Microtubule motors in mRNA localization:
The key to establishing polarity is to asymmetrically sort proteins to different regions of the cell. One way in which this is accomplished is via the process of mRNA localization. In this case, the mRNA that codes for a particular protein is specifically localized within the cell. Once localized, the mRNA is translated into protein. Thus, for these genes, the localization of the mRNA ultimately determines the localization of the protein. Recent studies have revealed that this mechanism of protein sorting is much more prevalent than initially thought.
The goal of our research is to determine the mechanism by which mRNAs that are destined for localization are identified by the cell and coupled to motor proteins for transport. The model organism that we use for these studies is Drosophila melanogaster (fruit fly). The Drosophila model provides us with the unique opportunity to study the process of mRNA localization in the context of the whole organism, rather than using isolated cells in culture. In addition, the wide array of genetic and molecular tools available for use with Drosophila will facilitate deeper and more rapid discovery.
In general, the mRNAs we study are transported on microtubules by motors such as Dynein and Kinesin (illustrated on the right). Microtubules are a type of cytoskeletal filament and can be thought of as the "roads" of our cells on which motor protein (cars) transport cargo. The example shown in this figure is mRNA cargo. However, proteins and even organelles can be transported by motors. Defects in motor based transport is associated with numerous neurological disorders.
oskar mRNA localization:
oskar mRNA (osk) is localized at the posterior pole of the Drosophila oocyte. As a consequence of this localization, Oskar protein is only found at the oocyte posterior. Oskar protein is required for establishing the anterior-posterior polarity of the oocyte and future embryo. If this process does not occur correctly, the oocyte will lack polarity and the resulting embryo will not survive.
Initial studies revealed that the localization of osk mRNA required microtubules and the plus-end directed microtubule motor, Kinesin-1. However, the adaptor that linked osk mRNA to Kinesin was not known. Using a proteomic purification strategy, we identified a novel isoform of Tropomyosin1 (referred to as Tm1C/I) as this unknown Kinesin adaptor. We used a genome editing strategy called CRISPR-Cas9 to specifically delete the Tm1C/I isoform from the Drosophila genome. In contrast to wild-type flies, osk mRNA was completely delocalized in flies that were null for Tm1C/I (adjacent figure) (publication).
A similar finding was published in a concurrent study by Imre Gáspár and Anne Ephrussi (link). Their study also examined the dynamics of osk mRNA in flies lacking Tm1C/I.
Somewhat surprisingly, another motor, Dynein, also co-localizes with osk mRNA at the posterior pole. This was apparent from studies published many years ago. However, whether Dynein was actively involved in the localization of osk mRNA was not known. For many years, this question could not be addressed because Dynein is required for specification of the oocyte. Mutants in Dynein components never specify an oocyte. Thus, the localization of osk mRNA, which takes places within the oocyte, could not be studied. We recently overcame this limitation using an inducible RNAi approach. Put simply, we were able to deplete Dynein after the oocyte was specified. These studies revealed that the efficient localization of osk mRNA requires Dynein in addition to Kinesin (publication). Our recent studies suggest that the adaptor protein, Egalitarian, is most likely responsible for linking Dynein to osk mRNA (publication).
Endocytosis:
Endocytosis is the process by which cargoes are internalized and sorted within the cell. In addition to mRNA localization, endocytic trafficking also contributes to the establishment of polarity. Our lab has recently shown that the Dynein motor complex performs an essential function in endocytic maturation. When Dynein is inhibited, the oocyte contains fewer yolk granules (YG; panel A vs B). Yolk granules are the end product of the endocytic pathway in oocytes. Instead, internalized cargo become trapped in enlarged endocytic vesicles (red circles in panels B and C) (publication). Our current efforts are focused on determining the precise mechanism by which Dynein functions in the endocytic pathway.
In order for cargo to be trafficked through the endocytic pathway, the membrane on endocytic vesicles has to become curved. This is a thermodynamically unfavorable process. Protein containing a domain known as a BAR domain are known to aid in this process. BAR domain proteins generate either membrane tubules or protrusions when over-expressed in cells. Our lab is currently studying the functions of a protein called Sh3px1. This protein contains a unique kind of BAR domain. When Sh3px1 is over-expressed in cells, it dramatically alters cell morphology. Some cells form long tubules (panel B), whereas other cells form long membrane protrusions (panel C)(publication). We are currently exploring the role of Sh3xp1 in development and tissue morphogenesis.
