Special feature: Lord of the Z ring

This feature appeared in the May/June 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.

Cell division is an essential part of biological growth and survival. Associate Professor Elizabeth Harry of the Institute for the Biotechnology of Infectious Diseases (IBID) at the University of Technology, Sydney, and ASM Frank Fenner Award recipient for 2009, is primarily interested in how bacteria tackle this process – how bacterial cells divide and what regulates it to happen at the right place and at the right time.

Despite recent advances, many questions remain about this essential cell function in bacteria.

Harry has long been fascinated by how bacteria divide so rapidly and faithfully – firstly, because it is so fundamentally interesting as a biological phenomenon, and secondly, as a platform to develop novel antibacterials. Towards the second aim, Harry’s group has identified proteins important in cell division that may be targeted by novel antibiotics with new modes of action, and less susceptible to the wiles of microbial resistance.

This work was conducted in collaboration with Sydney biotech company Tyrian Diagnostics as part of its infectious disease biomarker discovery program.

For the Frank Fenner Award Lecture at ASM 2009 in July, Harry will present an historical overview of cell division in bacteria and then discuss how our understanding of it has changed with the advent of better ways to see inside these tiny critters. Harry is certainly well qualified to speak on such a topic, being instrumental in introducing some of these “better ways”.

During her postdoctoral years at Harvard University working on bacterial sporulation, Harry developed an innovative, fluorescence-based technique for viewing bacterial morphology in much greater detail: basically as the only way to address the questions that the group there wanted to answer.

“Of course, fluorescence microscopy had been around for a long time, but was used mainly for eukaryotic cells, which are relatively large compared to bacteria,” Harry says. Protein localisation at any subcellular level in bacteria and viruses was done by immunoelectron microscopy, which is slow to do and fraught with technical issues.

“So in 1995, we developed immunofluorescence protocols similar to those used in yeast, and were amazed by what we found,” she says. “What was commonly believed to be ‘an amorphous bag of enzymes’ turned out to be highly organised at the level of protein localisation.”

The new technique allowed bacterial-cell interiors to be visualised and some proteins to be localised at a subcellular level for the first time, and a lot of subsequent work was and is reliant on this methodological advance.

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Lord of the ring

Illuminating this unchartered microscopic landscape generated an information explosion in bacterial cell biology, according to Harry, particularly in the area of cell division. Using immunofluorescence microscopy, she quickly discovered that all processes in bacteria actually involve proteins being in a particular place in the cell at a particular time.

“If this is not the case, everything falls down, and this in itself was really interesting and exciting to work out,” she says. It became clear that, as for eukaryotic cells, protein localisation is crucial for the integrity of vital processes in bacteria including cell division, DNA replication and cell shape maintenance.

Harry’s work and others’ subsequently revealed that all the division proteins in bacteria go to the place where the cell is going to divide, she says. “They go there in a certain sequence and they depend on other proteins to get there – so we started to get a picture of the assembly procedure and apparatus.”

It also revealed cytoskeletal proteins in bacteria that were thought never to exist. In fact it was only five or six years ago that another group identified an actin homologue in bacteria, published in Cell.

As with all organisms, bacteria must divide at the right place and the right time to ensure that each progeny cell contains the full complement of genetic material. The earliest event in bacterial cell division is the formation of a helical structure called a Z ring, which forms on the inside of the cell membrane at midcell.

The Z ring is composed of the tubulin-like cytoskeletelal protein, FtsZ, which was in fact cloned from Bacillus subtilis by Harry during her PhD at the University of Sydney in 1989.

Once the Z ring is formed, FtsZ recruits other division and accessory proteins, and together they drive the segregation of the two replicated chromosomes into two new cells.

Although only one of many proteins now known to participate in cell division, FtsZ remains the most intensively studied. This is because FtsZ is an essential and highly conserved protein in bacteria, and also because it represents a strong candidate for targeting by novel antibacterials.

Under the control of as yet unknown signals, FtsZ undergoes polymerisation, assembling first into protofilaments that subsequently associate laterally to form bundles or sheets. These bundles assemble into the helical Z ring structure, which appears highly dynamic, continually remodelling itself both before and during cell division. The timing of assembly and the positioning of the Z ring must be precisely regulated, although how this happens at a molecular level remains largely a mystery.

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A new force in visualising bacteria

Recently, Harry’s work revealed a new assembly mechanism for Z ring formation that involves cell cycle-mediated remodelling of FtsZ polymers. Their current studies are examining this remodeling process and what controls it in finer detail, including identifying other proteins that are involved.

“We suspect that some of these will be involved prior to ring formation, while others act after the ring forms,” she says. “We know from microscopy of live bacteria in real time that some of the protein assembly happens later – this is one area I would really like to study.”

The capability of viewing the dynamics of these processes in real time with the explosion of live cell imaging in cell biology is another huge advance, she says. “We can now look at where certain proteins are over time. We actually see the FtsZ helix move into the forming Z ring.”

A recent development in microscopy has even further enabled ‘seeing’ bacteria. Super-resolution microscopy (SRM), which was the 2008 Nature method of the year, breaks the diffraction barrier using lasers and fluorescence.

It thus achieves three- to four-fold higher resolution than is theoretically possible with light microscopy. Harry and her team started playing with an SRM instrument through their connections with Leica Microsystems, including at Leica’s headquarters in Mannhein, Germany.

Leica call its version a STED (Stimulated Emission Depletion) microscope. Using STED to look at the Z ring in more detail has already proven incredibly informative, according to Harry, with the results just submitted for publication.

This new advance will certainly help bacteriologists work out what is really happening in their microbe of choice.

Coordination is the key

The second focus of Harry’ presentation at ASM will be her fundamental research into the division site, that is, how does the Z ring know where to go. “We believe that cells control this timing and positioning very precisely.”

This information is important because if the cell doesn’t get the place and the time right, cells might divide without the DNA being copied faithfully and the daughter cells will end up dying because they don’t have the correct DNA.

Landmark work published in 1999 by Harry revealed a connection between DNA replication and the position of the Z ring. “Now we have identified a novel control mechanism for this connection and for coordinating the two processes,” she says.

“This has been a big question in the bacterial world for a long time. A lot is obviously known about cell cycles with regard to DNA processing in eukaryotic cells, but nothing similar was known in bacteria.

“What we have found is sort of like a cell cycle checkpoint that blocks midcell Z ring assembly during the very early stages of DNA replication.” Targeting molecules found to participate in this mechanism could potentially stop cells multiplying and thus prevent infections worsening.

This soon-to-be published work is quite juicy for Harry and her group because it questions the long-held assertion that only two mechanisms control division site positioning in bacteria.

“We now have this really good evidence of a third mechanism that might be essential for cell survival. What we also showed is that if you knock out these other two mechanisms, FtsZ and the Z ring faithfully find midcell, proving that there must be an alternative way to do that, and that will be by our mechanism of coordinating cell division with DNA replication.”