The recycling endosome (RE) is a nexus for membrane transport in both polarized and nonpolarized cells. In polarized cells, endocytic traffic originating on both the apical and basolateral surfaces passes through the recycling endosome. Basolateral secretory traffic and retrograde traffic bacterial toxins and endogenous TGN38/46 transit the RE. Additionally, the RE acts as a storehouse for both membrane and receptors. Up to 70% of constitutively recycled receptors, such as the transferrin receptor, are located in the RE. Similarly, the neuronal AMPA receptor is stored in the RE for recruitment during long term potentiation. The RE is a convergence point for all of these pathways, and so faces a major challenge: how to rapidly and correctly sort traffic along each pathway. It has been assumed that sorting is accomplished through selective inclusion into transport vesicles. Indeed, sorting of (tyrosine based) basolateral traffic through interaction with the clathrin adapter ?1-B has been directly demonstrated. However, we observe that fidelity of basolateral sorting at the RE exceeds 95%, an unusually high figure for a single sorting step. We have observed that the RE is divided into apical and basolateral subdomains that precede incorporation into transport vesicles. We have also observed that these subdomains, as well as retrograde traffic to the TGN are dependent upon the function of PI 3-Kinase. We hypothesize that phosphatidylinositol 3-phosphates regulate membrane traffic and sorting within the recycling endosome. We will aim to 1: Determine which phosphatidylinositol 3-phosphates are active in the recycling endosome. We will test for the presence of these inositol lipids through direct biochemical identification, as well as through fluorescent biological probes. We will further use functional studies of membrane traffic to examine the effects of pharmacological disruption of these lipids at the RE. We will use rapalog targeting of a 3-phosphatase to the RE to directly link phosphatidylinositol 3-phosphates to RE traffic. 2: Examine how phosphatidylinositol 3-phosphate binding EHD proteins are involved in retrograde through the RE. We will determine if EHD proteins are directly involved in retrograde RE exit to the TGN 3: Determine if signaling through phosphatidylinositol 3-phosphate pathways can occur from receptors transiting the RE. We will use insulin receptor as a model growth receptor and take advantage of the recycling itinerary of the insulin receptor to target activated kinase receptor to the RE. We will artificially recruit AKT to the RE using rapalog constructs to examine if signaling can potentially be activated at the RE.
As epithelial cells become cancerous, they lose their normal polarized morphologies. They also lose regulation governing cell growth and begin to multiply rapidly. We have demonstrated that the recycling endosome is the critical organelle for sorting of apical from basolateral traffic, and is thus the key site for maintaining cell polarity. We now present preliminary data suggesting that a major cell signaling pathway, the PI3 Kinase pathway may be active at the recycling endosome as well. We will investigate whether PI3 Kinase signaling is activated in the recycling endosome. This work will provide a mechanistic link between the loss of cell polarity and loss of regulation over growth in cancer cells. Gaining this knowledge will allow development of new therapies and diagnostic assays which target the recycling endosome in precancerous and cancerous cells.
| McKenzie, Jenna E; Raisley, Brent; Zhou, Xin et al. (2012) Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi. Traffic 13:1140-59 |
