Observing Xenopus laevis oocyte plasma membrane by atomic force microscopy

Orsini, F.; Santacroce, M.; Arosio, Paolo; Sacchi, V.F.

doi:10.1016/j.ymeth.2009.12.002

Cited by 7 publications

(7 citation statements)

References 28 publications

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“…Because this tendency to a fourfold symmetry has only been observed in membrane samples prepared from transfected oocytes (cf. membranes from untransfected oocytes in Figure b and also Schillers et al , ; Lau et al , ; Orsini et al ., , Santacroce et al , ), we tentatively interpret the visualized structures as higher‐order orthogonal arrays of AQP4‐M23 proteins. Higher spatial resolution remained elusive on these membranes although preventing a more definite identification.…”

Section: Resultsmentioning

confidence: 53%

“…Over the past years, there have been several AFM studies of the plasma membrane in X. laevis oocytes (Schillers et al , , ; Lau et al , , Schillers, ; Orsini et al ., , Santacroce et al , ), although thus far of insufficient resolution to identify native or heterologous membrane proteins based on their topography in the membrane. Here, we report on further steps in this direction.…”

Section: Introductionmentioning

confidence: 99%

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Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4

Orsini

Santacroce

Cremona

et al. 2014

J of Molecular Recognition

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Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes.

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Section: Resultsmentioning

confidence: 53%

Section: Introductionmentioning

confidence: 99%

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4

Orsini

Santacroce

Cremona

et al. 2014

J of Molecular Recognition

Self Cite

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“…In particular, the possibility to obtain high-resolution three-dimensional (3D) reconstructions of single molecules in quasi-physiological environment is one of the key aims of such a microscopy technique. So far, an operating protocol to isolate and study transmembrane proteins was proposed for specialized systems, such as red blood cells and frog oocytes (Orsini et al, 2010;Schillers et al, 2001Schillers et al, , 2004, and some attempts have also been made for more general purpose eukaryotic cells (Wang et al, 2010), but a definite protocol to attain single-molecule visualization in native plasma membrane has not yet been established. Cell surface investigation by AFM is integrative to biochemical, fluorescence, and electron microscopy methods, and can elucidate the structural properties of proteins on cell membranes, which are of paramount importance on the cell physiology.…”

Section: Introductionmentioning

confidence: 99%

“…To exploit the AFM resolution capabilities, it is mandatory to have a clean, flat, and mechanically stable surface to work on. So far, an operating protocol to isolate and study transmembrane proteins was proposed for specialized systems, such as red blood cells and frog oocytes (Orsini et al, 2010;Schillers et al, 2001Schillers et al, , 2004, and some attempts have also been made for more general purpose eukaryotic cells (Wang et al, 2010), but a definite protocol to attain single-molecule visualization in native plasma membrane has not yet been established.…”

Section: Introductionmentioning

confidence: 99%

Visualization of single proteins from stripped native cell membranes: A protocol for high-resolution atomic force microscopy

Marasini

Jacchetti

Moretti

et al. 2013

Microsc. Res. Tech.

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Atomic force microscopy (AFM) proved to be able to obtain high-resolution three-dimensional images of single-membrane proteins, isolated, crystallized, or included in reconstructed model membranes. The extension of this technique to native systems, such as the protein immersed in a cell membrane, needs a careful manipulation of the biological sample to meet the experimental constraints for high-resolution AFM imaging. In this article, a general protocol for sample preparation is presented, based on the mechanical stretch of the cell membrane. The effectiveness for AFM imaging has been tested on the basis of an integrated optical and AFM approach and the proposed method has been applied to cells expressing cystic fibrosis transmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possibility to identify and analyze single proteins in the plasma membrane.

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“…By providing images at nano-resolution, AFM is assisting researcher to get a better understanding of spermatozoa factor that contribute to a successful artificial insemination procedure. Thus improving ART successful rate (Orsini et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Albumin-V: Role in Bovine Spermatozoa Viability, Motility and Membrane Elasticity

Ibrahim¹,

Johor²,

JOthman³

et al. 2013

JAS

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Semen preservation is an important process to ensure semen quality is sufficient for assisted reproductive technology. Studies have showed that suppression of free radicals, crystal formation and regulation of extracellular osmotic pressure is essential to ensure survivability. Past studies have indicated that utilizing protein already existent in the body could be used as a free radical scavenger and osmotic regulator -a potential preservation agent. Thus, the objective of this study was to determine the effect of different albumin-V concentrations on pooled bovine spermatozoa. Pooled cryopreserved semen straws were thawed and incubated in different concentrations of albumin-V (0, 0.2, 0.4, 0.6, 0.8, 1.0 mg albumin-V/ml Bio-excel ® extender) at 4°C. Sperm motility and viability were analyzed at 0, 2, 24 and 48 hours using Makler Chamber. Results indicated that motility and viability were noticeably reduced against time except for sperm incubated in 0.4 mg/ml. Membrane elasticity study using atomic force microscope (AFM) was then conducted on 0, 0.4, and 1.0 mg/ml 48 hours spermatozoa. We had observed that sperm membrane in the 0.4 mg/ml had a lower elasticity value (1.7 ± 0.6 kPA) compared to fresh semen (5.5 ± 1.0 kPA), 0 mg/ml (5.5 ± 0.1 kPA) and 1.0 mg/ml (5.3 ± 0.9 mg/ml). It is very likely that the motility and viability reduction was partly due to the changes in spermatozoa membrane elasticity. We conclude that albumin-V is a potential molecule for 4°C semen preservation; varying albumin concentration will have an influence on the spermatozoa membrane and affect the overall outcome of semen quality. Further study is required to elucidate the function and mechanism of albumin-V in preservation and its direct effect on ART outcome.

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Observing Xenopus laevis oocyte plasma membrane by atomic force microscopy

Cited by 7 publications

References 28 publications

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4

Visualization of single proteins from stripped native cell membranes: A protocol for high-resolution atomic force microscopy

Albumin-V: Role in Bovine Spermatozoa Viability, Motility and Membrane Elasticity

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