The formation of transport vesicles at the endoplasmic reticulum (ER) is a generic phenomenon in eucaryotic cells. Due to the transient, sequential, and energy-dependent binding of peripheral membrane proteins (COPII) and the subsequent assembly of COPII complexes the ER membrane is deformed and a cargo-loaded bud/vesicle emerges. As of yet, the spatiotemporal aspects underlying the assembly of COPII-coated buds are as poorly explored as the concomitant emergence of 'ER exit sites' (ERES) of which ~100 copies are typically observed in mammalian cells.
The project aims at elucidating the dynamic aspects of COPII vesicle formation and ERES morphogenesis. To this end, fluorescently tagged proteins/lipids will be monitored with advanced light microscopy (e.g. fluorescence correlation spectroscopy) on the (almost) single molecule level in vitro and in vivo. Here, we will use giant unilamellar vesicles of dedicated lipid composition and purified COPII proteins; we will compare our insights to observations in transiently/stably transfected cell lines. Special attention will be given to the scaffolding role of lipids and the influence of non-equilibrium events. The obtained quantitative data on the binding kinetics of COPII proteins, the diffusion and interactions of lipids/proteins under various conditions, and the overall change in membrane morphology will be used to formulate and computationally implement a predictive mesoscopic model that unifies the experimental findings and yields a deeper understanding of the spatiotemporal aspects of COPII vesicle and ERES formation.
Eucaryotic cells are highly compartmentalized by membrane-engulfed organelles but little is known about the fundamental self-organization principles that govern their dynamic biogenesis. The endoplasmic reticulum (ER) and the Golgi apparatus (GA) are prominent examples of highly dynamic organelles with an intriguing morphological complexity: while the ER is a reticular network of membranes, the GA consists of a stack of flattened membrane cisternae. Both organelles disintegrate at least in part before cell division and are re-established afterwards to form the secretory pathway where cargo proteins (e.g. morphogens) are post-translationally modified. Indeed, the transport of biosynthetic cargo from the ER to the GA via membrane-engulfed intermediates (e.g. vesicles) is the primary cause for the emergence of a GA. Yet, how the concerted action of lipids and proteins drives the biogenesis of the GA has remained elusive: Why/how does a GA form de novo (e.g. after mitosis), and how does it dynamically attain and maintain its stack structure? To address these fundamental questions of organelle biogenesis, we will use a Systems Biology approach that combines experimental and theoretical/computational techniques. Using advanced light microscopy, we will quantify the binding kinetics and transport coefficients of proteins, lipids, and larger structures (e.g. transport intermediates) in different cell lines (polarized/unpolarized) and in vitro. To highlight various facets of GA biogenesis, cells will be perturbed by drugs, interfering RNA, or by removing parts of the GA via micro-surgery. Using giant unilamellar vesicles (GUVs) made from artificial lipids and/or cell-derived material with purified cytoplasm/proteins, we will complement the in vivo data and aim at constructing an in vitro secretory pathway, e.g. by GA transplantation into a GUV. Based on the quantitative experimental data and fundamental physical and mathematical principles, we will formulate a predictive, computational model for the self-organization of the GA that triggers a cycle between experiment and theory.