(The FASEB Journal. 1999;13:S248-S250.)
© 1999 FASEB
ER transport on actin filaments in squid giant axon: implications for signal transduction at synapse
GEORGE M. LANGFORD1
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755, USA
1Correspondence: Department of Biological Sciences, Dartmouth College, 6044 Gilman Laboratory, Hanover, NH 03755, USA.
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ABSTRACT
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The smooth endoplasmic reticulum (S-ER) is transported on actin
filaments in the giant axon of the squid. The identity of the myosin
motors that transport S-ER in the squid giant axon has been determined.
Our recent studies have shown that the motor for movement of S-ER
vesicles on actin filaments is Myosin-V (1)
. These
findings grew out of a series of studies that began with the initial
observation that vesicles in the giant axon of the squid move on both
microtubules and actin filaments (2)
. These initial
studies documented the ability of individual vesicles to move from
microtubules to actin filaments and led to the development of the dual
filament model of vesicle transport (3
, 4)
. The model
proposes that long-range movement of vesicles occurs on microtubules
and short-range movement on actin filaments. S-ER vesicles were
identified as the major population of vesicles in the axon that use
myosin-V for movement on actin filaments. The S-ER is the primary site
of calcium storage, and it regulates the local cytosolic calcium
concentration. Calcium release from the S-ER in neurons couples
electrical excitation to signal transduction cascades. The signaling
cascades triggered by the release of calcium from S-ER in dendritic
spines are postulated to initiate the cellular mechanisms that lead to
learning and memory.—Langford, G. M. ER transport on actin filaments
in squid giant axon: implications for signal transduction at synapse.
Key Words:
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INTRODUCTION
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THE MOVEMENT OF vesicles from the neuronal cell body
to the dendrites and the axon requires molecular motors. We have shown
that fast axonal transport in the giant axon of the squid involves both
microtubules and actin filaments. Video microscopy was used to observe
the movement of individual vesicles from microtubules to actin
filaments (2)
. The ability of vesicles to move on both
microtubules and actin filaments provides a mechanism for vesicle
transport in actin-rich regions of the cell where microtubules are
absent, such as the axon terminal and dendritic spines. We have been
able to identify vesicles of the smooth ER (S-ER) as one major type of
vesicle that moves on actin filaments and microtubules.
S-ER vesicles were isolated from the axoplasm of the squid giant axon,
and their movement on actin filaments was reconstituted in
vitro (1)
. Electron micrographs of the isolated S-ER
vesicles revealed that they are interconnected by thin (20–25 nm)
membrane tubules (Fig. 1
). Immunofluorescence microscopy of samples stained with an antibody to
the ER resident protein, protein disulfide isomerase, provided positive
identification of the isolated vesicle as S-ER (1)
. The
movement of the isolated S-ER vesicles on endogenous actin filaments
was documented by video microscopy (1
, 5)
. Myosin-V and
kinesin colocalized on the S-ER vesicles as demonstrated by immuno-gold
electron microscopy (6)
. The gold-labeled antibodies for
the two different motors overlapped on the surface of vesicles,
suggesting a potential interaction between these two motors
(1)
. Recent findings with the yeast 2-hybrid assay have
confirmed that myosin-V and kinesin interact directly with each other.
Huang et al. (7)
showed that the globular tail domain of
myosin-V and the rod-tail domain of kinesin bind to each other in the
yeast 2-hybrid assay. The fragments of the myosin-V tail that
interacted with the distal rod domain of the kinesin heavy chain were
those that spanned the AF-6/cno homology domain of the myosin globular
tail. These data provide strong evidence for the direct interaction of
the tail domains of these two motors, and they may explain the
mechanism by which transport of S-ER vesicles on microtubules and actin
filaments are coordinated.
Purified squid brain myosin V was identified by analysis of amino acid
sequences of 12 tryptic peptides representing the head, neck, and tail
regions of the molecule (1)
. Reverse
transcriptase-polymerase chain reaction was used to clone a 630 base
pair fragment in the head domain of the protein and a 515 base pair
fragment in the tail domain (8)
. The sequences of the
cloned fragments showed homology to the other members of the myosin V
family. An additional 4,000 base pair fragment of squid myosin V cDNA
was cloned yielding 85% of the gene sequence. Sequence alignment of
the class V myosins revealed high conservation in the head domain of
the molecule, while alignment in a portion of the tail domain showed
less identity but a high level of similarity. Phylogenetic analysis
revealed that squid myosin V is more like vertebrate than
Drosophila or yeast myosin V. The demonstrated role of
squid myosin V as a vesicle motor in the giant axon plus the data on
the phylogenetic relationships of squid myosin V to vertebrate myosin V
strengthens the argument that myosin V functions as a vesicle motor in
vertebrate neurons.
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ANTIBODY-INHIBITION OF VESICLE TRANSPORT ON ACTIN FILAMENTS
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An antibody (
QLLQ) that reacted strongly to purified squid
brain myosin V, and the equivalent band in axoplasm (1)
was generated to a synthetic peptide in the globular tail domain of the
protein. The antibody did not bind to squid brain myosin II that
sometimes copurifies with myosin V. Therefore, the antibody exhibited
specificity for squid myosin V and was tested for its ability to
inhibit actin-based vesicle movement. The addition of the antibody to
axoplasm reduced motile activity on actin filaments by 84–98%
compared with samples treated with control antibodies (1)
.
In three separate trials at an antibody concentration of 0.75 mg/ml,
actin based motility was reduced by 84%, while there was no difference
observed in the level of microtubule based motile activity in the
experimental preparation. At concentrations of 1 mg/ml, the inhibition
was 98%. To demonstrate that the inhibition was not a result of
precipitation of myosin V by the antibody, three trials were conducted
using Fab fragments of the QLLQ antibody. Fab fragments of the
antibody inhibited vesicle transport by 95%. For these experiments,
each axon was divided in half, and one half of the axoplasm was treated
with the myosin V antibody and the other half was treated with the
control antibody. This protocol insured that each axon used in these
experiments was active after dissection. The mechanism by which the
QLLQ antibody inhibits actin-based vesicle transport in axoplasm has
not been established. The antibody was made to a peptide sequence in
the tail of the myosin near the putative membrane docking site;
therefore, it could potentially interfere with the docking of the
myosin to the vesicle.
An antibody raised against bacterially expressed head domain of chicken
myosin V, Myo-V head (9)
, was also tested for its
ability to inhibit movement. This antibody was previously shown to
block myosin V motor activity in a sliding filament assay
(10)
. In squid axoplasm, the Myo-V head inhibited
actin-based movement by 98% (1)
.
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CONCLUSIONS AND SUMMARY
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These results demonstrate that squid myosin V is the motor
responsible for the movement of vesicles on actin filaments in squid
axoplasm. This is the most conclusive evidence to date that class V
myosins are directly involved in vesicle transport in neurons. In
support of these results are the recent studies showing that Purkinje
cells in the brains of rats and mice carrying the dilute mutation, a
myosin V defect, failed to localize S-ER to dendritic spines
(11)
. Collectively, these recent studies strongly argue
for myosin-V’s role as an important S-ER vesicle motor in neurons. A
loss of its function could lead specifically to loss of calcium
signaling in the dendrites and axons.
Localization of the S-ER at the synapse is required for efficient
signal transduction in neurons. S-ER is the principal site of storage
of calcium in neurons, and release of calcium postsynaptically from the
S-ER has been shown to trigger signal transduction cascades
(12)
. Two different second-messenger pathways involving
inositol 1,4,5-trisphosphate (IP3) and calcium—through calcium-induced
calcium release (CICR)—have been shown to regulate the release of
calcium from the S-ER. IP3 mobilizes calcium from distinct regions of
the S-ER by IP3 receptors, while the influx of extracellular calcium
through voltage-sensitive calcium channels mobilizes calcium from
various regions of the S-ER by the ryanodine receptor (i.e., CICR)
(13)
. These spatially distinct but interconnected regions
of the S-ER function as discrete units (14)
. CICR has been
shown to be important in the presynaptic terminal where action
potential-mediated calcium influx initiates the release of calcium that
leads to synaptic vesicle fusion and secretion of neurotransmitter.
Release of calcium from the S-ER through IP3 receptors has been shown
to be very important in the postsynaptic spines of dendrites where the
release of S-ER stored calcium triggers signal transduction cascades
(15
, 16)
. The latter are thought to lead to most forms of
long-term synaptic potentiation and long-term depression, the putative
cellular mechanisms of learning and memory.
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ACKNOWLEDGMENTS
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This work was supported by a grant from the National Science
Foundation and the National Institutes of Health (9506279 and
9842948).
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REFERENCES
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ABSTRACT
INTRODUCTION
