Chemistry Reference
In-Depth Information
motility with puri
ed proteins has been a very powerful approach toward under-
standing the necessary components and their interactions. With molecular motors
attached to the microscope slide, the appropriate cytoskeletal
filament can be shown
to translate in semblance to their relativemotion in a cell. This approach is termed the
gliding
lament assay and is a commonmethod of documenting direction, velocity
and regulation of all three molecular motors [5, 6]. Strongly labeled
fluorescent actin
filaments or microtubules can be visualized and their motions tracked with a
standard epi-
filament is attached to the microscope
slide or otherwise immobilized, then the motor can be manipulated by attaching it to
a small polymer (e.g. lucite or polystyrene) bead that serves as an easily visualized
marker for motion and can also be used as a handle for manipulating the motor or
applying a force. Bead assays on laser traps are discussed in detail elsewhere in this
book.
The present chapter emphasizes methods to visualize single molecular motors in
actionwithout attaching large cargos. Themost common strategy is to label themotor
with a
fluorescent probe and detect the individual
fluorophore in vitro in a geometry
that reconstitutes the motor activity. Methods for labeling protein components by
extrinsic
fluorophores or expressing them coupled to green
fluorescent protein or its
derivatives have received enormous attention because observing ensembles of these
fluorescent markers, their locations, dynamics and interactions is a predominant
approach in cell biology [7]. Techniques for attaching
uorescent tags and the
essential step of testing for functionality after labeling have been described in detail
in many contexts [8, 9]. Small organic
uorophores, auto-
uorescent proteins, such
as GFP, and semiconductor quantum dots have been used as markers. For motor
research on individual molecules, the labeled component is usually visualized in a
gliding
lament assay or while it is translating on a cytoskeletal
lament track that has
been immobilized in the microscope. Techniques to detect single molecular motors
or motions of their cargos in live cells are discussed towards the end of the chapter.
Single
fluorescent probes have been visualized at much higher precision than the
diffraction limit of classical optical microscopy and their functionally relevant
translational and rotational motions have been recorded in vitro. Sub-pixel localiza-
tion of single probes has been generalized into methods to obtain images of non-
motor cellular structures at much
finer resolution than previously thought possible.
In this way, the single-molecule imaging techniques
first described with regard to
molecular motors are having broader impact in cell biology.
uorescence microscope. If the
3.2
Methods
3.2.1
Detection of Single Fluorophores
A macromolecule is labeled with a
fluorescent probe, checked that it still functions
normally, and placed in an assay that demonstrates its activity. An example is a
