Chemistry Reference
In-Depth Information
biochemical transitions and used for communication between the two heads. These
mechanisms require two-headedmotors tomaintain contact with the
filament and to
communicate intramolecular strain. Thus, processive motility of a single-headed
myosin V construct, reported in an optical trap assay [72], should be con
rmed by
single
fluorophore imaging.
There are several levels ofmyosinVregulationnecessary to determine activity, cargo
binding, and cooperation with other motor proteins. CaM is involved inmany cellular
signaling cascades in which Ca
2þ
binding to CaM enables it to interact with
downstream effector proteins, such as kinases [83]. The CaM binding IQ motifs in
myosins are unusual because they serve a structural/mechanical role as the lever arm
and they interact stronglywithCaMin the Ca-free state. The CaMsubunitsmay also be
involved in regulation of motility. In the absence of Ca
2þ
, themyosin Vmolecule folds
into a triangular shape with the globular tail domain interacting with the head, thus
preventing it from associating with actin [84
-
87]. Moderate Ca
2þ
(1
M) or cargo
binding causes the molecule to open into the active form. At higher [Ca
2þ
], one or
more CaM subunits dissociate from their IQ domains, again suppressing motility,
either by diminishing actin binding or by structurally weakening or kinking the lever
arm [88, 89].
Dissociation of the CaM subunits is measured biochemically in bulk samples
[86, 88], but the ensemble experiments cannot address whether each molecule
homogeneously loses the same number of molecules or there is a distribution of
lost subunits. Single molecule imaging resolves this type of question by allowing the
number of dissociating subunits to be counted for individual myosin V molecules.
When CaM subunits were dissociated at high [Ca
2þ
] and then replaced with
fluorescent labeled CaM [90], most of the molecules lost two of the 12 CaM light
chains. Processivity measurements on single
fluorescent labeled molecules were
consistent with a model in which dissociation of one CaM subunit is suf
cient to
terminate a processive series of steps [91]. Whether these effects of Ca
2þ
are part of
the normal cellular regulatory pathways is not known, although other myosins are
dynamically modulated by Ca
2þ
[92, 93]. Other modes of myosin V regulation have
been found in ensemble cell biological studies [94, 95], but they have not been
approached yet by single molecule imaging.
m
3.3.2
Myosin II
The absence of processivity in muscle myosin has caused single molecule imaging
studies of myosin II to lag behind those of myosin V. In the presence of ATP, myosin
II dissociates rapidly from actin, and the proportion of the ATPase cycle occupied in
actin-bound states ismuch lower than formyosinV. In normal activity, only one of the
myosin heads binds to actin at a time. Thus it has been dif
cult to image single
myosin II molecules during normal function. Cy3-labeled ATP molecules were
visualized in TIRFmicroscopy when they bound to Cy5-labeledmyosinmolecules [3].
Free
fluorescent nucleotide molecules near the re
ecting surface of the TIRF
microscope diffuse rapidly in the aqueous medium causing delocalization of
