G protein activation is prerequisite for functional coupling between Galpha/Gbetagamma and tubulin/microtubules

Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. Interestingly, recent results suggest that G proteins also interact with microtubules and participate in cell division and differentiation. It has been shown earlier that both alpha and betagamma subunits of G proteins modulate microtubule assembly in vitro. Since G protein activation and subsequent dissociation of alpha and betagamma subunits are necessary for G proteins to participate in signaling processes, here we asked if similar activation is required for modulation of microtubule assembly by G proteins. We reconstituted Galphabetagamma heterotrimer from myristoylated-Galpha and prenylated-Gbetagamma, and found that the heterotrimer blocks Gi1alpha activation of tubulin GTPase and inhibits the ability of Gbeta1gamma2 to promote in vitro microtubule assembly. Results suggest that G protein activation is required for functional coupling between Galpha/Gbetagamma and tubulin/microtubules, and supports the notion that regulation of microtubules is an integral component of G protein mediated signaling.     

Glc7-Reg1 phosphatase signals to Yck1,2 casein kinase 1 to regulate transport activity and glucose-induced inactivation of Saccharomyces maltose permease

The Saccharomyces casein kinase 1 isoforms encoded by the essential gene pair YCK1 and YCK2 control cell growth and morphogenesis and are linked to the endocytosis of several membrane proteins. Here we define roles for the Yck1,2 kinases in Mal61p maltose permease activation and trafficking, using a yck1delta yck2-2(ts) (yck(ts)) strain with conditional Yck activity. Moreover, we provide evidence that Glc7-Reg1 phosphatase acts as an upstream activator of Yck1,2 kinases in a novel signaling pathway that modulates kinase activity in response to carbon source availability. The yck(ts) strain exhibits significantly reduced maltose transport activity despite apparently normal levels and cell surface localization of maltose permease protein. Glucose-induced internalization and rapid loss of maltose transport activity of Mal61/HAp-GFP are not observed in the yck(ts) strain and maltose permease proteolysis is blocked. We show that a reg1delta mutant exhibits a phenotype remarkably similar to that conferred by yck(ts). The reg1delta phenotype is not enhanced in the yck(ts) reg1delta double mutant and is suppressed by increased Yck1,2p dosage. Further, although Yck2p localization and abundance do not change in the reg1delta mutant, Yck1,2 kinase activity, as assayed by glucose-induced HXT1 expression and Mth1 repressor stability, is substantially reduced in the reg1delta strain.     

Live cell detection of caspase-3 activation by a Discosoma-red-fluorescent-protein-based fluorescence resonance energy transfer construct

A probe consisting of Discosoma red fluorescent protein (DsRed) and enhanced yellow fluorescent protein (EYFP) linked by a 19-amino-acid chain containing the caspase-3 cleavage site Asp-Glu-Val-Asp was developed to monitor caspase-3 activation in living cells. The expression of the tandem construct in mammalian cells yielded a strong red fluorescence when excited with 450- to 490-nm light or with a 488-nm argon ion laser line as a result of fluorescence resonance energy transfer (FRET) from donor EYFP to acceptor DsRed. The advantage over previous constructs using cyan fluorescent protein is that our construct can be used when excitation wavelengths lower than 488nm are not available. To validate the construct, murine HT-22 hippocampal neuronal cells were triggered to undergo CD95-induced neuronal death. An increase in caspase-3 activity was demonstrated by a reduction of FRET in cells transfected with the construct. This was manifested by a dequenching of EYFP fluorescence leading to an increase in EYFP emission and a corresponding decrease in DsRed fluorescence, which correlated with an increase in pro-caspase-3 processing. We conclude that CD95-induced caspase-3 activation in HT-22 cells was readily detected at the single-cell level using the DsRed-EYFP-based FRET construct, making this a useful technology to monitor caspase-3 activity in living cells.     

Phenotypic anchoring of arsenic and cadmium toxicity in three hepatic-related cell systems reveals compound- and cell-specific selective up-regulation of stress protein expression: implications for fingerprint profiling of cytotoxicity

Exposure of cells to toxic chemicals is known to up-regulate the expression of a number of stress proteins (SPs), including metallothionein (MT) and members of the heat shock protein (HSP) family, and this response may allow the development of a fingerprint profile to identify mechanisms of toxicity in an in vitro toxicology setting. To test this hypothesis, three hepatic-derived cell culture systems (rat hepatoma FGC4 cell line, rat hepatocytes, human hepatoma HepG2 cell line) were exposed to cadmium (as CdCl2) and arsenic (as NaAsO2), two compounds believed to exert their toxicity through an oxidative stress mechanism, under conditions of phenotypic anchoring defined as minimal and mild toxicity (approximately 5 and 25% reduction in neutral red uptake, respectively). The expression of six SPs--MT, HSP25/27, HSP40, HSP60, HSP70, and HSP90--was then determined by ELISA. Expression of four of these SPs--MT, HSP25/27, HSP40 and HSP70--was up-regulated in at least one experimental condition. However, the patterns of expression of these four SPs varied across the experimental conditions, according to differences in toxicant concentration and/or level of toxicity, cell-type and toxicant itself. This lack of uniformity in response of a focussed set of mechanistically defensible targets suggests that similar problems may emerge when using more global approaches based on genomics and proteomics, in which problems of redundancy in targets and uncertain mechanistic relevance will be greater.     

PRENATAL GENETIC TESTING KITS SOLD AT YOUR LOCAL PHARMACY: PROMOTING AUTONOMY OR PROMOTING CONFUSION?

Research groups around the world are developing non‐invasive methods of prenatal genetic diagnosis, in which foetal cells are obtained by maternal blood test. Meanwhile, an increasing number of genetic tests are sold directly to the public. I extrapolate from these developments to consider a scenario in which PNGD self‐testing kits are sold directly to the public. Given the opposition to over‐the‐counter genetic tests and the continuing controversy surrounding PNGD, it is reasonable to expect objections to PNGD self‐testing kits. I focus on one potential objection, that PNGD self‐testing kits would undermine the autonomy of potential test subjects. More specifically, that ‘direct to the public’ PNGD would fail to ensure that consumers exercise autonomy in the following PNGD‐related choices:

• ? Should I use PNGD?
• ? Based on the results of the PNGD test, should I continue or terminate my pregnancy?

Under the current system, PNGD is provided by health care practitioners, who are required to counsel women both before and after the test. In contrast, ‘direct to the public’ PNGD would allow women to make their PNGD‐related decisions outside the context of the health care system. I compare these two decision‐making contexts, arguing that the health care system is not unequivocally better at promoting the autonomy of potential test subjects. Therefore the promotion of autonomy does not constitute a strong argument against such test kits. Other objections may be more persuasive, so I do not offer an overall assessment of the acceptability of ‘direct to the public’ PNGD.     

Molecular basis of inherited skin-blistering disorders, and therapeutic implications

Epidermolysis bullosa (EB) and associated skin-fragility syndromes are a group of inherited skin diseases characterised by trauma-induced blistering of the skin and mucous membranes. Mutations in at least 14 distinct genes encoding molecular components of the epidermis or the dermal-epidermal junction (DEJ) can cause blistering skin diseases that differ by clinical presentation and severity of the symptoms. Despite great advances in discerning the genetic basis of this group of diseases, the molecular pathways leading to symptoms are not yet fully understood. Unravelling these pathways by molecular analysis of the structure and in vitro assessment of functional properties of the human proteins involved, combined with genetic models in lower organisms, should pave the way for specific cures for inherited skin fragility.     

The competent sentinel node: an association with an axillary presentation and an occult or a small primary invasive breast carcinoma

The concept of the sentinel node describes a primary or sentinel lymph node (SLN), which exists and through which tumour cells from a primary tumour in a particular location must first travel to spread to a particular regional lymph node group. In this series we present three patients presenting with a pathological axillary node associated with either an occult or very small primary breast cancer. In each case the primary tumour was found to have metastasised to the palpable node, however despite the significant enlargement of this node, no other axillary nodes were found to be affected on axillary node clearance. This has led us to postulate that the SLN in some cases contains unique characteristics that enable it to prevent further spread of the tumour up the lymphatic chain. Hence the term the competent sentinel node.     

An ultrastructural readout of fluorescence recovery after photobleaching using correlative light and electron microscopy

Fluorescence recovery after photobleaching (FRAP) provides an important quantitative readout of the mobility of fluorescently tagged structures in live tissue. Here we present a protocol for visualizing FRAP signal at the ultrastructural level, permitting the nature of recovered fluorescence signal to be studied at greater resolution than afforded by conventional light microscopy. Specifically we use FRAP, fixation, photoconversion and correlative light and electron microscopy (CLEM) to examine the ultrastructural organization of mobile FM1-43-labeled vesicles in synapses of cultured hippocampal neurons. At photobleached synapses, the FRAP signal can be visualized as photoconverted electron-dense vesicles. The combination of FRAP and CLEM provides a powerful tool for examining the specific localization of imported vesicles in relation to synaptic architecture. Moreover, with the increasing availability of photoconvertible fluorophores, this approach should be readily applicable to other systems where an ultrastructural characterization of FRAP signal is desirable. After cultures are prepared and ready to use, this protocol takes 2-3 days.     

Use of time-lapse imaging and dominant negative receptors to dissect the steroid receptor control of neuronal remodeling in Drosophila

During metamorphosis, the reorganization of the nervous system of Drosophila melanogaster proceeds in part through remodeling of larval neurons. In this study, we used in-vitro imaging techniques and immunocytochemistry to track the remodeling of the thoracic ventral neurosecretory cells. Axons of these neurons prune their larval arbors early in metamorphosis and a larger, more extensive adult arbor is established via branch outgrowth. Expression of EcR dominant negative constructs and an EcR inverted repeat construct resulted in pruning defects of larval axon arbors and a lack of filopodia during pruning, but showed variable effects on outgrowth depending on the construct expressed. Cells expressing either UAS-EcR-B1(W650A) or UAS-EcR-A(W650A) lacked filopodia during the outgrowth period and formed a poorly branched, larval-like arbor in the adult. Cells expressing UAS-EcR-B1(F645A), UAS-EcR-B2(W650A) or UAS-IR-EcR (core) showed moderate filopodial activity and normal, albeit reduced, adult-like branching during outgrowth. These results are consistent with the role of activation versus derepression via EcR for successive phases of neuronal remodeling and suggest that functional ecdysone receptor is necessary for some, but not all, remodeling events.