scholarly journals Protein flexibility is required for vesicle tethering at the Golgi

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Pak-yan Patricia Cheung ◽  
Charles Limouse ◽  
Hideo Mabuchi ◽  
Suzanne R Pfeffer
Keyword(s):  
Coiled Coil ◽  
Rab Gtpases ◽  
Force Microscopy ◽  
Vesicle Tethering ◽  
Proximity Ligation ◽  
Atomic Force ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Specific Sequences

The Golgi is decorated with coiled-coil proteins that may extend long distances to help vesicles find their targets. GCC185 is a trans Golgi-associated protein that captures vesicles inbound from late endosomes. Although predicted to be relatively rigid and highly extended, we show that flexibility in a central region is required for GCC185’s ability to function in a vesicle tethering cycle. Proximity ligation experiments show that that GCC185’s N-and C-termini are within <40 nm of each other on the Golgi. In physiological buffers without fixatives, atomic force microscopy reveals that GCC185 is shorter than predicted, and its flexibility is due to a central bubble that represents local unwinding of specific sequences. Moreover, 85% of the N-termini are splayed, and the splayed N-terminus can capture transport vesicles in vitro. These unexpected features support a model in which GCC185 collapses onto the Golgi surface, perhaps by binding to Rab GTPases, to mediate vesicle tethering.

scholarly journals GCC185 plays independent roles in Golgi structure maintenance and AP-1–mediated vesicle tethering

2011 ◽  
Vol 194 (5) ◽  
pp. 779-787 ◽  
Author(s):  
Frank C. Brown ◽  
Carmel H. Schindelhaim ◽  
Suzanne R. Pfeffer
Keyword(s):  
Coiled Coil ◽  
Retrograde Transport ◽  
Vesicle Tethering ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Transport Vesicle ◽  
Golgi Structure ◽  
Clathrin Adaptor ◽  
Golgi Network

GCC185 is a long coiled-coil protein localized to the trans-Golgi network (TGN) that functions in maintaining Golgi structure and tethering mannose 6-phosphate receptor (MPR)–containing transport vesicles en route to the Golgi. We report the identification of two distinct domains of GCC185 needed either for Golgi structure maintenance or transport vesicle tethering, demonstrating the independence of these two functions. The domain needed for vesicle tethering binds to the clathrin adaptor AP-1, and cells depleted of GCC185 accumulate MPRs in transport vesicles that are AP-1 decorated. This study supports a previously proposed role of AP-1 in retrograde transport of MPRs from late endosomes to the Golgi and indicates that docking may involve the interaction of vesicle-associated AP-1 protein with the TGN-associated tethering protein GCC185.

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.

Targeting Breast Cancer Cells with G4 PAMAM Dendrimers and Valproic Acid Derivative Complexes

2020 ◽  
Vol 20 (15) ◽  
pp. 1857-1872
Author(s):  
Alberto M. Muñoz ◽  
Manuel J. Fragoso-Vázquez ◽  
Berenice P. Martel ◽  
Alma Chávez-Blanco ◽  
Alfonso Dueñas-González ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Cell Lines ◽  
Water Solubility ◽  
Md Simulations ◽  
Pamam Dendrimer ◽  
Pamam Dendrimers ◽  
Force Microscopy ◽  
Micromolar Concentration ◽  
Atomic Force

Background: Our research group has developed some Valproic Acid (VPA) derivatives employed as anti-proliferative compounds targeting the HDAC8 enzyme. However, some of these compounds are poorly soluble in water. Objective: Employed the four generations of Polyamidoamine (G4 PAMAM) dendrimers as drug carriers of these compounds to increase their water solubility for further in vitro evaluation. Methods: VPA derivatives were subjected to Docking and Molecular Dynamics (MD) simulations to evaluate their affinity on G4 PAMAM. Then, HPLC-UV/VIS, 1H NMR, MALDI-TOF and atomic force microscopy were employed to establish the formation of the drug-G4 PAMAM complexes. Results: The docking results showed that the amide groups of VPA derivatives make polar interactions with G4 PAMAM, whereas MD simulations corroborated the stability of the complexes. HPLC UV/VIS experiments showed an increase in the drug water solubility which was found to be directly proportional to the amount of G4 PAMAM. 1H NMR showed a disappearance of the proton amine group signals, correlating with docking results. MALDI-TOF and atomic force microscopy suggested the drug-G4 PAMAM dendrimer complexes formation. Discussion: In vitro studies showed that G4 PAMAM has toxicity in the micromolar concentration in MDAMB- 231, MCF7, and 3T3-L1 cell lines. VPA CF-G4 PAMAM dendrimer complex showed anti-proliferative properties in the micromolar concentration in MCF-7 and 3T3-L1, and in the milimolar concentration in MDAMB- 231, whereas VPA MF-G4 PAMAM dendrimer complex didn’t show effects on the three cell lines employed. Conclusion: These results demonstrate that G4 PAMAM dendrimers are capableof transporting poorly watersoluble aryl-VPA derivate compounds to increase its cytotoxic activity against neoplastic cell lines.

scholarly journals Osteoblast-Like Cell Behavior on Porous Scaffolds Based on Poly(styrene) Fibers

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Andrada Serafim ◽  
Romain Mallet ◽  
Florence Pascaretti-Grizon ◽  
Izabela-Cristina Stancu ◽  
Daniel Chappard
Keyword(s):  
Interference Microscopy ◽  
Porous Scaffolds ◽  
Allogenic Bone ◽  
Force Microscopy ◽  
Atomic Force ◽  
Dry Spinning ◽  
Bone Trabeculae ◽  
Large Bone

Scaffolds of nonresorbable biomaterials can represent an interesting alternative for replacing large bone defects in some particular clinical cases with massive bone loss. Poly(styrene) microfibers were prepared by a dry spinning method. They were partially melted to provide 3D porous scaffolds. The quality of the material was assessed by Raman spectroscopy. Surface roughness was determined by atomic force microscopy and vertical interference microscopy. Saos-2 osteoblast-like cells were seeded on the surface of the fibers and left to proliferate. Cell morphology, evaluated by scanning electron microscopy, revealed that they can spread and elongate on the rough microfiber surface. Porous 3D scaffolds made of nonresorbable poly(styrene) fibers are cytocompatible biomaterials mimicking allogenic bone trabeculae and allowing the growth and development of osteoblast-like cellsin vitro.

scholarly journals Nanostructural and mechanical changes in the sclera following proteoglycan depletion

2018 ◽  
Vol 2 (2) ◽  
pp. 14-17
Author(s):  
Zhuola Zhuola ◽  
Steve Barrett ◽  
Yalda Ashraf Kharaz ◽  
Riaz Akhtar
Keyword(s):  
Mechanical Properties ◽  
Collagen Fibril ◽  
Enzymatic Degradation ◽  
Ocular Tissues ◽  
Force Microscopy ◽  
Nanomechanical Properties ◽  
Atomic Force ◽  
Fibril Morphology

The mechanical properties of ocular tissues, such as the sclera, have a major impact on healthy eye function, and are governed by the properties and composition of the microstructural components. For example, biomechanical degradation associated with myopia occurs alongside a reduction of proteoglycans (PGs). In this study, the role of PG degradation in the nanomechanical properties of the porcine sclera is explored. In-vitro enzymatic degradation of PGs was conducted with α-amylase and chondroitinase ABC enzymes. Collagen fibril morphology and nanomechanical stiffness were measured with atomic force microscopy (AFM). The elastic modulus of the tissue was reduced in all enzyme-treated samples relative to controls. In addition, collagen fibril organization was disrupted by PG depletion. Our data demonstrate that PGs play an important role in determining not only the mechanical properties at these length scales, but also collagen fibril arrangement.

scholarly journals Visualization by atomic force microscopy of tobacco mosaic virus movement protein–RNA complexes formed in vitro

2001 ◽  
Vol 82 (6) ◽  
pp. 1503-1508 ◽  
Author(s):  
O. I. Kiselyova ◽  
I. V. Yaminsky ◽  
E. M. Karger ◽  
O. Yu. Frolova ◽  
Y. L. Dorokhov ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Structural Conversion ◽  
Force Microscopy ◽  
Rna Transcripts ◽  
Atomic Force ◽  
Rna Complexes

The structure of complexes formed in vitro by tobacco mosaic virus (TMV)-coded movement protein (MP) with TMV RNA and short (890 nt) synthetic RNA transcripts was visualized by atomic force microscopy on a mica surface. MP molecules were found to be distributed along the chain of RNA and the structure of MP–RNA complexes depended on the molar MP:RNA ratios at which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1 to 60–100:1 resulted in an increase in the density of the MP packaging on TMV RNA and structural conversion of complexes from RNase-sensitive ‘beads-on-a-string’ into a ‘thick string’ form that was partly resistant to RNase. The ‘thick string’-type RNase-resistant complexes were also produced by short synthetic RNA transcripts at different MP:RNA ratios. The ‘thick string’ complexes are suggested to represent clusters of MP molecules cooperatively bound to discrete regions of TMV RNA and separated by protein-free RNA segments.

scholarly journals Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

2021 ◽  
Author(s):  
Kazuto Yoshimi ◽  
Kohei TAKESHITA ◽  
Noriyuki Kodera ◽  
Satomi Shibumura ◽  
Yuko Yamauchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Speed ◽  
Dna Helicase ◽  
Type I ◽  
Loop Formation ◽  
Force Microscopy ◽  
Atomic Force ◽  
Target Strand ◽  
Target Dna

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

scholarly journals In vitro evaluation of microbial adhesion on the different surface roughness of acrylic resin specific for ocular prosthesis

2018 ◽  
Vol 12 (02) ◽  
pp. 176-183 ◽  
Author(s):  
Agda Marobo Andreotti ◽  
Cecília Alves De Sousa ◽  
Marcelo Coelho Goiato ◽  
Emily Vivianne Freitas da Silva ◽  
Cristiane Duque ◽  
...  
Keyword(s):  
Surface Roughness ◽  
Staphylococcus Epidermidis ◽  
Microbial Growth ◽  
Acrylic Resin ◽  
Microbial Adhesion ◽  
Force Microscopy ◽  
Atomic Force ◽  
Colony Forming Units ◽  
Ocular Prostheses

ABSTRACT Objective: The purpose of this study was to evaluate the influence of surface roughness in biofilm formation of four microorganisms (Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans) on acrylic resin surface of ocular prostheses. Materials and Methods: Acrylic resin samples were divided into six groups according to polishing: Group 1200S (1200 grit + silica solution); Group 1200; Group 800; Group 400; Group 120 and Group unpolished. Surface roughness was measured using a profilometer and surface images obtained with atomic force microscopy. Microbial growth was evaluated after 4, 24, and 48 hours of incubation by counting colony-forming units. Statistical Analysis Used: For roughness, it was performed 1-way ANOVA and parametric Tukey test α5% (P ≤ 0.05). For CFU data found, it was applied Kruskal-Wallis and Mann-Whitney tests. Results: Group 120 and 400 presented the highest roughness values. For S. epidermidis and S. aureus, Group 1200S presented the lowest values of microbial growth. For E. faecalis at 4 hour, microbial growth was not observed. C. albicans did not adhere to the acrylic resin. Except for Group 1200S, different surface roughnesses did not statistically interfere with microbial adhesion and growth on acrylic surfaces of ocular prostheses. Conclusions: The roughness did not interfere with the microbial adhesion of the microorganisms evaluated. The use of silica decreases significantly microbial growth.

scholarly journals The extruded non-template strand determines the architecture of R-loops

2019 ◽  
Vol 47 (13) ◽  
pp. 6783-6795 ◽  
Author(s):  
Yeraldinne Carrasco-Salas ◽  
Amélie Malapert ◽  
Shaheen Sulthana ◽  
Bastien Molcrette ◽  
Léa Chazot-Franguiadakis ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Stability ◽  
Three Dimensional ◽  
Dna Breaks ◽  
Yeast Gene ◽  
Physical Constraints ◽  
Force Microscopy ◽  
Template Strand ◽  
Atomic Force

Abstract Three-stranded R-loop structures have been associated with genomic instability phenotypes. What underlies their wide-ranging effects on genome stability remains poorly understood. Here we combined biochemical and atomic force microscopy approaches with single molecule R-loop footprinting to demonstrate that R-loops formed at the model Airn locus in vitro adopt a defined set of three-dimensional conformations characterized by distinct shapes and volumes, which we call R-loop objects. Interestingly, we show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.

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