Our cryoEM images of the dividing Caulobacter cells show that in early stages of cell division, the inner and outer membranes constrict simultaneously, initially maintaining the 30-nm separation seen in regions distant from the constriction. As cell division progresses, the IM constricts faster, creating a growing distance between the inner and outer membranes near the division plane. Fission of the inner membrane creates a cell containing two inner membrane-bound cytoplasmic compartments surrounded by a single continuous outer membrane.
The constriction in each membrane becomes remarkably small (as small as 60 nm in diameter) before unknown terminal events effect complete closure. These highly constricted membranes are also tightly bent, with a radius of curvature of 20 nm or less. The bent membrane (inner membrane in Fig. ) may break spontaneously and then reseal around the cytoplasm of the nascent daughter cell, with a significantly larger radius of curvature (inner membranes in Fig. ). In other words, the final fission event may occur simply because the two-surface configuration is a lower energy state for the lipid membrane structure. After inner and outer membrane fission, we did not observed any evidence for a residual “scar” in either membrane (Fig. ).
Since the constriction of the IM occurs earlier and well separated spatially from the lagging OM constriction, distinct molecular processes must control inner and outer membrane constriction in the later stages of cell division. Early constriction of the inner membrane is effected by the FtsZ ring and associated proteins (13
). Separate protein structures controlling late constriction of the outer membrane have not yet been identified.
The FLIP experiments show that both inner membrane and periplasmic proteins are able to freely diffuse past the site of constriction throughout most of the constriction process. The FtsZ ring and associated proteins form a ring-like structure attached to the inner membrane at the constriction site (13
). The mode of attachment to the membrane is unknown. It is also unknown whether the FtsZ ring is a continuous ring. It is possible that the FtsZ ring is continuous but only attached to the inner membrane at discrete points. Alternatively, there may be breaks in the ring that allow membrane proteins to diffuse through. FtsZ is known to turn over rapidly, which may cause transient breaks in the ring. The slow diffusion of membrane and periplasmic proteins through the division site observed for a small fraction of the FLIP experiments could be due to the very small dimension of the remaining connection as the compartments near separation in these cells (Fig. ), or the constriction machinery may congest the connection as the constriction nears separation.
The measured diffusion coefficient for the inner membrane protein PleC-EYFP is D
= (12 ± 2) × 10−3
). This is roughly 200 times smaller than the D
value of (2,500 ± 600) × 10−3
/s measured by fluorescence recovery after photobleaching for a cytoplasmic protein of similar mass (GFP fused to a maltose-binding protein domain, 72 kDa) in E. coli
). Our results reported here for the FLIP experiments are consistent with slower diffusion of proteins in the inner membrane than in the cytoplasm. All the cytoplasmic EGFP in a cell could be bleached with a laser focused at one end of the cell for 5 s (10
). The membrane-bound EGFP, however, required localized bleaching for from 30 s (for PilA-EGFP) to 240 s (for CC2909-EGFP) to completely bleach the cell independent of the laser intensity. This indicates that the membrane-bound EGFP fusion proteins took longer to diffuse from the far end of the cell into the laser beam than did the cytoplasmic EGFP.
Electron microscope images show that in E. coli
and B. subtilis
, the inner membrane and the cell wall invaginate together, forming a septum, and the outer membrane constricts later (5
). Figure shows the progression of Caulobacter
cell division schematically based on the images in Fig. with an approximately consistent scale. For comparison, Fig. shows a schematic of progression of E. coli
cell division (adapted with permission from Fig. 32 in the work of Burdett and Murray [6
]). (The exterior blebs and interior mesosomes seen in the E. coli
images are probably artifacts of the fixation and slicing procedures involved at the time in producing the EM images on which the diagram in Fig. is based. We have not found published cryoEM images of either E. coli
or B. subtilis
cytokinesis.) The differences between the septum-forming division process in E. coli and the constrictive division process in Caulobacter
are quite clear. In E. coli
, inner membrane constriction also precedes outer membrane constriction, but the constriction of the inner membrane involves physical association of the two inner membranes of the nascent daughter cells to form the septal disk. Later, after closure of the septum, the E. coli
outer membrane constricts between the two inner membrane surfaces and separates the cell. In other EM images of E. coli
, it appears that the inner and outer membranes invaginate together (4
). The geometric differences in the cell constriction and separation process between Caulobacter, E. coli
, and B. subtilis
suggest there are differences at the molecular level between the Caulobacter
terminal cell constriction and closure mechanisms compared to the septum-forming bacteria. The spatial separation of the Caulobacter
inner and outer membrane constrictive rings will facilitate labeling and identification of the distinct proteins involved in IM and OM constriction.