Jamie White

Photobleaching GFP reveals protein dynamics inside live cells

Cell biologists have used photobleaching to investigate the lateral mobility of fluorophores on the cell surface since the 1970s. Fusions of green fluorescent protein (GFP) to specific proteins extend photobleaching techniques to the investigation of protein dynamics within the cell, leading to renewed interest in photobleaching experiments. This article revisits general photobleaching concepts, reviews what can be learned from them and discusses applications illustrating the potential of photobleaching GFP fusion proteins inside living cells.

OSM-11 facilitates LIN-12 Notch signaling during Caenorhabditis elegans vulval development.

Notch signaling is critical for cell fate decisions during development. Caenorhabditis elegans and vertebrate Notch ligands are more diverse than classical Drosophila Notch ligands, suggesting possible functional complexities. Here, we describe a developmental role in Notch signaling for OSM-11, which has been previously implicated in defecation and osmotic resistance in C. elegans. We find that complete loss of OSM-11 causes defects in vulval precursor cell (VPC) fate specification during vulval development consistent with decreased Notch signaling. OSM-11 is a secreted, diffusible protein that, like previously described C. elegans Delta, Serrate, and LAG-2 (DSL) ligands, can interact with the lineage defective-12 (LIN-12) Notch receptor extracellular domain. Additionally, OSM-11 and similar C. elegans proteins share a common motif with Notch ligands from other species in a sequence defined here as the Delta and OSM-11 (DOS) motif. osm-11 loss-of-function defects in vulval development are exacerbated by loss of other DOS-motif genes or by loss of the Notch ligand DSL-1, suggesting that DOS-motif and DSL proteins act together to activate Notch signaling in vivo. The mammalian DOS-motif protein Deltalike1 (DLK1) can substitute for OSM-11 in C. elegans development, suggesting that DOS-motif function is conserved across species. We hypothesize that C. elegans OSM-11 and homologous proteins act as coactivators for Notch receptors, allowing precise regulation of Notch receptor signaling in developmental programs in both vertebrates and invertebrates.

Spatial partitioning of secretory cargo from Golgi resident proteins in live cells

BackgroundTo maintain organelle integrity, resident proteins must segregate from itinerant cargo during secretory transport. However, Golgi resident enzymes must have intimate access to secretory cargo in order to carry out glycosylation reactions. The amount of cargo and associated membrane may be significant compared to the amount of Golgi membrane and resident protein, but upon Golgi exit, cargo and resident are efficiently sorted. How this occurs in live cells is not known.ResultsWe observed partitioning of the fluorescent Golgi resident T2-CFP and fluorescent cargo proteins VSVG3-YFP or VSVG3-SP-YFP upon Golgi exit after a synchronous pulse of cargo was released from the ER. Golgi elements remained stable in overall size, shape and relative position as cargo emptied. Cargo segregated from resident rapidly by blebbing into micron-sized domains that contained little or no detectable resident protein and that appeared to be continuous with the parent Golgi element. Post-Golgi transport carriers (TCs) exited repeatedly from these domains. Alternatively, entire cargo domains exited Golgi elements, forming large TCs that fused directly with the plasma membrane. However, domain formation did not appear to be an absolute prerequisite for TC exit, since TCs also exited directly from Golgi elements in the absence of large domains. Quantitative cargo-specific photobleaching experiments revealed transfer of cargo between Golgi regions, but no discrete intra-Golgi TCs were observed.ConclusionsOur results establish domain formation via rapid lateral partitioning as a general cellular strategy for segregating different transmembrane proteins along the secretory pathway and provide a framework for consideration of molecular mechanisms of secretory transport.

GFP: an illuminating tool: Green Fluorescent Proteins (Methods in Cell Biology, Vol. 58), edited by Kevin F. Sullivan and Steve A. Kay

Academic Press, 1999.

Multicolour imaging of post-Golgi sorting and trafficking in live cells

64.95 (386 pages)ISBN 0 12 676075 6Within the past four years, variants of the green-fluorescent protein (GFP) have become standard tools in cell biology. When Prasher and colleagues first cloned the gene for GFP from the jellyfish Aequorea victoria in 19921xPrasher, D.C. et al. Gene. 1992; 111: 229–233Crossref | PubMed | Scopus (1425)See all References1 it was not obvious that GFP would become so useful that it would warrant its own volume of Methods in Cell Biology. Initial problems with dim fluorescence, poor expression and lack of spectral variants have now been solved, making GFP an effective fluorescent tag for most cloned proteins. GFP is remarkably inert; fusions to GFP are quite often functional or at least localize properly despite an additional 237 amino acids of polypeptide sequence. GFP is also remarkably resistant to photobleaching, allowing long-term time-lapse microscopy studies. This book covers most of the broad range of applications cell biologists have found for GFP, with chapters from many of the labs that were involved in overcoming the initial problems. It is a comprehensive guide to GFP, useful both for someone starting to work with GFP and for those with considerable experience with GFP who want to see how others are using it.The style is consistent with the traditional format of Methods in Cell Biology; the book is organized by chapters on a particular topic, some quite specific, each with detailed protocols, descriptions of techniques and advice from the authors – all the helpful and sometimes critical details that are never published in research papers owing to space constraints. At the end of each chapter are extensive references that make a useful guide for further reading. The disadvantage of this format is that Green Fluorescent Proteins is far from concise. There is considerable overlap because each chapter was written separately – often on topics that use similar techniques.The range of topics is broad, beginning with three chapters that cover the biophysics and structural basis of GFP fluorescence and quantitative imaging with GFP, which provide a basic foundation for the more applied chapters that comprise the rest of the book. The majority of these chapters describe strategies for generating GFP-fusions and techniques for time-lapse imaging a variety of biological systems, from yeast to flies to tissue-culture cells. Other chapters cover single-molecule experiments with GFP, fluorescence resonance energy transfer (FRET), photobleaching techniques, multicolour imaging with GFP variants and cell sorting based on GFP fluorescence. The final chapter discusses practical considerations for setting up an imaging facility – computers, backup systems, image processing, space and ventilation.In general, Green Fluorescent Proteins is quite comprehensive, but there were several additional things that would have been useful to include. Two techniques could have been described more completely. The first is a method to tag proteins indirectly with GFP based on noncovalent heterodimerization of GFP and cytoplasmic structural proteins2xKatz, B.Z. et al. Biotechniques. 1998; 25: 298–302304PubMedSee all References2. Dimerization is mediated by a modified leucine-zipper protein spacer. This method seems to allow tagging of structural proteins whose assembly might be hindered by direct fusion to GFP. The second is a complete description of the recently characterized two-colour double-labelling technique based on sequential excitation of the cyan (optimized W7) and yellow (optimized 10C) spectral variants of GFP3xSee all References3. Spectral variants and double labelling are discussed briefly and rather speculatively in several chapters, but a thorough characterization of an effective technique is required. The method using cyan and yellow variants is the only technique that is amenable to long-term time-lapse imaging and photobleaching studies. In addition, movies of protein dynamics visualized with GFP are often quite dramatic and always informative, so I would have liked to have seen an accompanying CD-ROM with movies and images (such as Ref. 4xSee all ReferencesRef. 4). Finally, although some might feel it outside the scope of the book, a chapter on alternatives to GFP and future improvements in visualizing specific proteins in live cells using fluorescent probes would have provided valuable perspective and interesting directions for future experiments.Green Fluorescent Proteins fills an important niche as a detailed guide to using GFP variants to solve a variety of problems in cell biology. When people come to me with questions about GFP, I now refer them to this book.