Using light to see and control membrane traffic
Highlights
► GSDIM exploits dyes’ metastable dark states and is compatible with organic dyes. ► Chemical or light pulse-chase methods enable non-intrusive study of membrane traffic. ► Optogenetic approaches use light to control cellular machinery in space and time.
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.
Section snippets
Seeing better using dyes’ dark side
The laws of diffraction have limited the resolution of light microscopes to approximately half the wavelength of light or ∼0.2 μm [5]. But many objects in cells are much smaller. Cisternae of the Golgi are arranged like a stack of pancakes spaced <100 nm apart and synapses are packed with ∼30 nm vesicles. One-way nanoscopes can improve the resolving power by an order of magnitude is by exploiting dye switching between bright and dark states [6] (see Figure 1). In one technique, called stochastic
Imaging pulse-chase experiments
Pulse-chase experiments can reveal dynamics that are hard, if not impossible to obtain by viewing the ensemble state. In this regard, a variety of protein tagging strategies can provide direct control over the biology of the membrane traffic to affect a pulse. For example, by tagging cargo with homodimerization domains (e.g. F36M [32] and more specific recent variants [33]), the cargo can be forced to aggregate in the ER. Subsequent addition of a cell permeable drug will release the cargo as a
Controlling protein interaction with drugs and light
Nanoscopes can show in much finer spatial detail where (and in live cells when) molecules are localized. While a specific localization may implicate a given cellular function (sometimes quite strongly, especially when the spatial–temporal resolution is high) it does not per se prove a functional requirement. Here the standard molecular approach is to knockout or knockdown the protein of interest and then query the system. However, a problem, which is particularly pronounced in membrane traffic,
Seeing ahead
Looking ahead we project that novel probes will play a key enabling role in membrane traffic – from seeing sharper to driving interactions in space and time. Right now the temporal resolution of superresolution microscopy is still low, but with brighter probes and faster cameras this should improve. Likely there is no one size fits all – rather the nanoscope will need to match the application [4]. Ongoing developments are highly interdisciplinary as nanoscopes are not merely new optical devices
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was supported by a NIH New Innovator Award to D.T. (DP2-OD002980-01). We thank M. Juette, T. Gould, J. Bewersdorf, M. Lopalco and J. Fölling for their comments.
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