scholarly journals Mechanism of how augmin directly targets the γ-tubulin ring complex to microtubules

2018 ◽  
Vol 217 (7) ◽  
pp. 2417-2428 ◽  
Author(s):  
Jae-Geun Song ◽  
Matthew R. King ◽  
Rui Zhang ◽  
Rachel S. Kadzik ◽  
Akanksha Thawani ◽  
...  
Keyword(s):  
Cell Shape ◽  
Insect Cells ◽  
The Other ◽  
Negative Stain ◽  
In Vitro Reconstitution ◽  
Ring Complex ◽  
Negative Stain Electron Microscopy ◽  
The Right ◽  
And Function

Microtubules (MTs) must be generated from precise locations to form the structural frameworks required for cell shape and function. MTs are nucleated by the γ-tubulin ring complex (γ-TuRC), but it remains unclear how γ-TuRC gets to the right location. Augmin has been suggested to be a γ-TuRC targeting factor and is required for MT nucleation from preexisting MTs. To determine augmin’s architecture and function, we purified Xenopus laevis augmin from insect cells. We demonstrate that augmin is sufficient to target γ-TuRC to MTs by in vitro reconstitution. Augmin is composed of two functional parts. One module (tetramer-II) is necessary for MT binding, whereas the other (tetramer-III) interacts with γ-TuRC. Negative-stain electron microscopy reveals that both tetramers fit into the Y-shape of augmin, and MT branching assays reveal that both are necessary for MT nucleation. The finding that augmin can directly bridge MTs with γ-TuRC via these two tetramers adds to our mechanistic understanding of how MTs can be nucleated from preexisting MTs.

scholarly journals Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Michal Wieczorek ◽  
Shih-Chieh Ti ◽  
Linas Urnavicius ◽  
Kelly R. Molloy ◽  
Amol Aher ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Self Assembly ◽  
The Self ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Partial Cone ◽  
Ring Complex ◽  
Microtubule Networks ◽  
And Function

The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge” (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the holocomplex.

scholarly journals Micropropagation of Mini Orchid Hybrid Phalaenopsis “Sogo Vivien”

2016 ◽  
Vol 1 (1) ◽  
pp. 45 ◽  
Author(s):  
Exsyupransia Mursyanti ◽  
Aziz Purwantoro ◽  
Sukarti Moeljopawiro ◽  
Endang Semiarti
Keyword(s):  
Cell Shape ◽  
Histological Analysis ◽  
The Other ◽  
Mass Propagation ◽  
Flower Stalk ◽  
Shoot Segment ◽  
Plant Body ◽  
Pot Plant ◽  
Variegated Plant

Phalaenopsis “Sogo Vivien” is an orchid hybrid with mini size plant body, and exhibits numerous beautiful pink flowers, that is ideal as ornamental pot plant. Some plants of this orchid exhibit variegated leaves that improve the beauty of the plant, not only because of the flower but also as attracted leaves. This orchid has high economical value, but mass propagation of this orchid has not established yet. An effective method to propagate both the normal and variegated plants is worth to be generated. The objective of this research was to produce a large number of P. “Sogo Vivien” plants, including the variegated plants. The method used seeds from self pollinating variegated plant, and flower stalk nodes. The seeds were sown on three various medium: VW, NP and MS, and flower stalk nodes were planted on VW + BA 10 mg l-1 + active carbon. The results showed that the best medium for in vitro culture of P. “Sogo Vivien” was NP medium, in which all seeds could grew into plantlets. Most plantlets emerged from the seeds were non variegated, only one plantlet out of 1344 seeds was variegated (0.007%). Although all emerged plantlets from flower stalk exhibited variegated leaves. Particularly, the plantlets arised from the second and third basal nodes of flower stalk showed the highest growth rate than that from the other nodes. Histological analysis showed that at 11-13 days after shoot segment plantation on NP medium, the shape of apical cells in the nodes was changed, then followed by the change of cell shape in the basal part of the nodes, produced bipolar pattern, then gradually developed into shoot. These results suggest that mass propagation could be achieved using seed culture, but to get the variegated phenotypes, the second and third nodes of flower stalk from variegated plant were the best explants to be used.

OPTIMUM CULTURE CONDITIONS FOR THE PRODUCTION OF PROLACTIN FROM RAT ANTERIOR PITUITARY ORGAN CULTURES

1970 ◽  
Vol 65 (3) ◽  
pp. 466-476 ◽  
Author(s):  
Richard R. Gala
Keyword(s):  
Stainless Steel ◽  
Salt Solution ◽  
Anterior Pituitary ◽  
Culture Conditions ◽  
The Other ◽  
Organ Cultures ◽  
Hormone Production ◽  
Factors Influencing ◽  
And Function

ABSTRACT Factors influencing prolactin production and anterior pituitary (AP) tissue survival in vitro were investigated. Media 199, NCTC-109, MB752/1 and Trowells-T8 were compared for their ability to support the rat AP in vitro and found to be similar, although Trowells-T8 appeared to be less well suited for this tissue. Culturing AP in Hanks balanced salt solution (BSS) resulted in a lower prolactin production and tissue survival than in Medium 199; nevertheless, there was a tenfold net synthesis of prolactin. Streptomycin and penicillin at a level of 200 units each/ml and nystatin at a level of 50 units/ml were found to control the growth of microorganisms without any deleterious effects on the AP. Amphotercine B at a level of 25 μg/ml, on the other hand, was incompatible with AP tissue survival and function. When pituitary fragments weighed 2.8 mg or more, prolactin production and tissue survival were decreased relative to AP fragments which weighed 1.2 mg or less. Culture of pituitary tissue completely submerged in medium was not compatible with optimum hormone production or AP survival. Pituitary pieces supported by stainless steel platforms with filter paper wicks resulted in better tissue survival and significantly greater prolactin production that when cultured under similar conditions but without a wick.

scholarly journals Tendon-derived biomimetic surface topographies induce phenotypic maintenance of tenocytes in vitro

2020 ◽  
Author(s):  
Aysegul Dede Eren ◽  
Aliaksei Vasilevich ◽  
E. Deniz Eren ◽  
Phanikrishna Sudarsanam ◽  
Urandelger Tuvshindorj ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Shape ◽  
Flat Surface ◽  
Flat Surfaces ◽  
Tissue Culture Polystyrene ◽  
Surface Topographies ◽  
And Function ◽  
Shape Changes

AbstractThe tenocyte niche contains biochemical and biophysical signals that are needed for tendon homeostasis. The tenocyte phenotype is correlated with cell shape in vivo and in vitro, and shape-modifying cues are needed for tenocyte phenotypical maintenance. Indeed, cell shape changes from elongated to spread when cultured on a flat surface, and rat tenocytes lose the expression of phenotypical markers throughout five passages. We hypothesized that tendon gene expression can be preserved by culturing cells in the native tendon shape. To this end, we reproduced the tendon topographical landscape into tissue culture polystyrene, using imprinting technology. We confirmed that the imprints forced the cells into a more elongated shape, which correlated with the level of Scleraxis expression. When we cultured the tenocytes for seven days on flat surfaces and tendon imprints, we observed a decline in tenogenic marker expression on flat but not on imprints. This research demonstrates that native tendon topography is an important factor contributing to the tenocyte phenotype. Tendon imprints therefore provide a powerful platform to explore the effect of instructive cues originating from native tendon topography on guiding cell shape, phenotype and function of tendon-related cells.

scholarly journals II Rapporto sullo stato di attuazione della Legge 40/2004 recante “Norme in materia di procreazione medicalmente assistita” anche in confronto con le altre nazioni europee (aprile 2009)

2009 ◽  
Vol 58 (4) ◽  
Author(s):  
Carlo Casini
Keyword(s):  
Assisted Reproduction ◽  
Present Report ◽  
The Other ◽  
Human Beings ◽  
Right To Life ◽  
Medically Assisted Reproduction ◽  
Italian Law ◽  
The Law ◽  
The Right

Il contributo propone all’attenzione il II Rapporto sull’applicazione della legge italiana in materia di “procreazione medicalmente assistita” (PMA) (Legge 40/2004), elaborato nell’aprile 2009 all’indomani della quarta relazione ministeriale del 25 marzo 2009 sullo stesso tema. La particolarità del presente Rapporto consiste nell’indagare su uno dei due obiettivi della legge 40/2004: tutelare il diritto alla vita del concepito. Infatti, mentre è alta la concentrazione sull’obiettivo di “superare la sterilità e la infertilità”, lo scopo di difendere il diritto alla vita del concepito considerato dalla stessa legge “soggetto” al pari degli altri soggetti coinvolti nelle procedure di PMA, è troppo spesso dimenticato. Il Rapporto vuole proprio rimediare a tale dimenticanza e si chiede: di quanti nuovi esseri umani è stata evitata la distruzione per effetto della legge? Poiché, come si legge nel contributo, è chiaro che le tecniche di PMA per il fatto stesso di procreare “in vitro” espongono alla morte gli embrioni anche quando vengono trasferiti nelle vie genitali della donna (ed è questa, infatti, una delle principali riserve etiche nei confronti delle tecniche di PMA), l’indagine si occupa solo di vedere come è stato tutelato il diritto alla vita degli embrioni non trasferiti, cioè non destinati alla nascita, dalla Legge 40/2004. Emerge che l’effetto più benefico della legge è quello di aver evitato nel solo triennio 2005-2006-2007 la possibile formazione soprannumeraria di embrioni e la conseguente possibile distruzione, diretta o dovuta alla crioconservazione, di oltre 120.000 embrioni. La seconda parte della ricerca è condotta confrontando anche i dati di altri Paesi europei e dimostra che il rispetto dei limiti posti a tutela del diritto alla vita hanno anche garantito meglio la salute della donna e non hanno diminuito la percentuale del “successo”. Come è noto la recente sentenza n. 151/2009 della Corte Costituzionale ha gravemente compromesso la legge proprio nel punto di maggiore sensibilità nei confronti della tutela concreta del diritto alla vita del concepito. L’auspicio è che i dati raccolti in questo II rapporto vengano comunque presi in seria considerazione. ---------- The contribution proposes the II Report of April 2009 on the application of the Italian Law on “medically assisted reproduction” (PMA) (Law 40/2004). The peculiarity of the present Report consists of investigating one of the two purposes of the Law 40/2004: to protect the right to life of newborns. In fact, if, on one hand, attention to the purpose of “overcoming sterility and infertility” the Law considers, on the other hand the purpose of defending the right to life of newborn, that is considered as “subject” just like the others subjects involved in PMA procedures from the same law, is too often forgotten. The Report intends to remedy for this forgetfulness and it asks: how many new human beings have been saved thanks to the Law? It makes clear that the most beneficent effect of the Law is that, in the period 2005-2007, it has avoided the possible production of excess embryos and the consequent possible suppression – direct or due to the cryoconservation – of over 120.000 embryos. The second part of the search is conducted also comparing data of other European Countries and it shows that the respect of the imposed limits for protecting the right to life has also guaranteed the woman’s health and it has not reduced the outcome percentage.

scholarly journals The embryonic linker histone dBigH1 alters the functional state of active chromatin

2020 ◽  
Vol 48 (8) ◽  
pp. 4147-4160 ◽  
Author(s):  
Paula Climent-Cantó ◽  
Albert Carbonell ◽  
Milos Tatarski ◽  
Oscar Reina ◽  
Paula Bujosa ◽  
...  
Keyword(s):  
Functional State ◽  
Early Embryo ◽  
Active Chromatin ◽  
Linker Histones ◽  
In Vitro Reconstitution ◽  
Transcription In Vitro ◽  
And Function ◽  
Female Specific ◽  
Zygotic Genome

Abstract Linker histones H1 are principal chromatin components, whose contribution to the epigenetic regulation of chromatin structure and function is not fully understood. In metazoa, specific linker histones are expressed in the germline, with female-specific H1s being normally retained in the early-embryo. Embryonic H1s are present while the zygotic genome is transcriptionally silent and they are replaced by somatic variants upon activation, suggesting a contribution to transcriptional silencing. Here we directly address this question by ectopically expressing dBigH1 in Drosophila S2 cells, which lack dBigH1. We show that dBigH1 binds across chromatin, replaces somatic dH1 and reduces nucleosome repeat length (NRL). Concomitantly, dBigH1 expression down-regulates gene expression by impairing RNApol II binding and histone acetylation. These effects depend on the acidic N-terminal ED-domain of dBigH1 since a truncated form lacking this domain binds across chromatin and replaces dH1 like full-length dBigH1, but it does not affect NRL either transcription. In vitro reconstitution experiments using Drosophila preblastodermic embryo extracts corroborate these results. Altogether these results suggest that the negatively charged N-terminal tail of dBigH1 alters the functional state of active chromatin compromising transcription.

scholarly journals The structure of microtubule ends during the elongation and shortening phases of dynamic instability examined by negative-stain electron microscopy

1990 ◽  
Vol 96 (4) ◽  
pp. 571-582
Author(s):  
J.R. Simon ◽  
E.D. Salmon
Keyword(s):  
Electron Microscopy ◽  
Dynamic Instability ◽  
Large Fraction ◽  
Nucleation Site ◽  
Homogeneous Population ◽  
Negative Stain ◽  
Negative Stain Electron Microscopy ◽  
Self Assembled ◽  
Elongation Phase

Microtubules (MTs) are dynamic polymers that can exist in phases of elongation and rapid-shortening at steady-state. These phases have been observed in vitro and in living cells, and this property of MTs has been termed ‘dynamic instability’. The purpose of this study was to use negative-stain electron microscopy (EM) to test if there are structural differences between the ends of MTs in the elongation and shortening phases, which could provide insight into the mechanisms of dynamic instability. MTs in the elongation phase were obtained by seeding either highly purified porcine brain tubulin (PC-tubulin) or tubulin containing microtubule-associated proteins (MTP), from isolated Tetrahymena axonemes. The results are that, in addition to intact cylindrical MTs, a significant fraction of the tubulin polymer in the elongation phase occurred as sheets of parallel protofilaments, as found in previous investigations with self-assembled MTs. Therefore, sheet formation is an intrinsic property of MT assembly that does not depend on the tubulin purity or the method of nucleation. Also, since sheets lack helical symmetry, at least a fraction of tubulin polymers seeded from axonemes did not assemble by helical addition of tubulin dimers to the ends, an assumption often made in mathematical models of dynamic instability. Sheets and intact MTs that were seeded from isolated axonemes, emanated both from the intact MT wall of the axoneme A-subfiber and from the incomplete wall of the B-subfiber. Therefore, axoneme seeds do not provide a homogeneous nucleation site for tubulin growth, or produce a homogeneous population of tubulin polymers under our conditions. Previous evidence has indicated that MT disassembly can occur by a segmental release of tubulin oligomers from the ends and at sites along the length of MTs. However, these studies were performed with MTP, and disassembly was induced by cold depolymerization. We examined MT shortening under conditions that closely represent shortening via dynamic instability, namely isothermal dilution at 37 degrees C of self-assembled MTs. This was compared with the morphology of cold-disassembled MTs. The cold-depolymerization of MTs composed of MTP showed rings and protofilament curls as previously observed using similar methods. Surprisingly, cold-depolymerization of MTs assembled from PC-tubulin induced not only shortening, but also the opening of a large fraction of MTs into sheets, suggesting that the MT lattice contains a cold-labile seam. Under conditions that mimic stochastic shortening, MTs were intact, closed cylinders with ends that were approximately blunt. Therefore, rapid shortening occurs at the ends of the MT, without a long-range disruption of the MT wall. In conclusion, MTs in the elongation phase can have highly irregular ends and need not elongate by a helical assembly process. Conversely, MTs in the shortening phase can have relatively blunt, even ends and can depolymerize in a relatively uniform fashion.

scholarly journals P911 3D echo imaging of tricuspid valve: new look at the tricuspid valve

2020 ◽  
Vol 21 (Supplement_1) ◽  
Author(s):  
H S A Abdelgawad ◽  
M Shehata ◽  
M Abdelnabi ◽  
A Almaghrabi ◽  
R Abayazeed ◽  
...  
Keyword(s):  
Tricuspid Valve ◽  
Three Dimensional ◽  
Individual Case ◽  
The Other ◽  
Volume Data ◽  
Road Map ◽  
Dimensional Echocardiography ◽  
Three Dimensional Echocardiography ◽  
The Right ◽  
And Function

Abstract Introduction "" En face"" visualisation of the tricuspid valve (TV) leaflets and commissures is not possible using two-dimensional transthoracic echocardiography (TTE) unless the right ventricle (RV) is dilated via parasternal short axis or subcostal views. With the aid of three dimensional echocardiography (3DE) , complete assessment of the TV apparatus including the leaflets, annulus and subvalvular apparatus can be easily accomplished. Aim To use different modes of three dimensional (3D TTE) to assess TV in three standard views in different diseases. Methods : 6 patients with different tricuspid valve diseases (mean age is 29 ± 18 years, 3 were women)were studied.We used three-dimensional echocardiography to acquire zoomed mode and multi-beat, full-volume data sets of the TV to assess the leaflets and commissures. .Moreover, tricuspid valve area(TVA) was assessed by direct planimetry of the valve orifice at the level of the leaflets tips at end diastolic frame in cases of TV stenosis. Results Direct visualization of the leaflets motion and number, commissures and TVA were achieved in all patients.Tricuspid valve posterior leaflet prolapse was seen in two patients (figures A, B).2 patients had tricuspid valve stenosis ; one showed characteristic commissural fusion in a rheumatic heart (3D TVA= 1.2 cm2 )(Figure D) and the other has a peculiar commissural sparing in a carcinoid pathology (3D TVA = 1.9 cm2) (Figure E) .One patient revealed rudimentary anterior leaflet suggesting unguarded TV orifice (Figure F). Regarding the numerical variations: one patient got 4 leaflets (Figure C) while another one had 2 leaflets only. Conclusion Simultaneous visualization of the three TV leaflets is limited by using 2DE.On the other hand, RT-3DE allows better understanding of the anatomy and mechanisms of the TV diseases .Comprehensive assessment of the morphology and function using RT-3DE can provide a valuable road map to guide the intervention according to each individual case. Abstract P911 Figure. Different TV morpholgies.

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