A contributing factor to a wide spectrum of diseases is compromised cell polarity. Cells establish polarity by localizing cargo to distinct cellular sites. Central to this process are the molecular motors and cytoskeletal filaments such as microtubules and actin. Motors are protein complexes that bind cargo, and as suggested by their name, utilize energy in the form of ATP hydrolysis to transport cargo to a particular site within the cell. A fundamental gap in our understanding of this process is the mechanism by which motors are able to recognize and bind their respective cargo in the complex and crowded environment of the cell. It is generally assumed that adaptor proteins, by binding directly to cargo and tethering cargo to motor complexes, fulfill this critical role. However, for most cargo that we know to be actively transported by microtubule motors, the identity of the adaptor is unknown. The goal of this proposal is to address this gap in knowledge using the Drosophila egg chamber as a model. The central hypothesis of this application is that Egalitarian (Egl) and Tropomyosin1C (Tm1C) are cargo adaptors for the Dynein and Kinesin1 motors respectively. Several mRNAs have been shown to localize within specific regions of the Drosophila oocyte. These include native transcripts such as oskar, bicoid and gurken as well as transposon mRNAs such as TAHRE and I factor.
In Aim1 of this application, we test the hypothesis that Egl links these mRNAs to Dynein. We also propose to determine the specific protein interactions that tether the Egl/mRNA complex to the Dynein motor. Over the past decade, several transposons have been identified that localize within the oocyte. In addition, localization elements from these transposons are bound by Egl, suggesting that they are linked to motor complexes via this adaptor.
In Aim2 of this application, we hypothesize that oocyte localization represents an adaptive property of transposons that facilitates their propagation to subsequent generations. We suggest that transposons have evolved mechanisms to highjack the host localization machinery and we propose a genome-wide analysis to test this hypothesis. The posterior localization of oskar mRNA requires Kinesin heavy chain (Khc), but not its canonical adaptor, Kinesin light chain (Klc). In the previous funding cycle, we demonstrated that a novel isoform of Tropomyosin1, referred to as Tm1C, fulfills this role as the Khc adaptor.
In Aim3 of this application, we propose to define the mechanism by which Tm1C functions in the oskar pathway. Furthermore, our results suggest that Tm1C is expressed and present in a complex with Khc in somatic tissues. We hypothesize that this adaptor links additional unknown cargoes with the Kinesin1 motor. Therefore, an additional goal of this aim is to identify these unknown somatic cargoes bound by Tm1C.
Defective cell polarity underlies numerous diseases such as cancer, kidney diseases, and several neurological disorders. A key factor for polarity establishment is the motor-driven localization of RNAs, proteins and vesicles to distinct sites within the cell. In order to develop better treatments for cell-polarity based diseases, it is important to gain a mechanistic understanding of how cells establish polarity, and the role of motors proteins in this process.
