Examining the processes that deform bacterial membranes to initiate cell division - ABSTRACT Cell division is an essential process for all organisms; thus, understanding the proteins and mechanisms underlying the division machinery is essential for understanding how to design antibiotics to target this process. Exerting the force needed to constrict pressurized bacteria in half is a primary function of the division machinery. Still, despite decades of study, the underlying physical mechanisms exerting this force remain unknown. Dividing cells is an energetically costly process, especially for bacteria, given their high internal turgor. The first - most energetically costly - step of cell division is the initial invagination of the membrane, deforming it from a flat surface into an inward bend, working against the membrane tension. How this initial deformation occurs in the cell remains unclear. In vitro studies have shown that membrane-attached FtsZ filaments can deform liposomes from the inside. This demonstrates that FtsZ can exert membrane-deforming force, but these deformations occur even when FtsZ cannot hydrolyze GTP. Rather, the liposome membranes only deform inward when the FtsZ filaments become laterally associated and condense together. These deformations share a commonality with every membrane-deforming system studied in eukaryotes: the local crowding of proteins. Protein crowding is known to deform membranes due to different effects, including: 1) the high concentration of amphipathic helices bends adjacent lipid headgroups apart, and 2) the crowding of proteins (even GFP) on membranes results in entropic repulsion between proteins, causing the membrane to bend towards the proteins. While it has been demonstrated that FtsZ filament crowding deforms membranes in vitro, we wish to 1) examine if FtsA/FtsZ filament condensation in vivo works to overcome the membrane tension to initiate division and 2) if and how certain properties within these filaments modulate the ability of filaments to deform membranes. To examine FtsZ condensation in vivo, we utilize our recent finding that when B subtilis lacks proteins that bundle FtsZ filaments, the filaments never condense into a ring, and cell division never initiates. To test if filament crowding works against membrane tension to bend membranes, we will first develop ways to both measure and modulate membrane tension in B. subtilis. Next, we will examine if the amount of FtsZ bundling protein needed to initiate division scales with the cell’s membrane tension, as expected if condensation works to deform membranes. We will then examine this relationship in vitro, seeing if FtsA/FtsZ filaments can deform liposomes of defined tensions with different amounts of FtsZ binding proteins. Next, we will examine how 3 features of FtsA/FtsZ filaments affect their ability to deform the A) cell membrane and B) liposomes under different tensions: 1) FtsA’s amphipathic helix, 2) the disordered linker within FtsZ, and 3) the total amount of FtsA/FtsZ polymer in the cell. Overall, this proposal will A) test a novel mechanism for how the bacterial division machinery could bend the membrane to initiate cell division and B) examine what features of FtsA/FtsZ filaments contribute to deforming the membranes.
