In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex

2008 ◽  
Vol 412 (2) ◽  
pp. 367-378 ◽  
Author(s):  
Rosemary S. Mcandrew ◽  
Bradley J. S. C. Olson ◽  
Deena K. Kadirjan-Kalbach ◽  
Cecilia L. Chi-Ham ◽  
Stanislav Vitha ◽  
...  
Keyword(s):  
Density Gradient ◽  
Gradient Centrifugation ◽  
Biochemical Properties ◽  
Molar Ratio ◽  
Plastid Division ◽  
Bacterial Cell Division ◽  
Dynamic Regulation ◽  
Interacting Protein ◽  
Z Ring

FtsZ1 and FtsZ2 are phylogenetically distinct homologues of the tubulin-like bacterial cell division protein FtsZ that play major roles in the initiation and progression of plastid division in plant cells. Both proteins are components of a mid-plastid ring, the Z-ring, which functions as a contractile ring on the stromal surface of the chloroplast IEM (inner envelope membrane). FtsZ1 and FtsZ2 have been shown to interact, but their in vivo biochemical properties are largely unknown. To gain insight into the in vivo biochemical relationship between FtsZ1 and FtsZ2, in the present study we investigated their molecular levels in wild-type Arabidopsis thaliana plants and endogenous interactions in Arabidopsis and pea. Quantitative immunoblotting and morphometric analysis showed that the average total FtsZ concentration in chloroplasts of 3-week-old Arabidopsis plants is comparable with that in Escherichia coli. FtsZ levels declined as plants matured, but the molar ratio between FtsZ1 and FtsZ2 remained constant at approx. 1:2, suggesting that this stoichiometry is regulated and functionally important. Density-gradient centrifugation, native gel electrophoresis, gel filtration and co-immunoprecipitation experiments showed that a portion of the FtsZ1 and FtsZ2 in Arabidopsis and pea chloroplasts is stably associated in a complex of ∼200–245 kDa. This complex also contains the FtsZ2-interacting protein ARC6 (accumulation and replicatioin of chloroplasts 6), an IEM protein, and analysis of density-gradient fractions suggests the presence of the FtsZ1-interacting protein ARC3. Based on the mid-plastid localization of ARC6 and ARC3 and their postulated roles in promoting and inhibiting chloroplast FtsZ polymer formation respectively, we hypothesize that the FtsZ1–FtsZ2–ARC3–ARC6 complex represents an unpolymerized IEM-associated pool of FtsZ that contributes to the dynamic regulation of Z-ring assembly and remodelling at the plastid division site in vivo.

scholarly journals Subcellular organization of neurophysins, oxytocin, [8−lysine]-vasopressin and adenosine triphosphatase in porcine posterior pituitary lobes

1973 ◽  
Vol 132 (3) ◽  
pp. 361-371 ◽  
Author(s):  
J. C. Pickup ◽  
C. I. Johnston ◽  
S. Nakamura ◽  
L. O. Uttenthal ◽  
D. B. Hope
Keyword(s):  
Density Gradient ◽  
Adenosine Triphosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Posterior Pituitary ◽  
Neurosecretory Granules ◽  
Sucrose Density Gradient Centrifugation ◽  
Lysine Vasopressin ◽  
Neurophysin Ii

Posterior pituitary lobes from young pigs were fractionated by differential and sucrose-density-gradient centrifugation. The distributions of oxytocin and [8-lysine]-vasopressin were measured by bioassay and the distributions of neurophysin-I and -II by radioimmunoassays specific for each of these two proteins. Most of the hormone and neurophysin applied to the density gradient was localized in particles with the density expected of neurosecretory granules. However, the neurosecretory granules were separated into two bands (D and E). A close statistical correlation between the distributions of [8-lysine]-vasopressin and neurophysin-I, and of oxytocin and neurophysin-II on the gradients, suggested that in vivo porcine neurophysin-I binds [8-lysine]-vasopressin within one population of granules and porcine neurophysin-II binds oxytocin within another type of granule. However, there was no significant separation of oxytocin and vasopressin in fractions D and E. The molar ratios of hormones and neurophysins indicated that there was insufficient of either neurophysin to bind the [8-lysine]-vasopressin in the granule fractions or in the whole gland. Polyacrylamide-gel electrophoresis showed that only bands corresponding in mobility to porcine neurophysins-I, -II and -III were present in large amounts in the whole gland and in the granule fractions. The component with the mobility of neurophysin-III was, however, relatively enriched in whole young glands and granule fractions compared with adult gland extracts. It is suggested that the vasopressin that cannot be assigned to neurophysin-I may occur in (a) vesicles containing vasopressin but no neurophysin, (b) vesicles containing vasopressin and a protein that cannot be quantified by the radioimmunoassays used, such as porcine neurophysin-III, or (c) normal vasopressin–neurophysin granules which have accumulated extra vasopressin. Band E of the gradient was rich in adenosine triphosphatase activity, whereas band D possessed very little of this enzyme.

scholarly journals Lateral interactions between protofilaments of the bacterial tubulin homolog FtsZ are essential for cell division

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Fenghui Guan ◽  
Jiayu Yu ◽  
Jie Yu ◽  
Yang Liu ◽  
Ying Li ◽  
...  
Keyword(s):  
Cell Division ◽  
Bacterial Cell ◽  
Dynamic Structure ◽  
Living Cells ◽  
Lateral Interactions ◽  
Bacterial Cell Division ◽  
Z Ring ◽  
Crystallographic Structures ◽  
Order Structures

The prokaryotic tubulin homolog FtsZ polymerizes into protofilaments, which further assemble into higher-order structures at future division sites to form the Z-ring, a dynamic structure essential for bacterial cell division. The precise nature of interactions between FtsZ protofilaments that organize the Z-ring and their physiological significance remain enigmatic. In this study, we solved two crystallographic structures of a pair of FtsZ protofilaments, and demonstrated that they assemble in an antiparallel manner through the formation of two different inter-protofilament lateral interfaces. Our in vivo photocrosslinking studies confirmed that such lateral interactions occur in living cells, and disruption of the lateral interactions rendered cells unable to divide. The inherently weak lateral interactions enable FtsZ protofilaments to self-organize into a dynamic Z-ring. These results have fundamental implications for our understanding of bacterial cell division and for developing antibiotics that target this key process.

scholarly journals Separation of Degranulated Platelets from Normal Platelets

1977 ◽  
Author(s):  
P. Cieslar ◽  
J.P. Greenberg ◽  
M.A. Packham ◽  
R.L. Kinlough-Rathbone ◽  
J.F. Mustard
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Adenine Nucleotide ◽  
Thrombus Formation ◽  
Gradient Centrifugation ◽  
Rabbit Platelets ◽  
Fraction I

Platelets degranulated by thrombin (TDP) can be recovered, are effective in hemostasis and survive normally upon infusion into rabbits. Two approaches to determine whether platelets have been degranulated in vivo are: (1) measurement of circulating released materials; (2) detection of circulating degranulated platelets. We have used arabino-galactan (Stractan II) density gradient centrifugation to separate normal and degranulated platelets. The following distribution was obtained with washed rabbit platelets.The serotonin, PF4 and adenine nucleotide contents of the TDP were less than those of normal platelets and the TDP in fraction I had the lowest amounts. When TDP were labeled with 51cr and mixed with equal numbers of normal platelets, 85% of the platelets in fraction I were found to be TDP. 51Cr-TDP were injected into normal rabbits and reharvested after 18 hours. The greatest proportion of TDP was isolated in fraction I. Thus this method may make it possible to separate platelets that have lost their granule contents during participation in reversible thrombus formation in vivo.(* Visiting Fellow from the Faculty of Medicine, Charles University, Prague, Czechoslovakia.)

scholarly journals Trapping of a Spiral-Like Intermediate of the Bacterial Cytokinetic Protein FtsZ

2006 ◽  
Vol 188 (5) ◽  
pp. 1680-1690 ◽  
Author(s):  
Katherine A. Michie ◽  
Leigh G. Monahan ◽  
Peter L. Beech ◽  
Elizabeth J. Harry
Keyword(s):  
Ring Formation ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Bacterial Cell Division ◽  
Temporal Regulation ◽  
Division Site ◽  
Spiral Form ◽  
Z Ring

ABSTRACT The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.

scholarly journals Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Piotr Szwedziak ◽  
Qing Wang ◽  
Tanmay A M Bharat ◽  
Matthew Tsim ◽  
Jan Löwe
Keyword(s):  
Cell Division ◽  
Molecular Model ◽  
Three Dimensional ◽  
Bacterial Cell Division ◽  
Electron Cryomicroscopy ◽  
E Coli ◽  
Ftsz Ring ◽  
Z Ring

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.

scholarly journals Isolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy

2021 ◽  
Author(s):  
Christina Wichmann ◽  
Petra Rösch ◽  
Jürgen Popp
Keyword(s):  
Raman Spectroscopy ◽  
Bronchoalveolar Lavage ◽  
Density Gradient ◽  
Bronchoalveolar Lavage Fluid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Lavage Fluid ◽  
Biochemical Properties ◽  
Low Concentrations ◽  
A Cell

AbstractRaman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix.

scholarly journals The plastid division proteins, FtsZ1 and FtsZ2, differ in their biochemical properties and sub-plastidial localization

2005 ◽  
Vol 387 (3) ◽  
pp. 669-676 ◽  
Author(s):  
El-Sayed EL-KAFAFI ◽  
Sunil MUKHERJEE ◽  
Mahmoud EL-SHAMI ◽  
Jean-Luc PUTAUX ◽  
Maryse A. BLOCK ◽  
...  
Keyword(s):  
Cell Division ◽  
Higher Plants ◽  
Green Plant ◽  
Biochemical Properties ◽  
Plastid Division ◽  
Bacterial Cell Division ◽  
Topological Localization ◽  
Long Rod ◽  
Envelope Membranes ◽  
Chemical Cross Linking

Plastid division in higher plants is morphologically similar to bacterial cell division, with a process termed binary fission involving constriction of the envelope membranes. FtsZ proteins involved in bacterial division are also present in higher plants, in which the ftsZ genes belong to two distinct families: ftsZ1 and ftsZ2. However, the roles of the corresponding proteins FtsZ1 and FtsZ2 in plastid division have not been determined. Here we show that the expression of plant FtsZ1 and FtsZ2 in bacteria has different effects on cell division, and that distinct protein domains are involved in the process. We have studied the assembly of purified FtsZ1 and FtsZ2 using a chemical cross-linking approach followed by PAGE and electron microscopy analyses of the resulting polymers. This has revealed that FtsZ1 is capable of forming long rod-shaped polymers and rings similar to the bacterial FtsZ structures, whereas FtsZ2 does not form any organized polymer. Moreover, using purified sub-plastidial fractions, we show that both proteins are present in the stroma, and that a subset of FtsZ2 is tightly bound to the purified envelope membranes. These results indicate that FtsZ2 has a localization pattern distinct from that of FtsZ1, which can be related to distinct properties of the proteins. From the results presented here, we propose a model for the sequential topological localization and functions of green plant FtsZ1 and FtsZ2 in chloroplast division.

Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa

2010 ◽  
Vol 74 (6) ◽  
pp. 979-988 ◽  
Author(s):  
O. García-Álvarez ◽  
A. Maroto-Morales ◽  
M. Ramón ◽  
E. del Olmo ◽  
V. Montoro ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation

Microgravity Separation of Alginate Empty Capsules from Encapsulated Pancreatic Islets Using a Microfluidic System

2015 ◽  
Vol 15 (10) ◽  
pp. 7876-7880 ◽  
Author(s):  
Soojeong Shin ◽  
Young Je Yoo ◽  
Jong Wook Hong
Keyword(s):  
Pancreatic Islets ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Biomedical Application ◽  
Gradient Centrifugation ◽  
Previous Method ◽  
High Yield ◽  
One Pot ◽  
Optimal Separation

Although microencapsulated pancreatic islets have merits, such as ease of transplantation, viability and functionality improvement, and immune protection in vivo, the co-production of alginate empty capsules during the encapsulation of islets with alginate makes them unusable for biomedical application. In previous research, the removal of empty alginate capsules with high yield was achieved using density-gradient centrifugation. Here, we report advanced microgravity-based separation techniques in a microfluidic format for alginate empty capsules. The optimal separation conditions were mathematically evaluated using Stokes’ law and the separation of the encapsulation product was accomplished. A microfluidic chip was designed with two inlets and two outlets at different elevations to mimic the vertical percoll gradient in density-gradient centrifugation. The separation of alginate empty capsules using microgravitational force resulted in effective separation of encapsulated islets from alginate empty capsules with more than 70% efficiency. Moreover, no loss of encapsulated islets was expected because the process is a one-pot separation, unlike the previous method. This type of microgravitational particle separation could be used both for the fractionization of heterogeneous encapsulated cells and to remove empty capsules.

scholarly journals Assembly properties of bacterial tubulin homolog FtsZ regulated by the positive regulator protein ZipA and ZapA from Pseudomonas aeruginosa

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mujeeb ur Rahman ◽  
Zhe Li ◽  
Tingting Zhang ◽  
Shuheng Du ◽  
Xueqin Ma ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Species ◽  
Biochemical Properties ◽  
Gtpase Activity ◽  
Bacterial Cell Division ◽  
Contractile Ring ◽  
Positive Regulator ◽  
Regulator Protein ◽  
Z Ring

AbstractBacterial tubulin homolog FtsZ self-assembles into dynamic protofilaments, which forms the scaffold for the contractile ring (Z-ring) to achieve bacterial cell division. Here, we study the biochemical properties of FtsZ from Pseudomonas aeruginosa (PaFtsZ) and the effects of its two positive regulator proteins, ZipA and ZapA. Similar to Escherichia coli FtsZ, PaFtsZ had a strong GTPase activity, ~ 7.8 GTP min-1 FtsZ-1 at pH 7.5, and assembled into mainly short single filaments in vitro. However, PaFtsZ protofilaments were mixtures of straight and “intermediate-curved” (100–300 nm diameter) in pH 7.5 solution and formed some bundles in pH 6.5 solution. The effects of ZipA on PaFtsZ assembly varied with pH. In pH 6.5 buffer ZipA induced PaFtsZ to form large bundles. In pH 7.5 buffer PaFtsZ-ZipA protofilaments were not bundled, but ZipA enhanced PaFtsZ assembly and promoted more curved filaments. Comparable to ZapA from other bacterial species, ZapA from P. aeruginosa induced PaFtsZ protofilaments to associate into long straight loose bundles and/or sheets at both pH 6.5 and pH 7.5, which had little effect on the GTPase activity of PaFtsZ. These results provide us further information that ZipA functions as an enhancer of FtsZ curved filaments, while ZapA works as a stabilizer of FtsZ straight filaments.

Sign in / Sign up

Export Citation Format

Share Document