Investigating the in vivo dynamics of FtsZ and FtsZ-interacting proteins using vertical cell imaging by nanostructured immobilisation (VerCINI)

Abstract

Division is arguably one of the most difficult mechanistic tasks that cells face, requiring precise temporal and spatial regulation. Gram-positive bacteria also face an additional mechanistic problem - high turgor pressure. In order to successfully divide, Gram-positive organisms synthesise a peptidoglycan septum which cleaves the cell in two, yielding two daughter cells. Prior to cell division, the almost universally essential cytoskeletal tubulin homologue FtsZ polymerizes into a highly dynamic, ring-like band of short filaments at mid-cell- the Z-ring. The Z-ring is tethered to the plasma membrane via anchor proteins, following which the Z-ring recruits septal PG synthases forming the mature divisome. These synthases build the septum which partitions the cell in two. This work investigates the organisation and dynamics of the Z-ring in the Gram-positive model organism B. subtilis. In this thesis, I established a high-throughput approach of Vertical Cell Imaging by Nanostructured Immobilisation (VerCINI) in order to visualise the dynamics of the division machinery around the entirety of the division plane. Using this technique in combination with advanced fluorescence microscopy, I discovered that FtsZ filaments treadmill around the division plane in live bacteria, a phenomenon previously only described in vitro. Treadmilling is a type of motion whereby an asymmetric filament undergoes plus-end polymerization and minus-end depolymerisation. I investigated how the organisation and dynamics of filaments within the Z-ring develops over the cell cycle. My results indicate that FtsZ treadmilling is unstable in nascent Z-rings, but stabilizes during the transition to mature rings, before constriction has been initiated. This shows that both FtsZ filament assembly to midcell and FtsZ filament treadmilling dynamics are actively cell cycle regulated. I examined the dynamics of a number of key FtsZ interacting proteins (EzrA, SepF and DivIVA), observing a range of static and dynamic protein motions in ZIPs imaged, arguing against a single, monolithic divisome complex.