scholarly journals Structural Evidence that the P/Q Domain of ZipA Is an Unstructured, Flexible Tether between the Membrane and the C-Terminal FtsZ-Binding Domain

2002 ◽  
Vol 184 (15) ◽  
pp. 4313-4315 ◽  
Author(s):  
Tomoo Ohashi ◽  
Cynthia A. Hale ◽  
Piet A. J. de Boer ◽  
Harold P. Erickson
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
Transmembrane Domain ◽  
Persistence Length ◽  
Binding Domain ◽  
Globular Domain ◽  
Cell Division Protein ◽  
20 Nm ◽  
Rotary Shadowing

ABSTRACT The cell division protein ZipA has an N-terminal transmembrane domain and a C-terminal globular domain that binds FtsZ. Between them are a charged domain and a P/Q domain rich in proline and glutamine that has been proposed to be an unfolded polypeptide. Here we provide evidence obtained by electron microscopy that the P/Q domain is a flexible tether ranging in length from 8 to 20 nm and invisible in rotary shadowing electron microscopy. We estimated a persistence length of 0.66 nm, which is similar to the persistence lengths of other unfolded and unstructured polypeptides.

scholarly journals Microtubule Probe for Correlative Super-Resolution Fluorescence and Electron Microscopy

2018 ◽  
Author(s):  
Xiaohe Tian ◽  
Cesare De Pace ◽  
Lorena Ruiz-Perez ◽  
Bo Chen ◽  
Rina Su ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Synchronous Fluorescence ◽  
Live Cell ◽  
Fluorescence And Electron Microscopy ◽  
20 Nm ◽  
In Cells

We report a versatile cyclometalated Iridium (III) complex probe that achieves synchronous fluorescence-electron microscopy correlation to reveal microtubule ultrastructure in cells. The selective insertion of probe between repeated α and β units of microtubule triggers remarkable fluorescent enhancement, and high TEM contrast due to the presence of heavy Ir ions. The highly photostable probe allows live cell imaging of tubulin localization and motion during cell division with an resolution of 20 nm, and under TEM imaging reveals the αβ unit interspace of 45Å of microtubule in cells.

scholarly journals Binding sites of calmodulin and actin on the brain spectrin, calspectin.

1983 ◽  
Vol 97 (2) ◽  
pp. 574-578 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
H Ishikawa ◽  
M Kurokawa ◽  
K Morimoto ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Complex Formation ◽  
Binding Sites ◽  
Actin Binding ◽  
Calmodulin Binding ◽  
20 Nm ◽  
Labeling Method ◽  
The Brain ◽  
Brain Spectrin ◽  
Rotary Shadowing

We used rotary-shadowing electron microscopy to map the calmodulin-and actin-binding sites on the brain spectrin, calspectin (or fodrin). Calspectin dimers appeared as rods 110 nm long and joined in a head-to-head manner to form tetramers 220 nm long. We determined calmodulin-binding sites by a ferritin-labeling method combined with biotin-avidin complex formation. Ferritin particles were found to attach to the head parts of calspectin dimers at a position 10-20 nm from the top of the head. The number of the calmodulin-binding sites seemed to be only one for each dimer and two for each tetramer. In contrast, the actin-binding sites were localized at the tail ends of the calspectin molecules. The tetramers attached to muscle F-actin with their tail ends and often cross-linked adjacent filaments. The results are discussed in view of the analogy to the erythrocyte spectrin.

scholarly journals Localization of Cell Division Protein FtsQ by Immunofluorescence Microscopy in Dividing and Nondividing Cells ofEscherichia coli

1998 ◽  
Vol 180 (23) ◽  
pp. 6107-6116 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Mirjam E. G. Aarsman ◽  
Arend H. J. Kolk ◽  
Miguel Vicente ◽  
Nanne Nanninga
Keyword(s):  
Cell Division ◽  
Cytoplasmic Membrane ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Domain ◽  
Permissive Temperature ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Division Site ◽  
Periplasmic Domain ◽  
Cell Division Protein

ABSTRACT The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the γ form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.

scholarly journals Peptide Linkers within the Essential FtsZ Membrane Tethers ZipA and FtsA Are Nonessential for Cell Division

2019 ◽  
Vol 202 (6) ◽  
Author(s):  
Kara M. Schoenemann ◽  
Daniel E. Vega ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Membrane Binding ◽  
Globular Domain ◽  
Content Type ◽  
Linker Region ◽  
Intrinsically Disordered ◽  
Binding Domains

ABSTRACT Bacteria such as Escherichia coli divide by organizing filaments of FtsZ, a tubulin homolog that assembles into dynamic treadmilling membrane-associated protein filaments at the cell midpoint. FtsA and ZipA proteins are required to tether these filaments to the inner face of the cytoplasmic membrane, and loss of either tether is lethal. ZipA from E. coli and other closely related species harbors a long linker region that connects the essential N-terminal transmembrane domain to the C-terminal globular FtsZ-binding domain, and part of this linker includes a P/Q-rich peptide that is predicted to be intrinsically disordered. We found unexpectedly that several large deletions of the ZipA linker region, including the entire P/Q rich peptide, had no effect on cell division under normal conditions. However, we found that the loss of the P/Q region made cells more resistant to excess levels of FtsA and more sensitive to conditions that displaced FtsA from FtsZ. FtsA also harbors a short ∼20-residue peptide linker that connects the main globular domain with the C-terminal amphipathic helix that is important for membrane binding. In analogy with ZipA, deletion of 11 of the central residues in the FtsA linker had little effect on FtsA function in cell division. IMPORTANCE Escherichia coli cells divide using a cytokinetic ring composed of polymers of the tubulin-like FtsZ. To function properly, these polymers must attach to the inner surface of the cytoplasmic membrane via two essential membrane-associated tethers, FtsA and ZipA. Both FtsA and ZipA contain peptide linkers that connect their membrane-binding domains with their FtsZ-binding domains. Although they are presumed to be crucial for cell division activity, the importance of these linkers has not yet been rigorously tested. Here, we show that large segments of these linkers can be removed with few consequences for cell division, although several subtle defects were uncovered. Our results suggest that ZipA, in particular, can function in cell division without an extended linker.

scholarly journals Mapping of epitopes for monoclonal antibodies against human platelet thrombospondin with electron microscopy and high sensitivity amino acid sequencing.

1985 ◽  
Vol 101 (4) ◽  
pp. 1434-1441 ◽  
Author(s):  
N J Galvin ◽  
V M Dixit ◽  
K M O'Rourke ◽  
S A Santoro ◽  
G A Grant ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Human Platelet ◽  
High Sensitivity ◽  
Binding Domain ◽  
Peptide Chain ◽  
Dimensional Structure ◽  
Globular Domain ◽  
Heparin Binding ◽  
Heparin Binding Domain

A panel of monoclonal antibodies (Mab's) has been raised against human platelet thrombospondin (TSP). One Mab, designated A2.5, inhibits the hemagglutinating activity of TSP and immunoprecipitates the NH2 terminal 25 kD heparin binding domain of TSP (Dixit, V.M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Biochemistry, in press). Another Mab, C6.7, blocks the thrombin-stimulated aggregation of live platelets and immunoprecipitates an 18-kD fragment distinct from the heparin binding domain (Dixit, V. M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Proc. Natl. Acad. Sci. 82: 3472-3476). To determine the relative locations of the epitopes for these Mabs in the three-dimensional structure of TSP, we have examined TSP-Mab complexes by electron microscopy of rotary-shadowed proteins. The TSP molecule is composed of three 180-kD subunits, each of which consists of a small globular domain (approximately 8 nm diam) and a larger globular domain (approximately 16 nm diam) connected by a thin, flexible strand. The subunit interaction site is on the thin connecting strands, nearer the small globular domains. Mab A2.5 binds to the cluster of three small domains, indicating that this region contains the heparin binding domain and thus represents the NH2 termini of the TSP peptide chains. Mab C6.7 binds to the large globular domains on the side opposite the point at which the connecting strand enters the domain, essentially the maximum possible distance from the A2.5 epitope. Using high sensitivity automated NH2 terminal sequencing of TSP chymotryptic peptides we have ordered these fragments within the TSP peptide chain and have confirmed that the epitope for C6.7 in fact lies near the extreme COOH terminus of the peptide chain. In combination with other data, we have been able to construct a map of the linear order of the identified domains of TSP that indicates that to a large extent, the domains are arranged co-linearly with the peptide chain.

scholarly journals Morphology of Isolated Gli349, a Leg Protein Responsible for Mycoplasma mobile Gliding via Glass Binding, Revealed by Rotary Shadowing Electron Microscopy

2006 ◽  
Vol 188 (8) ◽  
pp. 2821-2828 ◽  
Author(s):  
Jun Adan-Kubo ◽  
Atsuko Uenoyama ◽  
Toshiaki Arata ◽  
Makoto Miyata
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Gel Filtration ◽  
Solid Surfaces ◽  
Membrane Protrusion ◽  
Molecular Image ◽  
Peptide Length ◽  
20 Nm ◽  
Molecular Images ◽  
Rotary Shadowing

ABSTRACT Several species of mycoplasmas rely on an unknown mechanism to glide across solid surfaces in the direction of a membrane protrusion at the cell pole. Our recent studies on the fastest species, Mycoplasma mobile, suggested that a 349-kDa protein, Gli349, localized at the base of the membrane protrusion called the neck, forms legs that stick out from the neck and propel the cell by repeatedly binding to and releasing from a solid surface, based on the energy of ATP hydrolysis. Here, the Gli349 protein was isolated from mycoplasma cells and its structure was analyzed. Gel filtration analysis showed that the isolated Gli349 protein is monomeric. Rotary shadowing electron microscopy revealed that the molecular structure resembles the symbol for an eighth note in music. It contains an oval foot 14 nm long in axis. From this foot extend three rods in tandem of 43, 20, and 20 nm, in that order. The hinge connecting the first and second rods is flexible, while the next hinge has a distinct preference in its angle, near 90 degrees. Molecular images revealed that a monoclonal antibody that can bind to the position at one-third of the total peptide length from the N terminus bound to a position two-thirds from the foot end, suggesting that the foot corresponds to the C-terminal region. The amino acid sequence was assigned to the molecular image, and the topology of the molecule in the gliding machinery is discussed.

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

1969 ◽  
Vol 27 ◽  
pp. 424-425
Author(s):  
Lee F. Ellis ◽  
Richard M. Van Frank ◽  
Walter J. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Interferon Inducer ◽  
Virus Particles ◽  
Staining Methods ◽  
Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

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