Amber Krauchunas

Education

• ​B.A. - Mount Holyoke College
• Ph.D - Cornell University
• Postdoctoral - Rutgers University

Research Interests

For all sexually reproducing species, the creation of a new organism requires the successful joining of an egg and sperm and the subsequent initiation of a developmental program within the egg. These events depend on 1) the activation of sperm to make them competent for fertilization, 2) signaling, recognition, binding, and fusion between the sperm and egg, and 3) activation of the egg to transition from a highly differentiated cell to a totipotent one-cell embryo capable of supporting development. A detailed understanding of fertilization and gamete activation will require knowing the full complement of molecules that act in these processes and evidence suggests that we are far from knowing the full repertoire of relevant molecules. Using C. elegans as a model, my lab uses a combined genetic and cell biology approach to investigate the molecular underpinnings of fertilization and gamete activation. Through this work, and complementary studies in other organisms, the identification of important molecules will no longer be a limiting factor in our knowledge of gamete activation and fertilization. In addition, both sperm and egg activation inform how coordinated, global cellular changes and cellular reprogramming can take place in the absence of transcription.

Current Projects

Sperm Activation

One of the challenges to understanding sperm activation in C. elegans is that many of the genes discovered thus far lack any identifiable protein domains. The spe-43 gene, which I characterized as a postdoc, encodes a protein with a single predicted transmembrane domain and a DX domain. The DX domain is defined by 6 conserved cysteine residues but has not been ascribed a specific function in SPE-43 or any other protein. There are seven other C. elegans genes predicted to encode DX domain containing proteins. Interestingly, expression of all of these genes is sperm-enriched or sperm-specific based on RNA-seq and microarray studies done by others (Ortiz et al., 2014; Reinke et al., 2004). Six of the seven are also predicted to encode single-pass transmembrane proteins, like spe-43. However, no phenotypic data has been reported for these genes. Projects include structure/function analysis of SPE-43 and determination of SPE-43 localization and studying the other DX domain containing genes to determine their role in sperm development, activation, and/or function.

Fertilization

At present we know of 10 sperm genes specifically required for fertilization in C. elegans. These discoveries have led us to propose the model of a "fertilization synapse" where fertilization is mediated by complex cis and trans interactions of proteins at the surface of the gametes rather than any single ligand-receptor pair (Krauchunas et al., 2016). Most recently, my identification of the SPE-36 protein as a secreted protein has broadened the types of proteins that contribute to the fertilization synapse. Interestingly, SPE-36 acts cell autonomously despite being secreted, suggesting interactions with other proteins keeps it localized to the sperm cell that produced it. Current projects are working towards visualizing SPE-36 localization with the aim of testing which other sperm genes are required for SPE-36 secretion and proper localization. Determining the relative localization and interdependence of these 10 sperm genes will be important in continuing to build the model of the fertilization synapse.

Egg Activation

Suppressor screens can be a powerful and targeted tool for the identification of interacting components in a pathway. We have a temperature-sensitive allele of egg-3 that now allows us to carry out a suppressor screen to specifically search for additional egg activation regulators/effectors. The egg-3 gene encodes a pseudophosphatase that is central to the execution of multiple events of egg activation in C. elegans and likely has roles beyond what has been initially characterized. Proteomic experiments will compliment the genetic screen to identify proteins that interact with EGG-3. Traditional pull-downs and proximity-labeling methods will be used to identify proteins that differentially interact with the wild type EGG-3, mutated EGG-3 at the restrictive temperature, and mutated EGG-3 at the permissive temperature. These sets of proteins will inform future hypotheses regarding the mechanisms of EGG-3 action at egg activation and critical changes of the egg cellular state to support development.