The last thirty years have been an "eye-opening" journey for microbial cell biologists, quite literally. I am talking, of course, about fluorescent labeling of individual cell components and the non‑invasive observation of their "behavior" in living bacterial cells. Just recently, I considered a video clip about Vibrio cholerae harpooning DNA. Now again a video clip, this time about In vitro assembly, positioning and contraction of a division ring in minimal cells − the notorious FtsZ ring − as the title of a recent paper by Kohyama et al. (2022) goes.
Petra Schwille's lab at the MPI für Biochemie in Martinsried, Germany, has set itself the ambitious goal of recapitulating the complex process of cell division in E. coli by its reconstruction in vitro. Their most recent paper describes the current achievement: functional establishment of the MinCDE oscillator in an artificial cell, a membrane-bound vesicle, that "shovels" newly synthesized FtsZ filaments and "ringlets" to the equator of the vesicle that then shows signs of constriction. All this within a little more than two hours of incubation, and stable over this time span (Figure 1 here is Supplementary Movie 8 of their paper).
What you see in the video clip, side by side, are the simultaneously filmed images of a vesicle in the light microscope (right) and the fluorescence excited by laser light inside this vesicle (left). The two E. coli proteins that you see moving within the vesicle are FtsZ in "green" and MinC in "magenta" (the caption to Figure 1 gives more details). Pay attention to the timer, which tells you that 2 hours and 15 minutes of observation are condensed into the time-lapse video of 40 seconds that you see in loop. The dynamic processes thus run considerably slower in real time! Also note the scale bar, which tells you that the diameter of the vesicle is ~40 µm, that is, roughly 10‑times that of an E. coli cell.
At 0:06 (h:min) into the video, you can see faintly shining "green" FtsZ filaments distributed randomly on the inner side of the vesicle membrane that become more prominently visible from 0:16 onwards. At around 0:25 you can see the specific growth mode of FtsZ filaments called "threadmilling": in a GTP-hydrolysis-dependent reaction, FtsZ monomers add to one end of the filament, while monomers detach from the other end. Growth or shrinkage of the filament is then determined by varying on/off rates (see here for a diagram).
After 0:16, you also see a coarse meshwork of FtsZ "ringlets" of varying diameters concomitant with a decrease in the number of filaments. Then, after 0:46, you see the vesicle wobble and a faintly shining cloud of "magenta" MinC. From 1:04 onwards you can see that the MinC protein oscillates between both halves of the vesicle, but the compression of the time-lapse prevents you from seeing the actual amplitude of the oscillation, which is close to 20 sec (see Figure 3, Bottom part). Keep in mind that you see a two-dimensional image of a 3D object. Thus, what appears as a "cloud" of MinC is actually the lining of the inside of the vesicle membrane by MinC rather than its diffuse distribution in the "cytoplasm" as explained below. In several instances you see in addition to the "cloud" a pronounced accumulation of MinC in "polar" regions of the vesicle.
Also after 1:04, most of the FtsZ "ringlets" grow to the diameter of the vesicle, and their fluorescence becomes brighter. This indicates a bundling of lengthwise aligned FtsZ filaments that finally results in the large "rings," all the while the individual filaments still threadmill. From 1:21 to the end of the video you see in the phase-contrast light microscopy (right panel) a slight indent, a constriction, that corresponds to the region where FtsZ rings successively accumulate (left panel).
An aside: you surely noticed the tiny greenish dot on the right side below the large vesicle. This is a tiny vesicle that you also see in Figure 2b, which shows that vesicle sizes in their experiments varied a lot.
What you can not see but need to know to appreciate the movie. The cell-free expression system of Kohyama et al. (2022) provided "invisible," that is untagged FtsA protein in addition to tagged, "green" FtsZ (see Figure 2a). Membrane-bound FtsA is instrumental in transiently attaching FtsZ filaments to the membrane, and without it more straight FtsZ filaments would have been randomly distributed in the vesicle's lumen. The various large FtsZ rings seen, for example at 1:27:20, would coalesce into one single compact ring as in real cells if ZapA were present. E. coli ZapA is known to cooperatively increase the spatial order of the filament network, but binds only transiently to FtsZ as sort of a crosslinker. However, ZapA was not included in the cell-free expression system, and Kohyama et al. (2022) explain that so far they could not obtain functional expression systems for not more than the 5 components indicated in Figure 2a.
You cannot "see" in the movie the untagged MinD and MinE proteins that were included in the expression system together with "magenta" tagged MinC (Figure 2a). To explain this better I give a brief introduction to the MinCDE system.
The MinCDE oscillator is a fascinating transient dynamic structure in growing E. coli cells. Guided by MinD, MinE forms a ring-like structure, the MinE ring, along the inner membrane at midcell, in the region of later constriction, septation, and cell division (Figure 3, Top). MinE can be visualized in elongating cells up to the point where the weakly indented midcell region becomes a prominent constriction, indicating its disassembly (Figure 3, compare b/b' with c/c'). Formation of the MinE ring is independent of FtsZ ring formation, and FtsZ and MinE rings appear rather to abut and not superimpose by microscopy. MinD binds to the inner membrane and forms large oligomeric patches together with MinC ("magenta" MinC in the Figure 1 movie). These patches form and dissolve in an oscillating manner in both cell halves dependent on their local concentrations (Figure 3, Bottom). MinD binds to both MinE and MinC but not simultaneously. This is what gets the oscillator started, and leads to MinE accumulation as ring-like structure at midcell while MinC accumulates in both polar regions (see here for a more detailed description of the oscillator).
It was already found in 1993 that FtsZ‑ring formation at midcell is suppressed in a Δmin mutant (FtsZ localizes randomly and eventually forms rings at sites close to the cell poles). Thus, MinCD patches alternating between both cell halves prevent the FtsA-mediated attachment of FtsZ filaments and result in their accumulation as FtsZ rings at midcell. This is an almost brazen but approximately correct summary of the current state of knowledge. And it is what you see in the video (Figure 1).
To conclude, I have to come back to a quip that Emanuele Severi had heard at a conference and shared on Twitter: "What is true for E. coli might be true for an elephant, but certainly not for Bacillus!" I know that Erin Goley and Yves Brun would add: "...and even less for Caulobacter." Yes, it's true, FtsZ rings form at midcell at the onset of cell division in both Bacillus and Caulobacter. Also most other coccoid or rod-shaped bacteria across the numerous phyla have ftsZ genes; even symbionts with reduced genomes like Buchnera aphidicola (0.65 Mb) and Stammera capleta (0.27 Mb) have them (if and how they form FtsZ rings during cell division is everyone's guess). But both Bacillus and Caulobacter lack the MinCDE oscillator and each have completely different mechanisms for positioning their Z rings at midcell. This is the issue with virtually all bacterial "model systems" for cellular processes in "model organisms" like E. coli: close and distant relatives tend to use very similar vocabulary but employ often drastically different syntax and grammar.
My personal attraction for microscopy movies goes back to a workshop in 1997 (Figure 4). We had brought together almost all the leading researchers working on the bacterial cell cycle at that time − many still do today. To name just a few "divisionists," Joe Lutkenhaus talked about the FtsZ ring, of course (see Pictures Considered #5), Yves Brun and James Gober about FtsZ in Caulobacter, Miguel Vicente and David Bramhill about FtsZ in E. coli, Keith Chater about cell septation in Streptomyces (see Pictures Considered #52), Jeff Errington and Alan Grossman about chromosome segregation in vegetative and sporulating Bacillus (see Subtle Bacillus subtilis in STC). And then there was Piet de Boer introducing the attendants to the MinE ring and the MinCDE oscillator in E. coli. Younger readers won't know: we didn't have regular PowerPoint presentations back then. Presenters had their manuscript and a stack of slides, hopefully shown in the right order from a manually operated projector ("next slide please"). At the end of his talk, Piet said rather casually that he could show to those interested a short movie on his laptop about the oscillation of MinC in living cells during the lunch break. Gosh! I could hardly take my eyes off Piet's screen, and, despite the rather poor quality of the video clip, Yves Brun and I confirmed to each other that this was a jaw‑dropping moment. I vividly remembered this episode when, twenty-five years later, I first saw the technically brilliant, beautiful MinC oscillations in the movie considered here.