W. J. Brad Hanna

Circadian photoreceptor organs inLimulus

SummaryThe circadian rhythm in the ERG amplitude of the lateral compound eye ofLimulus can be phase shifted either by general illumination or by illuminating combinations of the photoreceptor organs.1.For 15-min exposures, light confined to one lateral eye, or to the median ocelli, or to the ventral photoreceptor region resulted in the smallest phase shifts.2.Illuminating combinations of these organs produced larger shifts. The most effective combination tested included the median ocelli, the ventral photoreceptors, and one lateral eye. The phase shift resulting from illumination of this combination was only about one-half of the shift produced by general illumination.3.These results suggest that the circadian clock also receives light information from other, unidentified, photoreceptors located outside the prosoma.

Disrupted plasma membrane localization and loss of function reveal regions of human equilibrative nucleoside transporter 1 involved in structural integrity and activity.

Human Equilibrative Nucleoside Transporter 1 (hENT1) is an integral membrane protein that transports nucleosides and analog drugs across cellular membranes. Very little is known about intracellular processing and localization of hENT1. Here we show that disruption of a highly conserved triplet (PWN) near the N-terminus, or the last eight C-terminal residues (two hydrophobic triplets separated by a positive arginine) result in loss of plasma membrane localization and/or transport function. To understand the role of specific residues within these regions, we studied the localization patterns of N- or C-terminal deletion and/or substitution mutants of GFP-hENT1 using confocal microscopy. Quantification of GFP-hENT1 (mutant and wildtype) protein at the plasma membrane was conducted using nitrobenzylthioinosine (NBTI) binding. Functionality of the GFP-hENT1 mutants was determined by heterologous expression in Xenopus laevis oocytes followed by measurement of uridine uptake. Mutation of the proline within the PWN motif disrupts plasma membrane localization. C-terminal mutations (primarily within the hydrophobic triplets) lead to hENT1 retention within the cell (e.g. in the ER). Some mutants still localize to the plasma membrane but show reduced transport activity. These data suggest that these two regions contribute to the structural integrity and thus correct processing and function of hENT1.

Photoreceptor cells dissociated from the compound lateral eye of the horseshoe crab, Limulus polyphemus. I: Structure and ultrastructure

Isolated photoreceptors are desirable for whole-cell and patch-clamp studies of functional properties of visual processes that cannot be clearly analyzed when the photoreceptors are coupled. The retina of the compound lateral eye of the horseshoe crab, Limulus polyphemus, was dissociated into individual retinular cells using an enzyme pretreatment consisting of collagenase, papain, and trypsin, and a two-stage mechanical dissociation. These photoreceptors are functionally viable in an organ culture medium for up to 1 week and possess naked arhabdomeral and rhabdomeral segment membranes which are easily accessible for whole-cell recordings. A dissection technique was also developed whereby the retinal epidermis and neural plexus, as well as the second-order eccentric cells, could be separated from the ommatidia of the compound lateral eye in one simple step, providing viable isolated ommatidia attached to the cornea. The enzyme pretreatment used for dissociating the retina was then used to remove the individual ommatidia from the corneal cones. Hoffman modulation contrast microscopy was used to develop a reliable method for sorting and collecting viable isolated retinular cells for morphological and electrophysiological studies. Morphological analysis using light microscopy and scanning and transmission electron microscopy revealed that isolated retinular cells are morphologically nearly identical to retinular cells in situ. Isolated retinular cells possess a normal rhabdomere with no apparent loss of microvillar membrane as a result of the isolation process. Ommatidia can presently be isolated with up to six retinular cells possessing essentially normal structure and ultrastructure including thick rays of rhabdom. Isolated ommatidia possess naked A-segment membranes which are also well suited for whole-cell recording techniques.

Photoreceptor cells dissociated from the compound lateral eye of the horseshoe crab, Limulus polyphemus. II: Function

A combination of enzymatic digestions and mechanical disruption was used to isolate photoreceptor cells from the compound lateral eye of the horseshoe crab, Limulus polyphemus. The cells were maintained in a culture medium and tested for function using whole-cell and cell-attached patch configurations of the gigaseal technique. The cells dissociated from the eye generated spontaneous voltage and current bumps in the dark, and depolarized in a graded fashion to increasing intensities of light over several decades, producing responses similar to those of cells in vivo. Currents evoked during voltage clamp were similar to those in ventral photoreceptor cells of Limulus, although transient currents in the dark- and light-activated currents were smaller in isolated lateral eye cells, perhaps because of the slow speed and spatial nonuniformity of the clamp in these large cells. In addition to isolated cells, dissociation of the compound eye produced small clusters of cells and isolated ommatidia which were also tested for function. Comparison of the electrical characteristics of isolated cells with those of cells in small clusters and in their ommatidial matrix suggests that the electrical junctions normally connecting photoreceptor cells within an ommatidium are functional in the latter groups, but not in isolated cells. Cell-attached patches of rhabdomeral membrane of isolated cells contained light-activated channels, resembling those observed in ventral photoreceptor cells, but no voltage-activated channels. Similar patches of arhabdomeral membrane contained voltage-activated channels, but no light-activated channels. We conclude that this preparation is suitable for studies of processes involved in generating the light response in invertebrate photoreceptor cells.

Novel nuclear hENT2 isoforms regulate cell cycle progression via controlling nucleoside transport and nuclear reservoir

Nucleosides participate in many cellular processes and are the fundamental building blocks of nucleic acids. Nucleoside transporters translocate nucleosides across plasma membranes although the mechanism by which nucleos(t)ides are translocated into the nucleus during DNA replication is unknown. Here, we identify two novel functional splice variants of equilibrative nucleoside transporter 2 (ENT2), which are present at the nuclear envelope. Under proliferative conditions, these splice variants are up-regulated and recruit wild-type ENT2 to the nuclear envelope to translocate nucleosides into the nucleus for incorporation into DNA during replication. Reduced presence of hENT2 splice variants resulted in a dramatic decrease in cell proliferation and dysregulation of cell cycle due to a lower incorporation of nucleotides into DNA. Our findings support a novel model of nucleoside compartmentalisation at the nuclear envelope and translocation into the nucleus through hENT2 and its variants, which are essential for effective DNA synthesis and cell proliferation.