ARFs, GEFs, GAPs, and effectors
The ARFs are monomeric GTPases that regulate a range of essential cell functions. Included within the ARF family are three groups; the “true ARFs” (ARF1-6), the ARF-like proteins (ARLs (ARL1-6, 8-11, 13, 16), and the two SAR proteins. Both the ARF and SAR sub-families are very highly conserved and perform very similar/overlapping functions. The ARLs are more divergent with each typically serving distinct functions, though even these general statements have exceptions. They all operate as “molecular switches”, being interconverted between inactive (GDP-bound) and active (GTP-bound) conformations. The slow dissociation of bound GDP and low intrinsic rates of GTP hydrolysis necessitate the actions of GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating protein), to initiate and terminate signaling, respectively. Activated ARFs have increased affinity for effectors, resulting in a downstream biological output. This can be due to specific re-distribution of an effector (e.g., recruitment to a membrane), allosteric activation of effector enzymatic activity, conformational changes propagated to the effector, or a combination of such changes. Both ARF GAPs and effectors have high affinity for the activated GTPase and most, perhaps all, GAPs are also effectors. Despite being extensively studied, we still have only a limited understanding of what each ARF family GTPase does in cells, the extent to which they may share one or more activities, how they are regulated by GAPs and GEFs, how those GEFs and GAPs are regulated, how many different effectors each GTPase binds, and how those effectors perform their regulatory functions. Such questions are essential to a deep understanding of these families of essential cell regulators and clearly are best addressed by comparing and contrasting and studying the entire family.
Why the need for the ARF Consortium?
The detailed and comprehensive study of ~70 genes (or even just the ~30 GTPases) is clearly more than any one lab can manage. The diversity of processes influenced by these families of proteins is also outside the expertise of any one lab or even a small group of labs. At its heart, the Consortium is an effort to crowd source the work load and the expertise, and to work together towards the larger goal of understanding not just any one of these proteins but the family as a whole. Because these proteins regulate or can influence so many different processes and organelles it is critical to get input from researchers with expertise across a large swatch of cell biology.
A problem in the field is that we lack a fundamental knowledge of specificity in signaling by ARFs and their regulatory proteins. Many of the proteins in these families have very close paralogs in mammals, forming sub-families (ARF1-5, ARL5A/B, ASAP1-3, IQSEC1-3, etc.). Typically these are studied separately, despite the fact that there is often evidence of both shared and distinct functionalities. Recent work in our lab has revealed the likelihood of coordinate regulation of transcription within one such sub-family. Thus, studies of these smaller (typically 2-3 members) sub-families are likely to provide information on specificity and coordination of signaling. Importantly, published data may be compromised by mis-interpretation as a result of undetected changes in the level of a sub-family member in response to manipulation of another. Because we lack data for these and related questions, our model(s) for how any one of these proteins function in cells is weakened.
Which GEFs and GAPs work on which GTPases? Which GEFs/GAPs share substrates or binding partners? Which ones may compete for binding partners? When one protein has multiple effectors/actions can the level of expression or localization in the cell be rate limiting or dictate a particular cellular outcome(s)? Just the ARFs (ARF1-6) have >30 known effectors (defined here as a direct binding partner whose interaction leads to a biological response) and most of these were identified as binding to ARF1. As most of these effectors are expressed in all cells, how is specificity in output achieved?
Who can participate in the Consortium and what exactly are we asking the researchers in the Consortium to do?
Quite simply, anyone can participate and contribute in any way they wish. Participation can be as minimal as looking at our website and suggesting some improvements to it or to our overall goals and approaches. Or by correcting information in the databases posted. A more substantial involvement would be to send us protocols, plasmids or other reagents that may be shared (with appropriate attribution). We encourage the deposition of plasmids and cell lines in public repositories (e.g., Addgene or ATCC, respectively) and may be able to facilitate such contributions. We very much want your expertise in using the KO MEFs in assays routinely performed in your lab, and to deposit the obtained data in the Consortium database or make it available through publication. Please send your comments/information/questions to rkahn at emory dot edu) and let’s get going!
Why KOs? Why MEFs? Why not mice?
Why not use RNAi technologies to knockdown each target, as it is quicker and easier? Because knocking down a gene/protein is transient, typically incomplete, and in our experience often is difficult to interpret and misses important cellular effects. Since the earliest days of biomedical research it has been shown that removing the item under study allows the investigator to make sound conclusions as to the functions it serves when present. This is true whether we are speaking about organs in our body or proteins in our cells. KO cell lines will provide a consistent and stable resource to obtain a wealth of new information on each protein’s function.
MEFs are diploid, contact inhibited, and flat, with the latter making them very amenable for high resolution imaging. Many of the basic cell functions known to be regulated by ARFs (membrane traffic, cell division, ciliation, mitochondrial fusion, etc.) are best analyzed at the single cell level and often require high resolution imaging. In addition, the knockout mouse project (KOMP) and work in labs all over the world are contributing to the generation of animals with designed deletions in our target genes. These are also a rich source from which immortalized MEFs can be derived and these will clearly assist in our goal of obtaining KO lines for each of the ~70 targeted genes for further studies. As the collection of KO lines grows and researchers generate data on specific cellular defects resulting from those KOs, we will be positioned to better interpret results of animal studies as well as interpret or make predictions about the biological consequences of specific patient mutations.
Knockouts of any of the ~70 genes in mice in a tissue-specific fashion or complete KO has already and will continue to provide valuable information about their roles and functions in mammalian biology. Our goals here will complement such work and provide synergy and added strength to the animal data. We believe that gene knockouts are among the more readily interpretable of approaches to study gene/protein functions, and that our immortalized MEFs will provide invaluable data that will complement and extend the results obtained in animals. Because ARFs are so ancient, it is expected (and already shown in many cases) that they perform highly conserved functions in all cells, implying that any one cell type is likely to be illustrative of key roles in all cells. This is not to say that any specific GTPase may not have additional, more specialized roles in certain tissues, but those are best studied in animals. By focusing on one cell type (MEF), we understand that we will miss some functions of these ancient proteins, but the complexity of signaling by this group of 70 proteins requires that some limits must be placed on our immediate goals.
A deal you don’t want to miss!
If you want to generate in your lab a MEF KO in any of the ~70 genes listed here, but feel you lack the necessary CRISPR expertise, please contact Rick Kahn as I am offering to perform the front half of the KO (CRISPR guide design, cloning, transfection into MEFs, initial selection with puromycin, expansion, and freezing) in my lab. We would then send you pooled cells for cloning in your lab by limited dilution (the protocols are available below). This takes about one month and generates typically 20-40 clones which are then screened by DNA sequencing (may take another couple of weeks) for the presence of both alleles frameshifted to give the null line. Ideally, we want at least two KO clones from each of two different guides for at least 4 clones. We save and analyze all heterozygotes, though these are rare in our experience. We also save wild type clones coming through the CRISPR process as these (arguably) make another control (similar but different from original MEF line). Please let Rick Kahn know if you are interested in taking advantage of this opportunity and let him know the gene(s) you would like to target. Several other labs have already agreed to collaborate in this way so the genes are going fast. Don’t be late to the CRISPR Party!