Faculty Bibliography

In this commentary, Michelle Arbeitman et al., examine the topic of the Genetics of Sex as explored in this month's issues of GENETICS and G3: Genes|Genomes|Genetics. These inaugural articles are part of a joint Genetics of Sex collection (ongoing) in the GSA journals.

Background: Loss-of-function (LOF) mutations in FGFR1 are a frequent cause of congenital hypogonadotropic hypogonadism (CHH), a severe form of gonadotropin-releasing hormone (GnRH) deficiency, in males and females. They also predispose females to hypothalamic amenorrhea (HA), a milder and reversible form of GnRH deficiency associated with stress and/or energy deficits. FGF21 is an important metabolic regulator, which signals through a complex of FGFR1c with its co-receptor s-Klotho. Several lines of evidence support the hypothesis that mutations in KLB, which encodes s-Klotho, could also underlie CHH by compromising FGF21 signalling: 1) female Fgf21 transgenic(Tg) mice are resistant to high-fat diet and exhibit HH and infertility; 2) a CHH patient, obese with severe insulin-resistance carries a FGFR1 L342S mutation (1) that impairs FGF21 signalling in vitro. Methods: We screened 295 CHH patients for mutations in KLB and FGF21. The functionality of identified mutations were evaluated in vitro using cell-based reporter gene assays and expression assays, as well as in vivo using rescue experiments in C.elegans deficient of both worm KLB homologues. Klb deficient mice (Klb-/-) were evaluated for reproductive and metabolic phenotypes. Results: No mutations were identified in FGF21. We identified 9 heterozygous KLB mutations among 13/295 unrelated CHH patients (4%, 9 males and 4 females). Five patients harbor an identical KLB deletion (p.Phe777del) while the other mutations are missense. All mutations have a MAF<1% in EVS and 1000 genome database and are LOF in vitro and/or in vivo. Additional gene defects in CHH-associated genes were identified in 6/13 patients; these including 3 heterozygous FGFR1 mutations, consistent with an oligogenic model of inheritance. Notably, 10/13 subjects also exhibited metabolic defects, such as obesity, impaired fasting glucose, and/or severe dyslipidemia. Klb-/- mice are smaller in size than wild- type littermates. Female Klb-/- exhibit delayed sexual maturation and irregular estrous cycles, with reduced time spent in estrous. Further reproductive and metabolic phenotyping of the Klb-/-mice is underway. Conclusion: Loss-of-function KLB mutationsunderlie congenital GnRH deficiency. The delayed puberty phenotype of Klb-/- mice supports a role for KLB in reproduction. These findings highlight FGF21 as a probable important link between metabolism and reproduction

This interview chronicles the story of Sudhansu K. Dey in his journey from Calcutta, India to Kansas City, Kansas, establishing a research enterprise in the field of female reproduction. His research of over four decades has focused specifically on implantation biology using various model systems and reveling the impact of implantation on female reproductive medicine. This interview also reveals qualities of SK's character - his resolution, mentoring spirit, and humble nature - that contributed to his successes. SK is not shy to approach individuals for expertise or help, and in the same spirit, he is ready to offer his help to others irrespective of their positions or stature. He constantly attributes his success to the hard work of his laboratory members, the intellectual stimulation from his collaborators, and the support from his family. His ability to overcome challenges throughout his career is a reminder to students and junior investigators in the scientific community that each individual is endowed with talents and can accomplish their dreams if they pursue them. This interview tells the story of how he progressed from an inquisitive child to becoming a true servant to the cause of science and humankind.

Emerging data suggest that in polarized epithelial cells newly synthesized apical and basolateral plasma membrane proteins traffic through different endosomal compartments en route to the respective cell surface. However, direct evidence for trans-endosomal pathways of plasma membrane proteins is still missing and the mechanisms involved are poorly understood. Here, we imaged the entire biosynthetic route of rhodopsin-GFP, an apical marker in epithelial cells, synchronized through recombinant conditional aggregation domains, in live Madin-Darby canine kidney cells using spinning disk confocal microscopy. Our experiments directly demonstrate that rhodopsin-GFP traffics through apical recycling endosomes (AREs) that bear the small GTPase Rab11a before arriving at the apical membrane. Expression of dominant-negative Rab11a drastically reduced apical delivery of rhodopsin-GFP and caused its missorting to the basolateral membrane. Surprisingly, functional inhibition of dynamin-2 trapped rhodopsin-GFP at AREs and caused aberrant accumulation of coated vesicles on AREs, suggesting a previously unrecognized role for dynamin-2 in the scission of apical carrier vesicles from AREs. A second set of experiments, using a unique method to carry out total internal reflection fluorescence microscopy (TIRFM) from the apical side, allowed us to visualize the fusion of rhodopsin-GFP carrier vesicles, which occurred randomly all over the apical plasma membrane. Furthermore, two-color TIRFM showed that Rab11a-mCherry was present in rhodopsin-GFP carrier vesicles and was rapidly released upon fusion onset. Our results provide direct evidence for a role of AREs as a post-Golgi sorting hub in the biosynthetic route of polarized epithelia, with Rab11a regulating cargo sorting at AREs and carrier vesicle docking at the apical membrane.

In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips; including methods to perform electrical impedance spectroscopy; methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry; techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress; and methods to carry out high-resolution imaging of vesicular trafficking using light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers.

Accumulation of lipofuscin bisretinoids (LBs) in the retinal pigment epithelium (RPE) is the alleged cause of retinal degeneration in genetic blinding diseases (e.g., Stargardt) and a possible etiological agent for age-related macular degeneration. Currently, there are no approved treatments for these diseases; hence, agents that efficiently remove LBs from RPE would be valuable therapeutic candidates. Here, we show that beta cyclodextrins (beta-CDs) bind LBs and protect them against oxidation. Computer modeling and biochemical data are consistent with the encapsulation of the retinoid arms of LBs within the hydrophobic cavity of beta-CD. Importantly, beta-CD treatment reduced by 73% and 48% the LB content of RPE cell cultures and of eyecups obtained from Abca4-Rdh8 double knock-out (DKO) mice, respectively. Furthermore, intravitreal administration of beta-CDs reduced significantly the content of bisretinoids in the RPE of DKO animals. Thus, our results demonstrate the effectiveness of beta-CDs to complex and remove LB deposits from RPE cells and provide crucial data to develop novel prophylactic approaches for retinal disorders elicited by LBs.

Epithelial cells require apical-basal plasma membrane polarity to carry out crucial vectorial transport functions and cytoplasmic polarity to generate different cell progenies for tissue morphogenesis. The establishment and maintenance of a polarized epithelial cell with apical, basolateral and ciliary surface domains is guided by an epithelial polarity programme (EPP) that is controlled by a network of protein and lipid regulators. The EPP is organized in response to extracellular cues and is executed through the establishment of an apical-basal axis, intercellular junctions, epithelial-specific cytoskeletal rearrangements and a polarized trafficking machinery. Recent studies have provided insight into the interactions of the EPP with the polarized trafficking machinery and how these regulate epithelial polarization and depolarization.

The retinal pigment epithelium (RPE) comprises a monolayer of polarized pigmented epithelial cells that is strategically interposed between the neural retina and the fenestrated choroid capillaries. The RPE performs a variety of vectorial transport functions (water, ions, metabolites, nutrients and waste products) that regulate the composition of the subretinal space and support the functions of photoreceptors (PRs) and other cells in the neural retina. To this end, RPE cells display a polarized distribution of channels, transporters and receptors in their plasma membrane (PM) that is remarkably different from that found in conventional extra-ocular epithelia, e.g. intestine, kidney, and gall bladder. This characteristic PM protein polarity of RPE cells depends on the interplay of sorting signals in the RPE PM proteins and sorting mechanisms and biosynthetic/recycling trafficking routes in the RPE cell. Although considerable progress has been made in our understanding of the RPE trafficking machinery, most available data have been obtained from immortalized RPE cell lines that only partially maintain the RPE phenotype and by extrapolation of data obtained in the prototype Madin-Darby Canine Kidney (MDCK) cell line. The increasing availability of RPE cell cultures that more closely resemble the RPE in vivo together with the advent of advanced live imaging microscopy techniques provides a platform and an opportunity to rapidly expand our understanding of how polarized protein trafficking contributes to RPE PM polarity.

Most multicellular organisms show a physiological decline in immune function with age. However, little is known about the mechanisms underlying these changes. We examined Drosophila melanogaster, an important model for identifying genes affecting innate immunity and senescence, to explore the role of phagocytosis in age-related immune dysfunction. We characterized the localized response of immune cells at the dorsal vessel to bacterial infection in 1-week- and 5-week-old flies. We developed a quantitative phagocytosis assay for adult Drosophila and utilized this to characterize the effect of age on phagocytosis in transgenic and natural variant lines. We showed that genes necessary for bacterial engulfment in other contexts are also required in adult flies. We found that blood cells from young and old flies initially engulf bacteria equally well, while cells from older flies accumulate phagocytic vesicles and thus are less capable of destroying pathogens. Our results have broad implications for understanding how the breakdown in cellular processes influences immune function with age.