Chemistry Reference
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contains two heads. An additional large, multi-subunit 1.2-MDa complex, termed
dynactin (not shown), is required for most functions of cytoplasmic dynein [59].
3.3.1
Myosin V
Myosin V (Figure 3.8) is involved in transport of vacuoles and RNA
protein particles
in yeast vesicular cargoes in neurons, and pigment granules in melanocytes. Due to
its cellular roles, deletion ormisexpression of myosin V inmice and humans causes a
developmental neurological de
cit, with immune suppression and light pigmenta-
tion [60]. The term processivity refers to the feature of many DNA processing
enzymes and some molecular motors that take many productive mechanical steps
upon each diffusional encounter with their track. Myosin V exhibits very high
processivity (20
-
60 steps [61
-
63]), a very large step size (36 nm [27, 61]) and is
abundant enough to be isolated from brain tissue [64]. These characteristics have
causedmyosin V to be a very popular subject for optical trapping and singlemolecule
imaging studies, making it one of the best understood molecular motors.
Intheactin-activatedATPasecycleofmyosinV,thedissociationofADPfromthe
acto
-
myosin
-
ADP complex is very slow (15 s
1
) compared to that step in conven-
tional muscle myosin II (
-
200 s
1
). This slow rate implies that myosin V spends a
large proportion of the ATPase cycle attached to actin, leading investigators to
suspect that myosin Vmight exhibit processive motility [65]. This supposition was
con
rmed in a single-molecule imaging study by Ando and colleagues [62].
Fluorescent labeled CaM was introduced into the myosin Vmolecule by dissociat-
ing some of the endogenous calmodulin (CaM) subunits at high Ca
2 รพ
concentra-
tion. Actin
filaments were attached to the microscope slide surface using sparse
biotin
-
streptavidin linkages, and
fluorescent myosin V molecules added to the
sample chamber were observed by TIRF microscopy (Figure 3.9). Individual
>
fluorescent spots, representing single myosin V molecules, were seen to localize
with actin andmove along the
filament for several
m. Themyosin Vmolecules did
not readily dissociate from the actin and
fluorescence built up at the barbed ends of
the actin
filaments as the myosin molecules accumulated there. This direct
demonstration of processive motility con
rmed optical trapping experiments at
nearly the same time [66]. Further insightful experiments using single molecule
fluorescence imaging and optical trapping have elucidated many details of myosin
Vs stepping mechanism [67
-
72].
The initial application of FIONA was an investigation of myosin V [27]. Myosin V
molecules were labeled with rhodamine or Cy3 on one of the CaM subunits at low
stoichiometry tominimize the number ofmultiply labeledmolecules. The position of
the CaM was resolved to within a few nanometers during processive motility as
described above in Section 3.2.2. The labeled CaMwas found to move stepwise along
the actin
filament, and several classes of motion were detected: molecules with
fluorophores that took 74-nm steps, those that alternated between 52- and 23-nm
steps and those with alternating 42- and 33-nm steps. Such alternating large
and small steps are expected if the molecule translocates along actin using a
m
