Submitted by Yangnan Gu on
The nuclear envelope (NE) represents the hallmark of the eukaryotic cell. It evolved not only as a protective membrane for our heritable genetic material – DNA, but also a critical communication interface to transmit signals from the cell periphery to the genome. The NE harbors versatile proteins that perform essential cellular functions, including genome organization, signal transduction, cargo transport, etc. A myriad of genetic disorders in humans have been intimately linked with mutations in genes encoding NE constituents, and the spectrum of resulting diseases (e.g. muscle dystrophy, progeria – premature aging), collectively known as nuclear envelopathies, continues to expand. Despite the emerging role of the NE as a critical structural and signaling platform in eukaryotes, comprehensive knowledge of the composition and function of the NE is largely unknown in plants, which has greatly hampered our understanding of roles of the NE in key aspects of plant physiology.
Available evidence indicates that the plant NE proteome is largely distinct from animals and fungi. The extreme divergence of integral NE components demands direct approaches for identification. We have recently developed a subtractive proteomics approach, which enables NE membrane protein identification with unprecedented specificity in plants (News and Publications). We aim to build the first and most comprehensive inventory of NE proteins in plants!
We have identified more than a dozen new candidates for plant NE components using subtractive proteomics. The NE targeting of these candidates needs to be validated using fluorescence microscopy. The student will be involved in amplifying these genes from Arabidopsis and tagging them with yellow fluorescence protein (YFP). They will then make transiently expression of the YFP fusion genes in tobacco leaves followed by fluorescence microscopy to observe the subcellular localization of these YFP fusion proteins. The students will also transform those fusion constructs into Arabidopsis and generate transgenic plants for phenotype characterization and fluorescence imaging.
In addition, the student will determine the mutant phenotypes of these NE candidate genes. We will use CRISPR/Cas9 technology to knock out these NE genes in Arabidopsis and confirm their genotype using PCR and sequencing. Confirmed mutant lines will be planted and the student will be scheduled to observe and record phenotypes of mutants compared to wild-type plants once a week in the lab. Severe developmental defects in some mutants are expected due to the critical role of animal/yeast NE proteins in determining a variety of key cellular outcomes, such as survival and cell fate. This phenotype screen will potentially uncover the critical roles of NE components in regulating plant development. The student will also learn how to read and analyze references as a starting point for subsequent functional analysis of NE genes identified in the mutant screen.
Students with strong interests in plant biology, cell biology, and genetics will find the experience most rewarding. Excellent attention to detail and good record-keeping skills are necessary. Students with a flexible Spring 2023 class schedule are encouraged to apply.