The ER mediates some of the most fundamental processes within eukaryotic cells, including the biosynthesis of protein and lipid, folding and oligomerization of proteins, lipid detoxification, calcium regulation, nuclear envelope biogenesis and protein transport. We are using a variety of live cell imaging techniques (i.e., FRAP, FLIP, confocal time-lapse imaging, fluorescence protease protection, and photoactivation) combined with biochemical approaches to gain insight into the dynamic features of the ER that underlie its diverse functions. In one line of investigation, we are addressing whether proteins localized in the ER (e.g., misfolded proteins, protein translocation complexes, luminal markers and processing enzymes) are free to diffuse, immobilized to a scaffold or actively transported within the ER, and what conditions affect these dynamics.
In a second line of investigation, we are exploring what factors participate in the regulation of ER architecture and dynamics. Previous studies have implicated a variety of factors regulating organelle dynamics- including motor proteins to extend membrane elements out along the cytoskeleton, coat proteins for sorting and budding of membrane proteins into vesicles, and matrix proteins for tethering or stabilizing of membrane components. In our investigations of ER structure, we discovered an additional, unexpected mechanism for the regulation of ER architecture- that involving weak homotypic interactions between cytoplasmic domains of membrane proteins on apposing membranes (Snapp et al., J. Cell Biol. 2003). This mechanism was shown to be responsible for the dramatic remodeling of ER seen in response to elevated levels of specific resident proteins, in which the ER's branching tubule network transforms into a tightly stacked membrane array, termed organized smooth ER (OSER) (Fig. 1).
Fig. 1: OSER induction by overexpression of CytB5-GFP
The weak transient interactions between proteins on apposing membranes underlying OSER biogenesis (Fig. 2) provides a simple mechanism for the generation of these complex ER structures and could provide a general mechanism for the stack-like appearance of other organelles such as the Golgi apparatus, thylakoids in chloroplasts and the myelin sheath formed by Schwann cells around axons. This possibility is currently being explored by targeting dimerizing forms of GFP to the cytoplasmic tails of proteins residing in other organelles
Fig. 2: EM of OSER (S) near mitochondria (M)