Back to Cell Biology

Because you’ve just gotta love cells. And because this post is about a publication in The Journal of Cell Biology, published by the Rockefeller University Press. In the summer of 2014 I spent almost three months doing an undergraduate research programme at Cold Spring Harbor Laboratory in the lab of Lloyd Trotman and under the everyday supervision of Dawid G. Nowak. I mainly helped Dawid establish the CRISPR/Cas9 method in the lab to study several types of cancers, including lung and prostate cancer. The first story, in which we used CRISPR to knockout a potent oncogene called Myc, was published almost two years ago (Nowak et al, 2015). Now Dawid is the co-first author on a new paper studying a tumour suppressor protein called PTEN (Chen, Nowak, … Wang, … et al, 2017).

Here is an eLife-style digest of the manuscript. Tumours usually evolve when cells gain the function of so-called oncogenes and lose the function of one or more so-called tumour suppressor genes. One of the most frequently deleted or down-regulated tumour suppressors is a protein called PTEN. Some cancer types, including some types of lung and prostate cancer, do not always delete the two gene copies coding for the PTEN protein, but the levels of PTEN protein in those cancer cells is still kept low. Therefore we wanted to find out which pathways in cancer cells lower the PTEN protein levels. Knowing about this regulation could lead to the development of new therapies that aim at stabilising PTEN protein.

First, we used both mouse and human cancer cell lines to investigate the movement of PTEN between the cytoplasm and the nucleus. We hypothesised that PTEN might be protected from being degraded in the nucleus, since the enzymes that break proteins down are generally found in the cytoplasm. Biochemical experiments showed that PTEN was moved into the nucleus by a protein called importin-11. Next, and this is the experiment I performed, we deleted importin-11 using CRISPR/Cas9 and saw that PTEN abundance decreased, while active/phosphorylated Akt, an oncogene, increased:

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Western blot showing CRISPR/Cas9 deletion of importin-11 in human prostate cancer cell lines – copied directly from Fig. 2 of Chen, Nowak et al, 2017

Further experiments conducted in the cell lines supported the following model, in which PTEN is shuttled into the nucleus by importin-11 where it is protected from degradation by the ubiquitin ligase system:

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Model of PTEN shuttling: when importin-11 is present PTEN can “hide” in the nucleus (left), but when importin-11 is deleted/not functioning, PTEN accumulates in the cytoplasm where it can be targeted for degradation – copied directly from Fig. 4 of Chen, Nowak et al, 2017

Next we wanted to know whether this mechanism of keeping levels of PTEN low is also important for preventing tumours. When importin-11 was experimentally down-regulated in mice (the gene for importin-11 was not completely deleted but its mutation is said to be “hypomorphic”), the mice developed and eventually died from lung cancers, unlike the healthy control mice:

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Lesion-free survival curve of importin-11 mutant (red) versus control (black) mice – copied directly from Fig. 5 of Chen, Nowak et al, 2017

Similar results were also obtained for prostate tumours in mice. Lastly, we analysed publicly available data of human prostate cancer patients. Low levels of importin-11 (either by genetic deletion or low gene expression) correlated with higher rates of tumour recurrence, suggesting that importin-11 also acts as a tumour suppressor in some types of human cancer. Future experiments may involve conducting more sophisticated mouse experiments in which importin-11 is deleted in specific organs, together with the activation of known oncogenes. This work may also lead to studies that try to find ways of stabilising PTEN protein.


So that’s it. Publication number three! But I want to end on a slightly more philosophical/political note. Dawid, one of the two first authors, taught me a lot during that summer programme, has been supportive ever since, and I enjoy keeping in touch with him. At the moment he is looking for an independent research position – he is enthusiastic about science and very driven. He’s had interviews all over the place, both in Europe and North America. However, Dawid is Polish and is now having to re-think his options since neither the UK nor the USA seem particularly appealing places for him anymore. We live in a crazy world but I hope this won’t stop him from getting the lab he deserves, in the most tolerant place possible.


References:

Chen M, Nowak DG, Narula N, Robinson B, Watrud K, Ambrico A, Herzka TM, Zeeman ME, Minderer M, Zheng W, Ebbesen SH, Plafker KS, Stahlhut C, Wang VMY, Wills L, Nasar A, Castillo-Martin M, Cordon-Cardo C, Wilkinson JE, Powers S et al. (2017) The nuclear transport receptor Importin-11 is a tumor suppressor that maintains PTEN protein. The Journal of Cell Biology DOI: 10.1083/jcb.201604025

Nowak DG, Cho H, Herzka T, Watrud K, DeMarco DV, Wang VM, Senturk S, Fellmann C, Ding D, Beinortas T, Kleinman D, Chen M, Sordella R, Wilkinson JE, Castillo-Martin M, Cordon-Cardo C, Robinson BD, Trotman LC (2015) MYC Drives Pten/Trp53-Deficient Proliferation and Metastasis due to IL6 Secretion and AKT Suppression via PHLPP2. Cancer Discovery 5: 636-651

CRISPR Digest #13

It’s time to go back to some of the basic biology behind the whole CRISPR gene-editing hype. This week Cell and Molecular Cell published two nice papers on the why and how of CRISPR.

In one of my earliest posts on this blog, CRISPR Craze, I gave a brief overview of how CRISPR works in prokaryotes. I’ll reiterate here: bacteria and archaea have evolved a response against invading pathogens, often bacteriophages (viruses that infect bacteria), which has been compared to our mammalian immune system. In essence, CRISPR allows bacteria to recognise when a pathogen, and specifically its DNA, is infecting the cell again. During the first round of infection the bacterium incorporates parts of the pathogen’s DNA in its own genome and therefore keeps a record or memory of that invader. Then, during a second round of infection, the DNA can be transcribed into RNA by the bacterium, which is used as a “guide” to detect the invading DNA (since the RNA and DNA will be complementary). Additionally, the guiding RNA will bring/guide one (or several, depending on the exact type of system) so-called CRISPR-associated protein (Cas) to the invading DNA. The Cas protein(s) is then responsible for cleaving the pathogen’s DNA and thus thwarting the infection. CRISPR in a nutshell. Easy.

If you think about it, the findings from Pawluk et al., 2016 will not come as a surprise. First, bacteriophages infect bacteria. Second, bacteria evolve an intricate mechanism to defend themselves against the viruses and other, potentially harmful “mobile genetic elements”. So third – the logical conclusion – bacteriophages find a way to shut down the CRISPR defence. Pawluk et al. found that the Cas9 protein in the bacterial species Neisseria meningitidis can be inhibited by phage anti-CRISPR proteins:

anti-crispr

Schematic representation of the anti-CRISPR system, which can also be used in mammalian gene-editing system – image copied directly from Pawluk et al, 2016

In particular, Pawluk et al. discovered three acr genes in N. meningitidis, which code for the Acr proteins. The acr genes are incorporated into the bacterial genome but originally came from bacteriophages or mobile genetic elements. Biochemical experiments showed that the Acr proteins can bind directly to Cas9 and stop it from cutting DNA. Lastly, Pawluk et al. demonstrated that the Acr proteins can be expressed in mammalian cells to inhibit Cas9 activity there as well. This means that future CRISPR genome-editing experiments can be fine-tuned by switching off Cas9. Being able to turn Cas9 off may be especially important for future gene therapy treatments, since preventing Cas9 from being active for too long will reduce its off-target/side effects.

The second interesting paper for this digest, Patterson et al., 2016, investigated how bacterial cells regulate when their CRISPR system is active or not. The decision to have a fully active “immune system” or not is important because it is energetically costly to have the defence mechanism in place when there is little or no threat. Patterson et al. used a species of bacteria called Serratia to examine how the density of the bacterial population influences whether CRISPR is turned on or off. Many bacterial species use a system called quorum sensing to assess whether there are many other bacterial cells nearby. For example, Serratia cells produce and secrete a small chemical (of the homoserine lactone class), which, when present in sufficient quantities, can change which genes the bacterial cells express. When the population of cells is sparse the chemical does not reach a high enough concentration to have an effect. The experiments in this paper show that at high concentrations of the small chemical, and thus at a high cell density, Serratia cells de-repress the cas genes. In other words, when there are a lot of cells in one place they collectively switch on their immune system. This makes sense: infections spread more easily among humans in crowded places and it is similar in bacterial populations. Overall, these two papers are a beautiful demonstration of how “basic” research into highly relevant and applicable technologies are still, and will continue to be, important.

Lastly, since this is the last post before Christmas and the New Year, and possibly even until we say good-bye to President Obama, let me share this resource with you:

altmetric

Screenshot from the Altmetric website listing its top 100 most-discussed journal articles of 2016

Altmetrics are “metrics and qualitative data that are complementary to traditional, citation-based metrics” and track how much and in what form scientific research is being discussed. For example, a useful but very technical paper may get many citations in the scientific literature but might not be widely talked about by people outside that field. Other new papers, that may be controversial or have wide-ranging societal implications, will also be distributed in other ways (e.g. on Twitter, Facebook, Wikipedia and on blogs). So, for your festive reading, I recommend having a browse through Altmetrics’ 100 most-discussed articles from this year. Merry Christmas!

References:

Patterson AG, Jackson SA, Taylor C, Evans GB, Salmond GPC, Przybilski R, Staals RHJ, Fineran PC Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems. Molecular Cell 64: 1102-1108

Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR (2016) Naturally Occurring Off-Switches for CRISPR-Cas9. Cell 167: 1829-1838.e1829

New Scientist Live

This weekend the ExCeL centre in London hosted an event called New Scientist Live, which was aimed at the general public and invited speakers across various fields, including Brain & Body, Technology, Earth and Cosmos. Additionally, there were stands and interactive stations run by various scientific institutions from across the UK and Europe, including The Francis Crick Institute, the Royal Society of Biology and the European Space Agency, to name a few.

But, to be honest, I was already sold when I saw the giant bacterium (precise species is still a matter of debate; could be E. coli) hanging from the ceiling:

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Apart from this excellent demonstration of how cool cells are I want to write about two highlights.

  1. The talk by Molly Crockett on “What makes us moral?”
    Molly Crockett has a lab at the Department of Experimental Psychology, University of Oxford (but will be moving to Yale next year) where she and her research group study the neuroscience of “morality”. Dr Crockett’s talk was all-round excellent: from the clarity of her speaking, to the information on the slides, the science simplified enough to be understandable, yet retaining the references on the slides so that one can look up the original research (Crockett et al., 2014 and 2015, both open access!). The main finding of the 2014 paper was that people tend to be “hyperaltruistic”: when deciding whether to inflict painful electric shocks to oneself or another anonymous human being, the person deciding needed to be offered/paid more money to hurt another person. People also decided more slowly when the effects were to be felt by the other person rather than oneself. Importantly, and Dr Crockett emphasised this in her talk, these studies were conducted with real people and real electric shocks so that the results from their experiments might give us information about real life situations, as opposed to hypothetical ethical dilemmas. Possibly one of the most famous of these dilemmas is one in which a person needs to decide whether to save five people by actively sacrificing one, or to passively let five people die:moral-dilemmaIn the 2015 paper the authors then go on to test whether various drugs  – the antidepressant Citalopram, a selective serotonin re-uptake inhibitor and Levodopa, a dopamine precursor – can alter this moral decision making. Interestingly, the antidepressant reduced the overall number of electric shocks the deciders were giving out, both to themselves and to others. The hyperaltruism was preserved since deciders still gave fewer shocks to the receivers for the same amount of money. Levodopa, on the other hand abolished this hyperaltruistic effect:

    crockett-2015

    Bar charts showing the effects of citalopram and levodopa on harm aversion – copied directly from Crockett et al., 2015

    Obviously the talk and the papers go into much more detail, especially with the statistics used to evaluate these admittedly small effects. Lastly, it’s important to note that, as Dr Crockett pointed out, none of this means that researchers are working on, or should be working on, developing a “morality drug”…

  2. The science magazine Nautilus published by the MIT Press.
    Nautilus starts where the New Scientist stops, namely, where things get really interesting. To me, the New Scientist poses similar questions to the ones I might ask, but often fails to really answer them or provide a satisfactory explanation as to why there is no answer (yet). When I do read its articles they often leave me with more questions than before, which, of course, isn’t a bad thing. However, after reading a few articles of Nautilus it seems that this magazine is more thought-provoking: the articles are longer and maybe more on the creative side, but retain the references at the end, and the style of writing is more enjoyable to me. For instance, an article called “The Wisdom of the Aging Brain” by Anil Ananthaswamy discusses the possibility that there are neural circuits, or certain regions of the brain, that, with training and age, allow us to become wiser.
    So if any of my few readers is feeling particularly generous today then why not consider getting me the Sep/Oct edition…?

References:

Crockett MJ, Kurth-Nelson Z, Siegel JZ, Dayan P, Dolan RJ (2014) Harm to others outweighs harm to self in moral decision making. Proceedings of the National Academy of Sciences 111: 17320-17325

Crockett Molly J, Siegel Jenifer Z, Kurth-Nelson Z, Ousdal Olga T, Story G, Frieband C, Grosse-Rueskamp Johanna M, Dayan P, Dolan Raymond J (2015) Dissociable Effects of Serotonin and Dopamine on the Valuation of Harm in Moral Decision Making. Current Biology 25: 1852-1859

CRISPR Digest #12

I know what you’re all thinking. When is she finally going to post about CRISPR again? It’s been too long. Well, you’re absolutely right and I’m going to make up for it. Last week I glimpsed a short article on the Science News site discussing the first CRISPR-modified cabbage. The botanist Stefan Jansson at Umeå University in Sweden “cultivated, grew, and ate a plant that had its genome edited with CRISPR-Cas9”. This is obviously very fitting since one of the pioneers of the technology, Emmanuelle Charpentier, carried out some of the seminal work at the same university between 2009 and 2014.

To cheer you up at the end of the summer, here have a listen to a short radio report on the CRISPR cabbage served with garlic and pasta – it’s in Swedish but that makes it all the more charming.

http://sverigesradio.se/sida/embed?type=publication&id=6508845

On a slightly more serious note though, I wrote about CRISPR gene-editing in the context of HIV infection in a previous post, and want to follow up here. In the last paper I discussed (Kaminski et al, 2016), the authors showed, as a proof-of-principle, that it is possible to use the Cas9 protein to cut out the HIV genome from infected T cells’ genomes, at least in a model of HIV infection. However, following this promising result two papers published more recently (both Wang et al, 2016 – sadly not me) show that the same process actually generates HIV mutants that can become infectious again. In particular, when the Cas9 protein cuts the HIV DNA that is integrated in the human genome, the human cells try to repair the cut in a process called non-homologous end joining (NHEJ). This correction mechanism, however, is prone to making errors and can sometimes lead to the creation of HIV DNA sequences that can replicate again. These HIV DNA sequences could then potentially produce new virus particles that can replicate, start a new round of infection and are, of course, resistant to the original CRISPR/Cas9 targeting, since they now contain new mutations. Once again science proves to be more fickle than originally thought; it really shouldn’t surprise us anymore.

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Schematic showing how HIV can escape CRISPR/Cas9 editing – copied directly from Wang et al, 2016, Cell Reports

To return to and end on a more culinary note: not only has the world now seen CRISPR cabbage, but a report (Ren et al, 2016) published a couple of weeks ago demonstrated that the gene-editing technology also works in grapes, Chardonnay to be precise. The scientists modified the gene coding for the L-idonate dehydrogenase protein, which is involved in producing tartaric acid. So it is in theory possible to generate sweeter, or at least less acidic, grapes:

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Genome-edited Chardonnay plant – copied directly from Ren et al, 2016

References:

Kaminski R, Chen Y, Fischer T, Tedaldi E, Napoli A, Zhang Y, Karn J, Hu W, Khalili K (2016) Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing. Scientific Reports 6: 22555

Ren C, Liu X, Zhang Z, Wang Y, Duan W, Li S, Liang Z (2016) CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Scientific Reports 6: 32289

Wang Z, Pan Q, Gendron P, Zhu W, Guo F, Cen S, Wainberg Mark A, Liang C (2016) CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape. Cell Reports 15: 481-9

Wang G, Zhao N, Berkhout B, Das AT (2016) CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape. Mol Ther 24: 522-526

Synthetic biology

What I cannot create, I do not understand. – Richard Feynman

[Disclaimer: I am an amateur when it comes to synthetic biology, so if you spot mistakes in terminology or just sloppy expressions then please feel free to correct me.]

Synthetic biology is a topic that is being mentioned more and more frequently in scientific, but also mainstream, news. As with many things in life there is no one single, clean definition of this field of study. Broadly speaking, synthetic biology can either mean using existing building blocks (e.g. components of an organism’s DNA such as regulatory sequences or protein-coding sequences) to create new combinations, or to make up completely new biological building blocks (see, for example, the Editorial in Nature Methods, 2014 and a Q&A-style paper by Church et al., 2014).

Recently, a paper published in Science (Hutchison et al., 2016) made headlines: Craig Venter and his research team (or should I call it an army?) created the first fully synthetic cell. That is, they designed its genome, artificially synthesised its DNA and then transplanted it into existing cells, which subsequently lost their own genetic information, thus creating an entirely new type of cell. The cells were imaginatively called “JCVI-syn3.0” and look like this by scanning electron microscopy:

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JCVI-syn3.0 (scale bar: 0.2 µm) – image copied directly from Hutchison et al., 2016

Ignoring the fact that these cells were created almost entirely from scratch (the DNA did go into existing cells that already had a membrane etc.) in a Herculean effort, one of the interesting features of their genomes is that 17% of their genes have an unknown function. The cells have a minimal genome comprising 473 genes of which 83% can be classified into one of the following four functional groups: expression of genome information, preservation of genome information, cell membrane and cytosolic metabolism. So we do not even fully understand these simplest of all cells. This is in stark contrast to the (earlier) studies on bacteriophages (viruses that infect bacteria): here the so-called lambda phage is a good example of us understanding what each element of its genome does. An accessible account of how it works is given in Mark Ptashne’s 1986 book “A Genetic Switch”, which also, in some ways, paved the way for synthetic biology since it describes how knowledge of gene regulatory networks could be one day exploited to build new networks.

This means that Richard Feynman’s quote above does not hold completely true in the realm of synthetic biology. Funnily enough, when Venter & Co. first built semi-synthetic cells (Gibson et al., 2010) they used the four DNA codons (A, T, C, G) to incorporate small messages, the names of the authors of the paper and some quotes, including Feynman’s. However, according to this article in The Scientist they misquoted him as having written, “What I cannot build, I cannot understand.”

The example of JCVI-syn3.0 falls into the category of building something new from existing building blocks (genetic elements). However, other researchers are trying to expand the way cells work. For example, in a proof-of-principle paper Neumann et al., 2010 showed that they could expand the way ribosomes decode the famous genetic code. Normally, DNA in the form of messenger RNA is decoded in triplets (e.g. AUG represents a common start codon, which is translated into the amino acid methionine). Neumann et al. forced the evolution of a ribosome that can read the nucleotides in quadruplets. At the same time they managed to make these ribosomes translate the new codons into unnatural amino acids. This sort of approach may allow for the production of completely new proteins and molecules, which may, for example, have therapeutic applications. In fact, this has already been partially achieved for the anti-malarial drug artemisinin (see Paddon & Keasling, 2014 for a review).

Lastly, I would just like to add that part of my inspiration to read and write on the topic of synthetic biology came from a fellow PhD student, Aakriti Jain.  Until recently, Aakriti was an editor of the PLoS synthetic biology community, which aims to bring together scientists from different fields and allows them to communicate their research to a wider audience.

References:

Church GM, Elowitz MB, Smolke CD, Voigt CA, Weiss R (2014) Realizing the potential of synthetic biology. Nat Rev Mol Cell Biol 15: 289-294

Editorial (2014) Synthetic biology: back to the basics. Nature Methods 11: 463-463

Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang R-Y, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi Z-Q, Segall-Shapiro TH, Calvey CH et al. (2010) Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science 329: 52-56

Hutchison CA, III, Chuang R-Y, Noskov VN, Assad-Garcia N, Deerinck TJ, Ellisman MH, Gill J, Kannan K, Karas BJ, Ma L, Pelletier JF, Qi Z-Q, Richter RA, Strychalski EA, Sun L, Suzuki Y, Tsvetanova B, Wise KS, Smith HO, Glass JI et al. (2016) Design and synthesis of a minimal bacterial genome. Science 351: 1414-U73

Neumann H, Wang K, Davis L, Garcia-Alai M, Chin JW (2010) Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464: 441-444

Paddon CJ, Keasling JD (2014) Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development. Nat Rev Micro 12: 355-367