The development of NA⁺-dependent hexose transport in LLC-PK₁: a model for epithelial differentiation

Na+-dependent hexose transport, a secondary active transport system found in the small intestine and the proximal tubule of the kidney was used as a marker for epithelial differentiation in LLC-PK1, a cell line isolated from juvenile pig kidney by R. N. Hull. LLC-PK1 acquires Na^+-dependent hexose transport at confluence, when approximately 90% of the cells are quiescent, and transport levels increase continuously over time in culture. The appearance of this transport system can be stimulated by phosphodiesterase inhibitors, such as theophylline and MIX, and inhibited by TPA, a powerful tumor promoter. When the cells differentiate, the population acquires an increased capacity for Na+-dependent hexose transport, without a change in the rate constant for uptake. Several proposed mechanisms which could account for the observed increase in hexose transport levels were examined. The first possibility was that the efflux pathway for α-meG, a nonmetabolizable glucose analog specific for Na+ cotransport, declined over time in culture. Efflux was first order, with a rate constant of 0.48 hr-1, and was not altered by phlorizin, a competitive inhibitor of this transport system, or by cytochalasin B, an inhibitor of the facilitated diffusion transporter. Therefore, it was concluded that the primary pathway for efflux of α-meG in this cell line was via passive diffusion. The transporter was shown to be reversible. When ouabain was added to discharge the Na+ electrical gradient, the efflux rate constant increased to 0.65 h-1. This component could be transstimulated by adding a high concentration of nonradioactive extracellular α-meG and was inhibited to control levels by phlorizin. The rate constant for efflux remained the same at all levels of hexose transport, indicating that this was not the mechanism responsible for the development of Na+ cotransport capacity by these cells. Several mechanisms involving an increase in the influx component of the accumulation process were examined. An increase in the Na+ electrochemical gradient was ruled out by data from Kurt Amsler and Carolyn Shaffer, who showed that the gradient actually declined at confluence. The development of gap junctions between transporting and nontransporting cells might also generate an increased capacity for hexose transport but these cells had no gap junctions when examined by fluorescein dye transfer, electrical coupling and freeze fracture analysis. A method of separating transporting from nontransporting cells by density centrifugation in Percoll was developed in order to study the remaining two mechan-isms: an increase in the number of transporters per cell, and a progressive recruitment of nontransporting cells to a transporting population. Cells were incubated in Na-gluconate medium containing no K+, Cl-, or phosphate in order to inhibit volume regulation. In this medium, cells were unable to accumulate &alpha-meG. Attempts made to restore the electrical gradient by adding NaCl or NaCl plus bicarbonate were unsuccessful. When 2 mM (NH4)2SO4, which is transported by the Na,K-ATPase in place of K+, was added, Na+ cotransport levels were restored, indicating that the maintenance of the Na+ gradient was necessary for transport. In this medium, cells that took up α-meG were less dense and banded at a lighter density in Percoll than control cells. Phlorizin inhibited the movement of these cells on the gradient. A mixture of transporting and nontransporting cells could only be separated in the presence of &aplha;-meG. A timecourse of the development of Na+-dependent hexose transport conducted over a 20-day period showed a development of a transporting peak at a lighter density which increased in size over time while the nontransporting peak declined. Although it appeared that the cells were recruited gradually to a differentiating population, the radioactive α-meG curve was skewed to the left, which indicated that it took several days for the recruited cells to acquire all their transporters and reach their full capacity for α-meG transport. The question of whether this cell line undergoes terminal differentiation was also examined. Fractions of cells from two Percoll gradients—with and without α-meG—were replated under sterile conditions. Plating efficiencies and ability to incorporate thymidine were the same for transporting and nontransporting cells. Therefore, these cells do not undergo irreversible loss of growth potential as a prerequisite for differentiation. However, this experiment was only carried out for two cell cycles. Therefore, it does not rule out the possibility that these cells undergo terminal differentiation

Sox2 -Deficient Müller Glia Disrupt the Structural and Functional Maturation of the Mammalian Retina

Müller glia (MG), the principal glial cells of the vertebrate retina, display quiescent progenitor cell characteristics. They express key progenitor markers, including the high mobility group box transcription factor SOX2 and maintain a progenitor-like morphology. In the embryonic and mature central nervous system, SOX2 maintains neural stem cell identity. However, its function in committed Müller glia has yet to be determined

The Effect of Carboxyl-terminal Mutagenesis of G on Rhodopsin and Guanine Nucleotide Binding

The carboxyl terminus of G protein alpha subunits plays an important role in receptor recognition. To identify the amino acids that participate in this interaction, COOH-terminal mutants of alpha t (the transducin alpha subunit) were expressed in vitro and analyzed for their ability to interact with rhodopsin and to bind guanine nucleotide. Gly-348, the reported site of a beta turn, was replaced with other neutral amino acids without severely affecting rhodopsin binding. However, proline substitution abolished rhodopsin interaction, suggesting that flexibility is important at this site. A comparison between C347Y, which lost both rhodopsin and guanine nucleotide binding, and a mutant substituted with alpha q sequence (D346E/C347Y/G348N/F350V), in which guanine nucleotide binding was restored, implies that distinct motifs maintain the structure of the alpha subunit and are necessary for selective interaction with receptors. Surprisingly, mutants L344A, L349A, F350stop, and stop351A demonstrated a parallel loss of rhodopsin and guanine nucleotide binding. Altered profiles of L344A and F350stop on sucrose density gradients indicate that these mutants may undergo denaturation. The equivalent of alpha tL344A generated in alpha s and alpha i did not show such a severe loss of guanine nucleotide binding, revealing that the alpha t carboxyl terminus is unique in its susceptibility to changes in amino acid sequence

Employee Financial Wellness Programs Project: Comprehensive Report of Findings

Using insights from employers and employees to generate evidence on employee financial wellness programs (EFWPs), this research report illustrates findings from a mixed-methods study assessing the potential of these programs to increase the financial stability of American workers. The research team surveyed employers that offered or were interested in offering an EFWP and subsequently conducted in-depth interviews with a subsample of those employers to acquire a greater understanding of survey responses. Further, the research team conducted intensive case studies, examining the relationship between EFWP providers and their clients as well as the dynamics of program delivery. These case studies benefitted from administrative data on employees’ participation in their employer’s EFWP. Lastly, as part of a module within the Household Financial Survey of the Refund to Savings Initiative, the research team gathered individual-level survey data from low- and moderate-income employees to understand their interests in and experiences with EFWPs

Phosphorylation of GRK7 by PKA in cone photoreceptor cells is regulated by light

The retina specific G protein-coupled receptor kinases, GRK1 and GRK7, have been implicated in the shutoff of the photoresponse and adaptation to changing light conditions via rod and cone opsin phosphorylation. Recently, we have defined sites of phosphorylation by cAMP-dependent protein kinase (PKA) in the amino termini of both GRK1 and GRK7 in vitro. To determine the conditions under which GRK7 is phosphorylated in vivo, we have generated an antibody that recognizes GRK7 phosphorylated on Ser-36, the PKA phosphorylation site. Using this phospho-specific antibody, we have shown that GRK7 is phosphorylated in vivo and is located in the cone inner and outer segments of mammalian, amphibian and fish retinas. Using Xenopus laevis as a model, GRK7 is phosphorylated under dark-adapted conditions, but becomes dephosphorylated when the animals are exposed to light. The conservation of phosphorylation at Ser-36 in GRK7 in these different species (which span a 400 million-year evolutionary period), and its light-dependent regulation, indicate that phosphorylation plays an important role in the function of GRK7. Our work demonstrates for the first time that cAMP can regulate proteins involved in the photoresponse in cones and introduces a novel mode of regulation for the retinal GRKs by PKA

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals

Rhodopsin mutants discriminate sites important for the activation of rhodopsin kinase and Gt

The cytoplasmic loops of rhodopsin, the rod cell photoreceptor, play important regulatory roles in the activation of both rhodopsin kinase and the rod cell G protein, Gt. A number of studies have identified domains in rhodopsin that are important for the activation of Gt. However, less is known concerning the cytoplasmic regions that regulate phosphorylation of the photoreceptor by rhodopsin kinase. To identify regions that participate in these processes, a series of alanine mutations were generated in the three cytoplasmic loops of rhodopsin and transiently expressed in HEK-293 cells. Membranes prepared from these cells were reconstituted with the opsin chromophore, 11-cis-retinal, and characterized for their ability to undergo light-dependent phosphorylation by rhodopsin kinase and to catalyze GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) binding to Gt. We have identified mutants that fall into three distinct categories: 1) those that show altered phosphorylation but normal Gt activation, such as T62A/V63A/Q64A and R147A/F148A/G149A in Loops I and II, respectively; 2) mutants that have reduced ability to activate Gt but are phosphorylated normally, including T242A/T243A and V250A/T251A/R252A in Loop III; and 3) mutants that affect both phosphorylation and Gt activation, including A233G/A234G/A235G and A233N/A234N/A235N in Loop III. The use of these two assays in parallel have allowed us to distinguish the presence of distinct functional domains within the cytoplasmic loops which are specific for interaction with rhodopsin kinase or Gt

Rhodopsin Phosphorylation Sites and Their Role in Arrestin Binding

Rhodopsin, the rod cell photoreceptor, undergoes rapid desensitization upon exposure to light, resulting in uncoupling of the receptor from its G protein, transducin (Gt). Phosphorylation of serine and threonine residues located in the COOH terminus of rhodopsin is the first step in this process, followed by the binding of arrestin. In this study, a series of mutants was generated in which these COOH-terminal phosphorylation substrate sites were substituted with alanines. These mutants were expressed in HEK-293 cells and analyzed for their ability to be phosphorylated by rhodopsin kinase and to bind arrestin. The results demonstrate that rhodopsin kinase can efficiently phosphorylate other serine and threonine residues in the absence of the sites reported to be the preferred substrates for rhodopsin kinase. A correlation was observed between the level of rhodopsin phosphorylation and the amount of arrestin binding to these mutants. However, mutants T340A and S343A demonstrated a significant reduction in arrestin binding even though the level of phosphorylation was similar to that of wild-type rhodopsin. Substitution of Thr-340 and Ser-343 with glutamic acid residues (T340E and S343E, respectively) was not sufficient to promote the binding of arrestin in the absence of phosphorylation by rhodopsin kinase. When S343E was phosphorylated, its ability to bind arrestin was similar to that of wild-type rhodopsin. Surprisingly, arrestin binding to phosphorylated T340E did not increase to the level observed for wild-type rhodopsin. These results suggest that 2 amino acids, Thr-340 and Ser-343, play important but distinct roles in promoting the binding of arrestin to rhodopsin

Phosphorylation of GRK1 and GRK7 by cAMP-dependent Protein Kinase Attenuates Their Enzymatic Activities

Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K(m) and V(max) of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser(21) and GRK7 at Ser(23) and Ser(36) in vitro. These sites are also phosphorylated when FLAG-tagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca(2+)/calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases

Reevaluating the evidence for a Hadean-Eoarchean dynamo.

The time of origin of the geodynamo has important implications for the thermal evolution of the planetary interior and the habitability of early Earth. It has been proposed that detrital zircon grains from Jack Hills, Western Australia, provide evidence for an active geodynamo as early as 4.2 billion years (Ga) ago. However, our combined paleomagnetic, geochemical, and mineralogical studies on Jack Hills zircons indicate that most have poor magnetic recording properties and secondary magnetization carriers that postdate the formation of the zircons. Therefore, the existence of the geodynamo before 3.5 Ga ago remains unknown