Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Oct;1859(10):1911-1920.
doi: 10.1016/j.bbamem.2017.05.010. Epub 2017 May 23.

Topographic analysis by atomic force microscopy of proteoliposomes matrix vesicle mimetics harboring TNAP and AnxA5

Maytê Bolean  1 , Ivana A Borin  2 , Ana M S Simão  2 , Massimo Bottini  3 , Luis A Bagatolli  4 , Marc F Hoylaerts  5 , José L Millán  6 , Pietro Ciancaglini  7
Affiliations

Topographic analysis by atomic force microscopy of proteoliposomes matrix vesicle mimetics harboring TNAP and AnxA5

Maytê Bolean et al. Biochim Biophys Acta Biomembr. 2017 Oct.

Abstract

Atomic force microscopy (AFM) is one of the most commonly used scanning probe microscopy techniques for nanoscale imaging and characterization of lipid-based particles. However, obtaining images of such particles using AFM is still a challenge. The present study extends the capabilities of AFM to the characterization of proteoliposomes, a special class of liposomes composed of lipids and proteins, mimicking matrix vesicles (MVs) involved in the biomineralization process. To this end, proteoliposomes were synthesized, composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS), with inserted tissue-nonspecific alkaline phosphatase (TNAP) and/or annexin V (AnxA5), both characteristic proteins of osteoblast-derived MVs. We then aimed to study how TNAP and AnxA5 insertion affects the proteoliposomes' membrane properties and, in turn, interactions with type II collagen, thus mimicking early MV activity during biomineralization. AFM images of these proteoliposomes, acquired in dynamic mode, revealed the presence of surface protrusions with distinct viscoelasticity, thus suggesting that the presence of the proteins induced local changes in membrane fluidity. Surface protrusions were measurable in TNAP-proteoliposomes but barely detectable in AnxA5-proteoliposomes. More complex surface structures were observed for proteoliposomes harboring both TNAP and AnxA5 concomitantly, resulting in a lower affinity for type II collagen fibers compared to proteoliposomes harboring AnxA5 alone. The present study achieved the topographic analysis of lipid vesicles by direct visualization of structural changes, resulting from protein incorporation, without the need for fluorescent probes.

Keywords: Annexin V; Atomic force microscopy; Collagen; Matrix vesicles; Proteoliposomes; Tissue-nonspecific alkaline phosphatase.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
3D topographic AFM images of liposomes (1.5 mg.mL−1) samples composed by (A) DPPC (1000.00 × 1000.00 nm and y axis from 0 to 30.79 nm scales) and (B) 9:1 DPPC:DPPS (molar ratio) (1000.00 × 1000.00 nm and y axis from 0 to 84.50 nm scales).
Fig. 2
Fig. 2
AFM images of 9:1 DPPC:DPPS proteoliposomes (0.75 mg.mL−1) containing TNAP: (A) phase image; (B) 2D topographic profile; (C) Height analysis of domains formed by TNAP insertion on the surface of liposomes (graphics obtained from line 4); (D) 3D topographic profile and (E) zoomed detail at the surface of only one vesicle (472.03 × 472.03 nm and y axis from 0 to 28.96 nm scales).
Fig. 3
Fig. 3
AFM images in phase mode of 9:1 DPPC:DPPS proteoliposomes (0.75 mg.mL−1) containing AnxA5: (A) phase image; (B) 2D topographic profile; (C) Height analysis of domains formed by AnxA5 on the surface of liposomes (first graphic obtained from line 4 and second graphic obtained from line 8); (D) 3D topographic profile and (E) zoomed detail at the surface of only one vesicle (312.50 × 312.50 nm and y axis from 0 to 34.67 nm scales).
Fig. 4
Fig. 4
AFM images of 9:1 DPPC:DPPS proteoliposomes (0.75 mg.mL−1) containing AnxA5 + TNAP: (A) phase image with 2.0 μm scale bar; (B) phase image with 500 nm scale bar; (C) 2D topographic profile; (D) 3D topographic surface profile of only one vesicle (1.25 × 1.25 m and y axis from 0 to 21.92 nm scales) and (E) Height analysis of domains formed by both proteins in the surface of liposomes (graphic obtained from line 3).
Fig. 5
Fig. 5
Effect of AnxA5, TNAP and AnxA5 + TNAP on the binding percentage of 9:1 DPPC:DPPS-proteoliposome to type II collagen matrix, by fluorescence microscopy. The vesicles (450 μg/mL, lipid concentration incubated) were labeled with Rhodamine 6G (0.2 mol%) and the analysis as described in Material and Methods: White bar proteoliposomes harboring A5; Black bar proteoliposomes harboring TNAP and Striped bar proteoliposomes harboring TNAP + AnxA5. The asterisks indicate significant differences (p < 0.001).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bolean M, Simao AM, Favarin BZ, Millan JL, Ciancaglini P. The effect of cholesterol on the reconstitution of alkaline phosphatase into liposomes. Biophys Chem. 2010;152:74–79. - PubMed
    1. Wuthier RE. Lipids of matrix vesicles. Fed Proc. 1976;35:117–121. - PubMed
    1. Anderson HC. Mineralization by matrix vesicles Scan. Electron Microsc. 1984:953–964. - PubMed
    1. Thouverey C, Strzelecka-Kiliszek A, Balcerzak M, Buchet R, Pikula S. Matrix vesicles originate from apical membrane microvilli of mineralizing osteoblast-like Saos-2 cells. J Cell Biochem. 2009;106:127–138. - PubMed
    1. Wuthier RE, Lipscomb GF. Matrix vesicles: structure, composition, formation and function in calcification. Front Biosci. 2011;16:2812–2902. - PubMed

Publication types

MeSH terms

LinkOut - more resources