1 National Institute for Medical Research, London NW7 1AA, UK
2 MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT, UK
3 Department of Neuroscience, University of Edinburgh, Edinburgh EH8 9YL,
UK
4 Department of Pharmacology, UCL, London WC1E 6BT, UK
5 Department of Biomedical Sciences, Imperial College, London SW7 2AZ, UK
* Author for correspondence (e-mail: jmannev{at}nimr.mrc.ac.uk)
Accepted 16 May 2003
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Summary |
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Key words: TIRF, Evanescent field, Intracellular organelle motility, tPA, Rab27a, Weibel-Palade bodies, Cytoskeleton, Exocytosis, Nanotechnology
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Introduction |
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In the case of secretory organelles, the final destination is the plasma
membrane, where the organelles undergo exocytosis. Although the cytoskeleton
is involved in late transport steps and subsequent docking to and fusion with
the plasma membrane, most studies have focused on the role of the actin
cortex, which is thought to act as a barrier preventing organelle motion and
docking to the plasma membrane (Valentijn
et al., 1999). Less is understood about the role of microtubules
and their interaction with the actin cytoskeleton to regulate the transport
and motion of secretory organelles close to the plasma membrane.
Endothelial cells secrete by exocytosis several proteins that regulate
blood coagulation, blood flow and local immune responses. The fibrinolytic
tissue-type plasminogen activator (tPA) is found in small (0.1-0.2 µm
diameter) secretory vesicles (Emeis et al.,
1997; Schick et al.,
2001
). The pro-inflammatory adhesive protein von Willebrand factor
(vWF) is stored in unique large tubular organelles (1-3 µm long, 0.1-0.2
µm diameter) called Weibel-Palade bodies (WPb)
(van Mourik et al., 2002
;
Weibel and Palade, 1964
).
Disruption of microtubules blocks the regulated secretion of both tPA
(Santell et al., 1992
) and vWF
(Sinha and Wagner, 1987
;
Vischer et al., 2000
),
suggesting that the microtubule cytoskeleton plays a central role in the
processing and/or transport of tPA and vWF. WPb are members of the family of
lysosome-related organelles, which includes the pigment granules of
melanocytes (Marks and Seabra,
2001
). Studies in melanocytes indicate that pigment granules are
transported via microtubules to the cell periphery where they are trapped into
the actin cytoskeleton through the myosin Va-receptor Rab27a
(Hammer, III and Wu, 2002
;
Seabra et al., 2002
). However,
unlike pigment granules in melanocytes, nothing is known about the role of
microtubules and the actin cytoskeleton in regulating secretory organelle
dynamics in endothelial cells.
In this study, we have visualized WPb and tPA-containing vesicles in living endothelial cells by the expression of Rab27a-GFP (green fluorescent protein) and tPA-GFP, respectively. We have used Total Internal Reflection Fluorescence (TIRF) microscopy for three-dimensional single-particle tracking of fluorescent WPb and tPA vesicles, to quantify the movement of these morphologically distinct organelles and to study the role of the cytoskeleton and molecular motors in their transport and dynamics close to the plasma membrane.
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Materials and Methods |
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Cell culture
Primary human umbilical vein endothelial cells (HUVECs) were either
isolated and grown as previously described
(Carter et al., 1988) or
purchased as cryopreserved cells from pooled donors (ZHC-2101, TCS CellWorks,
Bucks, UK). No significant difference was observed between cells from the two
different sources. Custom cell chambers were assembled by gluing a
Teflon® frame to a glass slide. Cells were directly plated on
the chambers without any additional coating and cultured at 37°C in a 5%
CO2 atmosphere. Freshly isolated cells were grown in M199 medium
supplemented with 10% fetal calf serum (FCS), 10% newborn calf serum, 100 U/ml
penicillin and 100 µg/ml streptomycin. Cryopreserved cells were grown in
endothelial growth medium (ZHM-2953, TCS CellWorks).
Organelles and cytoskeleton labelling
Cells were microinjected with Rab27a-GFP or tPA-GFP cDNAs. Rab27a-GFP cells
were imaged live 48 hours after microinjection. tPA-GFP-expressing cells were
imaged live 4 hours after microinjection. Cells were fixed with 4%
paraformaldehyde for 15 minutes and permeabilized with 0.2% Triton X100 and
10% FCS for 10 minutes before staining. For microtubule labelling, 0.5%
glutaraldehyde (Sigma) was added during fixation. Primary antibodies were:
rabbit polyclonal anti-human vWF diluted 1/100 (A0082, Dako, Denmark) and rat
monoclonal anti -tubulin diluted 1/200 (clone YL1/2 MCAP77; Serotec,
Oxford, UK). Secondary antibodies were Rhodamine (TRITC)-conjugated donkey
anti-rat, Rhodamine (TRITC)-conjugated donkey anti-rabbit, Cy2-conjugated
donkey anti-rabbit (Jackson ImmunoResearch Labs, West Grove, PA). Actin was
stained with rhodamin-phalloidin (Sigma) diluted 1/500 for 45 minutes. Cells
were left in PBS for TIRF imaging. For colocalization experiments, cells were
fixed immediately after live imaging to minimize cell motion, cytoskeleton
deformation and fixation artefacts. A secretory organelle colocalized with a
cytoskeletal element when its fluorescence overlapped or was within one pixel
from the corresponding cytoskeletal structure.
Drug treatments
The microtubule cytoskeleton was depolymerized by incubating cells in
culture medium supplemented with 10 µM nocodazole (Sigma) for 1 hour at
37°C, 5% CO2. The cell shape and adhesion to the glass slide
remained satisfactory (data not shown). Nocodazole treatment selectively
disrupted the microtubule network but left the actin cytoskeleton intact (data
not shown). Microtubules repolymerized in 15 minutes after cells were washed
twice in culture medium. Actin filaments were depolymerized using 5 µM
latrunculin B (Calbiochem) for 10 minutes at 37°C, 5% CO2.
Under these conditions, few actin bundles remained visible in TIRF images,
whereas microtubules were not significantly affected (data not shown). At
higher concentrations or longer incubation times, dramatic shape changes and
loss of adhesion of the cell to the glass substrate occurred. Kinesin motors
were blocked with 10 µM aurintricarboxylic acid (ATA, Sigma) for 2 hours,
whereas myosin motors were inhibited using 10 mM 2,3-butanedione monoxime
(BDM, Sigma) for 30 minutes.
Dual-colour TIRF microscopy
Total internal reflection (reviewed by
Axelrod, 2001;
Steyer and Almers, 2001
;
Toomre and Manstein, 2001
) was
achieved at the glass slide/culture medium interface using a trapezoidal glass
prism. The refractive index of the prism and culture medium was
ni=nglass=1.52 and
nt=nculture medium
1.336,
respectively, giving a critical angle (
c) for total
internal reflection of
c=61.5°. Experiments
were carried out on an upright microscope (Axioplan, Zeiss, Oberkochen,
Germany) using either an Argon ion laser (excitation
=488 nm, 25 mW,
Melles-Griot, Carlsbad, CA) or a Nd:YAG laser (excitation
=532 nm, 50
mW, CrystaLaser, Reno, NV). The angle of incidence of the excitation light
could be adjusted and was fixed to 68-70°, above the critical angle. The
intensity profile of the evanescent wave is exponentially decaying:
I(z)=I0exp(-z/dP),
where z is the vertical distance,
I0=I(z=0) is the intensity at
the interface and dP is the penetration depth
given by:
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GFP (respectively rhodamine) fluorescence was excited by the Argon ion (resp. Nd:YAG) laser. Differences in beam radius and divergence were corrected by additional lenses to achieve the same spot size and position on the sample. Light from TIRF images was passed through a dichroic filter (505DRLP02 for GFP and Cy2 fluorescence or 560DRLP for rhodamine fluorescence, Omega Optical, VT) and an emission filter (530DF30 or 565ALP). Standard epifluorescence was achieved using a 100 W mercury lamp (excitation filters 485DF22 or 525DF45). The temperature in the cell chamber was maintained at 35-37oC by circulating temperature-controlled water around the prism. 5% CO2 in O2 was blown on the cell chamber through a collar around the objective.
Image acquisition
The light was collected with a 100x 1.0 NA water immersion objective
(Zeiss) driven by a piezo-electric focus drive (Physik Instrumente, Waldbronn,
Germany). Fluorescence images were magnified by a 0.5-2x optical zoom
(Zeiss), processed by an Argus 20 image processor (Hamamatsu Photonics K.K.,
Hamamatsu City, Japan) and collected with an intensified CCD camera (Remote
Head Darkstar, S25 Intensifier, Photonics Science, UK). Images were digitized
and stored in the memory of a Pentium III PC computer at a maximum rate of 25
frames/second by a frame grabber (IC-PCI 4Mb (AMVS), Imaging Technology, MA)
and then saved to disk. Image processing was carried out using Optimas 6.5
(Media Cybernetics L. P., Silver Spring, USA). Live time-lapse images of
GFP-labelled organelles were acquired at 0.5-2 frames/second with online two
frames averaging performed by the Argus-20 image processor. TIRF illumination
was limited to 200 frames to minimize photobleaching and phototoxicity.
Dual-colour images of fixed cells were acquired with 256 frames averaging. The
pixel size was 0.1092 µm (resp. 0.0870 µm) with a 1.6x (resp.
2x) optical zoom. The image size was typically 520x500 pixels.
Three-dimensional organelle tracking
Customized macros were written in the C++ based language ALI
(Analytical Language for Images) and run under Optimas 6.5. The raw images
(N frames, separated by the time interval ) were filtered
to enhance the visibility of the organelles. A low-pass Fast Fourier Transform
filter followed by a 3x3 pixels trimmed mean filter was applied to
remove nonuniform background. At the beginning of the sequence, a region of
interest (ROI) containing the organelle and a threshold above which the
organelle could be detected was defined. The `centre of grey' (centre of mass
weighted by pixel intensities) of the organelle was detected and the
fluorescence intensity of the organelle Ipart was
measured from the corresponding raw image in a 5x5 pixels box cantered
on the centre of grey. The local background fluorescence intensity
Ibkg was measured in another ROI close to the
previous one but containing no organelle. The 2D position of the centre of
grey yielded the x and y coordinates of the organelles,
whereas the z coordinate (relative to the initial position and
corrected for local background brightness variations) was given by:
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Long-range directed motions and short-range diffusive-like
motions
When visualized under the TIRF microscope, organelles displayed two types
of motions: long-range directed motions and short-range diffusive-like
motions. Long-range motions were defined as being >1 µm in distance and
with a maximum velocity >0.05 µm/second. All other motions were
considered as short-range motions. Long-range motions were identified by
thresholding and accumulating the images from a time-lapse sequence (e.g.
Fig. 3A) or using frame to
frame subtraction. Averaging an entire sequence enabled static organelles
undergoing short-range diffusive motions to be identified (e.g.
Fig. 4A).
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Data analysis
From the three-dimensional coordinates x(t),
y(t), z(t) of the organelle, the mean
squared displacement (MSD) travelled by the organelle during a time interval
t=n
was calculated according to:
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![]() | (1) |
![]() | (2) |
![]() | (3) |
Because the x-y spatial resolution of the microscope
(approx. 0.1 µm) is much lower than the vertical (z) resolution
(approx. 0.01 µm), three-dimensional MSD data are dominated by 2D
x-y motions. To specifically quantify displacements in the
z direction, the vertical MSD was calculated according to:
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The resolution of the TIRF microscope was estimated by tracking 0.1 µm diameter polystyrene beads (Molecular Probes, Eugene, OR) stuck to a glass slide in the same conditions as when cells were observed. The signal-to-noise ratio in the bead images was adjusted to match that in the organelle images. Motions of the immobilized beads were due to mechanical vibrations in the experimental set-up. The minimum detectable 3D diffusion coefficient was D=2.6±0.1 10-6 µm2/second (n=10), whereas the corresponding z-direction `diffusion coefficient' was Dz=1.2±0.2 10-7 µm2/second (n=10).
Fusion of secretory organelles with the plasma membrane
Secretion was stimulated by 100 µM histamine (Sigma). Cells were imaged
at 2 frames/second before and during stimulation. Individual fusion events
were analyzed by fitting a Gaussian distribution to the fluorescence image.
The diffusion coefficient of the fluorophore
Dfluo was deduced from a linear fit of the
distribution half-width Rfluo with time according
to: R2fluo(t) =
4Dfluo t + cst.
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Results |
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Fusion of Rab27a-GFP-positive organelles appeared as a fluorescent cloud
spreading away from the site of fusion after 100 µM histamine stimulation
(Fig. 2A). Fitting the
half-width Rfluo of the fluorescence distribution
with time (Fig. 2C) gives the
value for the diffusion coefficient of Rab27a-GFP in the plasma membrane:
Dfluo=0.24±0.16 µm2/second
(n=3). By contrast, exocytosis of tPA vesicles was observed as a
transient brightening of the vesicle followed by an exponential decay of its
fluorescence intensity (Fig.
2B,D) with a characteristic time =12.3±1.6 seconds
(n=13). Fusion of tPA vesicles was more frequent (n=29 in
eight cells) than fusion of Rab27a organelles (n=3 in ten cells).
|
WPb and tPA vesicle movements in resting HUVECs
Two types of motion were distinguished for WPb and tPA vesicles in resting
HUVECs: long-range directed motions and much slower short-range diffusive-like
motions (see Materials and Methods). Examples of time-lapse sequences are
given in Supplementary Material (see Movies 1, 2;
http://jcs.biologists.org/supplemental/).
Long-range directed motions
Approximately 22% of WPb within the evanescent field exhibited long-range
motions, corresponding to a frequency, defined as the number of long-range
motions per total number of organelles per unit time, of
1.0x10-3 per second. Long-range motions often involved rapid
changes or reversals in direction, changes in velocity and pauses between
consecutive runs. The average velocity during a long-range run was
0.16±0.02 µm/second and the maximum velocity was 0.54±0.04
µm/second (n=54). Run length and run duration averaged
7.2±0.8 µm and 70.5±10.1 seconds (n=54,)
respectively. A reduction in WPb velocity often correlated with an increase in
the fluorescence intensity of the organelle, indicating that it was moving
closer towards the plasma membrane, as seen in
Fig. 3B, where both sets of
data are plotted in time. During pauses between directed runs, we observed
complex behaviours of WPb, including rotations and oscillations (see Movies 3,
4;
http://jcs.biologists.org/supplemental/).
Rotations (e.g. Fig. 3C)
occurred in either clockwise or anticlockwise directions with the same average
frequency rot=0.268±0.036 rad/second
(n=5). In some cases, WPb exchanged their leading end without
reversing the direction of their motion, probably as a result of combined
rotation and translation. Oscillations
(Fig. 3D) occurred
preferentially during pauses and before direction reversals. The average
oscillation frequency was
osc=0.141±0.005
per second (n=6).
tPA vesicles also exhibited long-range motions. The frequency of long runs was greater than for WPb (2.2x10-3 per second), as were the average velocity, maximum velocity and average run length [0.67±0.07 µm/second, 1.48±0.13 µm/second and 8.2±1.1 µm (n=35), respectively]. As with WPb, the velocity of tPA vesicles often decreased when moving closer towards the plasma membrane (not shown).
Short-range diffusive-like motions
The majority (>70%) of WPb close to the plasma membrane undergo
diffusive-like short-range motions (e.g.
Fig. 4A). Plots of the
three-dimensional MSD as a function of the time interval t,
revealed three classes of diffusive-like behaviours: simple diffusion,
directed diffusion and restricted diffusion, occurring with approximately
equal frequency. MSD(
t) plots for individual WPb
(Fig. 4A) were fitted according
to Eqns 1-3 (see Materials and Methods). The diffusion coefficient D,
drift velocity v (directed diffusion), cage radius
Rcage and diffusion coefficient
Dcage (restricted diffusion) derived from these
fits are summarized in Table 1.
The averaged MSD plots are shown in Fig.
4B.
|
Similar results and orders of magnitude were obtained for tPA vesicles, except that in the case of caged diffusion, the cage was about five times more mobile than for WPb (data not shown).
Localization of the cytoskeleton and secretory organelles close to
the plasma membrane of HUVECs
In fixed cells, microtubules and actin filaments were found to extend into
the region illuminated by the evanescent field, coming closest in proximity to
the plasma membrane in peripheral regions of the cell
(Fig. 5). Actin formed stress
fibres with enhanced fluorescence at sites of focal adhesion contacts
(Fig. 5A). A more diffuse actin
staining was also visible over the cell footprint and was probably due to a
more random organization of short filaments in the actin cortex. Microtubules
were generally oriented radially, with tubulin fluorescence often greater on
peripheral microtubules, indicating that they come closer to the plasma
membrane in these regions (Fig.
5B). Quantification of dual-colour TIRF images (see Materials and
Methods) showed that 58.1±3.2% of WPb colocalized with microtubules
(n=15 cells) and 28.5±2.7% colocalized with actin fibres
(n=14 cells). Similar results were obtained for tPA-GFP vesicles
(data not shown). WPb clearly aligned with microtubules, particularly at the
cell periphery near microtubule plus-ends
(Fig. 5C). Superposition of
movies of WPb movements on images of the microtubule system after fixation
indicates that long-range motions of WPb frequently occur along microtubules
(Movies 5A,B;
http://jcs.biologists.org/supplemental/).
|
Long-range dynamics depend primarily on the microtubule
cytoskeleton
Microtubule disruption caused a significant decrease in the density (number
of organelles per µm2) of both populations of secretory
organelles (WPb: 16.9±7.0%, n=14; tPA vesicles:
39.8±14.7%, n=4), suggesting a role for the microtubule
cytoskeleton in the transport of both types of organelles to the cell
periphery. Disruption of microtubules almost completely abolished long-range
motions of WPb (Fig. 6A). In
nocodazole-treated cells, the frequency of WPb undergoing long-range motions
dropped from 1.0x10-3 to 8x10-5 per second
(Fig. 6B). For the few
remaining WPb undergoing long-range motion, the total displacement was just
above the arbitrary threshold for the definition of long-range motion, and the
average and maximum velocities of WPb were four times slower than in control
cells (Fig. 6B). Complex
dynamics such as oscillations and rotations seen in control cells were not
observed in nocodazole-treated cells. Long-range directed motions,
oscillations and rotations reappeared when microtubules repolymerized after
nocodazole washout (data not shown).
|
In the presence of the kinesin ATPase inhibitor aurintricarboxylic acid
(ATA) (Hopkins et al., 2000),
long-range motions were unidirectional and mostly directed towards the cell
centre (78.6%, n=14). Both the average and maximum velocities
significantly increased (P<0.04), whereas the total run length
decreased (Fig. 6B).
Disruption of actin filaments and inhibition of myosin motors by
2,3-butanedione monoxime (BDM) (Cramer and
Mitchison, 1995) had more subtle effects. Actin depolymerization
did not induce any significant change in organelle density for either WPb
(4.1±3.0%, n=4) or tPA vesicles (4.7±13.7%,
n=4). Latrunculin B and BDM treatments induced an increase in the
frequency of long-range motions of WPb and a decrease in the total run length
(Fig. 6B). The average velocity
increased significantly (P<0.04) on actin disruption and myosin
inhibition (Fig. 6B).
Qualitatively similar results were obtained for tPA-GFP vesicles, except that the average velocity in latrunculin B-treated cells was the same as in control cells (data not shown). Taken together, these results indicate that long-range motions are essentially microtubule-dependent, and that actin decreases their frequency and, in the case of WPb, their velocity.
Short-range diffusive motions are controlled by actin and microtubule
elements
Short-range motions of WPb were affected by actin and microtubule
depolymerization and by inhibition of kinesin and myosin motors (Figs
7,
8). We pooled data from all
three classes of diffusive-like behaviours (simple, directed and restricted
diffusion) (Fig. 7). On
treatment with nocodazole, the average diffusion coefficient decreased by a
factor 4.3, whereas it increased by a factor of 4.2 with latrunculin B
treatment (Fig. 7A). Effects of
cytoskeleton disruption also showed up on the averaged 3D MSD plots (obtained
by averaging all MSD plots together, as shown on
Fig. 7A, right panel). Blocking
kinesin motors activity by ATA induced a decrease in the diffusion coefficient
by a factor 2.7. By contrast, treatment with the myosin inhibitor BDM did not
change the value of the diffusion coefficient. Drug treatments had
qualitatively similar effects on the vertical diffusion coefficient
Dz (Fig.
7B). The z-direction averaged MSDz
plots from nocodazole- or latrunculin B-treated cells also clearly differed
from control cells (Fig. 7B,
right panel).
|
|
The distribution and characteristics of WPb diffusive motions in each class of diffusive-like behaviours (simple, directed and restricted diffusion) were affected by the disruption of microtubules or actin filaments (Fig. 8). Nocodazole treatment almost completely abolished restricted diffusive behaviours, and the proportion of directed diffusion increased compared with simple diffusion (Fig. 8A). The drift velocity v of directed diffusive motions did not change (not shown), whereas the diffusion coefficient of the cage (Dcage) and the cage radius (Rcage) strongly decreased (Fig. 8B). Latrunculin B treatment decreased the proportion of restricted diffusion in favour of simple diffusion. The drift velocity v increased by a factor of 2.5 (not shown), and the cage radius significantly increased (P<0.04) (Fig. 8B). Kinesin and myosin inhibition by ATA and BDM, respectively, did not significantly affect the distribution of diffusive motions among the three classes (Fig. 8A).
The effects of cytoskeleton disruption on the short-range motions of tPA vesicles differed in two respects from those on WPb movements (data not shown). Nocodazole treatment did not modify the average value of the diffusion coefficient and latrunculin B treatment slightly decreased the drift velocity of directed diffusion of tPA vesicles.
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Discussion |
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Immunohistochemical localization of WPb or tPA vesicles revealed a close association with microtubules (Fig. 5C and data not shown), and disruption of microtubules but not of the actin cytoskeleton decreased the density of WPb and tPA vesicles in close proximity to the plasma membrane. This indicates that a proportion of these organelles are transported to and/or maintained at the cell periphery via microtubule-dependent processes.
Actin interacts with microtubules during long-range transport of WPb
and tPA vesicles
WPb were seen to move continuously over distances of up to 20 µm (mean
run length approx. 7 µm). Long-range motions were saltatory and
bidirectional, with velocities in the range of 0.1-1.0 µm/second. These
characteristics are compatible with microtubule-driven motility observed in
vitro and in vivo (Howard,
2001). This was confirmed by the almost complete loss of
long-range motions when microtubules were disrupted by nocodazole
(Fig. 6). Moreover, nonspecific
inhibition of kinesin motors with the kinesin ATPase inhibitor
aurintricarboxylic acid (ATA) (Hopkins et
al., 2000
) strongly decreased the frequency of long-range motions
of WPb (Fig. 6B).
Microtubule-dependent transport of WPb to the plasma membrane is consistent
with the observation that microtubule disruption blocks vWF secretion
(Sinha and Wagner, 1987
;
Vischer et al., 2000
).
tPA vesicles moved three to four times faster than WPb and their run
lengths were, on average, longer by 1 µm. The motors responsible for the
motion of tPA vesicles and WPb may be different. However, another hypothesis
is that collective behaviours of motors control the motility of large
organelles (Welte et al.,
1998). Long-range transport of WPb may thus be slower due to pools
of microtubule motors with opposite polarity being active at the same time.
Indeed, blocking kinesin activity increased the velocity of WPb
(Fig. 6B), suggesting that
competition with dynein motors slows down motion on microtubules. The presence
of multiple motors may also give rise to the rotations and oscillations
displayed by WPb (Fig. 3C,D).
Oscillations frequently occurred during pauses or before direction reversals.
Bidirectionality and oscillations disappeared when kinesin ATPase activity was
blocked.
Actin and actin-based motors also play a role in the long-range motility of
WPb and tPA vesicles. Long-range motions of both organelles were more frequent
when actin was depolymerized, with about half the total run length. Similar
effects on the motion of WPb were observed when cells were treated with the
myosin ATPase inhibitor BDM (Cramer and
Mitchison, 1995). Disruption of the actin cytoskeleton or myosin
inhibition also increased the velocity of WPb
(Fig. 6B). These effects may be
due to a loss of a direct interaction with the actin cytoskeleton combined
with a reduction in physical restriction to movement imposed by friction of
the organelle in the viscous gel formed by the actin cortex. Pauses on
microtubules did occur preferentially at sites very close to the membrane,
where the actin cortex is thought to be denser. Actin and actin-based motors
may also prevent WPb from dropping off microtubules, thus increasing their
total run length. The finding that myosin motors slow down long-range motions
on microtubules supports a tug-of-war situation between actin and microtubule
motors (Gross et al., 2002a
;
Gross et al., 2002b
).
Restriction of short-range movements of WPb and tPA vesicles near the
plasma membrane
About 80% of WPb and tPA vesicles within the evanescent field appeared to
be almost immobile, undergoing short-range diffusive-like motions
(Fig. 4). The diffusion
coefficient D for WPb and tPA vesicles was of the order of
D10-4 µm2/second, as also found for
diffusive motions of chromaffin granules in chromaffin cells
(D
10-4-10-2 µm2/second)
(Oheim et al., 1999
;
Steyer and Almers, 1999
;
Steyer et al., 1997
), and PC12
cells (Han et al., 1999
;
Lang et al., 2000
). By
comparison, a 100 nm diameter bead diffusing in the cytoplasm would have a
diffusion coefficient 1000 times larger
(D=kT/(6
R)
10-1
µm2/second; taking the cytoplasm viscosity
6
water
6.10-3 kg/m/s). Thus,
diffusive motions of secretory organelles close to the plasma membrane are
severely restricted. The secretory organelles may be docked to the plasma
membrane or bound to cytoskeletal elements via complexes of molecular motors.
Their motion could also be physically but nonspecifically hindered by the
cortical actin gel.
The actin cytoskeleton and microtubules play opposing roles in
short-range motions of WPb and tPA vesicles close to the plasma membrane
In HUVECs, the 3D and vertical mobilities of WPb clearly increase following
actin depolymerization by latrunculin B
(Fig. 7). In other cell types,
conflicting results have been reported (Bi
et al., 1997; Johns et al.,
2001
; Lang et al.,
2000
; Oheim and Stühmer,
2000
). In the case of restricted diffusion, the diffusion
coefficient of the `cage' and the `cage' radius for both organelles increases
on actin depolymerization (Fig.
8B). The proportion of restricted diffusion is also reduced
compared with control cells (Fig.
8A), suggesting that actin not only decreases the mobility of the
organelles but also physically restricts their range of motion. Restricted
diffusion could thus represent motions within the cortical actin network
(Lang et al., 2000
), and the
increase in `cage' radius may reflect a widening of the network upon
latrunculin B treatment. Instead of acting as a barrier preventing the docking
of secretory organelles to the plasma membrane
(Johns et al., 2001
;
Rudolf et al., 2001
;
Valentijn et al., 1999
), the
actin cortex could actually promote docking and fusion of the organelles with
the plasma membrane by reducing their mobility and increasing the probability
of association of complementary docking complexes. Interestingly, blocking
myosin ATPase activity does not affect either the diffusion coefficient
(Fig. 7) or the proportion of
restricted diffusive motions compared with control cells
(Fig. 8A), further suggesting
that the cortical actin gel physically reduces organelle mobility at the
plasma membrane.
On microtubule depolymerization, we observed a decrease in three-dimensional and vertical diffusion coefficients (Fig. 7). The proportion of restricted diffusion behaviours is also strongly reduced compared with nontreated cells (Fig. 8A), suggesting that restricted diffusion may be due not only to physical hindrance by the actin cortex but also to a tethering of the organelles to microtubules via molecular motors. This is also consistent with the decrease in organelle density observed with nocodazole treatment. Interestingly, although actin plays a qualitatively similar role in the short-range mobility of tPA vesicles and WPb, we found that the role of microtubules was markedly different. The average diffusion coefficient of tPA vesicles was not modified when microtubules were depolymerized. As discussed above, a larger number of microtubule-based motors is probably recruited onto the surface of WPb, and this may increase their mobility. Consistent with this hypothesis, inhibition of kinesins by ATA reduces the average diffusion coefficient of WPb (Fig. 7) without affecting the distribution among the three classes of diffusive behaviours (Fig. 8A). These data show that microtubules increase the mobility of WPb near the plasma membrane, at least partly via the activity of kinesin motors.
Kinesin motors are supposed to act in the recruitment of secretory vesicles
during a step preceding an actin-dependent step
(Bi et al., 1997). Our results
show that short-range dynamics of WPb near the plasma membrane are regulated
not only by actin but also by microtubules and kinesin motors, indicating that
microtubules could also play a role in later stages, such as organelle docking
or fusion. Microtubules, probably via kinesin motors, counteract the confining
effect of actin by increasing short-range mobility of WPb at the plasma
membrane. Actin and microtubules may thus play opposing roles to fine-tune the
mobility and localize organelles at target sites on the plasma membrane.
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Acknowledgments |
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References |
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