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 proximity labeling-coupled label-free quantitative mass spectrometry (PL-LFQMS) approach, which enables NE membrane protein identification with unprecedented specificity in plants (News and Publications). We are now exploiting PL-LFQMS to profile the membrane protein landscape of the NE in model plant organism Arabidopsis. We aim to build the first and most comprehensive inventory of NE proteins in plants!
So far, we have identified more than a dozen new candidates for plant NE components using PL-LFQMS. The NE targeting of these candidates needs to be validated independently. We have amplified these genes from Arabidopsis and tagged them with yellow fluorescence protein (YFP). The student will be involved in transiently expression of the YFP fusion genes in tobacco leaves followed by fluorescence microscopy to observe the subcellular localization of these YFP fusion proteins. Due to public health ordinances, we will minimize the in-person parts of this project. It is a valuable experience in learning the heterologous transient protein expression system, which will be conducted in the lab while strictly adhering to guidelines from VCRO and CDC. The fluorescence microscopy can be trained and performed remotely in the Biological Imaging Facility at CNR thanks to the remote sampling and training protocols worked out by BIF staff in response to COVID-19.
In addition, the student will determine the mutant phenotypes of some NE candidate genes. We will order putative T-DNA insertion mutant lines for ABRC stock, and the student will learn remotely how to design primers for the amplification of T-DNAs from plant genomic DNA to confirm the mutant background. The genotyping process will be performed by lab essential personnel and showed to the students via video streaming. Confirmed mutant lines will be planted and the student will be scheduled to observe and record phenotypes of mutants comparing 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 critical roles of NE components in regulating plant development. The student will also learn how to read and analyze references as a starting point of subsequent functional analysis of NE genes identified in the mutant screen.
In the case of rising concerns about public health conditions, we will use a contingency plan and turn all the proposed in-person sessions to a remote mode, in which essential lab personnel will perform the experiments and video stream with the SPUR student to make sure the experimental principle and process can be understood by the student.
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 Fall 2020 class schedule are encouraged to apply.