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:

screen-shot-2017-02-12-at-16-20-15

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:

screen-shot-2017-02-12-at-16-20-37

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:

screen-shot-2017-02-12-at-16-20-50

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

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.

wang-2016

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:

screen-shot-2016-09-18-at-16-17-57

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

CRISPR Digest #11

By this point my interest in CRISPR research is apparently well known to the people around me: a friend shared a research article with me on Facebook by Kaminski et al. (2016) published earlier this month in the journal Scientific Reports. In this paper the researchers used the CRISPR/Cas9 genome-editing technology to eliminate the human immunodeficiency virus (HIV) genome from infected cells.

HIV is the causative agent of acquired immune deficiency syndrome (AIDS): the virus infects some of the so-called T cells in our immune system and integrates its genetic material into the DNA of the host cell. The infection causes a lot of T cells to die and eventually – over a period of years or often even decades – this makes the immune system less and less efficient. Therefore the causes of death of AIDS patients are usually infections, which would normally be fought off by a healthy immune system, or rare types of tumours. Currently there are efficient retroviral therapies available to treat HIV infection. However, these therapies do not remove the virus DNA from the patient, rather they keep the virus at bay (it is said to be “latent”). Therefore the treatment is usually lifelong and expensive.

Kaminski et al. mainly used a human cell line to test whether they could design guide RNAs that would specifically guide the Cas9 protein to the DNA sequences at either end of the HIV genome. The cell line is called 2D10 and has been well characterised and has a single HIV genome inserted at a known location, making it a good model to test their experimental tools. Since CRISPR is such a ubiquitous tool in the lab already a lot of the paper actually focusses on making sure that cutting out the HIV genome – which they manage successfully – does not have any unintended consequences. In particular, the researchers checked that Cas9 does not introduce mutations elsewhere in the host genome.

Having established these controls Kaminski et al. then go on to show that 2D10 cells with Cas9 and the guide RNAs (gRNAs) are less susceptible to a new HIV infection. To further test their system the researchers used human T cells from healthy individuals to show that these cells can also be made more resistant to infection when given the Cas9/gRNAs. Lastly, the paper shows that the technique can also target HIV DNA in human T cells from infected patients. However, here the Cas9 was not able to entirely excise the HIV genomes. Partly this can be attributed to the fact that human cells are much more heterogeneous than the 2D10 cell line: the virus will have integrated at different sites in the host genome in different T cells and there may also be several integration sites per cell.

This is an impressive study and a good step towards being able to treat patients with HIV using genome-editing technology, but there are still some shortcomings. To me one of the main problems seems to be the way in which the Cas9 and guide RNAs are delivered into the infected T cells: often this is done by putting the DNA that codes for the Cas9 protein and the gRNAs into a lentivirus, which belongs to the same group of viruses as HIV itself. The lentivirus would therefore itself integrate into the genome of the host cell and this might cause problems in itself, for example, by disrupting important genes. Furthermore, and the authors allude to this, not all HIV genomes are exactly the same and so for each patient one might have to design individual gRNAs.


Since we are on the topic of HIV/AIDS I would like to mention something another friend has brought to my attention. Some countries, such as the USA, Canada and France, have programmes to make so-called pre-exposure prophylaxis (PrEP) available to people who are HIV-negative but at high risk of contracting the infection. The National Health Service in England has recently released a statement explaining that it will no longer pursue this avenue although the once-daily pill has been shown to decrease the relative risk of becoming infected by over 90% in men who have sex with men (see, for example, Grant et al., 2010). The National AIDS Trust has therefore started a campaign for people to write to their local MPs so that this issue can be raised in parliament. And I did just that right now and realised that my MP is none other than Jeremy Corbyn.


On a slightly more upbeat note, here is a wallpaper design by Nature for their special CRISPR issue (downloaded directly from their website):

crispr wallpaper

And regarding the CRISPR patent dispute, there was a good News & Views article on the topic a few weeks ago and the take-home message is that it will probably take several years for it to be decided.

Lastly, happy Easter to all those who celebrate it in one way or another. Instead of (chocolate) eggs I will share with you a slightly abstract art image that I inadvertently took on the microscope a couple of months ago. With a little bit of imagination the organoids could be mistaken for Easter eggs.

organoids


References:

Grant  RM, Lama  JR, Anderson  PL, McMahan  V, Liu  AY, Vargas  L, Goicochea  P, Casapía  M, Guanira-Carranza  JV, Ramirez-Cardich  ME, Montoya-Herrera  O, Fernández  T, Veloso  VG, Buchbinder  SP, Chariyalertsak  S, Schechter  M, Bekker  L-G, Mayer  KH, Kallás  EG, Amico  KR, Mulligan  K, Bushman  LR, Hance  RJ, Ganoza  C, Defechereux  P, Postle  B, Wang  F, McConnell  JJ, Zheng  J-H, Lee  J, Rooney  JF, Jaffe  HS, Martinez  AI, Burns  DN, Glidden  DV (2010) Preexposure Chemoprophylaxis for HIV Prevention in Men Who Have Sex with Men. New England Journal of Medicine 363: 2587-2599

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

Of mimiviruses and making progress

It’s been so long since I’ve written anything about CRISPR that I feel completely rusty. Luckily, I spotted some interesting new research on an “immune system” found in giant viruses called mimiviruses. Levasseur et al. (2016) propose that mimiviruses – large viruses that can be seen with a light microscope and have a genome that is bigger than that of some bacteria – can defend themselves against so-called virophages. In analogy to the viruses that infect animals, bacteria can be infected by viruses called bacteriophages, and mimiviruses can be infected by virophages. Although generally viruses are regarded as non-living these giant viruses do possess some genes that code for proteins that help build the virus. Normally, viruses hijack the infected host cell’s machinery to replicate and are completely dependent on the host cell. Only last year a group reported (Ekeberg et al., 2015) using high power X-rays to study a single virus and being able to look inside the particle. There is a nice 3D reconstruction video in the Nature News & Views article.

One of the virophages that infects some mimivirus lineages, but not others, is called Zamilon. Levasseur et al. guessed that mimiviruses  might incorporate stretches of DNA from Zamilon and use them to recognise infecting virophages. This would be in complete analogy to many bacteria and archaea that protect themselves from their attackers using CRISPR – clustered regularly interspaced short palindromic repeats. The DNA “spacers” correspond to the sequences found in the invading DNA  of bacteriophages, for example. To test their idea, Levasseur et al. sequenced the genomes of 45 mimivirus strains and did indeed find a repeating region that corresponded to Zamilon DNA. They called it MIMIVIRE, for mimivirus virophage resistance element (the figure is copied directly from the paper):

Screen Shot 2016-03-05 at 21.30.06

Schematic of the mimivirus MIMIVIRE locus – copied directly from Levasseur et al., 2016

Nearby in the mimivirus genome the researchers also found (Cas-like) genes that might be involved in this defence mechanism: when those genes were genetically silenced the viruses that were normally resistant became sensitive to Zamilon infection. Furthermore, these Cas-like genes code for proteins that can bind and modify DNA and are therefore perfect candidates for destroying invading DNA. More experiments will need to be carried out to show exactly how the MIMIVIRE system works, but it is likely to be different from the CRISPR/Cas system. However, it is interesting to see how immune systems are pervasive in all domains of life (and almost life) and although I am no evolutionary biologist I think this may be an example of convergent evolution. [Thoughts on this would be appreciated!]


To end today’s post on a personal positive note: this year’s birthday present was being able to image the cells I am studying by electron microscopy. Electron microscopes are, in principle, similar to light microscopes, which most of us have used at school, except that they utilise electron beams instead of light rays as the source of illumination. Since electron beams have a much shorter wavelength (i.e. are higher in energy) than visible light they can visualise objects with much higher resolution, making it possible to get sharp images at greater magnification. Some of the images we – the extremely knowledgeable and helpful scientists at the electron microscopy facility in our institute and I – took were magnified 60,000 times!

Although I can’t share those images at the moment, here is a transmission electron micrograph of a normal pancreatic ductal cell (copied directly from here):

wt ductal cell

Pancreatic ductal cell

In the centre of the cell is the large nucleus, clearly defined by its double membrane. The darker patches within the nucleus are parts of the DNA that are more tightly compacted than others. In the cytoplasm (the area that isn’t the nucleus) there are some mitochondria, where a lot of the cell’s metabolism takes place, and so-called endocytic vesicles, membrane-bound compartments that are involved in recycling and housekeeping. At the top left are microvilli, short protrusions of the cell into pancreatic duct. Although I’m not sure I can spot it here, the images we took also revealed the rough endoplasmic reticulum where ribosomes sit and produce proteins. Can you believe I saw ribosomes?!

Lastly, and this goes out mainly to all the other PhD students, I’ve recently been finding it helpful and refreshing to relish the feeling that, as a student, there is always so much more to learn. To really bask in the glory of one’s own ignorance and then pester and listen to more knowledgeable and more experienced people to learn something new. For example, you can plan a decent experiment that has the proper controls and would give you some useful information. Then you tell your supervisor about it and s/he suggests doing that little extra something that suddenly turns the experiment into real science. On the one hand, I find these occurrences humbling. But, on the other hand, they are also exciting because I’m finding it easier and easier to recognise what makes a really good experiment and I can see, still beyond my reach but tantalisingly close, the potential of making that last mental leap myself. Needless to say that doesn’t at all mean that an experiment will technically work…


References:

Ekeberg T, Svenda M, Abergel C, Maia FRNC, Seltzer V, Claverie J-M, Hantke M, Jönsson O, Nettelblad C, van der Schot G, Liang M, DePonte DP, Barty A, Seibert MM, Iwan B, Andersson I, Loh ND, Martin AV, Chapman H, Bostedt C et al. (2015) Three-Dimensional Reconstruction of the Giant Mimivirus Particle with an X-Ray Free-Electron Laser. Physical Review Letters 114: 098102

Levasseur A, Bekliz M, Chabrière E, Pontarotti P, La Scola B, Raoult D (2016) MIMIVIRE is a defence system in mimivirus that confers resistance to virophage. Nature advance online publication