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

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.

Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis, properties, functions and biomimetic models

“…To understand how microdomains would affect the interaction of proteoliposomes with type II collagen fibers, which corresponds to the early MV activity during biomineralization, AFM images of proteoliposomes were recorded [228]. AFM images (Fig.…”

“…6E). A more complex microdomain structure was observed for the proteoliposomes harboring TNAP and AnxA5 concomitantly, resulting in a lower affinity for type II collagen fibers compared to exclusive proteoliposomes harboring AnxA5 alone [228]. …”

Lipid microenvironment affects the ability of proteoliposomes harboring TNAP to induce mineralization without nucleators

“…The ability of proteoliposomes to mimic the function and structure of biomembranes [22–27] makes them a convenient model to determine the factors of MV-mediated mineralization. Other researchers also aim to work with therapeutic mimetic cell to provide a source of cell-like assemblies, which exhibits the core structural or functional properties of their natural counterparts with broad envisioned applications in biomedicine.…”

Biophysical aspects of biomineralization

“…Biophysical and biochemical characterization of biomimetic models Biophysical tools applied to biomineralization studies A large number of studies have been carried out using proteoliposomes as chondrocyte-and osteoblast-derived MVs biomimetics (Andrade et al 2016;Bolean et al 2010Bolean et al , 2011Bolean et al , 2015Bolean et al , 2017Ciancaglini et al 2010;Favarin et al 2017;Simao et al 2010aSimao et al , b, 2013.…”