Essential Cell Division Protein FtsZ Assembles into One Monomer-thick Ribbons under Conditions Resembling the Crowded Intracellular Environment

Abstract

Experimental conditions that simulate the crowded bacterial cytoplasmic environment have been used to study the assembly of the essential cell division protein FtsZ from Escherichia coli. In solutions containing a suitable concentration of physiological osmolytes, macromolecular crowding promotes the GTP-dependent assembly of FtsZ into dynamic two-dimensional polymers that disassemble upon GTP depletion. Atomic force microscopy reveals that these FtsZ polymers adopt the shape of ribbons that are one subunit thick. When compared with the FtsZ filaments observed in vitro in the absence of crowding, the ribbons show a lag in the GTPase activity and a decrease in the GTPase rate and in the rate of GTP exchange within the polymer. We propose that, in the crowded bacterial cytoplasm under assembly-promoting conditions, the FtsZ filaments tend to align forming dynamic ribbon polymers. In vivo these ribbons would fit into the Z-ring even in the absence of other interactions. Therefore, the presence of mechanisms to prevent the spontaneous assembly of the Z-ring in non-dividing cells must be invoked.

Cooperative behavior of Escherichia coli cell-division protein FtsZ assembly involves the preferential cyclization of long single-stranded fibrils

“…Observations of thin FtsZ fibrils compatible with either a single-or double-stranded structure have been reported (4,6). Thicker multistranded filaments were observed more frequently after increasing protein concentration or lowering the pH (8,(12)(13)(14), suggesting that these conditions favor lateral contacts between FtsZ filaments that are important for assembly.…”

“…These observations suggest that the basic FtsZ polymer structure must be a multistranded filament, because cooperative assembly of linear polymers requires that two or more strands interact both laterally and longitudinally to form a helical or a two-dimensional͞three-dimensional array (23)(24)(25), as in the case of F-actin and microtubules (26). However, EM and scanning transmission microscopy images of the FtsZ preparations under the same solution conditions indicate that the protein is present partially or predominantly as single-stranded filaments (6,9,11). These observations leave open the question on how a single-stranded filament can assemble in a cooperative manner (2, 9, 11).…”

“…Centrifugation and light-scattering assays carried out by using FtsZ from different organisms indicate that FtsZ assembly proceeds as a highly cooperative process analogous to a first-order transition or condensation (6,8,10,21,22). According to these assays, there exists a critical protein concentration below which all of the protein is soluble; protein present in excess of this critical concentration is present in a distinct fraction that scatters light more intensely and is substantially more sedimentable than the soluble fraction.…”

“…Thicker multistranded filaments were observed more frequently after increasing protein concentration or lowering the pH (8,(12)(13)(14), suggesting that these conditions favor lateral contacts between FtsZ filaments that are important for assembly. Formation of larger FtsZ polymers (i.e., bundles, ribbons) in vitro is promoted by the polycation DEAEdextran or cationic phospholipids (5), high concentrations of calcium (7), glutamate (15), or ruthenium red (16), the presence of either of two proteins that stabilize the septal ring, ZipA (17) and ZapA (18), and high concentrations of inert polymers, mimicking the crowded bacterial cytoplasm (6). The strong tendency of FtsZ to form these multistranded polymers, together with the fact that at least some of these promoting agents are likely to be present in vivo, has led to the suggestion that the dynamic filament bundles or ribbons observed in vitro represent the basic structure that FtsZ adopts within the septal ring (6,19,20).…”

FtsZ and the division of prokaryotic cells and organelles

“…This bundling can be induced by several factors, such as Ca 2+ (REF. 21), macromolecular crowding 22 and the binding of partner proteins such as ZipA 23 (see FIG. 3).…”