Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Anatomy and Cell Biology

Supervisor

Lajoie, Patrick

Abstract

Pannexins (PANX) are a family of membrane glycoproteins comprised of 3 members: pannexin 1 (PANX1), pannexin 2 (PANX2), and pannexin 3 (PANX3). Oligomerized pannexin proteins form channels that allow for the passage of ions and small molecules. While PANX1 and PANX3 mostly localize to the cell surface, PANX2 remains intracellular within the endoplasmic reticulum (ER). Recently, a splice variant of PANX2 termed Panx2-202, was detected during development of murine skin. However, the localization and function of this shorter Panx2-202 isoform remains unknown. We found that Panx2-202 localizes to the ER in immortalized rat epidermal keratinocytes. We also found that expression of Panx2-202 in keratinocytes regulates mitochondria morphology. Collectively, we then hypothesized that Panx2-202 may regulate mitochondria morphology at the ER-mitochondria interface. We found that Panx2-202 colocalizes with split-GFP-based contact site sensor for the ER-mitochondria membrane. Lastly, Panx2 is a known substrate of caspase-3/7 cleavage at two residues in the C-terminal tail. These residues are present in Panx2-202, we presumed that the isoform also undergoes caspase-3/7 cleavage. Thus, we developed a novel tool to study C-terminal cleavage in living cells. Upon induction of apoptosis, the C-terminal tail of the Panx2-202 fused to a GFP tag containing a nuclear localization sequence was released from the ER membrane then trafficked to the nucleus. Overall, we defined the localization of Panx2-202 to the ER and identified a potential role in the regulation of mitochondria.

Summary for Lay Audience

Pannexins are a family of membrane proteins consisting of three members: pannexin 1 (PANX1), pannexin 2 (PANX2), and pannexin 3 (PANX3). Pannexins proteins form channels responsible for intracellular communication through the passage of small ions and molecules. These unique channels are characterized to play important roles in skin development as well as in various pathological diseases including a variety of cancers and osteoarthritis. The Panx2 gene encodes two versions of the Panx2 protein or what are called splice variants (PANX2 isoform 1 and PANX2 isoform 202). To date, no literature is available on the function of the isoform 202, or where it localizes within a cell. However, a recent study found expression of the Panx2 isoform 202 in the skin of developing mice. Therefore, we sought to define the localization and function of Panx2 isoform 202 in skin cells (keratinocytes). Using fluorescent fusion proteins, we were able to determine that Panx2 isoform 202 localizes to the endoplasmic reticulum (ER), an organelle responsible for protein synthesis and calcium regulation. In addition, when Panx2 isoform 202 is expressed in keratinocytes, we see a change in the shape of mitochondria. Lastly, we have preliminary data that suggests Panx2 isoform 202 may play a role in programmed cell death of keratinocytes. Collectively, we have defined a localization of Panx2 isoform 202 to the ER where it regulates mitochondria shape and have new data that suggests a potential role of this isoform in programmed cell death in the skin.

Download

Included in

Cell Biology Commons

Share

COinS