Stick, stretch, and scan imaging method for DNA and filaments

Abstract

Biomolecules and organelles usually undergo changes to their structure or form as a result of mechanical stretching or stimulation. It is critical to be able to observe these changes and responses, which trigger mechano-chemical coupling or signal transduction. Advanced techniques have been developed to observe structure and form during manipulation; however, these require sophisticated methods. We have developed a simple approach to observe fine structure after stretching without fluorophore labeling. DNAs or molecules on the cell surface were bound to magnetic microbeads, followed by stretching with a magnetic field. After fixing, staining, and drying, the samples were examined by scanning electron microscopy with no need to build a functional surface with complex processes. Straight DNAs were observed rather than random-walk-like loose polymers. In our cellular experiment, the magnetic beads were bound to a Jurkat cell and formed a rosette which was later stuck to the substrate. A 41.3 μm filament on the base of a filopodium was pulled out via integrin from a cell. Therefore, our method can reveal long structures up to hundreds of micrometers at nanometer resolution after stretching or twisting. Our approach could have wide applications in structure-function studies of biomolecules, and in mechanobiology and cell biology when diffraction cannot used.

Fig. 1. DNA stretching and imaging. (A) A DNA tethered to a bead and to the glass. (B) A DNA stretched by a magnetic force perpendicular to the glass surface. (C) DNA was manipulated to lie down on the glass. (D) SEM photograph of a DNA before stretching. Left, stretched DNA with M280 DynaBeads; right, stretched DNA with BeaverBeads of 5 μm diameter. (E) SEM images of stretched DNA with M280 DynaBeads. (F) SEM photographs of stretched DNA with BeaverBeads of 5 μm diameter.

Fig. 2. Steps for stretching cells via magnetic beads. (A) The magnetic beads were conjugated to peptides. (B) The bead and the cell formed a rosette during centrifugation. (C) The rosette was placed on peptide-coated glass and (D) stretched using permanent magnets.

Fig. 3. Rosette stretching under light microscope. Jurkat cells were bound to microbeads (A). Filopodia was stretched by the beads in the magnetic field for 2 h (B and C). The beads were aligned along the magnetic field.

Fig. 4. Fine structures observed by SEM. (A–D) Elongated filopodia were observed when the GRGDNP-coated bead was stretched by magnetic force for 5 h. The extension and thickness of the stretched filopodia varied. (E and F) A Jurkat cell was bound to beads coated with GRGDNP, without magnetic force, as a control. (G) A Jurkat cell was bound to the glass cover, without magnetic beads, as a control.