Simultaneous direct holographic fabrication of photonic cavity and graded photonic lattice with dual periodicity, dual basis, and dual symmetry.

For the first time, to the authors' best knowledge, this paper demonstrates the digital, holographic fabrication of graded, super-basis photonic lattices with dual periodicity, dual basis, and dual symmetry. Pixel-by-pixel phase engineering of the laser beam generates the highest resolution in a programmable spatial light modulator (SLM) for the direct imaging of graded photonic super-lattices. This technique grants flexibility in designing 2-D lattices with size-graded features, differing periodicities, and differing symmetries, as well as lattices having simultaneously two periodicities and two symmetries in high resolutions. By tuning the diffraction efficiency ratio from the SLM, photonic cavities can also be generated in the graded super-lattice simultaneously through a one-exposure process. A high quality factor of over 1.56 × 106 for a cavity mode in the graded photonic lattice with a large super-cell is predicted by simulations.

Holographic fabrication of 3D photonic crystals through interference of multi-beams with 4 + 1, 5 + 1 and 6 + 1 configurations.

In this paper, we are able to fabricate 3D photonic crystals or quasi-crystals through single beam and single optical element based holographic lithography. The reflective optical elements are used to generate multiple side beams with s-polarization and one central beam with circular polarization which in turn are used for interference based holographic lithography without the need of any other bulk optics. These optical elements have been used to fabricate 3D photonic crystals with 4, 5 or 6-fold symmetry. A good agreement has been observed between fabricated holographic structures and simulated interference patterns.

Digitally tunable holographic lithography using a spatial light modulator as a programmable phase mask.

In this paper, we study tunable holographic lithography using an electrically addressable spatial light modulator as a programmable phase mask. We control the phases of interfering beams diffracted from the phase pattern displayed in the spatial light modulator. We present a calculation method for the assignment of phases in the laser beams and validate the phases of the interfering beams in phase-sensitive, dual-lattice, and two-dimensional patterns formed by a rotationally non-symmetrical configuration. A good agreement has been observed between fabricated holographic structures and simulated interference patterns. The presented method can potentially help design a gradient phase mask for the fabrication of graded photonic crystals or metamaterials.

Assembly of an FtsZ mutant deficient in GTPase activity has implications for FtsZ assembly and the role of the Z ring in cell division.

FtsZ, the ancestral homologue of eukaryotic tubulins, assembles into the Z ring, which is required for cytokinesis in prokaryotic cells. Both FtsZ and tubulin have a GTPase activity associated with polymerization. Interestingly, the ftsZ2 mutant is viable, although the FtsZ2 mutant protein has dramatically reduced GTPase activity due to a glycine-for-aspartic acid substitution within the synergy loop. In this study, we have examined the properties of FtsZ2 and found that the reduced GTPase activity is not enhanced by DEAE-dextran-induced assembly, indicating it has a defective catalytic site. In the absence of DEAE-dextran, FtsZ2 fails to assemble unless supplemented with wild-type FtsZ. FtsZ has to be at or above the critical concentration for copolymerization to occur, indicating that FtsZ is nucleating the copolymers. The copolymers formed are relatively stable and appear to be stabilized by a GTP-cap. These results indicate that FtsZ2 cannot nucleate assembly in vitro, although it must in vivo. Furthermore, the stability of FtsZ-FtsZ2 copolymers argues that FtsZ2 polymers would be stable, suggesting that stable FtsZ polymers are able to support cell division.

Escherichia coli division inhibitor MinCD blocks septation by preventing Z-ring formation.

The min system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD was previously shown to inhibit division by preventing assembly of the Z ring (E. Bi and J. Lutkenhaus, J. Bacteriol. 175:1118-1125, 1993); however, this was questioned in a recent report (S. S. Justice, J. Garcia-Lara, and L. I. Rothfield, Mol. Microbiol. 37:410-423, 2000) which indicated that MinCD acted after Z-ring formation and prevented the recruitment of FtsA to the Z ring. This discrepancy was due in part to alternative fixation conditions. We have therefore reinvestigated the action of MinCD and avoided fixation by using green fluorescent protein (GFP) fusions to division proteins. MinCD prevented the localization of both FtsZ-GFP and ZipA-GFP, consistent with it preventing Z-ring assembly. Consistent with a direct interaction between FtsZ and the MinCD inhibitor, we find that increased FtsZ, but not FtsA, suppresses MinCD-induced lethality. Furthermore, strains carrying various alleles of ftsZ, selected on the basis of resistance to the inhibitor SulA, displayed variable resistance to MinCD. These results are consistent with FtsZ as the target of MinCD and confirm that this inhibitor prevents Z-ring assembly.

Topological regulation of cell division in E. coli. spatiotemporal oscillation of MinD requires stimulation of its ATPase by MinE and phospholipid.

Topological regulation of cell division in E. coli requires positioning a cell division inhibitor, MinC, at the poles of the cell, thus restricting the potential for division to midcell. This positioning is achieved through a rapid oscillation of MinC from pole to pole, a process requiring MinD and MinE. However, the mechanistic basis for this oscillation is not known. Here we report that MinE stimulates MinD ATPase activity, but only in the presence of phospholipid vesicles. Analysis of MinE mutants demonstrates that this stimulation is required for MinD oscillation and suggests that the level of stimulation determines the period of the oscillation. A model is presented in which the requirements for the MinD ATPase contribute spatial and temporal inputs that provide the mechanistic basis for the oscillation.

Analysis of MinC reveals two independent domains involved in interaction with MinD and FtsZ.

In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by the min system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts min function, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

Topological regulation of cell division in Escherichia coli involves rapid pole to pole oscillation of the division inhibitor MinC under the control of MinD and MinE.

Placement of the Z ring at midcell in Escherichia coli is assured by the action of the min system, which blocks usage of potential division sites that exist at the cell poles. This activity of min is achieved through the action of an inhibitor of division, MinC, that is activated by MinD and topologically regulated by MinE. In this study, we have used a functional GFP-MinC fusion to monitor the location of MinC. We find that GFP-MinC is a cytoplasmic protein in the absence of the other Min proteins. The addition of MinD, a peripheral membrane protein that interacts with MinC, results in GFP-MinC appearing on the membrane. In the presence of both MinD and MinE, GFP-MinC oscillates rapidly between the halves of the cell. Thus, MinC is positioned by the other Min products, but in a dynamic manner so that it is in position to inhibit Z ring assembly away from midcell.

The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization.

Positioning of the Z ring at the midcell site in Escherichia coli is assured by the min system, which masks polar sites through topological regulation of MinC, an inhibitor of division. To study how MinC inhibits division, we have generated a MalE-MinC fusion that retains full biological activity. We find that MalE-MinC interacts with FtsZ and prevents polymerization without inhibiting FtsZ's GTPase activity. MalE-MinC19 has reduced ability to inhibit division, reduced affinity for FtsZ, and reduced ability to inhibit FtsZ polymerization. These results, along with MinC localization, suggest that MinC rapidly oscillates between the poles of the cell to destabilize FtsZ filaments that have formed before they mature into polar Z rings.

Recruitment of ZipA to the division site by interaction with FtsZ.

ZipA is an essential cell division protein in Escherichia coli that is recruited to the division site early in the division cycle. As it is anchored to the membrane and interacts with FtsZ, it is a candidate for tethering FtsZ filaments to the membrane during the formation of the Z ring. In this study, we have investigated the requirements for ZipA localization to the division site. ZipA requires FtsZ, but not FtsA or FtsI, to be localized, indicating that it is recruited by FtsZ. Consistent with this, apparently normal Z rings are formed in the absence of ZipA. The interaction between FtsZ and ZipA occurs through their carboxy-terminal domains. Although a MalE-ZipA fusion binds to FtsZ filaments, it does not affect the GTPase activity or dynamics of the filaments. These results are consistent with ZipA acting after Z ring formation, possibly to link the membrane to FtsZ filaments during invagination of the septum.

Analysis of FtsZ assembly by light scattering and determination of the role of divalent metal cations.

FtsZ is an ancestral homologue of tubulin that polymerizes in a GTP-dependent manner. In this study, we used 90 degrees angle light scattering to investigate FtsZ polymerization. The critical concentration for polymerization obtained by this method is similar to that obtained by centrifugation, confirming that the light scattering is proportional to polymer mass. Furthermore, the dynamics of FtsZ polymerization could be readily monitored by light scattering. Polymerization was very rapid, reaching steady state within 30 s. The length of the steady-state phase was proportional to the GTP concentration and was followed by a rapid decrease in light scattering. This decrease indicated net depolymerization that always occurred as the GTP in the reaction was consumed. FtsZ polymerization was observed over the pH range 6.5 to 7.9. Importantly, Mg2+ was not required for polymerization although it was required for the dynamic behavior of the polymers. It was reported that 7 to 25 mM Ca2+ mediated dynamic assembly of FtsZ (X. -C. Yu and W. Margolin, EMBO J. 16:5455-5463, 1997). However, we found that Ca2+ was not required for FtsZ assembly and that this concentration of Ca2+ reduced the dynamic behavior of FtsZ assembly.

Organelle division: from coli to chloroplasts.

FtsZ, an ancestral homolog of eukaryotic tubulin, assembles into the cytokinetic Z ring that directs cell division in bacteria. Recent results indicate that FtsZ is also used for division by chloroplasts, though not by mitochondria.

FtsK is an essential cell division protein that is localized to the septum and induced as part of the SOS response.

The role of ftsK in the growth of Escherichia coli was examined by turning off its expression. This resulted in smooth filaments without constrictions, indicating that FtsK was required at an early step in septation. Consistent with this, FtsK was found to localize to the septum in 70% of the cells, indicating that it was recruited relatively early in this process. FtsK localization required the function of FtsZ and FtsA but not FtsI and FtsQ. Consistent with this, Z rings were present in FtsK-depleted filaments. Subcellular localization of FtsK confirmed that it was a membrane protein. Only the first 202 amino acids of FtsK were essential for its role in membrane localization, cell division and viability. The expression of ftsK increased as part of the SOS response, and increased expression of ftsK conferred increased resistance to DNA damage.

FtsI and FtsW are localized to the septum in Escherichia coli.

The localization of FtsI (PBP3), a penicillin-binding protein specifically required for cell division in Escherichia coli, was investigated by immunofluorescence microscopy and found to localize to the septum. The localization of FtsI was not observed in ftsZ or ftsA mutants, indicating that it was dependent on the prior localization of these proteins. Addition of furazlocillin, a specific inhibitor of FtsI, prevented localization of FtsI even though FtsZ and FtsA localization occurred. Interestingly, the localization of FtsN was also prevented by furazlocillin. FtsZ displayed limited localization in furazlocillin-treated cells, whereas it was efficiently localized in FtsI-depleted cells. FtsW, another essential cell division protein, was also localized to the septum.

Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia coli.

The bacterial cell division protein FtsZ assembles into the cytokinetic Z ring that directs cytokinesis in prokaryotes. In Escherichia coli the formation of the Z ring is prevented by induction of the cell division inhibitor SulA (SfiA), a component of the SOS response. Here we show that a MalE-SulA fusion that retains this inhibitory function in vivo inhibits the GTPase activity and polymerization of FtsZ in vitro. MalE-SulA10, which does not block Z ring formation in vivo, is unable to inhibit the GTPase activity and polymerization in vitro. Furthermore, FtsZ114, which is refractory to SulA in vivo, is not inhibited by MalE-SulA. These results indicate that SulA blocks Z ring formation by blocking FtsZ polymerization.

Dynamic assembly of FtsZ regulated by GTP hydrolysis.

FtsZ forms a cytokinetic ring, designated the Z ring, that directs cytokinesis in prokaryotes. It has limited sequence similarity to eukaryotic tubulins and, like tubulin, it has GTPase activity and the ability to assemble into various structures including protofilaments, bundles and minirings. By using both electron microscopy and sedimentation, we demonstrate that FtsZ from Escherichia coli undergoes a strictly GTP-dependent polymerization and the polymers disappear as the GTP is consumed. Thus, FtsZ polymerization, like that of tubulin, is dynamic and regulated by GTP hydrolysis. These results provide the basis for the dynamics of the Z ring and favor a model in which the Z ring is formed by a nucleation event.

The regulation of bacterial cell division: a time and place for it.

Temporal and spatial regulation of cell division assures that each daughter cell receives a copy of the chromosome. Within the past year, the application of fluorescence microscopy to the cell biology of bacteria has revealed an increasing number of proteins that are localized within the bacterial cell to carry out DNA segregation and cell division. The localization of these proteins implies the existence of positional information in the cell, but how this information is established is unknown.

Analysis of the interaction of FtsZ with itself, GTP, and FtsA.

The interaction of FtsZ with itself, GTP, and FtsA was examined by analyzing the sensitivity of FtsZ to proteolysis and by using the yeast two-hybrid system. The N-terminal conserved domain consisting of 320 amino acids bound GTP, and a central region of FtsZ, encompassing slightly more than half of the protein, was cross-linked to GTP. Site-directed mutagenesis revealed that none of six highly conserved aspartic acid and asparagine residues were required for GTP binding. These results indicate that the specificity determinants for GTP binding are different than those for the GTPase superfamily. The N-terminal conserved domain of FtsZ contained a site for self-interaction that is conserved between FtsZ proteins from distantly related bacterial species. FtsZ320, which was truncated at the end of the conserved domain, was a potent inhibitor of division although it expressed normal GTPase activity and could polymerize. FtsZ was also found to interact directly with FtsA, and this interaction could also be observed between these proteins from distantly related bacterial species.

Bacterial cytokinesis: let the light shine in.

Recent application of fluorescence microscopy to the study of the bacterial cell cycle has revealed the existence of a cytoskeletal element - once thought to occur only in eukaryotic cells - that mediates cytokinesis, and possibly another involved in chromosome segregation.