1.4 Midcell localization in Myxococcus xanthus
1.4.2 Pom cluster dynamics
Since PomZ seems to play a crucial role in positioning the cluster at midcell, it is important to understand its dynamics and how it interacts with the other components (Pom proteins, DNA) involved. PomZ is a ParA-like ATPase and its ATPase activity is stimulated synergistically by PomX, PomY and DNA. If PomZ is only in contact with non-specific DNA, it has an ATP turnover rate of (1.7±1.2) ATP/h, whereas this turnover increases to (34.0±1.3) ATP/h if PomX and PomY proteins are present.
PomZ variants (one specific amino acid substituted by another) can be used to infer in which state PomZ binds to the DNA and interacts with the PomX and PomY proteins.
Fluorescence images of cells with these variants indicate that PomZ binds only in the ATP-bound dimeric form to the nucleoid and recruits the PomXY cluster only in this state to the nucleoid (see Fig. 1.2E).
So far, we observed that PomX and PomY proteins form a cluster and PomZ is necessary for positioning this cluster at midcell, but how does the Pom cluster influence the Z-ring formation at midcell, which then constricts the cell? To investigate this question, experiments with both PomX or PomY proteins and FtsZ fluorescently labelled were performed. They show that the PomX and PomY proteins localize to midcell before FtsZ. Further experiments show that the Pom proteins also localize to midcell in the absence of FtsZ, indicating that the Pom cluster is positioned at midcell first and then recruits the Z-ring to this position. Furthermore, there is experimental evidence that PomY and PomZ interact with FtsZ and PomY in the Pom cluster is important for the recruitment of the Z-ring to the division site. However, the details of the recruitment process are not known and need to be investigated further.
A
C B
D ori
old pole new pole
ter
ori
old pole new poles
ter ter ori
old pole
Figure 1.3 Dynamics of the Pom cluster. (A) Time-lapse images of aM. xanthus cell with PomX labelled. PomX proteins accumulate in a cluster and this cluster moves from a position close to the cell poles to midcell in about 80 min. (B) Time-lapse images with a higher temporal resolution (PomY is labelled) for clusters starting off-center or at midcell and cells that lack PomZ proteins. If PomZ is absent, the cluster is stalled at its initial position, showing that PomZ is important for the positioning of the Pom cluster. (C) Mean square displacement curves of the cluster over time for cells with and without PomZ. A locus on the genome (tetO) shows only little motion, indicating that the cluster is not “piggybacked”
on the terminus region of the chromosome. (D) Sketch of chromosome segregation in M.
xanthus. DNA replication starts at the origin (ori) close to one of the cell poles. During chromosome segregation the duplicated origin moves across the cell until the chromosome is fully replicated. The cell then divides such that the terminus regions of the chromosome (ter) are close to the new cell poles. Subfigures (A-C) are taken from [2].
increases and then seems to reach a plateau. This is in accordance with the constrained movement of the clusters at midcell observed in the time-lapse experiments. In ∆pomZ mutants we do not distinguish between off-center and midcell clusters as no bias in the cluster’s movement is observed independent of the position of the cluster in the cell.
These clusters show a small MSD, in agreement with the observation that PomZ is important for the clusters mobility.
In order to explain the cluster’s movement from an off-center position to midcell, one could argue as follows: InM. xanthus the bacterial chromosome is oriented inside the cell such that the origin of replication, ori region (DNA sequence that signals
the start of replication) is close to the old pole and the terminus region, ter (stop sequence) is at the opposite site [102]. When the bacterial chromosome is segregated, the terminus moves from a position close to the cell pole to midcell [103] (see sketch, Fig. 1.3D). As this dynamics resembles the observed dynamics of the Pom cluster, one might hypothesize that the Pom cluster is just “piggybacked” on the terminus site of the chromosome. However, the terminus is a lot less mobile than Pom clusters starting from an off-center position (Fig. 1.3C), which speaks against such a mechanism. Other facts disfavoring this mechanism are that PomZ dimers bind, in their ATP-bound dimeric state, non-specifically to the nucleoid and the Pom cluster localizes at midcell when DNA replication is still ongoing.
B A
C
A B
-1 0 9 sec
PomZ-mCh
125 100
25 50 75
10 8 6 4 2 0 -2
time [sec]
intensity [%]
t1/2= 1.2±0.2 sec
125 100 75
25 50
10 8 6 4 2 0 -2
time [sec]
intensity [%]
t1/2= 1.7±0.4 sec
PomZ-mCh
-1 0 9 sec
PomZ-mCh
-1 0 1 60 sec
125 100
25 50 75
100 80 60 40 20 0
-20 time [sec]
intensity [%]
t1/2= 8.3±0.4 sec
-1 0 1 60 sec
PomZG62V-mCh
0 3 sec PomZK66Q-mCh
0 3 sec PomZD90A-mCh
0 3 sec PomZK268E-mCh
0 3 sec
PomZ-mCh
0 3 sec
C
PomZD90A-mCh
125 100
25 50 75
100 80 60 40 20 0 -20
intensity [%]
t1/2= 5.3±0.3 sec time [sec]
OEOE
Figure 1.4 PomZ is highly dynamic. (A) FRAP experiments of PomZ-mCh with a bleach spot over the cluster and over the nucleoid. On the right side, the fluorescence recovery curves of the regions indicated in the sketch of the cell are shown. (B) FRAP data for PomZ-mCh and PomZD90A-mCh overexpressed more than 50-fold. (C) Fluorescence signal of PomZ-mCh, the variants that cannot bind DNA (PomZK268E-mCh, PomZG62V-mCh, PomZK66Q-mCh) and the ATP hydrolysis mutant (PomZD90A-mCh) before and after bleaching for 3 s. The figure is taken from [2].
If the cluster is not “piggybacked” on a specific chromosome site, how do PomZ dimers lead to the movement of the cluster? To shed light on this question, the dynamics of the PomZ dimers was investigated. So far, we know that PomZ binds in its ATP bound dimeric state to the nucleoid and interacts with the PomXY cluster. A
common method to study the dynamics of proteins inside cells is FRAP, fluorescence recovery after photobleaching. Here, the fluorescence fusion protein (PomZ-mCh) is bleached with a laser in a small spot inside the cell and the recovery of the fluorescence signal at this spot is recorded. Such experiments are typically used to measure the diffusion constants of proteins and other rate constants (see section 2.2).
If PomZ is bleached over the cluster, the fluorescence intensity recovers quickly (half recovery time: t1/2 = (1.2±0.2) s) (Fig. 1.4A). This indicates that there is a fast turnover of PomZ dimers at the cluster. Already during bleaching with a laser pulse of 60 ms duration, the fluorescence signal of PomZ over the nucleoid outside of the bleaching spot is reduced (Fig. 1.4A), which shows that PomZ dimers are highly dynamic on the nucleoid. Fast dynamics of PomZ is also observed when PomZ-mCh is bleached over the chromosome instead of the cluster. After the 60 ms laser pulse, the fluorescence intensity on the chromosome of the site of the cluster where the bleach spot was located is drastically reduced (Fig. 1.4A).
To analyze diffusion of PomZ dimers on the nucleoid FRAP experiments of cells with PomZ-mCh and PomZD90A-mCh (mutant that does not hydrolyze ATP) overexpressed more than 50-fold were performed (Fig. 1.4B). First, we observe that the fluorescence intensity is high over the region where we expect the nucleoid and no cluster is visible although PomX and PomY proteins are present. Hence, we conclude that the capacity of PomZ dimers that can interact with the cluster at the same time is exceeded in these cells with PomZ overexpressed. Furthermore, if PomZ-mCh or PomZD90A-mCh is bleached (bleach spot over the nucleoid), the intensity recovers quickly, but more slowly compared to cells with less PomZ dimers in the cell. This can be attributed to the fact that PomZ dimers hinder each other in their movement because of crowding effects. PomZD90A binds in its ATP-bound dimeric state to the nucleoid, but cannot hydrolyze ATP. Experiments show that these proteins primarily localize over the nucleoid, indicating that detachment from the nucleoid is ATP hydrolysis dependent.
Since the ATPase activity of PomZ is stimulated by PomX, PomY and DNA, release of nucleoid-bound PomZ dimers mainly occurs at the Pom cluster for PomZ-mCh and no significant detachment from the nucleoid is expected for PomZD90A-mCh. Hence, the recovery of PomZD90A-mCh is mainly due to diffusion of PomZ proteins on the nucleoid instead of an exchange with the cytosol. In the case of PomZ-mCh, the fluorescence recovery is due to both diffusion on the nucleoid and exchange via the cytosol, thus explaining the slightly shorter recovery time. However, the recovery times for PomZ-mCh or PomZD90A-mCh overexpressed are very close, which suggests that the main factor that leads to the fluorescence recovery is fast diffusion of PomZ on the nucleoid in both cases.
If the dynamics of a protein is very fast, it is difficult to perform FRAP experiments, as a large fraction of the signal around the bleaching spot is gone after the laser pulse was applied (as observed for the PomZ dimers, see Fig. 1.4A). Hence, our collaboration partners performed the following bleaching experiments: They bleach at a spot over the nucleoid for 3 s and image the intensity distribution inside the cell before and after bleaching (Fig. 1.4C). For three variants of PomZ that cannot bind to the nucleoid (PomZK268E-mCh, PomZG62V-mCh and PomZK66Q-mCh) the intensity is drastically
reduced after 3 s showing that their dynamics in the cell is fast. Since they cannot bind to the nucleoid, they most likely diffuse in the cytosol, which is typically a fast process (the diffusion constant for Min proteins in the cytosol is on the order of 10 µm2/s, [104]).
Another interesting observation is that in cells with PomZD90A-mCh (not overexpressed) the proteins accumulate in a cluster and bleaching outside of the cluster does not change the intensity of the cluster significantly, whereas for PomZ-mCh the intensity is reduced a lot. This suggests that ATP hydrolysis is essential for the fast turnover of PomZ at the cluster and binding of nucleoid-bound PomZ dimers to the cluster occurs more frequently than detachment of PomZ dimers from the cluster such that they stay bound to the nucleoid, if this process occurs at all.