Materials Research Science and Engineering Center

Amber Jones

"Determining the Relationship Between Nervous Wreck and the Cell Membrane at Different Phosphoinositide Levels"

Amber B. Jones *, Steven J. DelSignore, Avital A. Rodal

Abstract

Nervous Wreck (Nwk), a neuronal F-Bar protein, plays a specific role in cell membrane curvature and deformation. The cell membrane contains these lipid components, Phosphoinositides (PIP), that have the capability of being phosphorylated or dephosphorylated by lipid kinases and phosphatases respectively. When phosphorylated, the cell membrane becomes more negative, and this negative charge recruits an electrostatic interaction between Nwk and the cell membrane.

Despite knowing this specific information, there is not much known on how Nwk gets off of the cell membrane. In order to answer this question, PIP2 levels were manipulated to make the cell membrane more or less negative in different experimental assays. One assay deals with purifying the phosphatase domain of OCRL and targeting a purified cell membrane with purified Nwk. The other methods include using Fluorescence Recovery After Photobleaching (FRAP) in larval neurons and S2 cells in order to determine the dynamics of the relationship between Nervous Wreck’s ability to bind to the cell membrane when exposed to different PIP concentrations.

When doing the phosphatase purification, the expression of the protein in E. Coli resulted in the protein remaining insoluble after trying two different protocols. Supplemental purification attempts are underway, using yeast rather than E. Coli, because of yeast’s potential success.

In a preliminary FRAP experiment, there was a localization of endogenously tagged Nwk in the synaptic bouton of the larval neuron. Nearly four minutes after Nwk was photobleached, there was hardly any recovery to original fluorescence, which suggests that the Nwk is tightly assembled to the cell membrane, and recovery would be limited. The original S2 cell experiment resulted in Nwk remaining on the cell membrane and the phosphatase remaining cytoplasmic.

In order to further continue this project, there is the plan to use the acquired information to develop an optogenetic tool for the lab’s use to alter membrane composition. By understanding the dynamics of this relationship, there is the potential to be able to engineer biological systems in such a way that can control the activation and deactivation of membrane deformation, and understand Nwk during in vivo processes like neuron growth.

Support: MRSEC REU