The Laboratory of Cellular Biophysics, The Rockefeller University, 1230 York Avenue, Box 304, New York, NY 10021, USA
* Author for correspondence (e-mail: simon{at}rockefeller.edu)
Accepted 22 November 2002
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Summary |
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Key words: Endocytosis, Clathrin, Dynamin, Cell migration, Total internal reflection fluorescence microscopy (TIR-FM), Evanescent-wave microscopy
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Introduction |
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Great strides have recently been made in the analysis of the different
factors involved in the production of clathrin-coated vesicles
(Takei and Haucke, 2001). The
presence and interaction of such components as the AP-2 adapter complex
(Benmerah et al., 1998
;
Kamiguchi et al., 1998
) and
the accessory proteins AP180 (Takei and
Haucke, 2001
), Eps15 (Benmerah
et al., 1998
; Benmerah et al.,
1999
) and Hip1R
(Engqvist-Goldstein et al.,
2001
) are beginning to be well characterized. Many of the proteins
that are involved in clathrin-mediated endocytosis have been identified and
cloned, their crystal structures have been determined
(ter Haar et al., 1998
;
Collins et al., 2002
), and
detailed models for the production and internalization of clathrin-coated
vesicles have been suggested (Schmid,
1997
; Takei and Haucke,
2001
; Kirchhausen,
2002
). However, numerous questions regarding the events and
interactions relevant to endocytosis remain unanswered. For example, it is not
clear whether activated receptors recruit AP-2 which results in the
polymerization of clathrin coat components, or if clathrin-coated pits are
preformed and recruit activated receptors. Additionally, the precise molecular
function(s) of the GTPase dynamin in endocytosis remains to be resolved
(Takei et al., 1995
;
Cao et al., 1998
;
McNiven et al., 2000
;
Ochoa et al., 2000
;
Schmid and Sorkin, 2002
;
Tsuboi et al., 2002
). Although
previous studies have focused on the neuron-specific dynamin1 isoform
(Tsuboi et al., 2002
), there
is also a functional role in endocytosis for the ubiquitously expressed
dynamin2. Dynamin2-enhanced green fluorescent protein (EGFP) colocalizes with
endogenous clathrin (Cao et al.,
1998
). Dominant negative mutants of dynamin1 and dynamin2 both
reduce endocytosis (Altschuler et al.,
1998
; Sun et al.,
2002
) and non-functional dynamin forms rings that wrap around the
neck of latent vesicles (Takei et al.,
1995
). These observations have led to the hypothesis that dynamin
associates with the neck of the budding vesicle to play a key role in membrane
fission.
The present studies have made use of simultaneous dual-color
total-internal-reflection fluorescence microscopy (TIR-FM) to characterize the
dynamics and interactions of components of the endocytosis machinery. In
TIR-FM, the fluorescence excitation is limited to a depth of 100 nm from
the cover slip. This both minimizes photodamage to the cell and maximizes the
signal over background of fluorophores at the cell surface
(Axelrod, 1981
;
Schmoranzer et al., 2000
;
Lampson et al., 2001
).
Previously, our laboratory has employed TIR-FM in the study of constitutive
exocytosis (Schmoranzer et al.,
2000
) and in an investigation of the endosomal recycling
compartment (Lampson et al.,
2001
). Using this approach to examine both the distribution of the
components of the endocytic machinery and the distribution of endocytic sites
allowed us to examine whether endocytosis is polarized or occurs uniformly in
migrating cells. Clathrin was expressed as plasma-membrane-associated spots,
which displayed apparently stochastic internalization. Whereas dynamin2, which
is endogenously expressed in these cells, colocalized with clathrin both prior
to and during internalization, the neuronal dynamin1 only appeared just prior
to the disappearance of the clathrin spots. Finally, contrary to our
expectations, clathrin-mediated endocytosis was polarized away from the
lagging edge of migrating cells, being concentrated towards the leading
edge.
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Materials and Methods |
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Cell culture and monolayer wounding assay
Madine Darby Canine Kidney (MDCK) cells were maintained in DMEM (Mediatech
Cellgro, VA) supplemented with 10% fetal bovine serum in a humidified 37°C
incubator with 5% CO2. Cells were plated onto sterilized glass
cover slips (Fisher Scientific, Atlanta, GA). When cells were transfected with
dsRed-clathrin, cells were plated at approximately confluence and transfected
during plating utilizing Lipofectamine 2000 (Invitrogen, Carlsbad, CA)
according to the supplier's directions. Cells co-transfected with
dsRed-clathrin and ECFP-Mem, or dsRed-clathrin and dynamin1-EGFP, were plated
between 75% and 95% confluence the day prior to transfection. In each case
where migrating cells were analyzed, when cells had reached or nearly reached
confluence, the monolayer was wounded with a scalpel; a circular region 1
cm in diameter was removed from the center of the monolayer. The morning after
wounding, some cells were microinjected with pEGFP-dynamin2 at 50 ng
µl-1 using continuous flow through the microinjection pipette.
Cells within the migrating front were imaged between 6 hours and 43 hours
after wounding.
Cell surface transferrin staining
Approximately 16 hours after wounding, cells were placed in serum-free DMEM
for 30 minutes in a 37°C incubator to chase out any cell-surface-bound
transferrin. Cells were then placed in Alexafluor488-transferrin (Molecular
Probes, Eugene, OR) diluted 1:100 in ice-cold PBS and incubated for 20 minutes
at 4°C. Finally, cells were rinsed once in PBS and fixed for 5 minutes in
4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA).
Image acquisition
TIR-FM was performed as previously described
(Schmoranzer et al., 2000;
Lampson et al., 2001
)
utilizing illumination through the microscope objective (Apo 60x NA
1.45; Olympus America, Melville, NY). All studies were performed with an
inverted epifluorescence microscope (IX-70, Olympus) placed within a
home-built temperature controlled enclosure set at 32°C for live cell
imaging. The optical configuration used to image dsRed-clathrin included
excitation with the 514 nm line of a tunable argon laser (Omnichrome, model
543-AP A01; Melles Griot, Carlsbad, CA) reflected off a polychroic mirror
(442/514pc). All filters and polychroic and dichroic mirrors were obtained
from Chroma Technologies (Brattleboro, VT). Emitted light was then collected
through a 560lp filter.
ECFP-Mem was imaged following excitation by the 442 nm line of a HeCd laser (Omnichrome, model 4056-S-A02), reflected off the same 442/514pc as above. The ECFP emission was collected utilizing a HQ485/40M band pass filter. Dynamin2-EGFP and Alexafuor488-transferrin were excited by the 488 nm line of the argon laser reflected off a dichroic mirror (498DCLP). EGFP and Alexafluor488 emission were collected through an emission band pass filter (HQ525/50M). When dsRed-clathrin and dynamin2-EGFP, or dsRed-clathrin and dynamin1-EGFP, were imaged simultaneously both fluorophores were excited with the 488 nm line of the same tunable argon laser as above reflected off the 498DCLP dichroic. Simultaneous image acquisition was performed utilizing an emission splitter (W-view, Hamamatsu Photonics, Hamamatsu City, Japan). The GFP/dsRed emissions were collected simultaneously through an emission splitter equipped with dichroic mirrors to split the emission (550DCLP). The GFP emission was then collected through an emission band pass filter (HQ525/50M) and the dsRed through an emission long pass filter (580lp). Fluorophore emission cross-talk from the green channel into the red and from the red into the green were both observed to be less than 1% in preliminary control experiments.
The camera utilized to acquire images was a 12-bit cooled CCD (ORCA-ER,
Hamamatsu Photonics, Bridgewater, NJ) with a resolution of 1280x1024
pixels (pixel size=6.45 µm x 6.45 µm). The camera and a mechanical
shutter (Uniblitz, Vincent Associates, Rochester, NY) were controlled by
MetaMorph (Universal Imaging, Downingtown, PA). Images were acquired utilizing
exposures times between 150 milliseconds and 300 milliseconds. For video
imaging, between 200 and 400 frames were streamed to memory on a PC during
acquisition and then saved to hard disk. The depth of the evanescent field was
typically 70-100 nm (Schmoranzer et
al., 2000
; Lampson et al.,
2001
). Analysis of video sequences and still frames was done with
MetaMorph and Excel (Microsoft, Redmond, WA). For the quantification of
dsRed-clathrin spot number and spot internalization a grid of squares (35
pixels x 35 pixels) was placed within each cell region and all of the
spots and disappearing spots within the squares were counted. Image noise was
reduced through background subtraction and digital brightness and contrast
adjustment in MetaMorph and Photoshop (Adobe Systems, San Jose, CA).
Dual-color processing
Dual-color image streams were acquired so that the separated channels
appear side by side on the camera chip. Regions of the same size were removed
from the whole field to yield separated image sequences. The two channels (GFP
and dsRed) were aligned by eye and then correlation coefficients were obtained
using automatic thresholding. For both
dsRed-clathrin/Alexafluor488-transferrin and dsRed-clathrin/dynamin2-EGFP
images, correlation coefficients were obtained following pixel shift of the
red image planes, one pixel at a time for ten pixels in each direction. Each
of the four resultant correlation coefficients for each pixel shift step was
then averaged.
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Results |
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|
In TIR-FM, the clathrin puncta showed similar behaviors to those previously
reported (Gaidarov et al.,
1999), including lateral movement
(Fig. 1B), disappearance
(Fig. 1C) and appearance or
formation (Fig. 1D; see
supplementary Movie 1,
http://jcs.biologists.org/supplemental).
However, TIR-FM permitted an analysis of the dynamics of clathrin motion and
disappearance at a level of sensitivity not previously attained
(Santini et al., 2002
).
Although most of the puncta were relatively static during the time course of
each sequence, a subpopulation underwent linear lateral movements parallel to
the plane of the membrane (Fig.
1B). The average lateral displacement quantified for sixteen
moving spots identified from three cells was 2.16±0.25 µm, and the
average lateral speed of these motile spots was 0.84±0.10 µm
second-1. This is greater than the speed of actin-based motility
for listeria (Goldberg, 2001
)
or vesicle-based movement (Merrifield et
al., 1999
) and is consistent with some of the rates of movement of
microtubules motors (Apodaca,
2001
) and myosin-V-based transport
(Tabb et al., 1998
).
The fluorescence of some clathrin puncta disappeared. In three cells, a
total of 1507 spots were counted and 237 of these disappeared within imaging
streams of 60.7±3.5 seconds. The average percentage of spots that
disappeared per stream was 14.4±5.4%. The average rate of disappearance
calculated for ten spots selected from the cell presented in
Fig. 1 was 17.5±4.1
seconds, although some spots disappeared in as little as 1.2 seconds
(Fig. 1E).
To characterize further the dynamics of dsRed-clathrin during endocytosis,
its fluorescence was monitored with either a second molecule involved in the
machinery of endocytosis (dynamin1 or dynamin2) or a cargo molecule
(transferrin). Transferrin has previously been used to study endocytosis in
MDCK cells, which are known to express functional transferrin receptor
endogenously (Fuller and Simons,
1986; Fialka et al.,
1999
). Cells were transfected with dsRed-clathrin and the cell
surface labeled with Alexafluor488-transferrin in the cold. Numerous
transferrin puncta (Fig. 2A)
colocalized with dsRed-clathrin (Fig.
2B). The colocalization of transferrin in dsRed-clathrin puncta
was quantified by the pixel shift analysis
(Fig. 2C). The correlation
coefficient between clathrin and transferrin was quantitatively reduced
following the deliberate misalignment of the two constituent single channel
images. The decrease in correlation coefficient caused by pixel shifting of
one channel in the overlay image demonstrates that the colocalization is not
due to the random alignment of isolated puncta. Additionally, the clathrin
fluorescence associated with a total of 150 transferrin spots (50 per cell)
was quantified and compared to the clathrin fluorescence within 150 regions of
equal size residing outside of transferrin spots
(Fig. 2D). This result confirms
the colocalization of clathrin and transferrin at the plasma membrane
(Fig. 2D), thus implicating
these puncta as functional in the clustering and endocytosis of substrates. A
population of clathrin spots did not contain appreciable transferrin
(Fig. 2). This suggests that
the contents of the cargo within each clathrin-coated pit might not be
identical.
|
Dynamin2 colocalizes with clathrin
(Takei et al., 1995) and
endocytosis is reduced in cells expressing mutant forms of dynamin2
(Altschuler et al., 1998
). To
examine the temporal dynamics of the interaction between clathrin and
dynamin2, we simultaneously imaged dsRed-clathrin
(Fig. 3A,D) and dynamin2-EGFP
(Fig. 3B,E). In
epifluorescence, there were some regions in which the two proteins colocalized
and others where they did not; in the juxtanuclear regions, there was a strong
dsRed-clathrin signal without an equivalent quantity of dynamin2
(Fig. 3A-C). However, in
TIR-FM, there were only plasma-membrane-based puncta of dsRed-clathrin, each
of which colocalized with dynamin2-EGFP
(Fig. 3D-F). As observed
between clathrin and transferrin (Fig.
2C), the colocalization of clathrin and dynamin2 was reduced
following pixel shift analysis (Fig.
3G). Additionally, the dynamin2-EGFP fluorescence present in 150
puncta from a total of three cells was observed to be linearly proportional to
the amount of dsRed-clathrin (Fig.
3H).
|
A number of the dsRed-clathrin/dynamin2-EGFP puncta were observed to disappear stochastically (Fig. 4A-D). In these puncta, the fluorescence of the dsRed-clathrin and the dynamin2-EGFP disappeared synchronously (Fig. 4), and the relative intensity of their fluorescence decreased proportionally in each punctum that disappeared (Fig. 4E). Therefore, it appears that the spatial correlation observed between clathrin and dynamin2 (Fig. 3) persists during the process of endocytosis.
|
The disappearance of the clathrin/dyanamin2 puncta could be the consequence of photobleaching, lateral movement or endocytosis. It is unlikely to be the consequence of photobleaching because the fluorescence intensity did not decrease in the regions surrounding the spots that disappeared. An example is shown in Fig. 4, where a punctum of clathrin (Fig. 4C) and dynamin (Fig. 4D) disappeared but the fluorescence in the neighboring region did not change. The decrease is unlikely to be the result of lateral motion, because the intensity in the area outside of the spots did not increase over time. Thus, the simultaneous disappearance of the clathrin and dynamin in the punctum is most like the result of an endocytic movement out of the plane of the evanescent field. Because the clathrin and dynamin2 are colocalized and are roughly proportional to each other both before and during endocytosis, there might be a relatively stable stoichiometric relationship between them.
The neuronal dynamin1 is 79% identical to dynamin2. To determine whether the two proteins behave similarly, MDCK cells were co-transfected with dsRed-clathrin and dynamin1-EGFP. As with dynamin2-EGFP, dynamin1-EGFP co-localized with many dsRed clathrin puncta (data not shown). However, in contrast to the behavior of dynamin2-EGFP (Fig. 4E), dynamin1-EGFP fluorescence increased just prior to internalization of dsRed-clathrin spots (Fig. 4F-H). This observation suggests that the behaviors of dynamin1 and dynamin2 prior to and during endocytosis are not identical, although the functional significance of this difference is not yet known.
The use of TIR-FM allows us to quantify the distribution of molecules involved in endocytosis (clathrin and dynamin1/2), endocytic cargo (transferrin) and endocytic events (the simultaneous disappearance of puncta of clathrin and dynamin). It has been proposed that endocytosis occurs at higher rates at the trailing edge of migrating cells. We used our techniques to assay whether there was a polarity of endocytic machinery, cargo or endocytic events in the basal membrane of MDCK cells during movement.
A monolayer of MDCK cells transfected with dsRed-clathrin was wounded with
a scalpel. Over a one-hour period, multiple cells expressing various levels of
dsRed-clathrin were observed by epifluorescence time-lapse microscopy to
migrate towards the area of monolayer wounding
(Fig. 5, supplementary Movie
2). Some shape changes were associated with the directed movement of these
cells, including the retraction of the lagging edge (see cell on the right
hand side of the field) as well as the extension of leading lamellae. The
migration velocity of these cells ranged between 10 µm hour-1
and 20 µm hour-1, similar to values derived from previous
studies of MDCK cell migration (Fenteany
et al., 2000; Sabo et al.,
2001
). MDCK cells expressing dynamin2-EGFP were observed to
migrate at the same rate in response to monolayer wounding (supplementary
Movie 3). These results indicate that MDCK cells migrate normally after
monolayer wounding despite the transient expression of dsRed-clathrin or
dynamin2-EGFP.
|
To quantify the distribution of endocytic machinery, cargo and fusion events, we imaged these cells with TIR-FM. At the start of each time-lapse series, an image was collected under epifluorescence, which was used to draw the cell boundaries and demarcate three regions (leading, middle and lagging) along the migratory trajectory of the cell (Fig. 6). Both the dsRed-clathrin signal (Fig. 6B) and the dynamin2-EGFP fluorescence (Fig. 6D) appeared to be weakest at the lagging edge and progressively stronger towards the leading edge of the migrating cells. Quantification of the relative fluorescence per unit area within the three regions of migrating cells revealed similar polarization of clathrin (Fig. 7A), dynamin2 (Fig. 7B) and transferrin (Fig. 7C) fluorescence from lowest values at the lagging edge to highest values at the leading edge. Thus, utilizing TIR-FM to evaluate the distribution of three markers for clathrin-mediated endocytosis revealed that each was concentrated away from the lagging edge.
|
|
The observation that the fluorescence of markers for clathrin-mediated
endocytosis was lowest at the trailing edge of migrating cells (Figs
6,
7) could be explained if the
trailing edge of the cells was not as close to the cover slip as the rest of
the cell (the excitatory field decreases exponentially with distance from the
cover slip). We tested this possibility by transfecting cells simultaneously
with dsRed-clathrin and a marker for the plasma membrane, palmitoylated
enhanced cyan fluorescent protein (ECFP-Mem)
(Jiang and Hunter, 1998). The
distribution of ECFP-Mem in TIR-FM images should reflect the contact zone
between the cell and the cover slip. In cells co-expressing ECFP-Mem
(Fig. 8A) and dsRed-clathrin
(Fig. 8B) ECFP-Mem was present
throughout the basal plasma membrane, whereas dsRed-clathrin was localized to
the cell middle and leading edge. Thus, the decreased dsRed signal at the
trailing edge is the result of less clathrin in this region and not a greater
distance between the plasma membrane and the cover slip. Calculation of the
relative dsRed-clathrin fluorescence per unit area divided by the relative
ECFP-Mem intensity per unit area clearly demonstrates that the polarized
distribution of dsRed-clathrin fluorescence is not due to variability in
distance between the plasma membrane and the cover slip throughout the cell
(Fig. 8C). Therefore, the
polarized distribution of dsRed-clathrin in the basal membrane is due to
differences in protein localization and not to differences in fluorophore
excitation.
|
The preceding results indicate that components of the endocytic machinery (clathrin and dynamin2) and an endocytic cargo (transferrin) are polarized in the plane of the plasma membrane towards the leading edge. To assay endocytic activity, we quantified the density of dsRed-clathrin puncta and frequency with which these puncta disappear. The density of dsRed-clathrin puncta (Fig. 8D) was highest in the leading edge and lowest in the trailing edge, similar to the distribution of the total dsRed-clathrin fluorescence (Fig. 7A, Fig. 8C). Furthermore, the frequency at which the dsRed-clathrin puncta disappeared was enhanced at the leading edge and deceased towards the lagging edge (Fig. 8E). These results suggest that clathrin-mediated endocytosis is polarized towards the leading edge in migrating cells, away from the lagging edge, in contrast to what had been expected.
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Discussion |
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The finding that the amount of dynamin2 associated with clathrin puncta is
linearly proportional to the amount of clathrin implies that there might be a
direct coupling between these two proteins in endocytic pits. It is possible
that the basis for the temporal, spatial and apparently stoichiometric
relationship between clathrin and dynamin2 could follow from the recently
proposed role for dynamin in actin organization
(McNiven et al., 2000;
Ochoa et al., 2000
;
Schmid and Sorkin, 2002
).
Although a role for dynamin in the remodeling of the actin cytosekeleton
during cell migration has been observed
(McNiven et al., 2000
), it is
not apparent whether this is based upon an affect on leading lamella
extension, guidance of transport vesicles through the cortical actin network
and/or actin based vesicle transport.
In addition to documenting the internalization of clathrin/dynamin2 spots
(Figs 1,
4), these results characterize
for the first time linear lateral motility of plasma membrane associated
clathrin puncta (Fig. 1).
Although some spots move laterally in the plane of the membrane and some are
internalized, a large majority of spots are stationary over the time periods
that were imaged (60 seconds). Until a simultaneous analysis of
receptor-ligand interactions and spot disappearance is performed, it will be
unclear to what extent preformed clathrin spots may wait for activated
receptors to cluster within them, or whether clathrin spots preferentially
form at the sites of clustered activated receptors and/or move laterally to
these same sites. Additionally, although the source of the motive force
responsible for the lateral motility of dsRed-clathrin spots is currently
unknown, the average rate measured (0.84±0.10 µm
second-1) is comparable to values previously measured for
microtubule motors (Apodaca,
2001
) and myosin-V-based transport
(Tabb et al., 1998
).
Although some of the data obtained in the present studies provide a clear
parallel with previous analyses of clathrin dynamics in living cells
(Fig. 1)
(Gaidarov et al., 1999), the
finding that the distributions of numerous markers for clathrin-mediated
endocytosis (clathrin, dynamin2 and transferrin) are polarized away from the
lagging edge in migrating cells (Figs
6,7,8)
represents a departure from models that suggest that cell migration requires
increased endocytosis near the trailing edge and increased exocytosis near the
leading edge (Bretscher, 1996
;
Palecek et al., 1996
;
Sheetz et al., 1999
). What,
then, is the role of endocytosis in cell migration? Is it to move and arrange
adhesion molecules or is it to reinternalize membrane and proteins functional
in membrane targeting and fusion (SNAREs etc.) following leading-edge
exocytosis? It is possible that the coupling of exocytosis and endocytosis at
the leading edge functions to adjust membrane tension to facilitate the
generation of motile force by actin polymerization
(de Curtis, 2001
;
Pollard et al., 2000
;
Watanabe and Mitchison, 2002
).
Alternatively, endocytosis at the leading edge might be important for the
internalization of chemokine, cytokine and growth factor receptors. These
questions await an integrated depiction of all of the constituent pathways
potentially involved in cell migration (signaling, exocytosis, endocytosis and
cytoskeletal organization).
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Acknowledgments |
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![]() |
Footnotes |
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After this manuscript was submitted, a study was published [Merrifield et al. (2002). Nat. Cell Biol. 4, 691-698] analyzing clathrin, dynamin1 and actin during endocytosis in 3T3 cells. Similarly to our results, they observed dyanamin1 associated with clathrin puncta just prior to endocytosis.
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschuler, Y., Barbas, S. M., Terlecky, L. J., Tang, K., Hardy,
S., Mostov, K. E. and Schmid, S. L. (1998). Redundant and
distinct functions for dynamin-1 and dynamin-2 isoforms. J. Cell
Biol. 143,1871
-1881.
Apodaca, G. (2001). Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton. Traffic 2,149 -159.[CrossRef][Medline]
Axelrod, D. (1981). Cell-substrate contacts illuminated by total internal reflection fluorescence. J. Cell Biol. 89,141 -145.[Abstract]
Bajno, L., Peng, X. R., Schreiber, A. D., Moore, H. P., Trimble,
W. S. and Grinstein, S. (2000). Focal exocytosis of
VAMP3-containing vesicles at sites of phagosome formation. J. Cell
Biol. 149,697
-706.
Benmerah, A., Bayrou, M., Cerf-Bensussan, N. and Dautry-Varsat,
A. (1999). Inhibition of clathrin-coated pit assembly by an
Eps15 mutant. J. Cell Sci.
112,1303
-1311.
Benmerah, A., Lamaze, C., Begue, B., Schmid, S. L.,
Dautry-Varsat, A. and Cerf-Bensussan, N. (1998). AP-2/Eps15
interaction is required for receptor-mediated endocytosis. J. Cell
Biol. 140,1055
-1062.
Bretscher, M. S. (1996). Moving membrane up to the front of migrating cells. Cell 85,465 -467.[Medline]
Cao, H., Garcia, F. and McNiven, M. A. (1998).
Differential distribution of dynamin isoforms in mammalian cells.
Mol. Biol. Cell 9,2595
-2609.
Carter, R. E. and Sorkin, A. (1998).
Endocytosis of functional epidermal growth factor receptor-green fluorescent
protein chimera. J. Biol. Chem.
273,35000
-35007.
Collins, B. M., McCoy, A. J., Kent, H. M., Evans, P. R. and Owen, D. J. (2002). Molecular architecture and functional model of the endocytic AP2 complex. Cell 109,523 -535.[Medline]
Conrad, M. E., Umbreit, J. N. and Moore, E. G. (1999). Iron absorption and transport. Am. J. Med. Sci. 318,213 -229.[Medline]
de Curtis, I (2001). Cell migration: GAPs
between membrane traffic and the cytoskeleton. EMBO
Rep. 2,277
-281.
Doxsey, S. J., Brodsky, F. M., Blank, G. S. and Helenius, A. (1987). Inhibition of endocytosis by anti-clathrin antibodies. Cell 50,453 -463.[Medline]
Engqvist-Goldstein, A. E., Warren, R. A., Kessels, M. M., Keen,
J. H., Heuser, J. and Drubin, D. G. (2001). The actin-binding
protein Hip1R associates with clathrin during early stages of endocytosis and
promotes clathrin assembly in vitro. J. Cell Biol.
154,1209
-1223.
Fenteany, G., Janmey, P. A. and Stossel, T. P. (2000). Signaling pathways and cell mechanics involved in wound closure by epithelial cell sheets. Curr. Biol. 10,831 -838.[CrossRef][Medline]
Fialka, I., Steinlein, P., Ahorn, H., Bock, G., Burbelo, P. D.,
Haberfellner, M., Lottspeich, F., Paiha, K., Pasquali, C. and Huber, L. A.
(1999). Identification of syntenin as a protein of the apical
early endocytic compartment in Madin-Darby canine kidney cells. J.
Biol. Chem. 274,26233
-26239.
Fuller, S. D. and Simons, K. (1986). Transferrin receptor polarity and recycling accuracy in `tight' and `leaky' strains of Madin-Darby canine kidney cells. J. Cell Biol. 103,1767 -1779.[Abstract]
Gaidarov, I., Santini, F., Warren, R. A. and Keen, J. H. (1999). Spatial control of coated-pit dynamics in living cells. Nat. Cell Biol. 1,1 -7.[CrossRef][Medline]
Goldberg, M. B. (2001). Actin-based motility of
intracellular microbial pathogens. Microbiol. Mol. Biol.
Rev. 65,595
-626.
Hopkins, C. R., Miller, K. and Beardmore, J. M. (1985). Receptor-mediated endocytosis of transferrin and epidermal growth factor receptors: a comparison of constitutive and ligand-induced uptake. J. Cell Sci. 3 (Suppl.),173 -186.
Jiang, W. and Hunter, T. (1998). Analysis of cell-cycle profiles in transfected cells using a membrane- targeted GFP. Biotechniques 24,349 , 350, 352, 354.[Medline]
Kamiguchi, H. and Lemmon, V. (2000). Recycling
of the cell adhesion molecule L1 in axonal growth cones. J.
Neurosci. 20,3676
-3686.
Kamiguchi, H., Long, K. E., Pendergast, M., Schaefer, A. W.,
Rapoport, I., Kirchhausen, T. and Lemmon, V. (1998). The
neural cell adhesion molecule L1 interacts with the AP-2 adaptor and is
endocytosed via the clathrin-mediated pathway. J.
Neurosci. 18,5311
-5321.
Kirchhausen, T. (2002). Clathrin adaptors really adapt. Cell 109,413 -416.[Medline]
Lamaze, C. and Schmid, S. L. (1995). Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase. J. Cell Biol. 129, 47-54.[Abstract]
Lampson, M. A., Schmoranzer, J., Zeigerer, A., Simon, S. M. and
McGraw, T. E. (2001). Insulin-regulated release from the
endosomal recycling compartment is regulated by budding of specialized
vesicles. Mol. Biol. Cell
12,3489
-3501.
McNiven, M. A., Kim, L., Krueger, E. W., Orth, J. D., Cao, H.
and Wong, T. W. (2000). Regulated interactions between
dynamin and the actin-binding protein cortactin modulate cell shape.
J. Cell Biol. 151,187
-198.
Merrifield, C. J., Moss, S. E., Ballestrem, C., Imhof, B. A., Giese, G., Wunderlich, I. and Almers, W. (1999). Endocytic vesicles move at the tips of actin tails in cultured mast cells. Nat. Cell Biol. 1,72 -74.[CrossRef][Medline]
Ochoa, G. C., Slepnev, V. I., Neff, L., Ringstad, N., Takei, K.,
Daniell, L., Kim, W., Cao, H., McNiven, M., Baron, R. et al.
(2000). A functional link between dynamin and the actin
cytoskeleton at podosomes. J. Cell Biol.
150,377
-389.
Palecek, S. P., Schmidt, C. E., Lauffenburger, D. A. and
Horwitz, A. F. (1996). Integrin dynamics on the tail region
of migrating fibroblasts. J. Cell Sci.
109,941
-952.
Pollard, T. D., Blanchoin, L. and Mullins, R. D. (2000). Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu. Rev. Biophys. Biomol. Struct. 29,545 -576.[CrossRef][Medline]
Raub, T. J. and Kuentzel, S. L. (1989). Kinetic and morphological evidence for endocytosis of mammalian cell integrin receptors by using an anti-fibronectin receptor beta subunit monoclonal antibody. Exp. Cell Res. 184,407 -426.[Medline]
Sabo, S. L., Ikin, A. F., Buxbaum, J. D. and Greengard, P.
(2001). The Alzheimer amyloid precursor protein (APP) and FE65,
an APP-binding protein, regulate cell movement. J. Cell
Biol. 153,1403
-1414.
Santini, F., Gaidarov, I. and Keen, J. H.
(2002). G protein-coupled receptor/arrestin3 modulation of the
endocytic machinery. J. Cell Biol.
156,665
-676.
Schmid, S. L. (1997). Clathrin-coated vesicle formation and protein sorting: an integrated process. Annu. Rev. Biochem. 66,511 -548.[CrossRef][Medline]
Schmid, S. L. and Sorkin, A. D. (2002). Days and knights discussing membrane dynamics in endocytosis: meeting report from the Euresco/EMBL Membrane Dynamics in Endocytosis, 6-11 October in Tomar, Portugal. Traffic 3,77 -85.[CrossRef][Medline]
Schmoranzer, J., Goulian, M., Axelrod, D. and Simon, S. M.
(2000). Imaging constitutive exocytosis with total internal
reflection fluorescence microscopy. J. Cell Biol.
149, 23-32.
Sheetz, M. P., Felsenfeld, D., Galbraith, C. G. and Choquet, D. (1999). Cell migration as a five-step cycle. Biochem. Soc. Symp. 65,233 -243.[Medline]
Sieczkarski, S. B. and Whittaker, G. R. (2002).
Dissecting virus entry via endocytosis. J. Gen. Virol.
83,1535
-1545.
Sun, T. X., van Hoek, A., Huang, Y., Bouley, R., McLaughlin, M.
and Brown, D. (2002). Aquaporin-2 localization in
clathrin-coated pits: inhibition of endocytosis by dominant-negative dynamin.
Am. J. Physiol. Renal Physiol.
282,F998
-F1011.
Tabb, J. S., Molyneaux, B. J., Cohen, D. L., Kuznetsov, S. A.
and Langford, G. M. (1998). Transport of ER vesicles on actin
filaments in neurons by myosin V. J. Cell Sci.
111,3221
-3234.
Takei, K. and Haucke, V. (2001). Clathrin-mediated endocytosis: membrane factors pull the trigger. Trends Cell Biol. 11,385 -391.[CrossRef][Medline]
Takei, K., McPherson, P. S., Schmid, S. L. and de Camilli, P. (1995). Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals. Nature 374,186 -190.[CrossRef][Medline]
ter Haar, E., Musacchio, A., Harrison, S. C. and Kirchhausen, T. (1998). Atomic structure of clathrin: a beta propeller terminal domain joins an alpha zigzag linker. Cell 95,563 -573.[Medline]
Tsuboi, T., Terakawa, S., Scalettar, B. A., Fantus, C., Roder,
J. and Jeromin, A. (2002). Sweeping model of dynamin
activity. Visualization of coupling between exocytosis and endocytosis under
an evanescent wave microscope with green fluorescent proteins. J.
Biol. Chem. 277,15957
-15961.
Watanabe, N. and Mitchison, T. J. (2002).
Single-molecule speckle analysis of actin filament turnover in lamellipodia.
Science 295,1083
-1086.