54 | L. M. Bond et al. Molecular Biology of the Cell
Myosin VI and its binding partner optineurin are
involved in secretory vesicle fusion at the plasma
Lisa M. Bond a, b , Andrew A. Peden a , John Kendrick-Jones c , James R. Sellers b , and Folma Buss a
aCambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom;
b Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, MD 20892, USA; and c MRC Laboratory of Molecular Biology, Cambridge CB2 OQH, United Kingdom
This article was published online ahead of print in MBoC in Press ( http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E10-06-0553 ) on December 9, 2010.
Address correspondence to: Folma Buss ( email@example.com ).
Abbreviations used: ALS, amyotrophic lateral sclerosis; ER, endoplasmic reticulum;
ERES, ER exit site; GalT, β-1,4-galactosyltransferase-1; GFP, green fl uorescent pro-
tein; PBS, phosphate-buffered saline; POAG, primary open angle glaucoma; RFP, red
fl uorescent protein; SEAP, secreted form of alkaline phosphatase; siRNA, small inter-
fering RNA; TGN, trans-Golgi network; TIRF, total internal refl ection fl uorescence.
© 2011 Bond et al. This article is distributed by The American Society for Cell Biol-
ogy under license from the author(s). Two months after publication it is available
to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License ( http://creativecommons.org/licenses/by-nc-sa/3.0 ).
“ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
ABSTRACT During constitutive secretion, proteins synthesized at the endoplasmic reticulum
(ER) are transported to the Golgi complex for processing and then to the plasma membrane
for incorporation or extracellular release. This study uses a unique live-cell constitutive secre-
tion assay to establish roles for the molecular motor myosin VI and its binding partner op-
tineurin in discrete stages of secretion. Small interfering RNA-based knockdown of myosin VI
causes an ER-to-Golgi transport delay, suggesting an unexpected function for myosin VI in
the early secretory pathway. Depletion of myosin VI or optineurin does not affect the number
of vesicles leaving the trans -Golgi network (TGN), indicating that these proteins do not func-
tion in TGN vesicle formation. However, myosin VI and optineurin colocalize with secretory
vesicles at the plasma membrane. Furthermore, live-cell total internal refl ection fl uorescence
microscopy demonstrates that myosin VI or optineurin depletion reduces the total number of
vesicle fusion events at the plasma membrane and increases both the proportion of incom-
plete fusion events and the number of docked vesicles in this region. These results suggest a
novel role for myosin VI and optineurin in regulation of fusion pores formed between secre-
tory vesicles and the plasma membrane during the fi nal stages of secretion.
Constitutive exocytosis is a fundamental cellular process governing
the transport of newly synthesized proteins to the cell surface for
insertion into the plasma membrane or secretion into the extracel-
lular environment. The secretory pathway involves a complex se-
quence of steps dependent on intracellular transport machinery. In
the fi rst stages of secretion, proteins synthesized by the ribosomes
of the rough endoplasmic reticulum (ER) are transported in vesicles
and larger tubular clusters from specifi c ER exit sites to the cis side
of the Golgi complex (Saraste and Svensson, 1991 ; Presley et al. ,
1997 ). After the proteins are moved through the different compart-
ments of the Golgi complex for posttranslational processing, they
are sorted at the trans side of the Golgi complex into tubular or
vesicular carriers and transported to the plasma membrane for
exocytosis (Griffi ths and Simons, 1986 ; Keller and Simons, 1997 ).
The release of secreted proteins into the extracellular space during
the fi nal stages of secretion requires fusion of juxtaposed phospho-
lipid bilayers of vesicular and plasma membranes to form an aque-
ous channel called a “fusion pore” that expands to permit full pro-
tein release (Jahn and Sudhof, 1999 ).
The intracellular transport system that coordinates the different
stages of the secretory process is driven by carrier proteins called
molecular motors. Transport by these motors can be divided into
the short-range movements of myosin molecular motors along actin
fi lament tracks and the comparatively faster and longer-ranged
movements of kinesin and dynein motors along microtubule tracks.
Within these three classes of motors lies the capacity for transport of
secretory cargo in all directions within the cell: kinesin proteins pri-
marily move cargo toward the plus ends of microtubules, dynein
proteins transport cargo toward the minus ends of microtubules,
University of Exeter
Received: Jul 2 , 2010
Revised: Oct 14 , 2010
Accepted: Oct 21 , 2010
MBoC | ARTICLE
Volume 22 January 1, 2011 Myosin VI and optineurin in secretion | 55
and specifi c myosin protein classes transport to either the plus or
the minus ends of actin fi laments. Cooperation between the three
classes of molecular motors is thereby the driving force in the
organization of traffi cking in secretory transport processes. In addi-
tion to this well-defi ned role in cargo transport, molecular motor
proteins have also been implicated in the organization of the secre-
tory pathway through regulation of vesicle tethering and budding
( Rudolf et al. , 2001 ; Egea et al. , 2006 ), cargo sorting and mainte-
nance of morphology at the Golgi complex (Donaldson and Lippin-
cott-Schwartz, 2000 ; Allan et al. , 2002 ; Sahlender et al. , 2005 ), and
vesicle docking and fusion pore formation at the plasma membrane
(Bhat and Thorn, 2009 ; Chung le et al. , 2010 ).
One such motor protein suggested to function in the secretory
pathway is myosin VI, which has the unique ability to transport cargo
toward the minus ends of actin fi laments ( Wells et al. , 1999 ). Myosin
VI localizes to vesicles in the perinuclear region at or around the
Golgi complex ( Buss et al. , 1998 ; Warner et al. , 2003 ) and to vesicles
close to the plasma membrane ( Sahlender et al. , 2005 ). Functional
studies using fi broblasts isolated from the myosin VI knockout
(Snell’s waltzer) mouse or myosin VI small interfering RNA (siRNA)
knockdown cells demonstrate a signifi cant reduction in the constitu-
tive exocytosis of a secreted form of alkaline phosphatase (SEAP)
from cells lacking myosin VI ( Warner et al. , 2003 ). Furthermore, my-
osin VI is required for the delivery of newly synthesized transmem-
brane proteins to the basolateral plasma membrane domain in po-
larized epithelial cells. In these cells, depletion of functional myosin
VI by overexpression of the dominant negative tail domain results in
a missorting of basolateral cargo such as vesicular stomatitis virus
G-protein (VSV-G) to the apical plasma membrane ( Au et al. , 2007 ;
Chibalina et al. , 2008 ). These data clearly establish a role for myosin
VI in secretion, but they provide no insight as to the specifi c involve-
ment of myosin VI throughout the stages of the secretory pathway.
The role of myosin VI in the secretory pathway is closely linked
to its interacting protein optineurin. Optineurin is a 67-kDa dimeric
protein that colocalizes with myosin VI in the perinuclear region and
on vesicles beneath the plasma membrane ( Sahlender et al. , 2005 ).
siRNA-mediated knockdown of optineurin decreases secretion of
SEAP and VSV-G in a similar manner to myosin VI depletion ( Sahl-
ender et al. , 2005 ; Au et al. , 2007 ; Chibalina et al. , 2008 ). Optineu-
rin not only binds to myosin VI but has also been shown to interact
with the GTPase Rab8 and with huntingtin, which are important
regulators of vesicle transport ( Huber et al. , 1993b ; Hattula and
Peranen, 2000 ; Caviston and Holzbaur, 2009 ). Recent implications
that defects in optineurin cause primary open angle glaucoma
(POAG) ( Rezaie et al. , 2002 ) and play a role in the pathogenesis of
amyotrophic lateral sclerosis (ALS) ( Maruyama et al. , 2010 ) stress
the importance of investigating its primary intracellular functions.
Although previous work has established a role for myosin VI and
optineurin in constitutive secretion of a variety of transmembrane
proteins and soluble luminal cargo in both polarized and unpolar-
ized cells, no available studies address the specifi c roles of these
proteins in the secretory pathway. Therefore, in this study, we inves-
tigate the exact roles of myosin VI and optineurin at each stage of
constitutive secretion using live-cell fl uorescence microscopy on a
HeLa cell line stably expressing a green fl uorescent protein (GFP)-
tagged reporter construct ( Gordon et al. , 2010 ). We are able to dem-
onstrate an unexpected role for myosin VI in the ER-to-Golgi trans-
port stage of secretion, because depletion of myosin VI leads to a
kinetic delay in cargo transport between the ER and Golgi complex.
Interestingly, our results further suggest that myosin VI and optineu-
rin are not required for transport carrier formation at the trans- Golgi
network (TGN). However, we demonstrate that myosin VI and
optineurin colocalize with secretory vesicles at the plasma mem-
brane. Furthermore, loss of myosin VI or optineurin dramatically re-
duces the number of cargo vesicles fusing with the plasma mem-
brane and increases both the number of incomplete fusion events
and the number of docked vesicles at the plasma membrane. Taken
together, these results suggest a role for myosin VI and optineurin in
the mechanical regulation of the fusion pore that opens to permit
protein release in the fi nal stages of the secretory pathway.
To examine the specifi c roles of myosin VI and optineurin in the se-
cretory pathway, we used a HeLa cell line stably expressing a GFP-
tagged reporter construct ( Gordon et al. , 2010 ). This reporter con-
struct contains multiple aggregation domains and remains arrested
in an aggregated form in the ER after synthesis. Binding of the small
ligand AP21998 (Ariad Pharmaceuticals, Cambridge, MA) to the ag-
gregation domains yields soluble protein, allowing a synchronous
pulse of GFP-tagged reporter construct to be transported from the
ER to the Golgi complex and then to the plasma membrane for re-
lease from the cell. This regulated secretion system permits visual-
ization of the main stages of constitutive secretion via live-cell time-
lapse fl uorescence microscopy.
Knockdown of myosin VI causes a kinetic delay in
To establish the exact role(s) of myosin VI and optineurin in the secre-
tory pathway, we fi rst investigated the involvement of these proteins
in the earliest transport-driven stage of secretion: the delivery of syn-
thesized proteins from the ER to the Golgi complex. For this study,
cells in our assay system were imaged using live-cell spinning disc
microscopy for a 30-min period immediately following AP21998 treat-
ment to monitor delivery of the GFP-tagged reporter molecule from
the ER into the Golgi region. The position of the Golgi complex was
visualized by expressing a red fl uorescent protein (RFP)-tagged β-1,4-
galactosyltransferase-I (GalT) construct. Still images of a video show-
ing movement of the reporter molecule from the ER into the Golgi
complex in a mock treated cell are shown in the top of Figure 1A . The
time-lapse images in the bottom of Figure 1A display the delay in
accumulation of cargo in the Golgi region observed in a cell in which
myosin VI has been depleted with a 5-d, double-hit siRNA knock-
down protocol. At each time point, signifi cantly less GFP-tagged re-
porter molecule is present in the Golgi complex in this myosin VI
knockdown cell, as compared with the mock cell in the top panel.
To quantify the rate of reporter construct transfer from the ER to
the Golgi complex, Volocity analysis software was used to track the
accumulation of fl uorescent reporter construct intensity in the re-
gion defi ned as the Golgi complex by the GalT-RFP marker pro-
tein. The fl uorescent intensity present in the Golgi region at each
time point was converted to a percentage of the total fl uorescent
intensity in the cell present before addition of AP21998 and plot-
ted versus time to the point of maximum Golgi fl uorescence. The
slope of this plot represents the rate of reporter construct transport
from the ER to the Golgi complex. This method was used to calcu-
late the rate of reporter construct transfer between ER and Golgi in
cells in which myosin VI or optineurin had been depleted by siRNA
transfection ( Figure 1C ). Optineurin SMARTpool knockdown cells
show no signifi cant difference in average ER-to-Golgi transport
rate when compared with mock cells ( Figure 1B ). However, knock-
down of myosin VI with a SMARTpool collection of four siRNA
primers or with distinct, individual siRNA primers consistently re-
sults in a reduction in ER-to-Golgi transport rate, as compared with
mock cells (38% overall average rate reduction) ( Figure 1B ).
56 | L. M. Bond et al. Molecular Biology of the Cell
The number of secretory vesicles
traveling out of the Golgi complex
is not changed, however, in myosin VI
or optineurin knockdown cells
To deter mine whether the delay in ER-to-
Golgi protein transfer after depletion of
myosin VI is propagated to the post-
Golgi stages of the secretory pathway
and whether myosin VI and optineurin
are required for vesicle formation at the
TGN, we next assayed the number of
vesicles leaving the Golgi complex in
mock versus myosin VI or optineurin
knockdown cells. For this analysis, we
took 1-min, confocal spinning disc vid-
eos of sections through the middle of
the cell at specifi c intervals between 25
and 60 min after AP21998 addition. Vesi-
cle tracking software designed at the
National Institutes of Health and Emory
University was used to quantify the num-
ber of individual vesicles present in 6 μm ×
6 μm regions between the trans- Golgi
and plasma membrane over the course of
these videos ( Figure 2 ). Com parison of
mock and siRNA SMARTpool knockdown
cells reveals that the depletion of myosin
VI or optineurin results in no signifi cant
change in the number of vesicles travel-
ing through these regions. This indicates
that the reduced transport rate between
the ER and Golgi complex in myosin VI
knockdown cells is not a rate-limiting
step in the overall secretory process and
does not reduce the number of cargo
vesicles leaving the trans side of the Golgi
complex. This enables us to study the
involvement of myosin VI and optineurin
in the post-Golgi stages of the pathway
independently of defects observed in the
early steps of the secretory pathway. Fur-
thermore, these results suggest that my-
osin VI and optineurin are unlikely to play
a major role in vesicle formation at the
TGN as previously speculated (Buss and
Kendrick-Jones, 2008 ).
Myosin VI and optineurin colocalize with secretory vesicles
at the plasma membrane
We examined the involvement of myosin VI and optineurin in the
later stages of the secretory pathway using total internal refl ection
fl uorescence (TIRF) microscopy, a technique that permits the selec-
tive illumination of the 100–200-nm region nearest the plasma
membrane at the base of the cell. Because the localization of op-
tineurin and myosin VI relative to vesicles in this proximity of the
plasma membrane has not previously been demonstrated, we used
TIRF microscopy with fi xed, immunofl uorescence-labeled cells to
examine the distribution of myosin VI and optineurin with respect to
secretory vesicles in the TIRF fi eld.
For these immunofl uorescence studies, HeLa cells stably express-
ing the GFP-tagged reporter construct were treated with AP21998
and fi xed 35 min after treatment. To examine the localization of
As a control, the same rate analysis study was conducted in
cells depleted of two distinct members of the SNARE family, a
group of proteins that mediate vesicle docking and fusion
( Figure 1C ). Knockdown of the vesicle-associated membrane
protein 7 (VAMP7), a post-Golgi SNARE, has no signifi cant effect
on ER-to-Golgi transport ( Figure 1B ), which indicates that the de-
lay in transport seen after a knockdown of myosin VI is not simply
a result of our siRNA knockdown protocol. Knockdown of the
ER-to-Golgi SNARE syntaxin5, however, completely inhibits ER-
to-Golgi transport ( Figure 1B ), suggesting that a knockdown of
myosin VI results in a kinetic delay rather than a complete block
in ER-to-Golgi transport. Overall, our results suggest an unex-
pected role for myosin VI in the transport of cargo between
the ER and the Golgi complex during the early stages of the
FIGURE 1: Rate of reporter molecule transport from the ER to the Golgi complex.
(A) Time-lapse images illustrating the accumulation of reporter molecule fl uorescence in the
Golgi region (as labeled with GalT-RFP) over the fi rst 10 min after AP21998 addition to a mock
cell (top row) or to a myosin VI knockdown cell (bottom row). (B) Rate of ER-to-Golgi reporter
molecule transfer in sets of two to eight knockdown cells as a percentage of mock. Knockdown
of myosin VI with SMARTpool primers (SP), individual primer 1, or individual primer 2 results in
48, 34, or 33% decreases in ER-to-Golgi transport rate, respectively (unpaired t test; n = 5, 4, 5
experimental sets; p = 0.005, p = 3 × 10 −8 , p = 3 × 10 −10 ). Knockdown of optineurin or VAMP7 has
no signifi cant effect on the rate of ER to Golgi transport (unpaired t test; n = 4, 6 experimental
sets; p = 0.3, p = 0.6), while a knockdown of syntaxin5 results in a 97% decrease in this rate
(unpaired t test; n = 6 experimental sets; p = 8 × 10 −8 ). Error bars represent standard error of the
mean (SEM). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (C) Western blot analysis of siRNA knockdown
of optineurin, myosin VI, VAMP7, and syntaxin5.
Volume 22 January 1, 2011 Myosin VI and optineurin in secretion | 57
fusion event can be observed as a sudden, punctate fl ash of light in
the TIRF fi eld. It is thus possible to quantify the number of individual
fusion events in a given cell by counting the number of punctate
fl ashes of light observed at the cell base using TIRF microscopy.
To determine the effects of an absence of myosin VI or optineu-
rin on vesicle fusion at the plasma membrane, we quantifi ed the
total number of fusion events in sets of mock or siRNA knockdown
cells monitored at specifi c, 5-min intervals between 25 and 60 min
after AP21998 treatment. Still images of videos taken in the TIRF
fi eld of mock, optineurin, myosin VI, or Rab8 knockdown cells are
shown in Figure 4A . Pink squares mark the sites of fusion events
during a 20-s time-lapse movie. When compared with mock cells,
knockdown of optineurin with a SMARTpool collection of four
siRNA primers results in a 50% average decrease in the total num-
ber of fusion events, whereas knockdown of myosin VI with SMART-
pool primers results in a 38% average decrease ( Figure 4B ). Sup-
plemental Movies 1–3 provide a visual demonstration of this
decrease in fusion events after a knockdown of myosin VI or
To control for possible off-target effects, we performed the same
study with individual siRNA primers for both optineurin and myosin
VI. Knockdowns with two individual primers for optineurin result in
very consistent 47% decreases in the total number of fusion events
( Figure 4B ). Similarly, knockdowns with two individual primers for
myosin VI result in consistent 34 and 37% decreases in the total
number of fusion events ( Figure 4B ). These data indicate that the
decrease in fusion events observed after a SMARTpool knockdown
of optineurin or myosin VI is not simply an off-target effect of the
We also measured fusion events at the plasma membrane in
Rab8 knockdown cells ( Figure 4C ) to assess whether this protein, an
optineurin-interacting protein and GTPase involved in post-Golgi
membrane traffi cking, is required for secretory vesicle fusion at the
plasma membrane. The results summarized in Figure 4B show that
loss of Rab8 expression has no signifi cant effect on the average
number of fusion events and so indicate that this Rab protein is not
required in the fi nal stages of the secretory pathway. Supplemental
Movies 1 and 4 provide a visual demonstration of this similar level of
fusion events in mock versus Rab8 knockdown cells.
An increased number of vesicles are docked
in the 100–200-nm TIRF fi eld in cells depleted
of myosin VI or optineurin
To test whether the decrease in fusion events after a knockdown of
optineurin or myosin VI results in an increase in the number of
docked vesicles present at the plasma membrane, we quantifi ed the
total area in the TIRF fi eld occupied by docked vesicles in sets of
10 mock and knockdown cells imaged at specifi c intervals between
25 and 60 min after AP21998 addition. The top panel in Figure 5A
presents representative images of the TIRF fi eld of mock and knock-
down cells. Confocal spinning disc images of the plasma membrane
at the base of sample mock and knockdown cells are presented
in the bottom panel as a reference point for the observation of
differences in vesicle content with higher x - y resolution.
Knockdown of optineurin results in an 83% increase in the area
of the TIRF fi eld occupied by docked vesicles and a knockdown of
myosin VI similarly results in a 70% increase in this area, as com-
pared with mock cells ( Figure 5B ). In Rab8 knockdown cells, no sig-
nifi cant increase in the area of the TIRF fi eld occupied by docked
vesicles is observed ( Figure 5B ). This increase in the relative propor-
tion of docked vesicles present within 100–200 nm of the plasma
membrane after a knockdown of myosin VI or optineurin suggests a
myosin VI with respect to GFP-tagged secretory cargo, fi xed cells
transfected with an mCherry-myosin VI construct were labeled with
a monoclonal antibody to GFP [cargo, Figure 3A (a)] and a polyclonal
antibody for mCherry [myosin VI construct, Figure 3A (b)]. The high
level of colocalization between myosin VI and cargo-containing
secretory vesicles at the plasma membrane [ Figure 3A (c)] directly
suggests a role for myosin VI in the later stages of the secretory
pathway. Depletion of optineurin by siRNA knockdown does not af-
fect this colocalization of myosin VI and secretory vesicles at the
plasma membrane (unpublished data). To examine the localization
of optineurin with respect to secretory cargo, fi xed cells were la-
beled with a monoclonal antibody to GFP [cargo, Figure 3B (a)] and
a polyclonal antibody to optineurin [optineurin, Figure 3B (b)]. This
low-level colocalization of optineurin with specifi c secretory vesicles
[ Figure 3B (c)] suggests a potential role for optineurin in the fi nal
stages of secretion.
The number of secretory vesicle fusion events at the
plasma membrane is decreased by a knockdown
of myosin VI or optineurin
To examine the function of the myosin VI and optineurin associated
with late-stage secretory cargo, we conducted TIRF microscopy ex-
periments with live cells. The use of TIRF to selectively illuminate the
100–200-nm region nearest the plasma membrane at the base of a
live cell increases the signal-to-noise ratio to such an extent that it is
possible to visualize the fusion of individual, cargo-fi lled secretory
vesicles with the plasma membrane ( Zenisek et al. , 2000 ). Such fu-
sion events represent the formation of a small, aqueous channel or
“fusion pore” between the membrane of a vesicle docked at the
plasma membrane and the plasma membrane itself (Jahn and
Sudhof, 1999 ). This fusion pore dilates to permit the release of se-
cretory proteins from the vesicle into the extracellular environment
(Sorensen, 2009 ). In our study, the rapid, extracellular release of the
fl uorescently tagged reporter construct during an individual vesicle
FIGURE 2: Number of vesicles traveling between the Golgi and
plasma membrane. (A) Illustration of vesicle numbering method. The
image displays a cell in which one 6 μm × 6 μm region between the
Golgi complex and the plasma membrane has been selected. For
analysis, the total number of vesicles present in regions of this size and
location were quantifi ed over the course of 1-min videos using vesicle
tracking software. (B) Average number of vesicles in 6 μm × 6 μm
regions between the Golgi and plasma membrane in mock, optineurin,
or myosin VI knockdown cells. Data represent averages of the total
number of vesicles in 6 μm × 6 μm regions placed in sets of fi ve cells
imaged at specifi c intervals between 25 and 60 min after the addition
of AP21998. There is no signifi cant difference in the number of vesicles
present in these regions after a knockdown of optineurin (unpaired t
test; n = 3 experimental sets; p = 0.9) or a knockdown of myosin VI
(unpaired t test; n = 3 experimental sets; p = 0.7). Error bars represent
SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
58 | L. M. Bond et al. Molecular Biology of the Cell
peak of the graph represents the formation
of a fusion pore with the vesicular mem-
brane (“fusion”), and the downward slope
of the graph represents the release of
the fl uorescent reporter molecule through
the expanded fusion pore. In such a full fu-
sion event, the average fl uorescent intensity
in the seconds (−5 to −1) immediately prior
to the fusion peak is characteristically higher
than the average fl uorescent intensity in the
seconds (+1 to +5) immediately following
the fusion peak, refl ecting the fact that
the fl uorescent reporter construct is fully
released from the fused vesicle.
Four percent of the vesicles in mock
cells exhibit a second type of fusion pat-
tern, in which a docked vesicle fl ashes
briefl y and then remains present and fi lled
with fl uorescent cargo beneath the plasma
membrane ( Figure 6B , images). The behav-
ior of these vesicles fi ts the profi le of the
“incomplete fusion events” previously ob-
served by patch amperometry or capaci-
tance recording ( Spruce et al. , 1990 ; Zhou
et al. , 1996 ; Albillos et al. , 1997 ). In these
incomplete fusion events, vesicles dock,
prime, form transient fusion pores with the
plasma membrane, and then close these
pores rather than expanding them for full
cargo release (Sorensen, 2009 ). Quantita-
tive modeling of the average fl uorescent
intensity in the area immediately surround-
ing an incomplete fusion event reveals a
characteristic peak shape ( Figure 6B , graph)
in which the average fl uorescent intensity
in the seconds (−5 to −1) immediately prior
to the fusion peak is lower than the average
fl uorescent intensity in the seconds (+1
to +5) immediately following the fusion peak, refl ecting an incom-
plete release of fl uorescent cargo. The color-coded boxes in Sup-
plemental Movies 1–4 highlight the appearance of full versus in-
complete fusion events as observed by live-cell TIRF microscopy.
To further characterize the exact role of myosin VI and optineurin
in the later secretory pathway, we assessed whether the decrease in
full fusion events at the plasma membrane after a knockdown of
myosin VI or optineurin is accompanied by a corresponding increase
in incomplete secretory vesicle fusion events. Still images of videos
taken in the TIRF fi eld of mock, optineurin, myosin VI, and Rab8
knockdown cells are shown in Figure 6A . Blue squares mark the sites
of full fusion events and red squares mark the sites of incomplete
fusion events during a 20-s time-lapse movie. For our study, we
quantifi ed the proportion of total vesicle fusion events that did not
proceed to a full release of the fl uorescent reporter molecule (“pro-
portion of incomplete fusion events”) in sets of cells monitored at
specifi c, 5-min intervals between 25 and 60 min after AP21998 treat-
ment ( Figure 6C ). Strikingly, cells in which optineurin has been
knocked down with a SMARTpool primer collection or with individ-
ual primers have an eight- to ninefold higher proportion of incom-
plete fusion events than mock cells. Cells in which myosin VI has
been knocked down with SMARTpool or individual primers also dis-
play a 1.6- to 1.7-fold increase in the proportion of incomplete fu-
sion events when compared with mock cells. Rab8 knockdown cells,
role for myosin VI and optineurin in the fi nal, vesicle fusion stages of
the secretory pathway.
The proportion of incomplete secretory vesicle fusion
events at the plasma membrane is increased in myosin VI
or optineurin knockdown cells
In our TIRF time-lapse movies, we observe two distinct types of exo-
cytic fusion events during the constitutive secretion of the reporter
molecule ( Figure 6 ). The primary type (96% of fusion events in mock
cells) can be visualized as a transient fl ash of spreading and then
disappearing fl uorescent light ( Figure 6B , images) and fi ts the pro-
fi le of the “full fusion events” described in previous studies of vesicle
fusion (Jahn and Sudhof, 1999 ; Steyer and Almers, 2001 ; Jackson
and Chapman, 2006 ; Sorensen, 2009 ). These full fusion events oc-
cur in a series of stages: the secretory vesicle docks at the plasma
membrane, the vesicle is primed as fusion machinery gathers, a
small fusion pore is formed as the vesicle membrane and plasma
membrane fuse to create an aqueous channel, and fi nally the fusion
pore expands to permit rapid release of vesicle contents from the
cell by diffusion (Jackson and Chapman, 2006 ). Quantitative model-
ing of the average fl uorescent intensity in the area immediately sur-
rounding a full fusion event reveals a characteristic peak shape ( Fig-
ure 6B , graph) in which the upward portion of the graph represents
the movement of the vesicle toward the plasma membrane, the
FIGURE 3: Colocalization of myosin VI and optineurin with secretory vesicles at the plasma
membrane. (A) Sample images of the TIRF fi eld at the base of a HeLa cell (a) stably expressing
the GFP-tagged reporter construct and (b) transfected with an mCherry-tagged myosin VI
construct. Cells were fi xed 35 min after AP21998 addition and labeled by immunofl uorescence
with a polyclonal antibody for mCherry and a monoclonal antibody to GFP. (c) A high level of
colocalization is visible between myosin VI and secretory vesicles in the TIRF fi eld containing the
reporter construct. Blue boxes highlight the region expanded for closer viewing in the top left of
each panel. Bar, 10 μm. (B) Sample images of the TIRF fi eld at the base of a fi xed HeLa cell (a)
stably expressing the GFP-tagged reporter construct and (b) labeled for endogenous optineurin.
Cells were fi xed 35 min after AP21998 addition and labeled by immunofl uorescence with a
polyclonal antibody to optineurin and a monoclonal antibody to GFP. (c) A low level of
colocalization is visible between optineurin and the secretory vesicles in the TIRF fi eld. White
arrows point out specifi c examples of colocalization and the blue boxes highlight the example
expanded in the top left of each panel. Bar, 10 μm.
Volume 22 January 1, 2011 Myosin VI and optineurin in secretion | 59
ing of a GFP-tagged reporter molecule from
the ER through the Golgi complex to the
plasma membrane, we are able to establish
roles for myosin VI and optineurin at dis-
crete stages of secretion. Specifi cally, our
results indicate an unexpected function
for myosin VI in ER to Golgi transport and
a role for both myosin VI and optineurin
in secretory vesicle fusion at the plasma
Role for myosin VI in ER-to-Golgi
The kinetic delay in ER-to-Golgi transport
visible after a knockdown of myosin VI
suggests a role for this protein in the early
stages of secretion. It is possible that my-
osin VI plays a novel mechanistic role in
this stage of the secretory pathway.
Though microtubule-based transport is
the classic mode of transport of COPII-
coated vesicles from ER exit sites (ERESs)
to the central Golgi complex ( Palmer
et al. , 2005 ), a complementary role for the
actin cytoskeleton in COPII vesicle trans-
port has been suggested (Disanza and
Scita, 2008 ). Myosin VI proteins moving
along such ERES-associated actin fi la-
ments could play a role in mediating
COPII exit from the ER, tethering COPII
vesicles near ERESs, or transporting these
vesicles in shorter-range movements
along actin fi laments before their longer-
range transport along microtubules. Alter-
natively, the kinetic delay that we note in
ER-to-Golgi transport may be an indirect
result of modifi cations in the actin cy-
toskeleton upon depletion of myosin VI.
Myosin VI has been implicated in the or-
ganization of the actin cytoskeleton
( Noguchi et al. , 2006 ), and the bidirec-
tional transport of cargo between ERESs
and the Golgi complex likely relies par-
tially on actin-based transport ( Lee et al. ,
2004 ). Whether direct or indirect, our
results suggest a novel infl uence of myosin VI on ER-to-Golgi
Golgi-to–plasma membrane transport
Interestingly, the number of vesicles budding and traveling away
from the TGN is not changed by a myosin VI knockdown, despite
the observed kinetic delay in ER-to-Golgi transport upon depletion
of myosin VI. This fi nding suggests that the moderate reduction in
transport rate between the ER and Golgi complex after myosin VI
depletion is not a rate-limiting factor in the overall secretory path-
way and so does not affect the transport levels between the Golgi
and the plasma membrane.
Myosin VI and optineurin are both associated with vesicular
structures in the perinuclear region around the Golgi complex and
have therefore been suggested to play a role in secretory vesicle
formation or cargo sorting at the TGN (Buss and Kendrick-Jones,
2008 ). Our results, however, do not support a critical role for myosin
however, show no signifi cant difference from mock cells in the pro-
portion of vesicle fusion events that do not proceed to completion.
Supplemental Movies 1–4 provide a visual demonstration of the in-
crease in incomplete fusion events after a knockdown of myosin VI
or optineurin and the unchanged proportion of incomplete fusion
events after a knockdown of Rab8. The decrease in full fusion events
and increase in incomplete fusion events observed after depletion
of optineurin or myosin VI directly suggests a role for these proteins
in the pore expansion stage necessary for full cargo release by se-
cretory vesicles at the plasma membrane.
Although a requirement for myosin VI and its interacting protein
optineurin in protein secretion has been established using a variety
of assays in different cell types, the exact molecular role of these
proteins in the exocytic pathway is unknown. Using a unique live-cell
secretion assay that allows visualization of the synchronized traffi ck-
FIGURE 4: Total number of vesicle fusion events at the plasma membrane. (A) Sample images
of the TIRF fi eld of mock and knockdown cells with pink squares marking the sites of fusion
events over the course of a 20-s movie. Bars, 10 μm. (B) Average total number of vesicle fusion
events observed by TIRF microscopy at the base of sets of cells monitored at specifi c, 5-min
intervals between 25 and 60 min after AP21998 addition. Knockdown of optineurin with
SMARTpool primers (SP), individual primer 1, or individual primer 2 results in a 50, 47, or 47%
decrease in the total number of vesicle fusion events, respectively (unpaired t test; n = 4 sets of
10 cells each [SP] or 4 sets of fi ve cells each [individual primers]; p = 9 × 10 −6 , p = 2 × 10 −5 , p = 1 ×
10 −5 ). Knockdown of myosin VI with SMARTpool primers (SP), individual primer 1, or individual
primer 2 results in a 38, 34, or 37% decrease in the total number of vesicle fusion events,
respectively (unpaired t test; n = 5 sets of 10 cells each [SP] or 4 sets of fi ve cells each [individual
primers]; p = 9 × 10 −5 , p = 7 × 10 −5 , p = 7 × 10 −5 ). Knockdown of Rab 8 has no signifi cant effect on
the total number of vesicle fusion events (unpaired t test; n = 3 sets of 10 cells each; p = 0.9).
Error bars represent SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (C) Western blot analysis of siRNA
knockdown of Rab8 with SMARTpool primers and of optineurin with individual primers 1 and 2.
60 | L. M. Bond et al. Molecular Biology of the Cell
FIGURE 5: Relative area of the TIRF fi eld occupied by vesicles. (A) Top, sample images of
vesicles in the TIRF fi eld at the base of mock, optineurin knockdown, myosin VI knockdown, and
Rab8 knockdown cells. Bottom, confocal images at the base of mock and knockdown cells as a
reference for vesicle content at a higher x - y resolution. Bar, 10 μm. (B) Relative average area of
the TIRF fi eld occupied by docked vesicles. Volocity analysis software was used to calculate the
total area in the TIRF fi eld occupied by fl uorescent vesicles in single plane images taken at
specifi c intervals between 25 and 60 min after AP21998 addition to mock and knockdown cells.
Knockdown of optineurin results in an 83% increase in the area occupied by vesicles as
compared with mock cells (unpaired t test; n = 3 sets of 10 cells; p = 0.008) and knockdown of
myosin VI similarly results in a 70% increase in the area occupied by vesicles (unpaired t test; n =
3 sets of ten cells; p = 0.04). A knockdown of Rab8, however, has no signifi cant effect on the
area in the TIRF fi eld occupied by vesicles (unpaired t test; n = 3 sets of 10 cells; p = 0.2). Bar,
SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
VI or optineurin in the basic mechanism of vesicle formation at the
TGN, because we do not observe any change in the number of
vesicles traveling from the TGN to the plasma membrane in myosin
VI or optineurin knockdown cells. The perinuclear localization of
myosin VI and optineurin may therefore be linked to a function in
cargo sorting rather than vesicle formation at the TGN. This hypoth-
esis is supported by previous fi ndings that myosin VI and optineurin
function with Rab8 in cargo sorting to the basolateral domain in
polarized epithelial cells and to the dendritic surface in neuronal
cells ( Huber et al. , 1993a ; Au et al. , 2007 ). Because unpolarized
HeLa cells do not sort cargo to specifi c
plasma membrane domains, such cargo
sorting defects could not be identifi ed for
the reporter molecule used in this study.
Role for optineurin and myosin VI in
fusion pore expansion at the plasma
Using TIRF microscopy, we have demon-
strated colocalization of myosin VI and op-
tineurin with secretory vesicles at the plasma
membrane and established that the ab-
sence of optineurin or myosin VI has a dra-
matic effect on the fi nal stages of the secre-
tory pathway. In myosin VI or optineurin
knockdown cells, we observe a 35–50% de-
crease in the number of secretory vesicles
fusing with the plasma membrane for pro-
tein release into the extracellular space.
This reduction in the total number of fusion
events is very similar to the previously re-
ported ∼50% decrease in constitutive se-
cretion of reporter molecules such as VSV-G
or SEAP in optineurin or myosin VI siRNA
knockdown cells or in fi broblasts isolated
from the myosin VI knockout mouse
( Sahlender et al. , 2005 ; Chibalina et al. ,
The fact that our observed reduction in
the total number of fusion events is accom-
panied by an increased number of docked
vesicles beneath the plasma membrane fo-
cuses the secretory defect arising from a
knockdown of myosin VI or optineurin to
the fi nal stages of the secretory pathway
(docking, priming, or vesicle fusion). Our
detailed examination of the fusion defect
demonstrates that knockdown of optineurin
or myosin VI results specifi cally in an in-
crease in the proportion of incomplete fu-
sion events. This result would appear to link
the defect to a change in the behavior of
the fusion pore formed between the vesicle
membrane and plasma membrane during
the fi nal stages of secretion. Because the
opening and closing of exocytic fusion
pores is believed to be a dynamic and re-
versible process (Jackson and Chapman,
2006 ), our observed increase in instances of
incomplete cargo release from fused vesi-
cles suggests that a knockdown of optineu-
rin or myosin VI either hinders the outward
expansion of fusion pores during exocytosis or increases the fre-
quency of reversible closure of these pores.
Such involvement of an actin-based molecular motor in fusion
pore mechanics is not without precedent. Myosin II has been shown
to maintain an open fusion pore in secretory epithelial cells and a
mutant form of myosin II hinders fusion pore expansion during se-
cretion in chromaffi n cells ( Neco et al. , 2008 ; Bhat and Thorn, 2009 ).
Related studies tie the role of myosin II in fusion pore mechanics
closely to its interaction with the actin cytoskeleton (Becker and
Hart, 1999 ; Doreian et al. , 2008 ; Berberian et al. , 2009 ), which has
Volume 22 January 1, 2011 Myosin VI and optineurin in secretion | 61
FIGURE 6: Full vs. incomplete fusion events at the plasma membrane. (A) Sample images of the
TIRF fi eld of mock and knockdown cells with locations of full fusion events during a 20-s movie
marked with blue squares and the incomplete fusion events with red squares. Bar, 10 μm.
(B) Representative profi les of average intensity vs. relative time for localized regions containing
full vs. incomplete fusion events. Before plotting, all intensities were corrected for
photobleaching and normalized to a peak value of 1. The images display the fusing vesicle at the
peak of the plot and at 1- and 5-s time points before and after this peak. (C) Average proportion
of incomplete vesicle fusion events observed by TIRF microscopy at the base of sets of cells
monitored at specifi c, 5-min intervals between 25 and 60 min after AP21998 addition.
Knockdown of optineurin with SMARTpool primers (SP) results in a ninefold increase in the
proportion of incomplete fusion events (unpaired t test; n = 4 sets of 10 cells; p = 2 × 10 −5 ) and
knockdown with individual primer 1 or individual primer 2 results in an eightfold increase
(unpaired t test; n = 4 sets of fi ve cells each; p = 6 × 10 −6 , p = 1 × 10 −6 ). Knockdown of myosin VI
with SMARTpool primers (SP), individual primer 1, or individual primer 2 results in 1.7-, 1.6-, and
1.7- fold increases in the percentage of incomplete vesicle fusion events, respectively (unpaired
t test; n = 5 sets of 10 cells each [SP] or 4 sets of fi ve cells each [individual primers]; p = 1 × 10 −4 ,
p = 0.003, p = 3 × 10 −5 ). Knockdown of Rab8 has no signifi cant effect on the proportion of
incomplete fusion events (unpaired t test; n = 3 sets of 10 cells; p = 0.4). Bar, SEM. *p ≤ 0.05,
**p ≤ 0.01, ***p ≤ 0.001.
itself been separately implicated in the regulation of fusion pore
expansion and contraction ( Larina et al. , 2007 ; Cingolani and Goda,
2008 ). Specifi cally, it has been suggested that myosin II and cortical
actin together promote the increased mem-
brane tension necessary to expand the fu-
sion pore for full content release ( Berberian
et al. , 2009 ). Membrane remodeling through
the interaction of myosin Ic and the actin
cytoskeleton has been similarly shown to
play a role in exocytic vesicle fusion in
mouse adipocytes ( Bose et al. , 2004 ) and in
compensatory endocytic vesicle formation
in Xenopus laevis (Sokac et al., 2006 ).
Though initiation of fusion at the plasma
membrane is closely tied to the functioning
of SNARE proteins, Sec1/Munc18 homo-
logues (SM proteins), and Rab proteins
(Jahn and Sudhof, 1999 ; Stenmark, 2009 ;
Jackson, 2010 ; Pieren et al. , 2010 ), the spe-
cifi c molecular mechanism regulating fusion
pore expansion and closure remains to be
determined. Several factors other than my-
osin II/myosin Ic and actin have also been
implicated in the regulation of fusion pore
mechanics, such as calcium concentration
( Elhamdani et al. , 2006 ), cholesterol levels
( Wang et al. , 2010 ), the SNARE-binding
proteins complexin II ( Archer et al. , 2002 )
and synaptotagmin ( Davis et al. , 1999 ; Rizo
et al. , 2006 ), and the GTPase dynamin
( Tsuboi et al. , 2002 ; Jaiswal et al. , 2009 ), but
the interaction between these factors on a
mechanistic level is presently unclear. Our
study adds to the list of likely players in
this process both optineurin and myosin VI.
The similar functional defects in secre-
tion after myosin VI or optineurin knock-
down (reduced secretion levels, increased
proportion of docked vesicles at the plasma
membrane, and increased number of in-
complete fusion events) suggest a coordi-
nated function for these proteins at the fu-
sion pore. As such, we propose a model
whereby the optineurin and myosin VI pres-
ent on secretory vesicles at the plasma
membrane function together in fusion pore
regulation ( Figure 7 ). Given the high level of
colocalization between myosin VI and se-
cretory vesicles in the TIRF fi eld, we suggest
that myosin VI is recruited at an earlier stage
to docked secretory vesicles, perhaps by
binding to PtdIns(4,5)P 2 , a phospholipid
known to function both in exocytosis (Wenk
and De Camilli, 2004 ) and in the recruitment
of myosin VI to clathrin-coated endocytic
vesicles ( Spudich et al. , 2007 ). On the basis
of the low-level colocalization of optineurin
with secretory vesicles in the TIRF fi eld, we
suggest that optineurin is recruited to vesi-
cles immediately prior to fusion (possibly by
the already present myosin VI) and binds to
a yet-to-be-determined member of the fu-
sion pore machinery. The functional connection between huntingtin,
a known binding partner of optineurin, and complexin II suggests
huntingtin as one possible link between optineurin and the fusion
62 | L. M. Bond et al. Molecular Biology of the Cell
gression of POAG, a disease often caused
by mutations in optineurin ( Joe et al. , 2003 ;
Kuchtey et al. , 2008 ). Patients with ALS, a
disease causally linked to improper op-
tineurin function, also demonstrate abnor-
mal secretion of growth hormone and insu-
lin-like growth factor ( Maruyama et al. , 2010 ;
Pellecchia et al. , 2010 ). Alteration in secre-
tion levels of brain natriuretic peptide has
been shown to mark disease progression in
hypertrophic cardiomyopathy, a disease
linked to mutations in myosin VI ( Pieroni et
al. , 2007 ). Furthermore, it has been sug-
gested that the overexpression of myosin VI
in prostate cancer cells enhances disease
progression by increasing secretion of pro-
teins such as vascular endothelial growth
factor ( Puri et al. , 2009 ). Our study of the
effects of myosin VI and optineurin on se-
cretion level modulation via fusion pore dy-
namics is therefore a new avenue for under-
standing the involvement of these proteins
in disease malignancy.
MATERIALS AND METHODS
The following antibodies were used: rabbit
polyclonal antibody to optineurin ( Sahlender
et al. , 2005 ); rabbit polyclonal antibody to
myosin VI ( Buss et al. , 1998 ), mouse monoclonal antibody to Rab8
(BD Transduction Laboratories, Franklin Lakes, NJ) , mouse mono-
clonal antibody to VAMP7 ( Gordon et al. , 2010 ), mouse monoclo-
nal antibody to syntaxin5 ( Williams et al. , 2004 ), rabbit polyclonal
antibody to Dab2 (Santa Cruz Biotechnology, Heidelberg, Ger-
many) , rabbit polyclonal antibody to calregulin (Santa Cruz Biotech-
nology), rabbit polyclonal antibody to DsRed (Clontech, Mountain
View, CA) , and mouse monoclonal antibody to GFP (Invitrogen,
Paisley, UK) .
Cell lines and expression constructs
The reporter construct secretion system was developed by Andrew
Peden from the RPD Regulated Secretion/Aggregation Kit (Ariad
Pharmaceuticals) as previously described ( Gordon et al. , 2010 ). In
short, eGFP (Clontech) was incorporated into the pC4S1-FM4-FCS-
hGH plasmid from this kit. The section of this plasmid containing the
eGFP, signal sequence, aggregation domains, furin cleavage site,
and human growth hormone was then transferred into a retroviral
vector (pQCXIP; Clontech). The resulting pQCXIP-S1-eGFP-FM4-
FCS-hGH construct was used to generate a stable secretion cell line
in HeLa-M cells. To initiate secretion during microscopy studies,
cells were treated with a 1-μM solution of AP21998, a ligand which
solubilizes the reporter molecule by interrupting the interaction of
its aggregation domains.
The GalT-mRFP plasmid used in this work was provided by G.
Patterson (Lippincott-Schwartz Lab, National Institute of Child
Health and Human Development, Bethesda, MD) .
Cell culture and transfection
The cell line used in this study was maintained at 37°C in
Dulbecco’s modifi ed Eagle medium (Invitrogen) supplemented
with 10% fetal calf serum (FCS), 2-mM L -glutamine, and penicillin/
streptomycin (Sigma-Aldrich, Dorset, UK) . To maintain selection
pore machinery ( Edwardson et al. , 2003 ). In light of our functional
TIRF studies, we suggest that that the fusion pore–bound optineurin
functions with the myosin VI previously recruited to docked secretory
vesicles to anchor the fusion pore to the actin cytoskeleton. The
movement of optineurin-bound myosin VI toward the inward-di-
rected minus ends of actin fi laments at the plasma membrane would
thereby provide the tension necessary for stabilization and expan-
sion of the fusion pore. Overall, we propose a model in which op-
tineurin and myosin VI serve as one interface between the fusion
pore regulatory machinery and the tension-providing modulations
of the actin cytoskeleton.
It is important to note that this model provides only one possibil-
ity for the function of myosin VI and optineurin in fusion pore forma-
tion. Given that myosin VI and optineurin colocalize with cargo-fi lled
vesicles at very different levels and that myosin VI and optineurin
knockdown cells show proportionally different increases in incom-
plete fusion events, it is also possible that these proteins function
separately in the mechanism of fusion pore expansion and closure.
Such independent functionality would explain the lack of effect of a
Rab8 knockdown on the number and type of secretory fusion events
at the plasma membrane, which suggests that the Rab8–optineurin–
myosin VI complex shown to be required for cargo sorting in polar-
ized epithelial cells ( Au et al. , 2007 ) does not function in fusion pore
The biomedical implications of the involvement of myosin VI and
optineurin in fusion pore dynamics are very interesting; recent work
points to the emerging fusion pore regulation machinery as a critical
control point for the regulation of release of the contents of secre-
tory vesicles (Thorn, 2009 ). It is possible, therefore, that the malig-
nant progression of diseases aggravated by mutations in optineurin
or myosin VI may stem from improper regulation of levels of secre-
tory cargo release. For example, secretory defects in myocilin and
angiopoietin-like 7 protein secretion have been linked to the pro-
FIGURE 7: Suggested model for involvement of optineurin and myosin VI in fusion pore
expansion. Fusion pore formation and regulation are presently tied to certain SNARE proteins
and Rab GTPases, Sec1/Munc18 homologues (SM proteins), the SNARE-binding proteins
complexin II and synaptotagmin, the GTPase dynamin, and the molecular motors myosin II and
myosin 1c. Our data suggest that the myosin VI and optineurin present on secretory vesicles at
the plasma membrane also function at the fusion pore. We propose that optineurin binds an
unidentifi ed protein associated with the fusion pore, in addition to the myosin VI previously
recruited to docked secretory vesicles. In this manner, a functional link is formed between the
fusion pore and the actin cytoskeleton associated with myosin VI. This link permits the use of the
tensile forces created by movement of myosin VI toward the inward-facing minus ends of actin
fi laments to stabilize or expand the fusion pore. In short, we propose that optineurin and myosin
VI regulate fusion pore dynamics by acting as a link between the fusion pore and the tensile
forces of the actin cytoskeleton.
Volume 22 January 1, 2011 Myosin VI and optineurin in secretion | 63
prepare protein samples for this process, mock and knockdown cells
were resuspended in urea/β-mercaptoethanol loading buffer (2%
SDS, 30% glycerol, 1 M β-mercaptoethanol, 6 M urea, 0.125 M Tris,
pH 6.8, and 0.01% bromophenol blue), lysed, and then boiled for
4 min. SDS–PAGE of protein samples was carried out on 10 or 12%
polyacrylamide gels as previously described (Matsudaira and Bur-
gess, 1978 ). A Dual Color Precision Plus Protein Standard (Biorad,
Bath, UK) was loaded at either end of the gel as a reference for com-
parison of molecular weights of protein samples. After electrophore-
sis, a semidry blotter was used to transfer proteins from the SDS-
polyacrylamide gel to a Protran nitrocellulose membrane with a pore
size of 0.45 μm (Schleicher and Schuell Bioscience, London, UK) .
The membrane was incubated in 10% wt/vol Marvel milk powder in
1× PBS to block nonspecifi c binding sites. Individual sections of
membrane containing specifi c proteins of interest were then incu-
bated with primary and then secondary antibodies diluted in 1% wt/
vol Marvel milk powder in 1× PBS. An enhanced chemiluminses-
cence reaction kit (GE Healthcare, Amersham, UK) was used to
detect protein bands in the membrane.
Spinning disc microscopy
For spinning disc microscopy studies, cells were grown on 25-mm
round coverslips (VWR International, Lutterworth, UK) and imaged
at 37ºC in CO 2 -independent medium (Invitrogen). Images were ob-
tained on a Zeiss Cell Observer SD microscope (Carl Zeiss) using a
63× or 100× lens and 2 × 2 binning. Images were acquired with a
Hamamatsu Photonics EM-CCD Digital Camera (Hamamatsu City,
Japan) and AxioVision imaging software (Carl Zeiss).
Volocity analysis software (Improvision, Cambridge, UK) was
used to quantify the rate of transfer of the reporter molecule from
the ER to the Golgi complex in spinning disc videos.
For live-cell TIRF microscopy studies, cells were grown on 25-mm
round coverslips (VWR International) and imaged at 37ºC in CO 2 -
independent medium (Invitrogen). Imaging was conducted on a
Zeiss TIRF 3 microscope (Carl Zeiss). During each TIRF experiment,
a 488-nm argon laser was directed at the coverslip at an angle
greater than the critical angle of the laser line. The resulting total
internal refl ection of the laser line created an evanescent fi eld in the
100–200-nm region of the sample closest to the coverslip.
Images were acquired with a 100× lens, Hamamatsu Photonics
EM-CCD digital camera (Hamamatsu Photonics), and AxioVision
Imaging Software (Carl Zeiss). Samples were imaged in 5-min inter-
vals at maximum speed (approximately 4 frames/s) at points 25, 32,
39, 46, and 53 min after the addition of 1 μM AP21998. Exactly two
cells were imaged at each time point to ensure that the distribution
of cells over the course of the imaging period was equivalent
between mock and knockdown data sets.
The total area covered by docked vesicles at the base of a given
cell was calculated from the fi rst frame of each movie using Volocity
Analysis software (Improvision). This software was also used to pro-
fi le fl uorescent intensity over time at the sites of individual full or
incomplete fusion events.
Vesicle tracking program
The number of vesicles traveling in 6 μm × 6 μm regions between
the trans- Golgi and the plasma membrane in spinning disc videos
was quantifi ed using a customized particle tracking program. This
program was modifi ed by C.A. Combs (Light Microscopy Core Fa-
cility at the National Heart, Lung, and Blood Institute) and R. Kling
(Ronn Kling Consulting, Warrenton, VA) from open-source particle
for cells containing the pQCXIP-S1-eGFP-FM4-FCS-hGH construct,
this medium was also supplemented with puromycin (1.66 μg/ml).
Transfection of the GalT-mRFP or myosin VI–mCherry plasmids
into tissue culture cells was executed according to the instructions
provided by the manufacturer of the FuGENE transfection reagent
(Roche Diagnostics, Welwyn Garden City, UK) . Prior to transfection,
cells were grown to 50% confl uence on a six-well tissue culture tray
(Greiner Bio-One, Stonehouse, UK) . Two micrograms of plasmid
DNA and 6 μl of FuGENE transfection reagent diluted in 95 μl
of Optimem I medium (Invitrogen) were used for each given trans-
fection in one well of the six-well tray.
For immunofl uorescence experiments, cells transfected 24 h prior
with the appropriate constructs were treated with AP21998 for
35 min, fi xed with 4% paraformaldehyde in phosphate-buffered sa-
line (PBS) for 20 min, permeabilized in 0.2% Triton X-100 in PBS for
5 min, and blocked in 1% bovine serum albumin in PBS for 30 min.
After blocking, cells were incubated with primary antibodies (poly-
clonal antibody to DsRed (Clontech) or polyclonal antibody to op-
tineurin; monoclonal antibody to GFP [Invitrogen]) and then with
secondary antibodies (goat anti–rabbit IgG coupled with Alexa 555;
goat anti–mouse IgG coupled with Alexa 488 [Invitrogen]). After
washing, cells were mounted on glass slides with Prolong Gold
Antifade Mounting Reagent (Molecular Probes, Invitrogen, United
Kingdom) and imaged on a Zeiss TIRF 3 microscope (Carl Zeiss,
Welwgn Garden City, UK) .
ON-TARGET plus SMARTpool mixtures of four individual primers
(Dharmacon, Cramlington, UK) were used for depletion of myosin
VI, optineurin, and Rab8. Individual primers to myosin VI (MVI 1,
5′-CAUUGUAUCUGGAGAAUCAUU-3′; MVI 2, 5′-ACAUUCU-
GAUUGCAGUGAAUU-3′) and optineurin (Optn 1, 5′-CUUCGAA-
CAUGAGGAGUUA-3′; Optn 2, 5′-CUAAUGGCCUUGAGUCAUG-3′)
were used to verify results and control for off-target effects.
For effi cient siRNA knockdown of myosin VI, optineurin, or Rab8,
we used a double-knockdown protocol in which cells were trans-
fected with siRNA primers on days 1 and 3 using Oligofectamine (In-
vitrogen) according to the manufacturer’s instructions. Before siRNA
knockdown, cells were grown to 70% confl uence in a six-well tray. For
each knockdown, siRNA primers were diluted in Optimem I and
transfected into individual wells of the six-well tray at a fi nal concen-
tration of 90 nM. The effectiveness of the knockdown was checked by
immunoblotting on day 4 and effi ciently depleted cells were imaged
on day 5. A similar double-knockdown protocol was used for deple-
tion of syntaxin5, but transfections were conducted with a single
Dharmacon siRNA primer (5′-GGACAUCAAUAGCCUCAAC-3′) at a
40-μM fi nal concentration using Lipofectamine 2000 (Invitrogen).
VAMP7 was depleted in a single-hit siRNA transfection protocol
with a single Dharmacon primer (5′-GAACCUCAAGCUCACUAUU-3′)
at a fi nal concentration of 90 nM using Oligofectamine. The effec-
tiveness of the knockdown was checked by immunoblotting on day
3 and effi ciently depleted cells were imaged on day 4.
Mock-transfected cells were treated with the same mixture
of Oligofectamine/Lipofectamine 2000 in Optimem I as siRNA-
transfected cells, but the solution used for these mock cells
contained no siRNA.
Immunoblotting was used to assess whether individual proteins had
been effi ciently depleted by our siRNA knockdown protocol. To
64 | L. M. Bond et al. Molecular Biology of the Cell
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mid, M. Gratian and M. Bowen (Cambridge Institute for Medical
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work was funded by the Wellcome Trust (F.B.), a scholarship from
the Winston Churchill Foundation of the United States (L.B.), and
an NIH-Oxford-Cambridge Ph.D. studentship (L.B.) and was sup-
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