Speaker: Christine Jacobs-Wagner
Department: Microbial sciences institute, dept. of molecular cellular and developmental biology
Subject: How to achieve cellular replication without fail: lessons from bacterial cells
Location: TU Delft, BN-seminar
Author: Maricke Angenent
A cell partitioning mechanism which does not involve any of the complicated processes such as cytoskeleton filament assembly, motor protein functioning and the participation of the actin proteins. That is a system we were introduced to by Christine Jacobs-Wagner, who is currently the principle investigator in the Jacobs-Wagner lab at the university of Yale. She started her seminar by giving a short outline on cell replication in general. Hereafter the presentation would consist of two distinct parts. Firstly the focus was on how to ensure that partition of cell compartments happens equally among daughter cells. The second part was supposed to be about how different processes are integrated, especially how the cell cycle function integrates cell metabolism. The latter topic would have been rather innovative as the cell cycle and metabolism are usually viewed separately. Unfortunately an hour proved too short to cover both of the subjects, so instead Christine set on answering some additional questions on the first subject.
Christine started by emphasizing the fact that replication is a truly remarkable process. As she said “the ability to replicate distinguishes the non-living organisms from the living”, which is the source of her fascination for the process. In her lab, bacterial replication in particular is analyzed, since replication is fast and it is an incredibly robust processes. Replication is a stochastic process, meaning that it is submissive to internal variations, yet the process seems to never fail. One of the proposed reasons for this observation is that various processes are highly integrated and often rely on intracellular organization. But how exactly is this spatial organization achieved?
Though diffusion is an effective means of molecular transport in bacteria, due to travel distances being small, this only leads to random distributions. This in itself is not a problem, however when you have a low copy number, the probability of finding all copies of a cellular compartment on one side of the cell considerably increases. Consequently, the chance to obtain unequal daughter cells also rises. Diffusion in bacteria is thus ineffective for partitioning cellular compartments in low count. A different separation machinery must be present in bacteria to partition single components. As Christine explained, this mechanism makes use of stochastic interactions that are transformed into active transport, which results in the spatial patterning of low count cell compartments. Based on experiments, it was concluded that when you have more than one copy in the cell, they are equidistantly distributed among the axis of the cell ensuring their propagation to the daughter cells. This distribution was analyzed for the plasmid system, as this system is best understood compared to others.
In this experiment, the mechanism of just a single plasmid was taken into account rather than of a cluster of plasmids, which you would normally encounter in real situations. Two proteins are of central importance in the partitioning mechanism: parA and par B. The former mediates active transport and binds to the chromosomal DNA as a dimer. The latter binds to a specific sequence of a plasmid, resulting in a plasmid rich region (or so called cargo). Bound parA interacts with the parB cargo, resulting in the oscillation of a single plasmid cluster over the nucleoid. What is interesting, is that parA does not form any cytoskeleton filaments, nor does it use any motor proteins, yet the oscillations point to a form of active transport. In modeling this process it is important to assume that chromosomal DNA is not static. Instead the DNA (and thus also when parA is bound) experiences intrinsic fluctuations, which is observed as “wiggling”. Now the plasmid cargo (=parB) diffuses until it encounters parA, where it activates hydrolyzation. Consequently the parA is released from the DNA and parB can go on to interact with a different parA-DNA complex. When plotting the movement of parB over time, you obtain evidence for oscillatory behavior, where the plasmid goes from one end of the nucleoid to the other end of the nucleoid. This movement usually follows the direction of the parA gradient as the chance for interactions is higher in an environment with a higher concentration of bound parA.
The above figure shows the interaction of two parB plasmids, resulting in an opposite spatial organization.
In practice not just one, but multiple plasmid cargo’s are present in the cell. All of them undergoing interactions with the DNA-bound parA. These cargo’s influence each other’s direction because as they get closer the surrounding will be depleted of parA making sure that the cargo’s will turn around to go to a more favorable surrounding (where more parA is present). This influence happens continuously and as a result a regular pattern arises in which the cargoes are aligned along the axis of the bacteria. The regular patterning leads to correct partitioning of low counts during replication.
So in a nutshell, Christine introduced us to a replication mechanism, simpler and easier to generate than the conventional system. A mechanism in which there is no need for cytoskeletal structures or motor proteins since the DNA fulfills a mechanical function instead. Only two factors are needed, factor A binding the DNA and factor B ensuring the lifetime of binding of A. This simple mechanism relies on random fluctuations in the chromosomal DNA and stochastic interactions between various plasmids, which leads to a perfectly organized, spatial pattern. Overall I thought the seminar was fascinating, especially because I had never heard of this alternative partitioning machinery before. In addition, Christine incorporated many expressions which we have just covered in our lectures on physical biology of the cell, showing me how the theoretical information is also really applied nowadays in the laboratories.