Using light to see and control membrane traffic

Cellular compartmentalization into discrete organelles is maintained by membrane trafficking including vesiculation and tubulation. Recent advances in superresolution imaging have begun to bring these small and dynamic events into focus. Most nanoscopes exploit, and are limited by, switching dyes ON and OFF. Using ground state depletion to switch dyes into long-lived dark states can exploit specific photophysical properties of dyes, such as redox potential or pK_a, and expand the repertoire of nanoscopy probes for multicolor imaging. Seeing is not enough, and new technologies based on homodimerization, heterodimerization and selective release can manipulate membrane trafficking in pulse-chase and light-controlled ways. Herein we highlight the utility and promise of these strategies and discuss their current limitations.

Introduction

A textbook model, or single snapshot, of a cell's compartments can appear deceivingly simple, with static, spatially restricted organelles of distinct composition. But cells are abuzz with activity, shuttling membranous vesicles between organelles in a process called membrane trafficking. This can be remarkably fast. Neurotransmission and hormone secretion occurs in milliseconds to seconds and extracellular nutrients can traffic through multiple endocytic organelles in minutes. The ability to actually see these processes in action using genetically encoded fluorescent proteins has profoundly affected the membrane trafficking field where fleeting transient intermediate states such as internalization of a receptor can now be seen and studied [1]. This in turn can inform trafficking defects in a panoply of diseases including microbial pathogenesis, diabetes, immune dysfunction and cancer.

History shows that understanding of the inner workings of the cell has been intimately tied with technological advances. Ultracentrifugation made it possible to separate organelles, electron microscopy gave spatial context, and molecular biology made it possible to readily manipulate proteins, especially in genetically tractable model systems such as yeast, C. elegans, and Drosophila. Innovations have continued to drive modern biological science, exemplified with Nobel prizes for RNA interference (RNAi) (2006) and green fluorescent protein (2008). Today powerful imaging approaches are accessible to most of the cell biology community – there are genetically encoded probes to tag nearly any protein of interest in multiple colors, knockdown strategies to functionally replace endogenous proteins with tagged variants, and image-based high-content assays to screen genomic or chemical libraries in living cells [2, 3]. But as powerful as these methods are, their shortcomings embolden new ‘what if’ questions. What if we can see at spatial resolution approaching electron microscopy, but in live cells? What if we can functionally knockdown (or displace) proteins not in days, but in seconds? What if we can control precisely in space where these interactions occur and in turn use this to control cellular processes? These are not just fanciful wishes. New developments in seeing and manipulating cells have spawned a surge of technological innovations that will probably converge and further accelerate biological discovery.

Herein, we highlight some of the advances in the last few years that have the potential to transform studies of membrane trafficking and cell dynamics. As a point of reference, in the last decade the ability of total internal reflection fluorescence microscopy (TIRFM) to selectively illuminate the cell surface has enabled hundreds of studies of exocytosis and endocytosis (see [4] and references therein). If successful, new ways of imaging cells with higher resolution and acutely controlling them should also have a major scientific impact. There are entire subdisciplines whereby if one could ‘merely’ see the process with high-enough spatial resolution, models that have been debated for decades could be directly proven. Do forward moving vesicles travel between cisternae of the Golgi, or not? Are there lipid rafts microdomains, and if so what is their size and composition? What is the spatial–temporal ordering of signal transduction processes that are typically shown as a web of arrows? Seeing will help, but controlling these interactions should be even more powerful.