Imaging and Molecular Motors - Single Molecule Dynamics in Life Science - page 73

the dynein - dynactin complex may participate directly in bidirectional transport of
cargos within cells. Whether this activity requires dynactin and how the directionality
and reversals are controlled are not yet known. Higher resolution studies of dynein
constructs and dual color imaging, such as shown for myosin V in Figures 3.10
and 3.12, may lead to an understanding of the stepping and control mechanisms of
this complex molecular motor. An approach toward studying the interactions among
the different molecular motor families is to construct arrays of cytoskeletal filaments
in vitro that introduce some of the complexity of cells. A few reports of this type of
study have been published [159, 160].
3.3.7
Single Molecule Intracellular Imaging
Dramatic technical advances in optical microscopy over the past 50 - 75 years,
including phase contrast, differential interference contrast, epi uorescence, and
confocal microscopy, the commercial availability of highly sensitive cameras, and the
improvement in speci c markers of cellular structures using organic uorescent
dyes and auto- uorescent proteins have made optical microscopy one of the core
research tools in cell biology [7, 9].Within the last few years, the techniques developed
for imaging single molecules have been applied to fluorescent structures within live
cells both to study how molecular motors operate in their native environment and to
improve spatial resolution of images in many other cell biological contexts. These
evolving methods have great potential for further improving light microscopic
imaging to understand molecular processes.
With brightly fluorescent vesicles containing dozens of GFP tags [161], quantum
dots endocytosed into vesicles in cells [162, 163], or optically dense melanin
transporting vesicles [164], the brightness or contrast of the particle relative to the
surrounding cytoplasm is high enough to resolve nanometer movements within
milliseconds. These vesicles display 8- or 35-nmdiscrete stepwisemotions when they
are transported by several microtubule-based or actin-based molecular motors.
Transport velocities up to 10-fold higher than those measured in vitro, were observed
in some of the experiments without alteration of the 8-nm stepwise character. These
results are puzzling, because increasing the number of motors carrying a cargo
in vitro does not generally increase the velocity of motility above that of the unloaded
single molecule [139] and it tends to reduce the observed step size due to the
smoothing effect of multiple unsynchronized working strokes [165]. Understanding
the high velocities and quantized step size of cargos observed in vivo will await further
detailed imaging studies.
Direct imaging of single molecules within cells, rather than their cargo, is
limited by auto uorescence of cellular components that reduces the contrast
between the target molecule and its surroundings. Several routes to increasing
the signal above background have been reported. A kinesin fused to three tandem
yellow fluorescent proteins is bright enough to observe within cultured cells [166].
TIRF illumination reduces the volume illuminated within the cell, but then