Cheryl M. Craft

Rb regulates proliferation and rod photoreceptor development in the mouse retina.

The retinoblastoma protein (Rb) regulates proliferation, cell fate specification and differentiation in the developing central nervous system (CNS), but the role of Rb in the developing mouse retina has not been studied, because Rb-deficient embryos die before the retinas are fully formed. We combined several genetic approaches to explore the role of Rb in the mouse retina. During postnatal development, Rb is expressed in proliferating retinal progenitor cells and differentiating rod photoreceptors. In the absence of Rb, progenitor cells continue to divide, and rods do not mature. To determine whether Rb functions in these processes in a cell-autonomous manner, we used a replication-incompetent retrovirus encoding Cre recombinase to inactivate the Rb1lox allele in individual retinal progenitor cells in vivo. Combined with data from studies of conditional inactivation of Rb1 using a combination of Cre transgenic mouse lines, these results show that Rb is required in a cell-autonomous manner for appropriate exit from the cell cycle of retinal progenitor cells and for rod development.

Mouse Cones Require an Arrestin for Normal Inactivation of Phototransduction

Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation establishes that a visual arrestin is required for normal cone inactivation. Arrestin-independent inactivation is 70-fold more rapid in cones than in rods, however. Dual arrestin expression in cones could be a holdover from ancient genome duplication events that led to multiple isoforms of arrestin, allowing evolutionary specialization of one form while the other maintains the basic function.

Photoreceptors of Nrl -/- mice coexpress functional S- and M-cone opsins having distinct inactivation mechanisms.

The retinas of mice null for the neural retina leucine zipper transcription factor (Nrl −/ −) contain no rods but are populated instead with photoreceptors that on ultrastructural, histochemical, and molecular criteria appear cone like. To characterize these photoreceptors functionally, responses of single photoreceptors of Nrl −/ − mice were recorded with suction pipettes at 35–37°C and compared with the responses of rods of WT mice. Recordings were made either in the conventional manner, with the outer segment (OS) drawn into the pipette (“OS in”), or in a novel configuration with a portion of the inner segment drawn in (“OS out”). Nrl −/ − photoreceptor responses recorded in the OS-out configuration were much faster than those of WT rods: for dim-flash responses t peak = 91 ms vs. 215 ms; for saturating flashes, dominant recovery time constants, τD = 110 ms vs. 240 ms, respectively. Nrl −/ − photoreceptors in the OS-in configuration had reduced amplification, sensitivity, and slowed recovery kinetics, but the recording configuration had no effect on rod response properties, suggesting Nrl −/ − outer segments to be more susceptible to damage. Functional coexpression of two cone pigments in a single mammalian photoreceptor was established for the first time; the responses of every Nrl −/ − cell were driven by both the short-wave (S, λmax ≈ 360 nm) and the mid-wave (M, λmax ≈ 510 nm) mouse cone pigment; the apparent ratio of coexpressed M-pigment varied from 1:1 to 1:3,000 in a manner reflecting a dorso-ventral retinal position gradient. The role of the G-protein receptor kinase Grk1 in cone pigment inactivation was investigated in recordings from Nrl −/ −/Grk1 − / − photoreceptors. Dim-flash responses of cells driven by either the S- or the M-cone pigment were slowed 2.8-fold and 7.5-fold, respectively, in the absence of Grk1; the inactivation of the M-pigment response was much more seriously retarded. Thus, Grk1 is essential to normal inactivation of both S- and M-mouse cone opsins, but S-opsin has access to a relatively effective, Grk1-independent inactivation pathway.

GRK1-Dependent Phosphorylation of S and M Opsins and Their Binding to Cone Arrestin during Cone Phototransduction in the Mouse Retina

The shutoff mechanisms of the rod visual transduction cascade involve G-protein-coupled receptor (GPCR) kinase 1 (GRK1) phosphorylation of light-activated rhodopsin (R*) followed by rod arrestin binding. Deactivation of the cone phototransduction cascade in the mammalian retina is delineated poorly. In this study we sought to explore the potential mechanisms underlying the quenching of the phototransduction cascade in cone photoreceptors by using mouse models lacking rods and/or GRK1. Using the “pure-cone” retinas of the neural retina leucine zipper (Nrl) knock-out (KO, -/-) mice (Mears et al., 2001), we have demonstrated the light-dependent, multi-site phosphorylation of both S and M cone opsins by in situ phosphorylation and isoelectric focusing. Immunoprecipitation with affinity-purified polyclonal antibodies against either mouse cone arrestin (mCAR) or mouse S and M cone opsins revealed specific binding of mCAR to light-activated, phosphorylated cone opsins. To elucidate the potential role of GRK1 in cone opsin phosphorylation, we created Nrl and Grk1 double knock-out (Nrl-/-Grk1-/-) mice by crossing the Nrl-/- mice with Grk1-/- mice (Chen et al., 1999). We found that, in the retina of these mice, the light-activated cone opsins were neither phosphorylated nor bound with mCAR. Our results demonstrate, for the first time in a mammalian species, that cone opsins are phosphorylated and that CAR binds to phosphorylated cone opsins after light activation.

KASH protein Syne-2/Nesprin-2 and SUN proteins SUN1/2 mediate nuclear migration during mammalian retinal development

Nuclear movement relative to cell bodies is a fundamental process during certain aspects of mammalian retinal development. During the generation of photoreceptor cells in the cell division cycle, the nuclei of progenitors oscillate between the apical and basal surfaces of the neuroblastic layer (NBL). This process is termed interkinetic nuclear migration (INM). Furthermore, newly formed photoreceptor cells migrate and form the outer nuclear layer (ONL). In the current study, we demonstrated that a KASH domain-containing protein, Syne-2/Nesprin-2, as well as SUN domain-containing proteins, SUN1 and SUN2, play critical roles during INM and photoreceptor cell migration in the mouse retina. A deletion mutation of Syne-2/Nesprin-2 or double mutations of Sun1 and Sun2 caused severe reduction of the thickness of the ONL, mislocalization of photoreceptor nuclei and profound electrophysiological dysfunction of the retina characterized by a reduction of a- and b-wave amplitudes. We also provide evidence that Syne-2/Nesprin-2 forms complexes with either SUN1 or SUN2 at the nuclear envelope to connect the nucleus with dynein/dynactin and kinesin molecular motors during the nuclear migrations in the retina. These key retinal developmental signaling results will advance our understanding of the mechanism of nuclear migration in the mammalian retina.

Ultrashort pulsed electric fields induce membrane phospholipid translocation and caspase activation: differential sensitivities of Jurkat T lymphoblasts and rat glioma C6 cells

Megavolt-per-meter electric pulses with durations shorter than charging time constants associated with external cell membrane dielectric properties can generate significant voltages on the membranes of intracellular structures. Nanosecond-duration, high-field (2-4 MV/m) pulses are not immediately lethal to cells and do not produce the conductive openings in the cytoplasmic membrane associated with long-pulse, low-field electroporation, but can induce profound physiological changes, including apoptosis (programmed cell death). We demonstrate rapid, non-destructive, field-dependent translocation of the plasma membrane inner leaflet phospholipid phosphatidylserine in Jurkat T lymphocytes, and we show that cells which exhibit a similar geometry in suspension, rat glioma C6 cells, are highly resistant to these pulses and respond differently even to much higher doses.

Taurine deficiency is a cause of vigabatrin-induced retinal phototoxicity

Although vigabatrin irreversibly constricts the visual field, it remains a potent therapy for infantile spasms and a third‐line drug for refractory epilepsies. In albino animals, this drug induces a reduction in retinal cell function, retinal disorganization, and cone photoreceptor damage. The objective of this study was to investigate the light dependence of the vigabatrin‐elicited retinal toxicity and to screen for molecules preventing this secondary effect of vigabatrin.

Nanosecond pulsed electric fields perturb membrane phospholipids in T lymphoblasts.

Nanosecond, megavolt‐per‐meter pulsed electric fields scramble the asymmetric arrangement of phospholipids in cell membranes without the permeabilization associated with longer, lower‐field pulses. A single 30 ns, 2.5 MV/m pulse produces perturbations consistent with phosphatidylserine (PS) externalization in Jurkat T lymphoblasts within milliseconds, polarized in the direction of the applied field, indicating an immediate interaction between membrane components and the electric field. This disturbance occurs only at the anode pole of the cell, supporting the hypothesis that the pulsed field drives the negatively charged PS head group toward the positive electrode, directly providing the energy for crossing the membrane dielectric barrier.

Pulse generators for pulsed electric field exposure of biological cells and tissues

This paper describes three pulse generators: a spark gap switched coaxial cable, a spark gap switched Blumlein, and a solid state modulator, developed for applying ultrashort electrical pulses to biological materials in culture. Research has shown that ultrashort pulsed electric fields can induce apoptosis in biological cells, and that pulses as short as 10 ns with field amplitude greater than 1 W/m cause membrane phospholipid rearrangement and activation of the effector enzymes of apoptosis. Pulses of very short duration use only tens of mJ per mL per pulse to induce apoptosis and other intracellular effects without causing thermal trauma. The pulse generators discussed here, each of a different topology, deliver ns pulsed electric fields (nsPEF) to cells in liquid suspension, and can be modified to drive electrodes for external, surgical, or endoscopic treatment of tissues in situ.

The arrestin superfamily: cone arrestins are a fourth family

Arrestins constitute a superfamily of regulatory proteins that down‐regulate phosphorylated G‐protein membrane receptors, including rod and cone photoreceptors and adrenergic receptors. The potential role of arrestin in color visual processes led us to identify a cDNA encoding a cone‐like arrestin in Xenopus laevis, the principle amphibian biological model system. Alignment of 18 deduced amino acid sequences of all known arrestins from both invertebrate and vertebrate species reveals five arrestin families. Further analysis identifies 7 variable and 4 conservative arrestin structural motifs that may identify potential functional domains. The adaptive evolutionary relationship of Xenopus cone arrestin to the arrestin gene tree suggests high intrafamily homology and early gene duplication events.