Jillian Coffey

Live Imaging of Caveolae and FSH-R on Cell Membranes Using Atomic Force Microscopy and Confocal Microscopy

Follicle-stimulating hormone (FSH) and its receptor (FSHR) have important roles in reproductive endocrinology for both females and males. Visualizing live cells containing the human FSHR in conditions that mimic their native environment can create new insight on the cell membrane and its dynamic nature. Our current research took a new approach to look at the cell surface using Atomic Force Microscopy (AFM). AFM generates nanoscale maps of cell topography. HEK293 cells expressing hFSHR were grown on petri dishes and then analyzed by AFM. After generating the map, the next step will be to correlate structures on the cell surface with a fluorescent marker for caveolae. To make the fluorescent marker, the gene for the protein caveolin was fused to the gene for a red fluorescent protein. Details of this construction will be described. We will test the expression of this fluorescent protein by confocal microscopy and determine if it is located in caveolae with hFSHR. Through topographic images of live cells on the AFM and fluorescent tagging of caveolin for confocal microscopy, we can determine how follicle-stimulating hormone receptors and caveolin respond to the binding of FSH. These findings may have implications for fertility enhancement or contraception.

Chelsea Curtis

Activation of p-38 Map Kinase by Alternate G protein-Coupled Receptor Pathways

When human follicle stimulating hormone (hFSH) binds to its receptor (hFSHR) it signals through the Gs alpha protein leading to an increase in cyclic AMP (via adenylyl cyclase) which activates protein kinase A (PKA). In addition to this canonical pathway, activation of both the p38 and p44/42 (ERK1) MAP kinases has also been demonstrated in response to FSH. Previous research in our lab has demonstrated that when cells are treated with the cholesterol depleting agent methyl beta-cyclodextrin it blocks FSH stimulation of adenylyl cyclase but not the activation of p38 map kinase. Surprisingly, when cells were treated with H89, an inhibitor of PKA, FSH induced p38 activation was unaffected. These findings contradict findings of other researchers who have shown that p38 activation is PKA ependent. Our results suggest an alternate pathway of activation for p38 that is PKA independent. The identification of the regulators in this alternate pathway could lead to new therapies for regulating reproduction.

Lucas First

Extraction and purification methods of cordycepin from the pharmaceutical mushroom Cordyceps sinensis and a comparison of potency between the wild and cultivated species

For 3,000 years, Traditional Chinese Medicine (TCM) including herbal medicine, acupuncture and dietary therapy has been used to treat patients in East Asia. The herbal supplement Cordyceps sinensis (CS) or “Dong Chong Xia Cao” is a parasitic fungus used as an aid to the immune system and to improve lung and kidney function. This highly sought after supplement is now readily available through cultivation, while the wild type remains very expensive and difficult to purchase outside of China.
Our goal was to compare “wild” cordyceps from the Tibetan plateau to cultivated American cordyceps. The active ingredient is believed to be the adenine derivative cordycepin. Extraction of cordycepin from wild CS is an important first step in examining the medicinal implications of this compound. In this study, we optimized previous successful extraction methods and then used liquid chromatography tandem mass spectrometry (LC-MS) to identify and quantify cordycepin from the extract of Chinese Cordyceps. We applied the same analytical methods to the American cultivated CS. We predicted that the potency of the American cultivated CS would be lower than that of the wild species. Future studies will focus on accurately quantitating the difference in cordycepin levels between cultivated and native CS.

Johanna Ryan

Nucleosome Positioning in Yeast

DNA wraps around small proteins called histones to facilitate packing tightly into chromosomes. The complex of a length of DNA (usually ~140 bp) wrapped around the histone octamer is called a nucleosome. In addition to structural function, nucleosomes act as epigenetic repressors. The yeast gene STE6, an a-factor transporter, is expressed in a-haploids and repressed in alpha-haploids. In Saccharomyces cerevisiae, the inactive copy of STE6 in alpha-cells is known to have nucleosomes on its promoter which are absent in a-cells. We optimized an assay to clearly demonstrate the presence or absence of nucleosomes over a small region of DNA, then used the assay to determine if the same pattern found in S. cerevisiae holds in the promoter of an analogous gene in S. bayanus, a related species of yeast. Studying these mechanisms of epigenetic regulation can lead to better understanding of basic processes of gene transcription control.

Danielle Steinmetz

A Multifaceted Investigation of the Localization of the Human Follicle Stimulating Hormone Receptor

Follicle stimulating hormone (FSH) is an anterior pituitary glycoprotein hormone responsible for follicular maturation in females and production of sperm-stabilizing proteins in males. Follicle Stimulating Hormone Receptor (hFSHR) is a G-protein coupled receptor located in granulosa cells in the ovaries and Sertoli cells in the testes. It is believed that hFSHR is preferentially located and internalized in lipid raft microdomains of the plasma membrane. Lipid rafts are areas of the cell membrane that demonstrate increased rigidity, resistance to detergents, and have a high percent composition of cholesterol and sphingolipids. It is proposed that hFSHR is associated with a specific class of lipid rafts known as caveolae; hFSHR contains the caveolin binding motif -ΦXΦXXXXΦXXΦ- in its 4th transmembrane domain. To verify if hFSHR resides in lipid rafts, a sucrose gradient fractionation experiment was performed and analyzed by western blot. hFSHR was observed to reside in the fractions associated with buoyant membrane components as predicted. To determine if hFSHR interacts with caveolin, four mutant receptors were created by altering the putative caveolin binding motif. Understanding hFSHR localization in the plasma membrane will lead to a greater understanding of its signaling pathways and the molecular basis of reproduction.

Honorary Members:

  • Jordan Pereira

Practicum Students

  • Megan Dondarski ’14
  • Ayon Ibrahim ’13