefinitely (many minutes). The RDC is invariably associated with a reduction in sensitivity to light by at least 100- fold. The RDC has a similar absolute magnitude, reversal potential, and voltage dependence to the light-induced current, suggesting that it is mediated by the same channels. The RDC is almost completely (> or = 98%) blocked by La3+ (10-20 microM) and is absent, or reduced and altered in the trp mutant (which lacks a La(3+)-sensitive light- dependent Ca2+ channel), suggesting that it is largely mediated by the trp-dependent channels. Power spectra of the steady-state noise in the RDC can be fitted by simple Lorentzian functions consistent with random channel openings. The variance/mean ratio of the RDC noise suggests the underlying events (channels) have conductances of approximately 1.5-4.5 pS in wild-type (WT), but 12-30 pS in trp photoreceptors. Nevertheless, the power spectra of RDC noise in WT and trp are indistinguishable, in both cases being fitted by the sum of two Lorentzians with a major time constant (effective "mean channel open time") of 1-2 ms and a minor component at higher frequencies (approximately 0.2 ms). This implies that the noise in the WT RDC may actually be dominated by non-trp- dependent channels and that the trp-dependent channels may be of even lower unit conductance.     

PIP2 hydrolysis and calcium release are required for cytokinesis in Drosophila spermatocytes

The role of calcium (Ca(2+)) in cytokinesis is controversial, and the precise pathways that lead to its release during cleavage are not well understood. Ca(2+) is released from intracellular stores by binding of inositol trisphosphate (IP3) to the IP3 receptor (IP3R), yet no clear role in cytokinesis has been established for the precursor of IP3, phosphatidylinositol 4,5-bisphosphate (PIP2). Here, using transgenic flies expressing PLCdelta-PH-GFP, which specifically binds PIP2, we identify PIP2 in the plasma membrane and cleavage furrows of dividing Drosophila melanogaster spermatocytes, and we establish that this phospholipid is required for continued ingression but not for initiation of cytokinesis. In addition, by inhibiting phospholipase C, we show that PIP2 must be hydrolyzed to maintain cleavage furrow stability. Using an IP3R antagonist and a Ca(2+) chelator to examine the roles of IP3R and Ca(2+) in cytokinesis, we demonstrate that both of these factors are required for cleavage furrow stability, although Ca(2+) is dispensable for cleavage plane specification and initiation of furrowing. Strikingly, providing cells with Ca(2+) obviates the need to hydrolyze PIP2. Thus, PIP2, PIP2 hydrolysis, and Ca(2+) are required for the normal progression of cytokinesis in these cells.     

Kinesin II is required for cell survival and adherens junction positioning in Drosophila photoreceptors

Photoreceptor morphogenesis requires specific and coordinated localization of junctional markers at different stages of development. Here, we provide evidence that Drosophila Klp64D, a homolog of Kif3A motor subunit of the heterotrimeric Kinesin II complex, is essential for viability of developing photoreceptors and localization of junctional proteins. Genetic analysis of mutant clones shows that absence of Klp64D protein in early larval eye disc does not affect initial differentiation, but results in abnormal nuclear position in differentiating photoreceptors. These cells eventually die in the pupal stage, indicating klp64D's role in cell viability. The function of Klp64D protein is cell type specific because the p35 cell death inhibitor can rescue cell death in cone cells but not photoreceptors. In contrast to early induction of mutant clones, late induction during third instar larval stage just prior to pupation allows survival of single‐ or few‐celled clones of klp64D mutant cells. Analysis of these lately induced clones shows that Klp64D function is essential for Bazooka (Par‐3 homolog) and Armadillo localization to the adherens junction (AJ) in pupal photoreceptors. These findings suggest that Kinesin II complex plays a cell type‐specific function in the localization of AJ and cell polarity proteins in the developing retina, thereby contributing to photoreceptor morphogenesis. genesis 48:522–530, 2010. © 2010 Wiley‐Liss, Inc.     

Drosophila dumpy is a gigantic extracellular protein required to maintain tension at epidermal-cuticle attachment sites

BACKGROUND: Growth and morphogenesis during development depend both on patterning genes, which assign positional information, and on genes that regulate mechanical forces. The dumpy gene of the fruit fly Drosophila melanogaster is an example of the latter class, with mutant phenotypes affecting size and shape of the limbs, thoracic cuticle, trachea and mouthparts. RESULTS: The genetically complex dumpy locus was found to span over 100 kb and encode a gigantic 2.5 MDa extracellular matrix protein. Dumpy represents an extreme form of modular protein evolution, containing 308 epidermal growth factor (EGF) modules, interspersed with a new module class, DPY, and terminating in a crosslinking zona pellucida domain and membrane anchor sequence. We determined the three-dimensional structure of the DPY module by nuclear magnetic resonance (NMR) spectroscopy and found that it forms a disulphide-stabilised beta sheet motif, capable of linking end-to-end with EGF modules to form a fibre. Consistent with its cuticle phenotypes, dumpy is expressed at several sites of cuticle-epidermal cell attachment, including the trachea and the muscle tendon cells, which mediate anchorage of the muscles to the cuticle. CONCLUSIONS: The dumpy gene encodes a gigantic extracellular molecule that we predict to be a membrane-anchored fibre of almost a micrometer in length. Insertion and crosslinking of this fibre within the cuticle may provide a strong anchor for the underlying tissue, allowing it to maintain mechanical tension at sites under stress. This would explain its contribution to tissue morphogenesis through its regulation of mechanical properties.     

A novel PIP2-binding protein acts to enhance TRP channel activity

Commentary to: Kim, AY, Tang, Z, Liu Q et al. Pirt, a phosphoinositide-binding protein, functions as a regulatory subunit of TRPV1. Cell 2008; 133:475-85.

Commentary to: Suh BC, Hille B. PIP2 is a necessary cofactor for ion channel function: how and why? Annu Rev Biophys 2008; 37:175-95.     

Polymerase epsilon is required to maintain replicative senescence

Replicative senescence is a permanent cell cycle arrest in response to extensive telomere shortening. To understand the mechanisms behind a permanent arrest, we screened for factors affecting replicative senescence in budding yeast lacking telomere elongation pathways. Intriguingly, we found that DNA polymerase epsilon (Pol ε) acts synergistically with Exo1 nuclease to maintain replicative senescence. In contrast, the Pol ε-associated checkpoint and replication protein Mrc1 facilitates escape from senescence. To understand this paradox, in which DNA-synthesizing factors cooperate with DNA-degrading factors to maintain arrest, whereas a checkpoint protein opposes arrest, we analyzed the dynamics of double- and single-stranded DNA (ssDNA) at chromosome ends during senescence. We found evidence for cycles of DNA resection, followed by resynthesis. We propose that resection of the shortest telomere, activating a Rad24(Rad17)-dependent checkpoint pathway, alternates in time with an Mrc1-regulated Pol ε resynthesis of a short, double-stranded chromosome end, which in turn activates a Rad9(53BP1)-dependent checkpoint pathway. Therefore, instead of one type of DNA damage, different types (ssDNA and a double-strand break-like structure) alternate in a "vicious circle," each activating a different checkpoint sensor. Every time resection and resynthesis switches, a fresh signal initiates, thus preventing checkpoint adaptation and ensuring the permanent character of senescence.     

Calcium influx through If channels in rat ventricular myocytes

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I_f)/neuronal (I_h) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I_h channels in neurons. This raises the possibility of Ca²⁺ permeation in I_f, the I_h counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca²⁺ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I_f in rat ventricular myocytes. We observed Ca²⁺ influx when HCN/I_f channels were activated. Ca²⁺ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I_f channel blocker, inhibited I_f and Ca²⁺ influx at the same time. Quantitative analysis revealed that Ca²⁺ flux contributed to 0.5% of current produced by the HCN2 channel or I_f. The associated increase in Ca²⁺ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I_f current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca²⁺ chelator), preactivation of I_f channels significantly reduced the action potential duration, and the effect was blocked by another selective I_f channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I_f channels had no effects on action potential duration. Our data extend our previous discovery of Ca²⁺ influx in I_h channels in neurons to I_f channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels