Suchergebnisse

During interphase of cycling cells, the surface of the nuclear envelope (NE), consisting of inner and outer nuclear membrane (I/ONM) fused at each nuclear pore complex (NPC) approximately doubles. This organelle growth requires homeostatic synthesis and delivery of lipids and proteins to maintain a fully functional NE and prepare for the next nuclear division. INM proteins reside specifically in the INM and carry out several essential functions. They constitute a heterogeneous class of transmembrane proteins and are delivered by the probably least understood cellular membrane trafficking pathway. How targeting of the different classes of INM proteins is achieved and how many trafficking and regulatory mechanisms exist is currently not well understood. One reason is that unlike for other trafficking pathways such as membrane secretion, it has so far been impossible to visualize INMP trafficking in live cells. To address this point in the first part of my project I have developed a novel INMP trafficking reporter system (named Target-INM) that allows the acute release of INM proteins from the ER to the INM. The system is based on trapping INM proteins in the ER with a cleavable retention domain that can be removed by acute activation of a protease. I applied this generic reporter strategy to a set of INM proteins (LBR, Lap2beta, Tor1AIP1, Man1 and Sun1) that represent the major transmembrane protein classes and could image and quantify their synchronous delivery from the ER to the INM in interphase with high spatial and temporal resolution. Exploiting this assay, I screened by siRNA knockdown and automated high resolution confocal time-lapse microscopy 96 candidate genes for their requirement in LBR targeting. These genes include nucleoporins, importins, lamins as well as NE and ER membrane proteins. I identified several genes that affect LBR INM targeting. Together with Antonio Politi, a postdoc in the lab, I developed a mathematical model of the INM protein targeting process that I used to fit the kinetic signatures of the different transport phenotypes and cluster the scoring genes into three major phenotypic classes providing evidences for the basic principle governing INM protein targeting. Comparing the genetic requirements for targeting of the different INMP protein classes should now put us in a position to define the number of molecularly distinct trafficking pathways from the ER to the INM.