ABSTRACT
The signaling mechanisms controlling internal calcium release at fertilization in animals are still largely unknown. Echinoderms, such as the sea star Patiria miniata, produce abundant and easily accessible sperm and eggs. In addition, eggs are naturally synchronized at the same cell cycle stage, collectively making these animals an attractive model to study the signaling proteins controlling fertilization. However, the lack of antibodies to identify proteins in this model system has slowed progress in identifying key signaling molecules. With the advances in mass spectrometry, we present a method for identifying tyrosine phosphorylated proteins binding to GST-tagged SH2 domains in sea star cell lysates for downstream mass spectrometry analysis.
Subject(s)
Protein Interaction Mapping/methods , Protein Interaction Maps , Starfish/physiology , Animals , Blotting, Western/methods , Electrophoresis, Polyacrylamide Gel/methods , Female , Fertilization , Male , Oocytes/cytology , Oocytes/metabolism , Spermatozoa/cytology , Spermatozoa/metabolism , Starfish/metabolism , src Homology DomainsABSTRACT
To provide a better understanding of the composition of the egg cell membrane, we describe a method in which proteins and peptides that are either naturally released by the egg or cleaved by sperm proteases can be collected, analyzed, and identified. Such molecules are captured and isolated from the surrounding seawater via biotinylation, before being concentrated by an affinity interaction and subsequently analyzed by western blotting and mass spectrometry.
Subject(s)
Egg Proteins/metabolism , Membrane Proteins/metabolism , Starfish/physiology , Animals , Biotinylation , Blotting, Western/methods , Cell Culture Techniques/methods , Cells, Cultured , Egg Proteins/isolation & purification , Electrophoresis, Polyacrylamide Gel/methods , Female , Fertilization , Membrane Proteins/isolation & purification , Ovum/metabolismABSTRACT
The explosion in the amount of genomic data has changed how we educate future biologists. Students are inundated with information and need to develop skills in how to observe and understand this data. This chapter describes a teaching laboratory for undergraduate Genetics students that uses a combination of bioinformatics and wet lab exercises to teach skills in applying genomic data to a real scientific question. Students identify a target protein and search for the encoding RNA in newly available sea star transcriptome databases. The students design primers against specific regions or domains in their target RNA and amplify these by reverse transcription PCR. The PCR reactions are analyzed by agarose gel electrophoresis. The goal of this laboratory is to provide an example of how bioinformatics can be used to solve a real biological problem.
Subject(s)
Gene Expression/genetics , Starfish/genetics , Animals , Computational Biology/methods , Databases, Genetic , Genomics/methods , RNA/genetics , Students , Transcriptome/geneticsABSTRACT
The starfish oocyte and egg offer advantages for use as a model system for signal transduction research. Some of these have been recognized for over a century, including the ease of procuring gametes, in vitro fertilization, and culturing the embryos. New advances, particularly in genomics, have also opened up opportunities for the use of these animals. In this chapter, we give a few examples of the historical use of the starfish for research in cell biology and then describe some new areas in which we believe the starfish can contribute to our understanding of signal transduction-particularly in fertilization.
