Laser Tweezers and Scissors to Study Cellular Motility and Biomechanics
The development of new molecular probes, gene fusion products, imaging technologies, and information transfer technology, are technologies that can be combined with micro-scale (and smaller) technologies to study and manipulate cells and tissues like never before.
Laser scissors are used to alter subcellular organelles involved in cell motility (microtubules, chromosome centromeres, centrosomes/centrioles). Laser tweezers are used to apply specific forces to structures of the mitotic spindle and cell cytoskeleton in order to study dynamic interactions and biomechanics in live cells. Fluorescence recovery after photobleaching (FRAP) is being used to examine the functional biochemistry of these structures. We are studying the mobility and turnover of specific chemical species (MAD1 and MAD2) at the cell kinetochore by FRAP (Shah et al, Cur. Biol., Jun 2004). We are continuing to study these molecules using fluorescence correlation spectroscopy (FCS), which permits the measurement of the change in molecular concentration at different regions of the cell during the cell cycle (Wang et al, J. Biomed. Optics., Mar-Apr 2004).
Laser Scissors and Tweezers to Study Neuronal Function
We have used a laser scissors beam to create small lesion in growing axons and demonstrated that (a) the damage heals, and (b) the growth cone of the damage axon, or the growth cone of a nearby axon, extends filopodia towards and actually touches the damage site, and in some cases the whole growth cone turns and moves towards the damage site. In addition, there is a temporary cessation of axonal transport while the damaged nerve heals (Wu et al., J. R. Soc. Interface 9: 535-547, 2012).
A circular polarized laser tweezers has been used to rotate birefringent micron-size particles adjacent to neuronal growth cone. The shear force created by the rotating particle causes the axon growth gone to grow in a specific “right” or “left” direction, which is related to the direction of rotation of the particle and the consequent fluid flow against the axon (Wu et al Nature Photonics 6: 62-67, 2012). Studies are underway to elucidate the receptor and molecular mechanisms of this response.
Laser Scissors and Fluorescent Gene-Fusion Proteins
One of the most exciting developments in cell and developmental biology over the past has been the creation of genetic fluorescent fusion proteins, as exemplified by green fluorescent protein (GFP). This has permitted visualization of structures in live cells not previously possible. A focus of our present and future research is to use fluorescent fusion proteins to visualize and study cell structures that can be manipulated with laser scissors and tweezers. We are already applying this combined systems approach to problems of cellular motility, particularly with respect to organelles of the mitotic spindle (kinetochores, centrosomes, microtubules; Botvinick et al, Biophysical Journal, Dec 2004). This approach will be used to study other organelles such as nucleoli, golgi, and the cell membrane.