Imaging Platforms for Measurement of Membrane Trafficking

Abstract

In this chapter we describe automated imaging methods used to measure the transport of an established membrane transport marker from the endoplasmic reticulum to the plasma membrane. The method is fast and significantly robust to be applied in systematic studies on a large scale such as genome‐wide screening projects. We further describe the use of software macros and plugins in Image J that allow the quantification of the kinetics of membrane transport intermediates in fluorescence microscopy time‐lapse sequences.

Introduction

Membrane traffic enables cells to distribute proteins, lipids, and carbohydrates between membrane compartments and is thus vital for many cellular processes such as cell growth, homeostasis, and differentiation. Genetic and biochemical approaches have been very efficiently applied in the past to identify and characterize individual molecular components involved in the regulation of membrane traffic in the secretory and endocytic pathways. Central to many of these studies has been the reconstitution of the particular transport step of interest in vitro using purified components. Although this has led to an enormous body of information on how membrane traffic is organized at the molecular level, such simplified in vitro systems are lacking important regulatory elements relating to the spatial organization that occurs in living cells. More recently, systematic approaches, such as organelle proteomics or yeast two hybrid screening, have attempted to identify structural and regulatory components of membrane traffic with the goal of reaching a more complete description of its molecular regulation (see for example Bell 2001, Calero 2002, Monier 2002). However, despite their great potential, these techniques have limitations, not least of which is their lack of demonstrating a functional involvement of the molecules identified in the particular trafficking step under investigation.

Functional microscope‐based assays in intact living cells with the potential for large‐scale analyses have been recently developed and applied to problems in membrane trafficking (Ghosh et al., 2000; Liebel 2003, Pelkmans 2005, Starkuviene 2004). These techniques provide single cell or even subcellular resolution. They promise, in combination with genome‐wide RNA interference (RNAi, Elbashir et al., 2001) or over‐expression strategies (Starkuviene et al., 2004), to help to reveal comprehensively the regulatory networks underlying membrane traffic in intact cells. Here we describe such assays used to quantitatively monitor secretory transport to the plasma membrane and the subcellular kinetics of transport carriers in time‐lapse series.