Lumen formation within unicellular, seamless tubes is essential in the development and function of the cardiovascular system. Small tubes, such as terminal vascular bed capillaries within the microcirculation, often form lumens by intracellular vesicle coalescence and membrane fusion with the leading edge of an invading apical domain. My long-term goal is to determine how polarized vesicle trafficking is regulated to ensure proper cell hollowing and tube formation in vivo. I will use the unicellular C. elegans excretory canal as a simple system to study this process, as it offers powerful genetic and cell biological tools, and has proven to utilize pathways conserved during vascular development and disease. Defects in nascent vessel lumen expansion lead to numerous vascular disorders, including myocardial infarction and stroke, and a molecular understanding of how lumens expand during vascular development and disease remains elusive. I anticipate that my findings will provide important insights to better understand how seamless capillaries within the vasculature are formed, with the hope of improving cardiovascular disease intervention. PAR proteins are conserved regulators of cell polarity that contribute to diverse cellular processes. In the excretory canal, PARs localize to the luminal membrane, where they co-localize with the vesicle tethering exocyst complex. Our lab recently showed that exocyst is required for vesicle fusion during lumen formation, and that PARs can induce asymmetry of exocyst proteins in embryos. Based on these findings, I hypothesize that PARs define where the lumen will form by recruiting exocyst and directing vesicle fusion to these sites. Using a method to acutely deplete proteins in specific cells developed in our lab, I will test this hypothesis in vivo by removing PAR and exocyst function in the canal.
The specific aims of my proposal are to: 1) Test the hypothesis that PAR proteins are required for lumen formation and/or maintenance; 2) Determine if the exocyst complex functions downstream of the PAR complex to mediate luminal vesicle recruitment; 3) Identify novel genes required to distinguish luminal from non-luminal surfaces in seamless tubes. First, I will generate conditional loss-of-function alleles for PARs to deplete their function in the canal and determine their role during lumenogenesis and exocyst recruitment. I will also use a conditional loss-of-function strategy to eliminate a core exocyst component, SEC-5, from the canal to determine its epistasis with respect to PARs by evaluating PAR localization. Finally, I will test candidate genes and undertake a genetic screen to uncover new genes required for luminal PAR and exocyst localization. My findings will greatly expand our understanding of the role for polarity cues and vesicle trafficking during cell hollowing. Understanding this process directly relates to many aspects of human health, including recovery from cardiovascular injury and ischemic disease. Thus the results of my studies will provide new insights into how nascent vessel growth can be restored as a means of improving current therapies of cardiovascular disease.
The proper formation of new blood vessels is critical for cardiovascular function and recovery from vascular injury and disease, such as in myocardial infarction and stroke. Without a complete molecular understanding of how nascent blood vessels form, effective therapies to re-vascularize diseased tissue have been limited. The aim of this research is to harness the genetics and cell biological tools in C. elegans to uncover determinants of tubule formation and lumen hollowing, which may provide an avenue for new therapeutic targets of cardiovascular injury and ischemic disease. !
