One ring to rule them all: The cohesin complex

By Johannes Buheitel, PhD

In my blog post about mitosis (, I explained some of the challenges a human cell faces when it tries to disentangle its previously replicated chromosomes (for an overview of the cell cycle, see also and segregate them in a highly ordered fashion into the newly forming daughter cells. I also mentioned a protein complex, which is integral for this chromosomal ballet, the cohesin complex. To recap, cohesin is a multimeric ring complex, which holds the two chromatids of a chromosome together from the time the second sister chromatid is generated in S phase until their separation in M phase. This decreases complexity, and thereby increases the fidelity of chromosome segregation, and thus, mitosis/cell division. And while this feat should already be enough to warrant devoting a whole blog post to cohesin, you will shortly realize that this complex also performs a myriad of other functions during the cell cycle, which really makes it “one ring to rule them all”.

Figure 1: The cohesin complex. The core complex consists of three subunits: Scc1/Rad21, Smc1, and Smc3. They interact to form a ring structure, which embraces ("coheses") sister chromatids.
Figure 1: The cohesin complex. The core complex consists of three subunits: Scc1/Rad21, Smc1, and Smc3. They interact to form a ring structure, which embraces (“coheses”) sister chromatids.

But let’s back up a little first. Cohesin’s integral ring structure is composed of three proteins: Smc1, Smc3 (Structural maintenance of chromosomes), and Scc1/Rad21 (Sister chromatid cohesin/radiation sensitive). These three proteins attach to each other in a more or less end-to-end manner, thereby forming a circular structure (see Figure 1; ONLY for the nerds: Smc1 and -3 form from long intramolecular coiled-coils by folding back onto themselves, bringing together their N- and C-termini at the same end. This means that these two proteins actually interact with their middle parts, forming the so-called “hinge”, as opposed to really “end-to-end”). Cohesin obviously gets its name from the fact that it causes “cohesion” between sister chromatids, which has been first described 20 years ago in budding yeast. The theory that the protein complex does so by embracing DNA inside the ring’s lumen was properly formulated in 2002 by the Nasmyth group, and much evidence supporting this “ring embrace model” has been brought forth over last decades, making it widely (but not absolutely) accepted in the field. According to our current understanding, cohesin is already loaded onto DNA (along the entire length of the decondensed one-chromatid chromosome) in telophase, i.e. only minutes after chromosome segregation, by opening/closing its Smc1-Smc3 interaction site (or “entry gate”). When the second sister chromatid is synthesized in S phase, cohesin establishes sister chromatid cohesion in a co-replicative manner (only after you have the second sister chromatid, you can actually start talking about “cohesion”). Early in the following mitosis, in prophase to be exact, the bulk of cohesin is removed from chromosome arms in a non-proteolytic manner by opening up the Smc3-Scc1/Rad21 interface (or “exit gate”; this mechanism is also called “prophase pathway”). However, a small but very important fraction of cohesin molecules, which is located at the chromosomes’ centromere regions, remains protected from this removal mechanism in prophase. This not only ensures that sister chromatids remain cohesed until the metaphase-to-anaphase transition, but also provides us with the stereotypical image of an X-shaped chromosome. The last stage in the life of a cohesin ring is its removal from centromeres, a tightly regulated process, which involves proteolytic cleavage of cohesin’s Scc1/Rad21 subunit (see Figure 2).

Figure 2: The cohesin cycle. Cohesin is topologically loaded onto DNA in telophase by opening up the Smc1-Smc3 interphase ("entry gate"). Sister chromatid cohesion is established during S phase, coinciding with the synthesis of the second sister. In prophase of early mitosis, the bulk of cohesin molecules are removed from chromosome arms (also called "prophase pathway") by opening up the interphase between Scc1/Rad21 and Smc3 ("exit gate"). Centromeric cohesin is ultimately proteolytically removed at the metaphase-to-anaphase transition.
Figure 2: The cohesin cycle. Cohesin is topologically loaded onto DNA in telophase by opening up the Smc1-Smc3 interphase (“entry gate”). Sister chromatid cohesion is established during S phase, coinciding with the synthesis of the second sister. In prophase of early mitosis, the bulk of cohesin molecules are removed from chromosome arms (also called “prophase pathway”) by opening up the interphase between Scc1/Rad21 and Smc3 (“exit gate”). Centromeric cohesin is ultimately proteolytically removed at the metaphase-to-anaphase transition.

As you can see, during the 24 hours of a typical mammalian cell cycle, cohesin is pretty much always directly associated with the entire genome (the exceptions being chromosomes arms during most of mitosis, i.e. 20-40 minutes and entire chromatids during anaphase, i.e. ~10 minutes). This means that cohesin has at least the potential to influence a whole bunch of other chromosomal events, like DNA replication, gene expression and DNA topology. And you know what? Turns out it does!

Soon after cohesin was described as this guardian of sister chromatid cohesion, it also became clear that there is just more to it. Take DNA replication for example. There is good evidence that initial cohesin loading is already topological (meaning, the ring closes around the single chromatid). That poses an obvious problem during S phase: While DNA replication machineries (“replisomes”) zip along the chromosomes trying to faithfully duplicate the entire genome in a matter of just a couple of hours, they encounter – on average – multiple cohesin rings that are already wrapped around DNA. Simultaneously, cohesin’s job is to take those newly generated sister chromatids and hold them tightly to the old one. Currently, we don’t really know how this works, whether the replisome can pass through closed cohesin rings, or whether cohesin gets knocked off and reloaded after synthesis. What we do know, however, is that cohesion establishment and DNA replication are strongly interdependent, with defects in cohesion metabolism causing replication phenotypes and vice versa.

Cohesin has also been shown to have functions in transcriptional regulation. It was observed quite early that cohesin can act as an insulation factor, blocking long-range promoter-enhancer association. Today we have good evidence showing that cohesin binds to chromosomal insulator elements that are usually associated with the CTCF (CCCTC-binding factor) transcriptional regulator. Here, the ring complex is thought to help CTCF’s agenda by creating internal loops, i.e. inside the same sister chromatid!

Studying cohesin has, of course, not only academic value. Because of its pleiotropic functions, defects in human cohesin biology can cause a number of clinically relevant issues. Since actual cohesion defects will cause mitotic failure (which most surely results in cell death), most of cohesin-associated diseases are believed to be caused by misregulation of the complex’s non-canonical functions in replication/transcription. These so-called cohesinopathies (e.g. Roberts syndrome and Cornelia de Lange syndrome) are congenital birth defects with widely ranging symptoms, which usually include craniofacial/upper limb deformities as well as mental retardation.

It is important to mention that cohesin also has a very unique role in meiosis where it not only coheses sister chromatids but also chromosomal homologs (the two maternal/paternal versions of a chromosome, each consisting of two sisters, which themselves are cohesed). As a reminder, the lifetime supply of all oocytes of a human female is produced before puberty. These oocytes are arrested in prophase I (prophase of the first meiotic division) with fully cohesed homologs and sisters, and resume meiosis one by one each menstrual cycle. This means that some oocytes might need to keep up their cohesion (between sisters AND homologs) over decades, which, considering the half-life of your average protein, can be challenging. This has important medical relevance as cohesion failure is believed to be the main cause behind missegregation of homologs, and thus, age-related aneuploidies, like e.g. trisomy 21.

After twenty years of research, the cohesin complex still manages to surprise us regularly, as new functions in new areas of cell cycle regulation come to light. Currently, extensive research is conducted to better understand the role of certain cohesin mutations in cancers such as glioblastoma, or Ewing’s sarcoma. And while we’re still far away from completely understanding this complex complex, we already know enough to say that cohesin really is “one ring to rule them all”.


The Phase That Makes The Cell Go Round


By Johannes Buheitel, PhD


There comes a moment in every cell’s life, when it’s time to reproduce. For a mammalian cell, this moment usually comes at a ripe age of about 24 hours, at which it undergoes the complex process of mitosis. Mitosis is one of the two main chromosomal events of the cell cycle. But in contrast to S phase (and also to the other phases of the cell cycle) it’s the only phase that is initiated by a dramatic change in the cell’s morphology that, granted, you can’t see with your naked eye, but definitely under any half-decent microscope without requiring any sort of tricks (like fluorescent proteins): Mitotic cells become perfectly round. This transformation however, as remarkable as it may seem, is merely a herald for the main event, which is about to unfold inside the cell: An elegant choreography of chromosomes, which crescendoes into the perfect segregation of the cell’s genetic content and the birth of two new daughter cells.


To better understand the challenges behind this choreography, let’s start with some numbers: A human cell has 23 unique chromosomes (22 autosomes and 1 gonosome) but since we’re diploid (each chromosome has a homolog) that brings us to a total of 46 chromosomes that are present at any given time, in (nearly) every cell of our bodies. Before S phase, each chromosome consists of one continuous strand of DNA, which is called a chromatid. Then during S phase, a second “sister” chromatid is being synthesized as a prerequisite for later chromosome segregation in M phase. Therefore, a pre-mitotic cell contains 92 chromatids. That’s a lot! In fact, if you laid down all the genetic material of a human cell that fits into a 10 micrometer nucleus, end to end on a table, you would wind up having with a nucleic acid string of about 2 meters (around 6 feet)! The challenge for mitosis is to entangle this mess and ultimately divide it into the nascent daughter cells according to the following rules: 1) Each daughter gets exactly half of the chromatids. 2) Not just any chromatids! Each daughter cell requires one chromatid of each chromosome. No more, no less. And maybe the most important one, 3) Don’t. Break. Anything. Sounds easy? Far from it! Especially since the stakes are high: Because if you fail, you die (or are at least pretty messed up)…


Anatomy of a mitotic chromosome
Anatomy of a mitotic chromosome

To escape this dreadful fate, mitosis has evolved into this highly regulated process, which breaks down the high complexity of the task at hand into more sizable chunks that are then dealt with in a very precise spatiotemporal manner. One important feature of chromosomes is that its two copies – or sister chromatids – are being physically held together from the time of their generation in S phase until their segregation into the daughter cells in M phase. This is achieved by a ring complex called cohesin, which topologically embraces the two sisters in its lumen (we’ll look at this interesting complex in a separate blog post). This helps the cell to always know, which two copies of a chromosome belong together, thus essentially cutting the complexity of the whole system in half, and that before the cell even enters mitosis.

Actual mitosis is divided into five phases with distinct responsibilities: prophase, prometaphase, metaphase, anaphase and telophase (cytokinesis, the process of actually dividing the two daughter cells, is technically not a phase of mitosis, but still a part of M phase). In prophase, the nuclear envelope surrounding the cell’s genetic content is degraded and the chromosomes begin to condense, which means that each DNA double helix gets neatly wrapped up into a superstructure. Think of it like taking one of those old coiled telephone receiver cables (that’s your helix) and wrapping it around your arm. So ultimately, chromosome condensation makes the chromatids more easily manageable by turning them from really long seemingly entangled threads into a shorter (but thicker) package. At this point each chromatid is still connected to its sister by virtue of the cohesin complex (see above) at one specific point, which is called the centromere. It is this process of condensation of cohesed sister chromatids that is actually responsible for the transformation of chromosomes into their iconic mitotic butterfly shape that we all know and love. While our butterflies are forming, the two microtubule-organizing centers of the cell, the centrosomes, begin to split up and wander to the cell poles, beginning to nucleate microtubules. In prometaphase, chromosome condensation is complete and the centrosomes have reached their destination, still throwing out microtubules like it’s nobody’s business. During this whole time, their job is to probe the cytoplasm for chromosomes by dynamically extending and collapsing, trying to find something to hold on to amidst the darkness of the cytoplasm. This something is a protein structure, called the kinetochore, which sits on top of each sister chromatid’s centromere region. Once a microtubule has found a kinetochore, it binds to it and stabilizes. Not all microtubules will bind to kinetochores though, some of them will interact with the cell cortex or with each other to gradually form the infamous mitotic spindle, the scaffold tasked with directing the remainder of the chromosomal ballet. Chromsomes, which are attached to the spindle (via their kinetochores) will gradually move (driven by motor proteins like kinesins) towards the middle region of the mother cell and align on an axis, which lies perpendicular between the two spindle poles. This axis is called the metaphase plate and represents a visual mark for the eponymous phase. The transition from metaphase to anaphase is the pivotal moment of mitosis; the moment, when sister chromatids become separated (by proteolytic destruction of cohesin) and subsequently move along kinetochore-associated microtubules with the help of motor proteins towards cell poles. As such a critical moment, the metaphase-to-anaphase transition is tightly safeguarded by a checkpoint, the spindle assembly checkpoint (SAC), which ensures that every single chromatid is stably attached to the correct side of the spindle (we’ll go into some more details in another blog post). In the following telophase, the newly separated chromosomes begin to decondense, the nuclear envelope reforms and the cell membrane begins to restrict in anticipation of cytokinesis, when the two daughter cells become physically separated.


Overview over the five phases of mitosis.
Overview over the five phases of mitosis (click to enlarge).

To recap, the process of correctly separating the 92 chromatids of a human cell into two daughter cells is a highly difficult endeavor, which, however, the cell cleverly deals with by (1) keeping sister chromatids bound to each other, (2) wrap them  into smaller packets by condensation, (3) attach each of these packets to a scaffold a.k.a. mitotic spindle, (4) align the chromosomes along the division axis, so that each sister chromatid is facing opposite cell poles, and finally (5) move now separated sister chromatids along this rigid scaffold into the newly forming daughter cells. It’s a beautiful but at the same time dangerous choreography. While there are many mechanisms in place that protect the fidelity of mitosis, failure can have dire consequences, of which cell death isn’t the worst, as segregation defects can cause chromosomal instabilities, which are typical for tissues transforming into cancer. In future posts we will dive deeper into the intricacies of the chromosomal ballet, that is the centerpiece of the cell cycle, as well as the supporting acts that ensure the integrity of our life’s code.


How Science Trumps Trump: The Future of US Science Funding


By Johannes Buheitel, PhD

I was never the best car passenger. It’s not that I can’t trust others but there is something quite unsettling about letting someone else do the steering, while not having any power over the situation yourself. On Tuesday, November 8th, I had exactly this feeling, but all I could do was to sit back and let it play out on my TV set. Of course, you all know by now, I’m talking about the past presidential election, in which the American people (this excludes me) were tasked with casting their ballots in support for either former First Lady and Secretary of State Hillary Clinton or real estate mogul and former reality TV personality Donald Trump. And for all that are bit behind on their Twitter feed (spoiler alert!): Donald Trump will be the 45th president of the United States of America following his inauguration on January 20th, 2017. Given the controversies around Trump and all the issues he stands for, there are many things that can, have been  and will be said about the implications for people living in the US but also elsewhere. But for us scientists, the most pressing question that is being asked left and right is an almost existential one: What happens to science and its funding in the US?

The short answer is: We don’t know yet. Not only has there been no meaningful discussion about these issues in public (one of the few exceptions being that energy policy question  by undecided voter-turned-meme Ken Bone), but, even more worryingly, there is just not enough hard info on specific policies from the future Trump administration to go on. And that means, we’re left to just make assumptions based on the handful of words Mr. Trump and his allies have shared during his campaign. And I’m afraid, those paint a dire picture of the future of American science.

Trump has not only repeatedly mentioned in the past that he did not believe in the scientific evidence around climate change (even going as far as calling it a Chinese hoax), but also reminded us of his position just recently, when he appointed  known climate change skeptic Myron Ebell to the transition team of the Environmental Protection Agency (EPA). He has furthermore endorsed the widespread (and, of course misguided) belief that vaccines cause autism. His vice president, Mike Pence, publicly doubted  that smoking can cause cancer as late as in 2000, and called evolution “controversial”.

According to specialists like Michael Lubell from the American Physical Society, all of these statements are evidence that “Trump will be the first anti-science president we have ever had.” But what does this mean for us in the trenches? The first thing you should know is that science funding is more or less a function of the overall US discretionary budget, which is in the hand of  the United States Congress, says  Matt Hourihan, director of the R&D Budget and Policy Program for the American Association for the Advancement of Science (AAAS). This would be a relief, if Congress wasn’t, according to Rush Holt, president of the AAAS, on a “sequestration path that […] will reduce the fraction of the budget for discretionary funding.” In numbers, this means that when the current budget deal expires next year, spending caps might drop by another 2.3%. Holt goes on to say that a reversal of this trend has always been unlikely, even if the tables were turned, which doesn’t make the pill go down any easier. Congress might raise the caps, as they have done before, but this is of course not a safe bet, and could translate to a tight year for US science funding.

So when the budget is more or less out of the hands of Donald Trump, what power does he actually possess over matters of research funding? Well, the most powerful political instrument that the president can implement is the executive order. But also this power is not unlimited and could for example not be used to unilaterally reverse the fundamentals of climate policy, said David Goldston from the Natural Resources Defense Council (NRDC) during a Webinar hosted by the AAAS shortly after the election. Particularly, backing out of the Paris agreement, as Trump has threatened to do, would take at least four years and requires support by Congress (which, admittedly, is in Republican hand). And while the president might be able to “scoop out” the Paris deal by many smaller changes to US climate policy, this is unlikely to happen, at least not to a substantial degree, believes Rush Holt. The administration will soon start to feel push-back by the public, which, so Holt during the AAAS Webinar, is indeed not oblivious about the various impacts of climate change, like frequent droughts or the decline of fisheries in the country. There was further consensus among the panelists that science education funding will probably not be deeply affected. First, because this matter usually has bipartisan support, but also because only about 10% of the states’ education funding actually comes from the federal budget.

So, across the board, experts seem to be a reluctantly positive. Whether this is just a serious case of denial or panic control, we don’t know, but even Trump himself has been caught calling for  “investment in research and development across a broad landscape of academia,” and even seems to be a fan of space exploration. Our job as scientists is now, to keep our heads high, keep doing our research to the best of our abilities but also to keep reaching out to the public, invite people to be part of the conversation, and convincing them of the power of scientific evidence. Or to say it with Rush Holt’s words: “We must make clear that an official cannot wish away what is known about climate change, gun violence, opioid addiction, fisheries depletion, or any other public issue illuminated by research.”


In the Life of a Cell

An introduction to the cell cycle


By Johannes Buheitel, PhD

Omnis cellula e cellula”. We all heard or read this sentence probably sometime during college or grad school and no, it’s not NYU’s university motto. This short Latin phrase, popularized by the German physician/biologist Rudolf Virchow, states a simple fact, which, however, represents a fundamental truth of biology: “All cells come from cells”. It’s so fundamental that we often take it for granted that the basis for all of those really interesting little pathways and mechanisms that we study is life itself; and, moreover, that life is not simply “created” from thin air but can actually only derive from other life. Macroscopically, you (and Elton John) might call this “the circle of life” but microscopically, we’re talking about nothing less than the cell cycle. But what is the cell cycle exactly? What has to happen when and how does the cell maintain this order of events?

The cell cycle’s main purpose is to generate two identical daughter cells from one mother cell by first, duplicating all its genetic content in order to get two copies of each chromosome (DNA replication), and then carefully distributing those two copies into the newly forming daughter cells (mitosis and cytokinesis). These two major chromosomal events take place during S phase (DNA replication) and M phase (mitosis), which during consecutive cycles alternate, separated by two “gap” phases (G1 between M and S phase and G2 between S and M phase; FYI: everything outside M phase is also sometimes also called interphase). It goes without saying that the temporal order of events, G1 to S to G2 to M phase, must be maintained at all times; just imagine trying to divide without previously having replicated your DNA! And not only the order is important, but each phase must also be given enough time to faithfully fulfill its purpose. How is this achieved?

If you want to boil it down, there are two main principles that drive the cell cycle: timely expression and degradation of key proteins and irreversible switch-like transitions, called checkpoints. So let’s try and get an overview over each of these principles.

Overview over the different phases of the cell cycle: G1 (“gap”) phase, S (“synthesis”) phase, G2 phase and M (“mitosis”) phase.

In the early eighties, a scientist called Tim Hunt performed a series of experiments, unknowing that these will turn into a body of work, which will ultimately win him a Nobel prize. For these experiments, he radioactively labeled proteins in sea urchin embryos (yes, you read correctly!) and stumbled across one that exhibited an interesting pattern of abundance over time in that it appeared and vanished in a fashion that was not only cyclic but also seemed to be in sync with the embryos’ division cycles. Dr. Hunt had just found the first member of a protein family, which later turned out to be one of the main drivers of the cell cycle: the cyclins. What do cyclins do? Cyclins are co-activators of cyclin-dependent kinases or Cdks, whose job it is to phosphorylate certain target proteins in order to regulate their function in a cell cycle-dependent fashion. Since Cdks are pretty much around all the time, they need the cyclins to tell them, when to be active and when not to be. There are a variety of different Cdks, which interact very specifically with various cyclins. For example, cyclin D interacts with Cdk4 and 6 to drive the transition from G1 to S phase, while a complex between cyclin B and Cdk1 is required for mitotic entry. This system allows for enough complexity to explain how the proper length of each phase is assured (slow accumulation of a specific cyclin until the respective Cdk can be fully activated), but also how the correct order of events is maintained; because it turns out that the expression of, say, the cyclin assigned to start replication (cyclin E) is dependent on the activity of the Cdk/cyclin complex of the previous phase (in this example: Cdk4/6-cyclin D) via phosphorylation-dependent regulation of transcription factors.

The second principle I was talking about, are checkpoints. A checkpoint is a way for the cell to take a short breath and check if things are running smoothly so far, and if they are not, to halt the cell cycle in order to give itself some time to either resolve the issue or, if that’s not working out, throw in the towel (i.e. apoptosis). Researchers describe more and more checkpoint-like pathways that react to different stimuli all over the cell cycle, but canonically, we distinguish three main ones: the restriction checkpoint at the G1 to S phase transition, the DNA damage checkpoint at the G2 to M phase transition and the spindle assembly checkpoint (SAC) during mitosis at the transition from metaphase to anaphase. What do these checkpoints look for, or in more technical words, what requirements have to be met in order for a checkpoint to become satisfied? The restriction checkpoint integrates a variety of internal and external signals, but is ultimately satisfied by proper activation of S phase Cdk complexes (see above). The DNA damage checkpoint’s main function is to give the cell time to correct DNA damage, which naturally occurs during genome replication but can also be introduced chemically or by ionizing radiation. Therefore, it remains unsatisfied as long as the DNA damage kinases ATM and ATR are active. Finally, the SAC governs one of the most intricate processes of the cell cycle: the formation of the mitotic spindle including proper attachment of each and every chromosome to its microtubules. After a checkpoint becomes satisfied, one or more positive feedback loops spring into action and effectively jump re-start the cell cycle.

As one can imagine, all of these processes must be exquisitely controlled to ensure the mission’s overall success. In future posts, we will explore those mechanisms in more detail and will furthermore discuss, how a handful of biochemical fallacies can have the potential to turn this wonderful circle of life into a wicked cycle of death.