scholarly journals Biparental Inheritance of γ-Tubulin during Human Fertilization: Molecular Reconstitution of Functional Zygotic Centrosomes in Inseminated Human Oocytes and in Cell-free Extracts Nucleated by Human Sperm

1999 ◽  
Vol 10 (9) ◽  
pp. 2955-2969 ◽  
Author(s):  
Calvin Simerly ◽  
Sara S. Zoran ◽  
Chris Payne ◽  
Tanja Dominko ◽  
Peter Sutovsky ◽  
...  
Keyword(s):  
Human Sperm ◽  
Calcium Sensitivity ◽  
Microtubule Assembly ◽  
Biparental Inheritance ◽  
Western Blots ◽  
Human Fertilization ◽  
Egg Extracts ◽  
Sperm Aster

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents γ-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. γ-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal γ-tubulin. Recruitment of maternal γ-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm “priming” induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.

scholarly journals Stathmin/Op18 Phosphorylation Is Regulated by Microtubule Assembly

2001 ◽  
Vol 12 (2) ◽  
pp. 437-448 ◽  
Author(s):  
Thomas Küntziger ◽  
Olivier Gavet ◽  
Valérie Manceau ◽  
André Sobel ◽  
Michel Bornens
Keyword(s):  
Feedback Loop ◽  
Somatic Cells ◽  
Network Control ◽  
Microtubule Assembly ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Mitotic Chromatin

Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of “mitotic chromatin.” Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals A novel dephosphorylation targeting chimera selectively promoting tau removal in tauopathies

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Jie Zheng ◽  
Na Tian ◽  
Fei Liu ◽  
Yidian Zhang ◽  
Jingfen Su ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Protein Phosphatase ◽  
High Efficiency ◽  
Microtubule Assembly ◽  
Hyperphosphorylated Tau ◽  
Phosphatase 2A ◽  
Dependent Learning ◽  
The Brain

AbstractIntraneuronal accumulation of hyperphosphorylated tau is a hallmark pathology shown in over twenty neurodegenerative disorders, collectively termed as tauopathies, including the most common Alzheimer’s disease (AD). Therefore, selectively removing or reducing hyperphosphorylated tau is promising for therapies of AD and other tauopathies. Here, we designed and synthesized a novel DEPhosphorylation TArgeting Chimera (DEPTAC) to specifically facilitate the binding of tau to Bα-subunit-containing protein phosphatase 2A (PP2A-Bα), the most active tau phosphatase in the brain. The DEPTAC exhibited high efficiency in dephosphorylating tau at multiple AD-associated sites and preventing tau accumulation both in vitro and in vivo. Further studies revealed that DEPTAC significantly improved microtubule assembly, neurite plasticity, and hippocampus-dependent learning and memory in transgenic mice with inducible overexpression of truncated and neurotoxic human tau N368. Our data provide a strategy for selective removal of the hyperphosphorylated tau, which sheds new light for the targeted therapy of AD and related-tauopathies.

scholarly journals Drosophila Stathmin: A Microtubule-destabilizing Factor Involved in Nervous System Formation

2002 ◽  
Vol 13 (2) ◽  
pp. 698-710 ◽  
Author(s):  
Sylvie Ozon ◽  
Antoine Guichet ◽  
Olivier Gavet ◽  
Siegfried Roth ◽  
André Sobel
Keyword(s):  
Nervous System ◽  
System Development ◽  
Nervous System Development ◽  
Microtubule Assembly ◽  
Nervous Systems ◽  
Germ Cell Migration ◽  
Numerous Cell ◽  
First Time

Stathmin is a ubiquitous regulatory phosphoprotein, the generic element of a family of neural phosphoproteins in vertebrates that possess the capacity to bind tubulin and interfere with microtubule dynamics. Although stathmin and the other proteins of the family have been associated with numerous cell regulations, their biological roles remain elusive, as in particular inactivation of the stathmin gene in the mouse resulted in no clear deleterious phenotype. We identified stathmin phosphoproteins inDrosophila, encoded by a unique gene sharing the intron/exon structure of the vertebrate stathmin andstathmin family genes. They interfere with microtubule assembly in vitro, and in vivo when expressed in HeLa cells. Drosophila stathmin expression is regulated during embryogenesis: it is high in the migrating germ cells and in the central and peripheral nervous systems, a pattern resembling that of mammalian stathmin. Furthermore, RNA interference inactivation ofDrosophila stathmin expression resulted in germ cell migration arrest at stage 14. It also induced important anomalies in nervous system development, such as loss of commissures and longitudinal connectives in the ventral cord, or abnormal chordotonal neuron organization. In conclusion, a single Drosophilagene encodes phosphoproteins homologous to the entire vertebrate stathmin family. We demonstrate for the first time their direct involvement in major biological processes such as development of the reproductive and nervous systems.

Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Perike Srikanth ◽  
Andrielle E Capote ◽  
Alsina Katherina M ◽  
Benjamin Levin ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

Directionality of microtubule assembly: an in vivo study with the ciliate Tetrahymena

1978 ◽  
Vol 33 (1) ◽  
pp. 227-234
Author(s):  
S.F. Ng
Keyword(s):  
Tetrahymena Thermophila ◽  
Recessive Gene ◽  
Microtubule Assembly ◽  
In Vivo Study ◽  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Posterior Portion ◽  
Temperature Sensitive Mutant

A temperature-sensitive mutant homozygous for the recessive gene molb in Tetrahymena thermophila offers opportunity for studying the direction of microtubule assembly in vivo. At 39 degrees C the mutant fails to divide properly; the 2 daughter animals remain attached and bend over each other. As revealed by protargol staining, the bending results in acute turning and breaking of some of the longitudinal microtubular bands close and parallel to the surface. Hence, 2 broken microtubular ends are available for study of the problem of directionality of microtubule assembly, by assessing which of the 2 ends regenerates. In most cases the posterior portion of the longitudinal microtubular band regenerates. The present study hence supports the conclusion based on in vitro observation in other systems that microtubule assembly is predominantly unidirectional.

Abstract P458: Myosin Light Chain Dephosphorylation By Ppp1r12c Promotes Atrial Hypocontractility In Atrial Fibrillation

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Srikanth Perike ◽  
Frederick Damen ◽  
Andrielle Capote ◽  
Katherina M Alsina ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Introduction: Atrial fibrillation (AF), is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 2.7 million to 6.1 million and is predictive to increase to 12.1 million in 2030. AF increases the chances of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. Objective: The overexpression of PPP1R12C, causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), decreasing atrial contractility. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluated the role of PP1c-PPP1R12C interaction in MLC2a de-phosphorylation we used Western blots, coimmunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The Overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, that cause a reduction in atrial contractility and increases AF inducibility. All these discoveries advocate that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

O-228 The SSRI antidepressant Sertraline inhibits CatSper calcium channels in human sperm

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
R Rahban ◽  
A Rehfeld ◽  
C Schiffer ◽  
C Brenker ◽  
D. Louise Egeberg Palme ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Human Sperm ◽  
Fertilization Process ◽  
Acrosomal Exocytosis ◽  
Depth Analysis ◽  
Sperm Penetration ◽  
Viscous Media ◽  
Voltage Activation

Abstract Study question Do Selective Serotonin Reuptake Inhibitor (SSRI) antidepressants affect the function of human sperm? Summary answer The SSRI-antidepressant Sertraline (e.g. Zoloft) inhibits the sperm-specific Ca2+ channel CatSper and affects human sperm function in vitro. What is known already In human sperm, CatSper translates changes of the chemical microenvironment into changes of the intracellular Ca2+ concentration ([Ca2+]i) and swimming behavior. CatSper is promiscuously activated by oviductal ligands, but also by synthetic chemicals that might disturb the fertilization process. It is well known that SSRIs have off-target actions on Ca2+, Na+, and K+ channels in somatic cells. Whether SSRIs affect the activity of CatSper is, however, unknown. Study design, size, duration We studied the action of the seven drugs belonging to the most commonly prescribed class of antidepressants, SSRIs, on resting [Ca2+]i and Ca2+ influx via CatSper in human sperm. The SSRI Sertraline was selected for in-depth analysis of its action on steroid-, prostaglandin-, pH-, and voltage-activation of human CatSper. Moreover, the action of Sertraline on sperm acrosomal exocytosis and penetration into viscous media was evaluated. Participants/materials, setting, methods The activity of CatSper was investigated in sperm of healthy volunteers, using kinetic Ca2+ fluorimetry and patch-clamp recordings. Acrosomal exocytosis was investigated using Pisum sativum agglutinin (PSA) and image cytometry. Sperm penetration in viscous media was evaluated using the Kremer test. Main results and the role of chance Four SSRIs increased [Ca2+]i, two out of which also attenuated ligand-induced Ca2+ influx via CatSper. In contrast, Sertraline decreased [Ca2+]i and almost completely suppressed ligand-induced Ca2+ influx via CatSper. Remarkably, the drug was about four-fold more potent to suppress prostaglandin- versus steroid-induced Ca2+ influx. Sertraline also suppressed alkaline- and voltage-activation of CatSper, indicating that the drug directly inhibits human CatSper. Finally, Sertraline suppressed ligand-induced acrosome reaction and sperm penetration into viscous media. Limitations, reasons for caution This is an in vitro study. Future studies have to assess the physiological relevance in vivo. Wider implications of the findings The off-target action of Sertraline on CatSper in human sperm might impair the fertilization process. In a research setting, Sertraline may be used to selectively inhibit prostaglandin-induced Ca2+ influx. Trial registration number CRU326

The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

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