Structures and Dynamics of the E. Coli FtsZ-Ring and its Associated Proteins during Cell Division Revealed by Superresolution Imaging

Membrane constriction is a prerequisite for cell division. The most common membrane constriction system in prokaryotes is based on the tubulin homologue FtsZ, whose filaments in E. coli are anchored to the membrane by FtsA and enable the formation of the Z-ring and divisome. The precise architecture of the FtsZ ring has remained enigmatic. In this study, we report three-dimensional arrangements of FtsZ and FtsA filaments in C. crescentus and E. coli cells and inside constricting liposomes by means of electron cryomicroscopy and cryotomography. In vivo and in vitro, the Z-ring is composed of a small, single-layered band of filaments parallel to the membrane, creating a continuous ring through lateral filament contacts. Visualisation of the in vitro reconstituted constrictions as well as a complete tracing of the helical paths of the filaments with a molecular model favour a mechanism of FtsZ-based membrane constriction that is likely to be accompanied by filament sliding.

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Three-Dimensional Superresolution Imaging of the FtsZ Ring during Cell Division of the Cyanobacterium Prochlorococcus

mBio ◽

10.1128/mbio.00657-17 ◽

2017 ◽

Vol 8 (6) ◽

Cited By ~ 8

Author(s):

Riyue Liu ◽

Yaxin Liu ◽

Shichang Liu ◽

Ying Wang ◽

Kim Li ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Cell Division ◽

Three Dimensional ◽

Plant Cells ◽

Flowering Plant ◽

Superresolution Microscopy ◽

Superresolution Imaging ◽

Ftsz Ring ◽

Cell Division Protein ◽

Photosynthetic Cells

ABSTRACT Superresolution imaging has revealed subcellular structures and protein interactions in many organisms. However, superresolution microscopy with lateral resolution better than 100 nm has not been achieved in photosynthetic cells due to the interference of a high-autofluorescence background. Here, we developed a photobleaching method to effectively reduce the autofluorescence of cyanobacterial and plant cells. We achieved lateral resolution of ~10 nm with stochastic optical reconstruction microscopy (STORM) in the sphere-shaped cyanobacterium Prochlorococcus and the flowering plant Arabidopsis thaliana. During the cell cycle of Prochlorococcus, we characterized the three-dimensional (3D) organization of the cell division protein FtsZ, which forms a ring structure at the division site and is important for cytokinesis of bacteria and chloroplasts. Although the FtsZ ring assembly process in rod-shaped bacteria has been studied extensively, it has rarely been studied in sphere-shaped bacteria. Similarly to rod-shaped bacteria, our results with Prochlorococcus also showed the assembly of FtsZ clusters into incomplete rings and then complete rings during cell division. Differently from rod-shaped bacteria, the FtsZ ring diameter was not found to decrease during Prochlorococcus cell division. We also discovered a novel double-Z-ring structure, which may be the Z rings of two daughter cells in a predivisional mother cell. Our results showed a quantitative picture of the in vivo Z ring organization of sphere-shaped bacteria. IMPORTANCE Superresolution microscopy has not been widely used to study photosynthetic cells due to their high-autofluorescence background. Here, we developed a photobleaching method to reduce the autofluorescence of cyanobacteria and plant cells. After photobleaching, we performed superresolution imaging in the cyanobacterium Prochlorococcus and the flowering plant Arabidopsis thaliana with ~10-nm resolution, which is the highest resolution in a photosynthetic cell. With this method, we characterized the 3D organization of the cell division protein FtsZ in Prochlorococcus. We found that the morphological variation of the FtsZ ring during cell division of the sphere-shaped cyanobacterium Prochlorococcus is similar but not identical to that of rod-shaped bacteria. Our method might also be applicable to other photosynthetic organisms. IMPORTANCE Superresolution microscopy has not been widely used to study photosynthetic cells due to their high-autofluorescence background. Here, we developed a photobleaching method to reduce the autofluorescence of cyanobacteria and plant cells. After photobleaching, we performed superresolution imaging in the cyanobacterium Prochlorococcus and the flowering plant Arabidopsis thaliana with ~10-nm resolution, which is the highest resolution in a photosynthetic cell. With this method, we characterized the 3D organization of the cell division protein FtsZ in Prochlorococcus. We found that the morphological variation of the FtsZ ring during cell division of the sphere-shaped cyanobacterium Prochlorococcus is similar but not identical to that of rod-shaped bacteria. Our method might also be applicable to other photosynthetic organisms.

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Faculty Opinions recommendation of The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1065743.524831 ◽

2007 ◽

Author(s):

Miguel Valvano

Keyword(s):

Cell Division ◽

Outer Membrane ◽

E Coli ◽

Membrane Invagination

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Life without Division: Physiology ofEscherichia coliFtsZ-Deprived Filaments

mBio ◽

10.1128/mbio.01620-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 18

Author(s):

Alicia Sánchez-Gorostiaga ◽

Pilar Palacios ◽

Rocío Martínez-Arteaga ◽

Manuel Sánchez ◽

Mercedes Casanova ◽

...

Keyword(s):

Solid Medium ◽

Membrane Integrity ◽

Test Tube ◽

Antimicrobial Compounds ◽

Liquid Form ◽

Altered Expression ◽

E Coli ◽

Ftsz Ring ◽

Altered Expression Pattern ◽

The Stability

ABSTRACTWhen deprived of FtsZ,Escherichia colicells (VIP205) grown in liquid form long nonseptated filaments due to their inability to assemble an FtsZ ring and their failure to recruit subsequent divisome components. These filaments fail to produce colonies on solid medium, in which synthesis of FtsZ is induced, upon being diluted by a factor greater than 4. However, once the initial FtsZ levels are recovered in liquid culture, they resume division, and their plating efficiency returns to normal. The potential septation sites generated in the FtsZ-deprived filaments are not annihilated, and once sufficient FtsZ is accumulated, they all become active and divide to produce cells of normal length. FtsZ-deprived cells accumulate defects in their physiology, including an abnormally high number of unsegregated nucleoids that may result from the misplacement of FtsK. Their membrane integrity becomes compromised and the amount of membrane proteins, such as FtsK and ZipA, increases. FtsZ-deprived cells also show an altered expression pattern, namely, transcription of several genes responding to DNA damage increases, whereas transcription of some ribosomal or global transcriptional regulators decreases. We propose that the changes caused by the depletion of FtsZ, besides stopping division, weaken the cell, diminishing its resiliency to minor challenges, such as dilution stress.IMPORTANCEOur results suggest a role for FtsZ, in addition to its already known effect in the constriction ofE. coli, in protecting the nondividing cells against minor stress. This protection can even be exerted when an inactive FtsZ is produced, but it is lost when the protein is altogether absent. These results have implications in fields like synthetic biology or antimicrobial discovery. The construction of synthetic divisomes in the test tube may need to preserve unsuspected roles, such as this newly found FtsZ property, to guarantee the stability of artificial containers. Whereas the effects on viability caused by inhibiting the activity of FtsZ may be partly overcome by filamentation, the absence of FtsZ is not tolerated byE. coli, an observation that may help in the design of effective antimicrobial compounds.

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Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli: an Interspecies Approach to the Study of Protein-Protein Interactions in Multiprotein Complexes

Journal of Bacteriology ◽

10.1128/jb.00462-08 ◽

2008 ◽

Vol 190 (18) ◽

pp. 6048-6059 ◽

Cited By ~ 17

Author(s):

Carine Robichon ◽

Glenn F. King ◽

Nathan W. Goehring ◽

Jon Beckwith

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Cell Division ◽

Protein Interactions ◽

Fluorescent Protein ◽

Bacterial Cell Division ◽

Protein Protein Interactions ◽

Positive Interaction ◽

E Coli ◽

Multiprotein Complexes

ABSTRACT Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

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Dissecting the Control Mechanisms for DNA Replication and Cell Division in E. coli

Cell Reports ◽

10.1016/j.celrep.2018.09.061 ◽

2018 ◽

Vol 25 (3) ◽

pp. 761-771.e4 ◽

Cited By ~ 21

Author(s):

Gabriele Micali ◽

Jacopo Grilli ◽

Jacopo Marchi ◽

Matteo Osella ◽

Marco Cosentino Lagomarsino

Keyword(s):

Cell Division ◽

Dna Replication ◽

Control Mechanisms ◽

E Coli

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Identification of putative Z-ring-associated proteins, involved in cell division in human pathogenic bacteriaHelicobacter pylori

FEBS Letters ◽

10.1002/1873-3468.12230 ◽

2016 ◽

Vol 590 (14) ◽

pp. 2158-2171 ◽

Cited By ~ 4

Author(s):

Mohammad Kamran ◽

Swati Sinha ◽

Priyanka Dubey ◽

Andrew M. Lynn ◽

Suman K. Dhar

Keyword(s):

Cell Division ◽

Associated Proteins ◽

Z Ring

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Of the Morphogenes that Make a Ring, A Rod and a Sphere in Escherichia Coli

Science Progress ◽

10.3184/003685007x216912 ◽

2007 ◽

Vol 90 (2-3) ◽

pp. 59-72 ◽

Cited By ~ 2

Author(s):

Medhatm Khattar ◽

Issmat I. Kassem ◽

Ziad W. El-Hajj

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Bacterial Cell ◽

Bacterial Cell Division ◽

Antibacterial Compounds ◽

E Coli ◽

The Past ◽

New Knowledge ◽

Fundamental Process ◽

Simple Question

In 1993, William Donachie wrote “The success of molecular genetics in the study of bacterial cell division has been so great that we find ourselves, armed with much greater knowledge of detail, confronted once again with the same naive questions that we set to answer in the first place”1. Indeed, attempts to answer the apparently simple question of how a bacterial cell divides have led to a wealth of new knowledge, in particular over the past decade and a half. And while some questions have been answered to a great extent since the early reports of isolation of division mutants of Escherichia coli2,3, some key pieces of the puzzle remain elusive. In addition to it being a fundamental process in bacteria that merits investigation in its own right, studying the process of cell division offers an abundance of new targets for the development of new antibacterial compounds that act directly against key division proteins and other components of the cytoskeleton, which are encoded by the morphogenes of E. coli4. This review aims to present the reader with a snapshot summary of the key players in E. coli morphogenesis with emphasis on cell division and the rod to sphere transition.

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High-resolution crystal structures of Escherichia coli FtsZ bound to GDP and GTP

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x20001132 ◽

2020 ◽

Vol 76 (2) ◽

pp. 94-102 ◽

Cited By ~ 2

Author(s):

Maria A. Schumacher ◽

Tomoo Ohashi ◽

Lauren Corbin ◽

Harold P. Erickson

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Cell Division ◽

High Resolution ◽

Crystal Structures ◽

Bacterial Cell ◽

Model System ◽

E Coli ◽

Z Ring ◽

Main Model

Bacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a main model system for the study of the Z-ring and the associated divisome, a structure has not yet been reported for E. coli FtsZ. To address this gap, structures were determined of the E. coli FtsZ mutant FtsZ(L178E) with GDP and GTP bound to 1.35 and 1.40 Å resolution, respectively. The E. coli FtsZ(L178E) structures both crystallized as straight filaments with subunits in the R conformation. These high-resolution structures can be employed to facilitate experimental cell-division studies and their interpretation in E. coli.

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