The bacterium Caulobacter crescentus divides asymmetrically as part of its normal life cycle. This asymmetry is regulated in part by the membrane-bound histidine kinase PleC, which localizes to one pole of the cell at specific times in the cell cycle. Here, we track single copies of PleC labeled with enhanced yellow fluorescent protein (EYFP) in the membrane of live Caulobacter cells over a time scale of seconds. In addition to the expected molecules immobilized at one cell pole, we observed molecules moving throughout the cell membrane. By tracking the positions of these molecules for several seconds, we determined a diffusion coefficient (D) of 12 ؎ 2 ؋ 10 ؊3 m 2 ͞s for the mobile copies of PleC not bound at the cell pole. This D value is maintained across all cell cycle stages. We observe a reduced D at poles containing localized PleC-EYFP; otherwise D is independent of the position of the diffusing molecule within the bacterium. We did not detect any directional bias in the motion of the PleC-EYFP molecules, implying that the molecules are not being actively transported.single molecule ͉ diffusion ͉ PleC ͉ enhanced yellow fluorescent protein T he inner membranes of bacterial cells contain proteins required for a wide variety of functions, including energy generation, solute transport, signaling, proteolysis, polar morphogenesis, chemotaxis, and cell division (1-3). The size of the diffusion coefficient (D) of these proteins in the membrane can affect their interactions with each other and with cytoplasmic proteins. For example, in Escherichia coli, the MinCDE system for locating the division plane is thought to require a difference in D between the membrane-associated and the cytoplasmic forms of the MinD and MinE proteins for its proper function (4-6). The D values of several cytoplasmic proteins have been measured in E. coli (7). Measurements of D for membrane proteins in eukaryotic cells, using fluorescence recovery after photobleaching (FRAP) (8), single gold bead tracking (9-11), and single-molecule tracking techniques (12, 13), have yielded values ranging from 5 ϫ 10 Ϫ3 to 500 ϫ 10 Ϫ3 m 2 ͞s. Each Caulobacter cell division produces a pair of distinct daughter cells (Fig. 1): a motile swarmer (SW) cell with a single flagellum located at a specific pole and a stalked (ST) cell possessing an adhesive holdfast at the end of the stalk, allowing it to attach to a surface (14, 15). The transmembrane histidine kinase PleC regulates polar organelle formation, motility, and asymmetric cell division in Caulobacter (16). PleC is a 90-kDa inner membrane protein, with four predicted transmembrane domains as obtained from TMPRED (www.ch.embnet.org͞ software͞TMPREDform.html). Cells with mutant PleC do not form stalks or pili and have paralyzed flagella (17)(18)(19). These mutant cells undergo symmetric cell division, producing two daughter cells of similar size, each possessing a paralyzed flagellum. By using conventional fluorescence microscopy, molecules of PleC were found to be localized to the flagellar pole of SW...

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Asymmetric cell division in Caulobacter crescentus yields daughter cells that have different cell fates. Compartmentalization of the predivisional cell is a critical event in the establishment of the differential distribution of regulatory factors that specify cell fate. To determine when during the cell cycle the cytoplasm is compartmentalized so that cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments, we designed a fluorescence loss in photobleaching assay. Individual cells containing enhanced GFP were exposed to a bleaching laser pulse tightly focused at one cell pole. In compartmentalized cells, fluorescence disappears only in the compartment receiving the bleaching beam; in noncompartmentalized cells, fluorescence disappears from the entire cell. In a 135-min cell cycle, the cells were compartmentalized 18 ؎ 5 min before the progeny cells separated. Clearance of the 22000 CtrA master transcriptional regulator molecules from the stalked portion of the predivisional cell is a controlling element of Caulobacter asymmetry. Monitoring of a fluorescent marker for CtrA showed that the differential degradation of CtrA in the nascent stalk cell compartment occurs only after the cytoplasm is compartmentalized.

Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed prefission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.The early stages of bacterial cytokinesis, involving septal ring assembly and constriction, are better understood than the terminal stages. In this work we investigate the late stages of cytokinesis in the gram-negative bacterium Caulobacter crescentus. We use fluorescence loss in photobleaching (FLIP) and cryoelectron microscope tomography (cryoEM) to determine (i) the geometry of the inner and outer membranes at various stages of cell division and (ii) whether the FtsZ ring and associated cell division machinery hinder diffusion of membranebound or periplasmic molecules through the division plane in predivisional cells.Caulobacter crescentus divides asymmetrically, producing two distinct daughter cells: the swarmer cell and the stalked cell (Fig. 1). These cells differ in their transcription programs, protein composition, and behavior (3,20,28,32). The daughter cells also differ in size; the nascent swarmer-cell compartment is about two-thirds as long as the nascent stalked-cell compartment. After cell division, swarmer cells undergo an initial motile phase, and then they differentiate into stalked cells; they lose their flagellum and pili, and DNA replication is initiated. The compartmentalization of the predivisional cell is an important step in the process of creating daughter cells with different cell fates. Immediately after cytoplasmic compartmentalization, the complement of cytoplasmic proteins in the two compartments can begin to diverge, launching the two daughter cells on different developmental paths. The physical compartmentalization of the Caulobacter cytoplasm well before cell division triggers a phosphotransport-based switch mechanism that initiates the differential regulation of development in the nascent daughter cells (19,21).Many proteins involved in cytokinesis are known in Escherichia coli and Bacillus subtilis (1,17,35) and to a lesser extent in Caulobacter crescentus...

Two recent papers report the de novo design of a functioning biological circuit using well‐characterized genetic elements.(1,2) Gardner et al. designed and constructed a genetic toggle switch while Elowitz and Leibler built an oscillating genetic circuit. Both circuits were designed with the aid of mathematical models. These papers demonstrate progress towards the unification of theory and experiment in the study of genetic circuits. Comparison of the predicted and observed behavior of the circuits, however, shows that the models explain only some of the circuits' properties. Further study of the observed behaviors not predicted by the model would lead to new insight into the properties of genetic networks. BioEssays 22:507–509, 2000. © 2000 John Wiley & Sons, Inc.