BioLinks Journal Club 2 - The Immune System during Mitosis

Paper: “Phosphorylation of cGAS by CDK1 impairs self-DNA sensing in mitosis”

Please read the introduction to this week’s research paper below, which includes the most crucial concepts and experiments involved. Don’t worry if you don’t understand everything in this summary - we will break it down over the course of the week. Please remember questions that come up from reading this or anything of particular interest you would like to discuss more on Monday!

How do cells protect their own DNA from the immune response during mitosis? When a cell is not dividing (interphase), self-DNA is compartmentalized in the nucleus, making it easier for cells to recognise DNA in the cytosol as pathogenic and differentiate this genetic material from our own. Further, cytosolic sensors are sensitive to particular molecular structures present in microbial but not human DNA called pathogen-associated molecular patterns (PAMPs). Similar to how our immune system can identify and create antibodies against foreign cells by distinct protein structures on their surfaces, PAMPs are a key factor for flagging non-host DNA. However, our cells are constantly dividing through mitosis, which presents an obstacle to this recognition method as the nuclear envelope, the crucial barrier separating self and microbial DNA, breaks down. Given that the continuation of this pathway through mitosis would cause our bodies to destroy our own genetic material, scientists speculate that this aspect of our innate immunity is suspended during division. But how? The mechanism behind this is explored in this paper. cGAS (cyclic GMP-AMP synthase) is one of these cytosolic DNA sensors for microbial DNA. When cGAS binds to DNA in the cytoplasm, it triggers a pathway that ultimately leads to the synthesis of cytokines, which are secreted immune signalling proteins. Given the key role of cGAS in inducing the immune response, this group investigates whether and how cGAS is inactivated during cell division. First, they confirmed that cGAS was inactivated during mitosis by imaging the location of cGAS throughout the cell cycle and comparing levels in the cytoplasm and near the cell’s chromosomes. They observed a rapid decline in cGAS levels in the cytoplasm and an increase of levels in the nucleus with the beginning of mitosis: cGAS levels in the cytoplasm were dynamically regulated to match the state of the nuclear envelope. As cGAS is a cytosolic sensor, this result suggests its usual function does not continue in the nucleus and corroborates the idea that DNA sensing in the cytoplasm may be shut down when there is no barrier between self and foreign DNA. But does cGAS translocation to chromosomes during mitosis actually prevent DNA sensing-triggered immune response? When cells were arrested during mitosis, levels of key intermediate products of the cGAS immune pathway were measured to be at almost untraceable levels in the dividing cells. These results suggested that chromosome bound cGAS cannot perform its usual role, however this had to be confirmed as it was possible that the DNA sensing mechanism was inactive simply because the mitotic DNA itself was incapable of eliciting an immune response. To test this, rodent lung cells were injected with human mitotic and non-mitotic cells and the activity of the cGAS pathway was tested. The similar levels of DNA sensing activity in cells injected with mitotic and non-mitotic products suggested that it was the binding of cGAS to chromosomes that caused suspension of the immune response. Once in the nucleus, what prompts the cGAS pathway’s inability to sense DNA? The addition of a phosphate group to a molecule, or phosphorylation can completely alter the 3D structure of a compound, thus often causing inactivation or activation. Given this and previous studies showing that cGAS phosphorylation suppresses its enzymatic capabilities, the group hypothesized that cGAS was selectively phosphorylated during mitosis, leading to its inactivation. They performed an experiment that indicated that there were 2 sites of phosphorylation in cGAS’ genome, S291 and S305, that were highly present in mitotic but not in non-mitotic equivalent cells. To test the viability of S305 phosphorylation as responsible for the observed phenomenon, they mutated the site to mimic phosphorylation and found that a key reporter in the cGAS pathway was unable to be activated. Furthermore, the location of cGAS’ S305 in the domain of the enzyme where the substrate binds suggests that the inactivation of cGAS occurs because S305 phosphorylation does not allow the enzyme to have catalytic effect (enzymes have specific shapes that fit lock and key with their substrates). If phosphorylation of cGAS inactivates it during mitosis, exit from the mitotic cycle likely involves the reversal of the phosphate group addition (dephosphorylation). What controls the phosphorylation and dephosphorylation of cGAS? Kinases are enzymes responsible for phosphorylating molecules. Cyclin-Dependent Kinases (CDKs) are particularly important kinases for cell cycle regulation, controlled by the levels of cyclins within cells. When certain concentrations of cyclins are reached in cells, they bind to enough CDKs to activate their enzymatic capabilities and cause cellular response, usually the phosphorylation of a key cell cycle enzyme that allows mitotic progression. As activation and inactivation of cGAS was established in previous experiments to follow the oscillation of the cell cycle, they found through a series of tests (more on this on Monday!) that CDK1 and its partner cyclin, cyclin B, levels were responsible for controlling the timely phosphorylation and dephosphorylation of cGAS at the entry and exit of mitosis respectively. Phosphatases, opposite in function to kinases like CDK1, are enzymes that dephosphorylate molecules. In the case of cGAS, it was found that a particular phosphatase, PP1 is involved. While PP1 was kept inactive by CDK1 until the end of the mitotic phase, it dephosphorylates cGAS upon mitotic exit, reactivating its DNA sensing capabilities as the nuclear envelope was reestablished. The topic of this paper, cGAS inactivation during mitosis, is a demonstration of the power of post-translational modifications like phosphorylation. The carefully timed addition and removal of phosphate groups completely alters the state of proteins, and this data suggests that phosphorylation is at least partially responsible for preventing inflammatory response to our own DNA whenever our cells divide, which for skin cells can be once a day! This paper also brings up a new intriguing question of whether mitotic cells are more susceptible to infection, or how mitotic cells prevent viral DNA infection if the usual cytosolic DNA sensor cGAS is inactivated during that period. We will discuss the exact pathway through which cGAS operates, break down the paper's figures and take a section by section closer look at strategies for reading and the experiments involved on Monday. For Friday, we will dive into some of the common research techniques utilized in this study. Again, come prepared with any questions you may have!