Cell Biology

Dozens of Mycoplasma species belonging to the class Mollicutes bind to solid surfaces through the organelle formed at a cell pole and glide in its direction by a unique mechanism. In Mycoplasma mobile, the fastest gliding species in Mycoplasma, the force for gliding is generated by ATP hydrolysis on an internal structure. However, the spatial and temporal behaviors of the internal structures in living cells were unclear. High-speed atomic force microscopy (HS-AFM) is a powerful method to monitor the dynamic behaviors of biomolecules and cells that can be captured while maintaining their active state in aqueous solution. In this protocol, we describe a method to detect their movements using HS-AFM. This protocol should be useful for the studies of many kinds of microorganisms.

Graphic abstract:

Scannnig Mycoplasma cell

Dense networks of amyloid nanofibrils fabricated from common globular proteins adsorbed to solid supports can improve cell adhesion, spreading and differentiation compared to traditional flat, stiff 2D cell culture substrates like Tissue Culture Polystyrene (TCPS). This is due to the fibrous, nanotopographic nature of the amyloid fibril networks and the fact that they closely mimic the mechanical properties and architecture of the extracellular matrix (ECM). However, precise cell responses are strongly dependent on the nanostructure of the network at the cell culture interface, thus accurate characterization of the immobilized network is important. Due to its exquisite lateral resolution and simple sample preparation techniques, Atomic Force Microscopy (AFM) is an ideal technique to characterize the fibril network morphology. Thus, here we describe a detailed protocol, for the characterization of amyloid fibril networks by tapping mode AFM.

The growing plant cell wall is comprised of long, thin cellulose microfibrils embedded in a hydrated matrix of polysaccharides and glycoproteins. These components are typically constructed in layers (lamellae) on the inner surface of the cell wall, i.e., between the existing wall and the plasma membrane. The organization of these components is an important feature for plant cell growth and mechanics. To directly visualize the nano-scale structure of the newly-deposited surface of primary plant cell walls without dehydration or chemical extraction, a protocol of cell wall preparation for AFM imaging the most recently-synthesized cell wall surface in aqueous solutions was developed. Although the method was developed for onion scale epidermal peels, it can also be adapted to other organs, such as Arabidopsis hypocotyls, as well as ground samples of cell walls from the leaf petioles or hypocotyls of Arabidopsis and cucumber, maize coleoptiles and onion parenchyma. Potential artifacts of AFM imaging of plant cell walls are also discussed.

The free-living soil nematode Caenorhabditis elegans has become an iconic experimental model animal in biology. This transparent animal can be easily imaged using optical microscopy to visualise its organs, tissues, single cells and subcellular events. The epicuticle of C. elegans nematodes has been studied at nanoscale using transmission and scanning (SEM) electron microscopies. As a result, imaging artefacts can appear due to embedding the worms into resins or coating the worms with a conductive gold layer. In addition, fixation and contrasting may also damage the cuticle. Conventional tapping mode atomic force microscopy (AFM) can be applied to image the cuticle of the dried nematodes in air, however this approach also suffers from imaging defects. Ideally, the nematodes should be imaged under conditions resembling their natural environment. Recently, we reported the use of PeakForce Tapping AFM mode for the successful visualisation and numerical characterisation of C. elegans nematode cuticle both in air and in liquid (Fakhrullina et al., 2017). We imaged the principal nematode surface structures and characterised mechanical properties of the cuticle. This protocol provides the detailed description of AFM imaging of liquid-immersed C. elegans nematodes using PeakForce Tapping atomic force microscopy.

Hydroxyproline-rich glycoproteins (HRGPs) are major protein components in dicot primary cell walls and generally account for more than 10% of the wall dry weight. As essential members of the HRGP superfamily, extensins (EXTs) presumably function in the cell wall by assembling into positively charged protein scaffolds (Cannon et al., 2008) that direct the proper deposition of other wall polysaccharides, especially pectins, to ensure correct cell wall assembly (Hall and Cannon, 2002; Lamport et al., 2011a). Extensins are recalcitrant to purification as they are rapidly cross-linked into a covalent network after entering the cell wall but there exists a short time window in which newly synthesized extensin monomers can be extracted (Smith et al., 1984; Smith et al., 1986) by salt elution. A detailed protocol for extraction of extensin and other wall structural proteins has been described earlier (Lamport et al., 2011b). The protocol elaborated here provides an approach to studying the self-assembly of extensins and potentially of other cell wall components in vitro using AFM.