Superresolution microscopy for microbiology

Coltharp, Carla; Xiao, Jie

doi:10.1111/cmi.12024

Cited by 115 publications

(87 citation statements)

References 83 publications

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“…It is often the case that bacterial nanomachines are assembled at specific sites within the bacterial envelope (38)(39)(40). For example, some bacteria deploy their flagellar or type IV protein secretion machines at their poles, and many bacteria deploy components of their cell division machine at the midplate (41)(42)(43).…”

Section: Resultsmentioning

confidence: 99%

Visualization and characterization of individual type III protein secretion machines in live bacteria

Zhang

Lara‐Tejero

Bewersdorf

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

103

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Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the Salmonella type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in Salmonella mutants we obtained major insights into the machine's assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines.bacterial nanomachines | protein secretion | superresolution microscopy | bacterial pathogenesis | Salmonella virulence T ype III secretion systems are remarkable molecular machines with the capacity to inject multiple bacterial proteins into eukaryotic cells (1, 2). These machines play an essential role in the pathogenic or symbiotic relationships between the bacteria that encode them and their respective hosts. Consequently, type III secretion systems are rapidly emerging as targets for the development of novel therapeutic and prevention strategies with the potential to combat several important infectious diseases (3-5). The components of type III secretion machines are highly conserved across bacterial species although the effector proteins that they deliver are customized for the specific bacteria that harbor them (1, 6, 7). The entire type III secretion machine or injectisome is ∼40 nm in width and ∼150 nm in length and is organized in defined substructures (Fig. 1A). The bestcharacterized substructure is the needle complex, which is a multi-ring cylindrical structure embedded in the bacterial envelope with a needle-like extension that projects several nanometers from the bacterial surface. The needle complex is traversed by a narrow channel that serves as the conduit for the proteins that travel this secretion pathway (8). The needle is capped at its end by the tip complex, which is thought to coordinate the activation of the secretion machine upon contact with target cells (9, 10). Because the needle complex can be isolated in a manner suitable for single-particle cryo-electron microscopy, its organization at the quasi-atomic level is known (11-18). Passage of the secreted proteins through the inner membrane requires the export apparatus, which is composed of a group of poly-topic inner membrane proteins located at the center of the needle complex base (19). Finally, several cytoplasmic proteins form a large complex known as the...

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Section: Resultsmentioning

confidence: 99%

Visualization and characterization of individual type III protein secretion machines in live bacteria

Zhang

Lara‐Tejero

Bewersdorf

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

103

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“…Nowadays, mitochondrial analysis by live-cell microscopy represents the most direct method to retrieve authentic information about mitochondrial shape, position and content. Development of superresolution microscopy techniques has allowed imaging of fluorescently labeled cells and organelles at unprecedented levels of resolution [206][207][208][209][210][211][212]. For example, using three-dimensional (3D) structuredillumination microscopy (SIM) resolution was doubled relative to a wide-field fluorescence microscope yielding values of 120 nm (lateral) and 360 nm (axial).…”

Section: Fluorescence Microscopymentioning

confidence: 99%

Regulation and Quantification of Cellular Mitochondrial Morphology and Content

Tronstad¹,

Nooteboom²,

Nilsson³

et al. 2014

CPD

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Running title: Regulation and quantification of mitochondrial morphology and content. Word count: 15,454 (total)Characters: 107,091 (including spaces); 92,067 (excluding spaces) Keywords: mitochondrial biogenesis, mitochondrial dynamics, mitochondrial degradation, living cells, TMRM, microscopy, segmentation, image analysis.Page 2 of 37 ABSTRACTMitochondria play a key role in signal transduction, redox homeostasis and cell survival, which extends far beyond their classical functioning in ATP production and energy metabolism. In living cells, mitochondrial content ("mitochondrial mass") depends on the cell-controlled balance between mitochondrial biogenesis and degradation. These processes are intricately linked to changes in net mitochondrial morphology and spatiotemporal positioning ("mitochondrial dynamics"), which are governed by mitochondrial fusion, fission and motility. It is becoming increasingly clear that mitochondrial mass and dynamics, as well as its ultrastructure and volume, are mechanistically linked to mitochondrial function and the cell. This means that proper quantification of mitochondrial morphology and content is of prime importance in understanding mitochondrial and cellular physiology in health and disease. This review first presents how cellular mitochondrial content is regulated at the level of mitochondrial biogenesis, degradation and dynamics. Next we discuss how mitochondrial dynamics and content can be analyzed with a special emphasis on quantitative live-cell microscopy strategies.

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“…How the divisome components are disposed after the closure of the septum is another question that will also need additional research. Techniques such as high-resolution microscopy (97) and in vitro reconstruction procedures (98) are now available to investigate these questions. The answers will clarify both how the process of septation is initiated and how it finishes.…”

Section: Additional Questionsmentioning

confidence: 99%

In the Beginning, Escherichia coli Assembled the Proto-ring: An Initial Phase of Division

Rico

Krupka²,

Vicente

2013

Journal of Biological Chemistry

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Cell division in Escherichia coli begins by assembling three proteins, FtsZ, FtsA, and ZipA, to form a proto-ring at midcell. These proteins nucleate an assembly of at least 35 components, the divisome. The structuring of FtsZ to form a ring and the processes that effect constriction have been explained by alternative but not mutually exclusive mechanisms. We discuss how FtsA and ZipA provide anchoring of the cytoplasmic FtsZ to the membrane and how a temporal sequence of alternative protein interactions may operate in the maturation and stability of the proto-ring. How the force needed for constriction is generated and how the proto-ring proteins relate to peptidoglycan synthesis remain as the main challenges for future research. Overview of SeptationCell division is the last event in the bacterial cell cycle. It takes place by producing a rigid transversal septum after the growth processes fully duplicate the contents of the cell. Septation is always preceded by segregation of the duplicated nucleoids, ensuring that each of the daughter cells contains a fully replicated chromosome. Specialized molecules control the positioning of the division machinery at midcell, preventing any harmful fragmentation that septum progression could cause on unsegregated nucleoids.The division machinery, the divisome, establishes a complex interacting network together with DNA, lipids, and peptidoglycan components. It comprises a variable number of proteins, depending on the bacterial species. These proteins have redundant multiple functions and connections serving as safeguards to complete division and to guarantee the efficiency and versatility of the process. Altogether, an efficient division process allows the proliferation of bacteria under a variety of conditions, and it is one of the reasons for their success in the environment. Free-living bacteria, such as Escherichia coli, have more elaborated divisomes, whereas those that need to grow as intracellular parasites, as such Mycoplasma genitalium, have streamlined ones (1, 2).The structure of the E. coli divisome as revealed by immunofluorescence images of dividing cells is called the division ring (3). For its assembly, three essential proteins, FtsZ, FtsA, and ZipA, are first gathered at midcell, forming a proto-ring attached to the inner membrane ( Fig. 1) (4). In the proto-ring structure, the cytoplasmic GTPase FtsZ is anchored to the membrane by its interaction with ZipA and FtsA. ZipA is a bitopic protein containing a transmembrane domain (5). FtsA is a protein of the actin family that associates with the membrane by a short amphipathic helix (6, 7). The assembly of the FtsZ ring is dependent on a continuous energy supply. The FtsZ ring requires either ZipA or FtsA (8). Although it can be formed in the presence of either of them, division is affected unless both are present (9). A maturation period takes place after the protoring is assembled and before the rest of the divisome proteins become incorporated (10). During maturation, the proto-ring assembly is not stable...

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Superresolution microscopy for microbiology

Cited by 115 publications

References 83 publications

Visualization and characterization of individual type III protein secretion machines in live bacteria

Visualization and characterization of individual type III protein secretion machines in live bacteria

Regulation and Quantification of Cellular Mitochondrial Morphology and Content

In the Beginning, Escherichia coli Assembled the Proto-ring: An Initial Phase of Division

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