 |
INTRODUCTION |
The cytoskeleton plays a pivotal role in activating
2 integrin molecules by restraining the integrin
molecules on the cell membrane in an inactivated state (1-3). Upon
stimulation by PKC1
activation, the cytoskeleton constraint is released and integrin is
activated. Both actin and microtubule filaments are involved in
constraining integrin molecules. The PKC signal is most likely transduced to cytoskeleton by MacMARCKS protein, a member of the MARCKS
(myristoylated alanine-rich protein kinase C substrate) family of PKC
substrates (4-6), because mutation or lack of this protein
prevents PKC-stimulated molecular mobility of integrin (2), as well as
ligand binding by integrin (7-9). In addition, MARCKS protein, the
homologue of MacMARCKS, has been shown to be involved in cell spreading
(10, 11). MacMARCKS protein contains an N-terminal membrane-targeting
domain that is myristoylated at its N-terminal glycine residue. The
effector domain, in the center part of the protein, contains the PKC
phosphorylation sites, two serine residues at positions 93 and 104.
Interaction between MacMARCKS and dynamitin, a subunit of dynactin, has
been observed (12). Dynactin (reviewed in Refs. 13-16), a complex of
proteins, is an important regulator of dynein, a
microtubule-dependent minus end-directed motor protein
(17). The dynactin complex has been suggested as a "molecular
bridge" connecting dynein to vesicle membranes (13, 18) and as an enhancement to the processivity of dynein (19). Overexpression of wild type dynamitin (20) or the addition of excess amounts of
recombinant full-length dynamitin (21) breaks the dynactin complex,
whereas overexpression of the conserved N terminus of dynamitin
is not sufficient to break the dynactin complex but is sufficient to disturb the Golgi and endosomes (22).
Because dynamitin overexpression dissociates dynein from the mitotic
kinetochore (20) and Golgi membrane (23), it has been proposed as a
link between dynein and its cargo.
However, interactions between motor proteins and membranes are usually
reversible, and one or more regulatory mechanisms may exist. The
interaction between dynamitin and MacMARCKS may aid the reversible
link, because MacMARCKS is a membrane-bound protein. Both the membrane
association of MacMARCKS and MacMARCKS-dynamitin interaction are
regulated by PKC-mediated phosphorylation (12, 24). This interaction
may provide a link between microtubules and cell spreading (12).
Interaction between MacMARCKS and dynamitin has been shown in
vitro by coimmunoprecipitation from total lysate (12). However,
whether these two proteins truly interact in living cells and the
spatial and temporal information of such interaction remain unknown.
Therefore, we decided to examine whether these two proteins are in
proximity in cells, using the fluorescent resonance energy transfer
(FRET) method.
FRET is a quantum mechanical phenomenon of radiationless energy
transfer from a donor molecule to an acceptor molecule via an
induced-dipole interaction. Two conditions must be satisfied for FRET
to occur. First, two fluorophores must have the proper spectral
overlap, i.e. the emission spectrum of the first fluorophore (donor) must overlap with the excitation spectrum of the second (acceptor) fluorophore. Second, the spatial distances and the relative
orientation of the two fluorophores' transition dipoles must be
sufficiently close. The efficiency of FRET depends on the inverse sixth
power of the distance R between the donor and acceptor.
Thus, the distance should not be more than 10 nm. By labeling proteins
with fluorophores, FRET can be used to determine the proximity of
two proteins. Cyan fluorescent protein (CFP) and yellow fluorescent
protein (YFP) greatly enhance the use of FRET. This pair of proteins
has been widely used to obtain information both about the existence of
FRET and the localization of FRET in live cells, including the
interaction of calmodulin and calmodulin-binding peptide (25), calpain
activation (26), and oligomerization (clustering) of
-factor
receptor (27).
In this paper, we report that when a chimera of MacMARCKS-YFP and a
chimera of CFP-dynamitin were expressed in RAW macrophages and in 293 epithelial cells, FRET was observed between these two proteins. When
phorbol ester was added to disrupt the interaction of these two
proteins (28), FRET disappeared. These data suggest that MacMARCKS and
dynamitin indeed interact in the cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
RAW macrophage cells and 293 epithelial cells
were purchased from ATCC (Manassas, VA). cDNA encoding MacMARCKS
(4) and dynamitin (12) were obtained as described. Plasmids pECFP-C1 and pEYFP-N1 were purchased from CLONTECH.
Prokaryotic expression vector pGEX-4T-2 and glutathione-Sepharose 4B
were purchased from Amersham Pharmacia Biotech. pProEX Hta was
purchased from Life Technologies, Inc. Rabbit anti-MacMARCKS antibody
was generated in collaboration with Dr. S. Slivka (Tenabe
Laboratory, La Jolla, CA) against His-MacMARCKS fusion protein
and was affinity-purified as described (29). Goat anti-dynamitin
antiserum was generated using purified GST fusion protein of dynamitin
in Ferrell Farms (Oklahoma City, OK). Other secondary antibodies were
purchased from Jackson Immunological Laboratory (West Grove, PA).
SuperFect transfection reagents were purchased from Qiagen. All other
routine chemicals were purchased from Sigma.
Expression of CFP-Dynamitin and MacMARCKS-YFP and Preparation of
Cells for FRET--
cDNA encoding MacMARCKS was amplified by
polymerase chain reaction with the 5'-primer, 5'-TCC TTC GGA TCC ATG
GGC AGC CAG AGC TCT AAG GCT-3', and the 3'-primer, 5'-GCC TTG GGA TCC
CAC TCA TTC TG-3', using wild type MacMARCKS in pcDNA II as
template (4). The primers contained a BamHI site for easy
cloning into the pEYFP-N1 upstream of YFP. The term "MacMARCKS-YFP"
indicates that YFP is at the C terminus of MacMARCKS. Dynamitin or its
mutant cDNA were amplified by polymerase chain reaction with the
5'-primer, 5'-GGA CTC GGA TCC CCG GAA TTC-3', and the 3'-primer, 5'-CTG
TTC TCC GGA TCC CAA ATG-3', using wild type dynamitin or
MacMARCKS-binding domain deletion (MBD) mutant in pGEX plasmid as
template (12). The primers contained a BamHI site for easy
cloning into pECFP-C1 downstream of CFP. The term "CFP-dynamitin"
indicates that the CFP is at the N terminus of dynamitin. The sequences
of all polymerase chain reaction products were confirmed before use.
The MacMARCKS plasmid (1 µg) and dynamitin plasmid (1 µg) described
above were cotransfected into 106 RAW cells or into 5 × 105 293 cells using the SuperFect liposome method as
described in the manufacturer's instructions (Qiagen). The RAW cells
were analyzed by FRET and Western blot 15 h after transfection,
and the 293 cells were analyzed by FRET and Western blot 20 h
after transfection.
Expression of Fusion Proteins in Escherichia coli--
cDNA
encoding CFP-dynamitin containing a blunt end at its 5' and
SalI sticky end at its 3' were inserted downstream of GST into pGEX-4T-2 cut with SmaI site at the upstream end and
XhoI at the downstream end. cDNA encoding MacMARCKS-YFP
containing EcoRI site at its 5' end and NotI at
its 3' end were inserted into pProEX HTa plasmid downstream of the His
tag by using matching restriction sites. After being transformed into
the DH5
strain, expression of the fusion proteins was induced by 0.1 mM isopropyl-1-thio-
-D-galactopyranoside at
30 °C for 3 h. The GST-CFP-dynamitin fusion protein was
purified using glutathione-Sepharose 4B beads as described (30). The His6 fusion protein of MacMARCKS-YFP was purified using
Ni/NTA-Sepharose as described in the manufacturer's manual (Qiagen).
In Vitro Binding between CFP-Dynamitin and
MacMARCKS-YFP--
The His6 fusion proteins of
MacMARCKS-YFP (2 µg each) were incubated with glutathione-Sepharose
(50 µl of 50% slurry) conjugated with GST fusion proteins of
CFP-dynamitin in 1 ml of phosphate-buffered saline in Eppendorf tubes
for 1 h at 4 °C. Meanwhile, glutathione-GST beads were used as
negative control. After three washes, Sepharose beads were transferred
to fresh tubes and washed again. The GST-dynamitin and the
dynamitin-bound MacMARCKS were then subjected to SDS-polyacrylamide gel
electrophoresis (9%) and transferred to an Immobilon membrane. MacMARCKS was detected by immunoblotting with the anti-MacMARCKS antibody. The GST-dynamitin fusion proteins and GST on the Sepharose beads were detected by Coomassie blue staining of the same membrane.
Spectrofluorometer Study of FRET--
The spectrofluorometric
study was carried out on an SLM 8000 spectrofluorometer. RAW cells
(5 × 106) were transiently transfected with both
CFP-dynamitin and MacMARCKS-YFP. After 20 h, the culture was
stimulated with or without 100 nM PMA for 15 min. The cells
were then scraped off the plate and lysed in 0.5% Triton X-100 for 5 min at room temperature. The emission spectra of CFP-dynamitin
and MacMARCKS-YFP in lysate were then measured using an excitation
wavelength of 425 nm, which was chosen to maximize the excitation of
CFP and minimize the direct excitation of YFP. The spectrofluorometer
was set at the ratio mode of 2 nm wavelength intervals, the emission
bandpass was 2 nm at 5 s integration.
FRET Measurement and Calculation--
With an ideal pair of
fluorophores, the emission spectrum of the donor should overlap largely
with the excitation spectrum of the acceptor and absolutely should not
overlap with the emission spectrum of the acceptor. At the same time,
the excitation spectrum of the acceptor absolutely should not overlap
with the excitation wavelength of the donor. However, CFP and YFP
showed certain overlapping spectra that needed to be corrected
(Fig. 1). We used three sets of filters in our FRET experiments. The
CFP filter set was for observing the location and intensity of
CFP-dynamitin: excitation filter 440 ± 21 nm, a dichroic beam
splitter of 455 nm DRLP, and an emission filter of 480 ± 30 nm.
The YFP filter set was for the MacMARCKS-YFP: excitation filter
500 ± 25 nm, a dichroic beam splitter of 525 nm DRLP, and an
emission filter of 545 ± 35 nm. The FRET was observed by exciting
cells at the CFP wavelength and detecting at the YFP emission
wavelength. Thus, the FRET filter set contained the excitation filter
at 440 ± 20 nm, a dichroic beam splitter of 455 nm DRLP, and an
emission filter of 535 ± 25 nm.
When cells was observed under the FRET filter set, a certain amount of
the CFP emission leaked through the FRET filter, and a certain amount
of YFP was directly excited by the FRET excitation wavelength (Fig.
1). To eliminate these two contributions,
the following calculation was used,
|
(Eq. 1)
|
where IFRET is the intensity of FRET with
the FRET filter set of CFP/YFP-coexpressed cells and
CCFP and CYFP are the
contributions from CFP and YFP, respectively, because of overlapping
spectra. The contribution of these two could not be directly measured
in cells coexpressing the YFP and CFP. However, they can be measured in
cells expressing only CFP or YFP. The contribution of YFP and CFP to
FRET in coexpressed cells is proportional to that in single expression
cells (31).
|
(Eq. 2)
|
|
(Eq. 3)
|
The right side of the equation can be obtained experimentally
and was determined to be 30.84% CFP and 13.97% YFP with our equipment. Thus,
|
(Eq. 4)
|
where ICFP is the intensity of CFP
observed under the CFP filter set in coexpressing cells and
IYFP is the intensity of YFP observed under the
YFP filter set in coexpressing cells.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 1.
A, diagrams showing cDNA constructs
used. These are MacMARCKS-YFP in pEYFP-N1, CFP-dynamitin in pECFP-C1,
CFP-MBD in PECFP-C1, His-MacMARCKS-YFP in pProEx HTa, and
GST-CFP-dynamitin in pGEX-4T-1. B, excitation and emission
spectra of CFP and YFP. The arrow indicates the excitation
wavelength used in spectral study of FRET.
|
|
In our experiments, the cells were cultured on coverslips overnight.
Fifteen hours after transfection with both cDNAs encoding CFP-dynamitin and MacMARCKS-YFP, a coverslip was placed face down in a
steel chamber on a heated stage in Dulbecco's modified Eagle's medium without serum, buffered with 20 mM HEPES at
pH 7.2. The bottom of the chamber was an 0.15-mm thick glass that
allowed the use of a 100× object lens. We first measured the FRET with the FRET filter, then the ICFP with the CFP
filter, and finally the IYFP with the YFP filter
under the exact same exposure time using a CCD camera (Optronic DE750).
The FRET was then calculated by
|
(Eq. 5)
|
To obtain information about the localization of FRET in a cell,
the FRET value of each pixel of the total 307,200 pixels was calculated
individually using a computer program written in-house and based on the
above formula.2 The
calculated FRET value was then plotted pixel by pixel so that a net
image of FRET could be presented.
In addition, because FRET intensity is a function of the expression
level of CFP and YFP, it is not possible to compare FRET intensity
between cells expressing different levels of CFP and YFP. To compare
FRET between cells, FRETN (normalized FRET) was introduced (31)
as follows.
|
(Eq. 6)
|
In a sense, FRETN has the same meaning as the equilibrium
constant in a chemical reaction.
|
(Eq. 7)
|
Therefore, the larger the FRETN, the higher the ratio of complex
between CFP-dynamitin and MacMARCKS-YFP.
 |
RESULTS |
In Vitro Binding between CFP-Dynamitin and
MacMARCKS-YFP--
Although a previous report has shown that
His6-tagged MacMARCKS binds to GST-tagged dynamitin on
glutathione-Sepharose beads (12), it was not known whether the
fluorescent tag would affect the binding between these two proteins. To
verify that attaching fluorescent protein to dynamitin and to MacMARCKS
would not affect their binding, we first assayed the binding between
purified the GST-CFP-dynamitin fusion protein the and His-MacMARCKS-YFP
fusion protein. We used the same method as previously described (12) except that this time a YFP was tagged at the C terminus of MacMARCKS and a CFP was inserted between the N terminus of dynamitin and the C
terminus of GST. Fig. 2 shows that the
His-MacMARCKS-YFP protein was specifically retained on the
GST-CFP-dynamitin beads but not on the GST beads, even though the GST
was used in excess amounts. Therefore, tagging fluorescent protein on
dynamitin and on MacMARCKS does not inhibit their ability to bind to
each other.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 2.
GST-CFP-dynamitin binds to
His-MacMARCKS-YFP. His-MacMARCKS-YFP was incubated with
Sepharose-4B beads conjugated with GST-CFP-dynamitin (A) or
GST only (B). After washing off unbound MacMARCKS, the beads
were boiled and SDS-polyacrylamide gel electrophoresis was performed
followed by Western blot with anti-MacMARCKS antibody. Only
GST-CFP-dynamitin bound MacMARCKS.
|
|
Expression of CFP-Dynamitin and MacMARCKS-YFP in RAW and 293 Cells--
Once we determined that CFP-dynamitin and MacMARCKS-YFP
interact in vitro, we then packaged the cDNA, encoding
each of them into the cytomegalovirus-driven expression vectors and
transiently expressed them in RAW cells. The expression levels
of CFP-dynamitin and MacMARCKS-YFP were determined both by
immunoblotting with antibodies against MacMARCKS and dynamitin (Fig.
3) and by surveying with a microscope for
cells expressing both blue and green fluorescence. By Western blot,
MacMARCKS-YFP in transfected cells migrated at a molecular mass
of about 67 kDa, roughly equal to the total of the apparent molecular
mass of MacMARCKS (42 kDa) and YFP (26 kDa). In untransfected cells, no
corresponding band was observed (Fig. 3). The cotransfected dynamitin
was also expressed in these cells; CFP-dynamitin (76 kDa) migrated at
the total apparent molecular mass of dynamitin (50 kDa) and CFP (26 kDa). Again, no corresponding band was seen in untransfected cells
(Fig. 3). With microscopy, we found that ~20% of the RAW cells
expressed both proteins. All transfected cells expressed both
fluorescent proteins, and we did not find any cells expressing only one
of the transfected constructs. With regard to the ratio of the two
proteins in the transfected cells, MacMARCKS-YFP was approximately
equal to CFP-dynamitin in RAW cells as determined by their fluorescent
intensity quantitated by a CCD camera.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Expression of CFP-dynamitin and MacMARCKS-YFP
fusion proteins in RAW cells. 2 × 106 RAW cells
were transfected with a mixture of MacMARCKS cDNA and dynamitin
cDNA (+cDNAs) as described under "Experimental
Procedures." The total lysate was subjected to SDS-polyacrylamide gel
electrophoresis followed by Western blot with antibodies against
MacMARCKS and dynamitin and compared with that of nontransfected cells
( cDNA).
|
|
When we attempted to culture the transfected cells for more than
2 weeks under the G418 selection pressure, all transfected cells
eventually died, a result that agreed with a report that overexpression
disrupts normal cell division (20).
Characterization of Fluorescent Spectrum of the FRET between
MacMARCKS-YFP and CFP-Dynamitin--
The FRET assay under a microscope
observes only the increase in YFP emission. To be sure that an
increased emission at the YFP emission peak indeed results from
authentic FRET, one must show a simultaneous decrease in CFP emission
and an increase in YFP emission. Therefore, we first examined the
fluorescent spectral properties of the
MacMARCKS-YFP·CFP-dynamitin complex using a spectrofluorometer. Because portions of these two proteins exist as a
complex in lysate from untreated cells but not in cells treated with
PMA (12), we therefore studied the MacMARCKS-YFP·CFP-dynamitin complex in RAW cells cotransfected with cDNAs encoding
MacMARCKS-YFP and CFP-dynamitin. These cells presumably contain a
mixture of unbound MacMARCKS-YFP, CFP-dynamitin, and bound
MacMARCKS-YFP·CFP-dynamitin.
Because the light scattering caused by intact cells in solution
interfered with the measurement, 1% Triton X-100 was used to dissolve
the cells, and the solution was measured for the emission spectrum
excited at 425 nm. The excitation wavelength was selected at the blue
side of the CFP excitation wavelength to avoid excessive cross-excitation of YFP (Fig. 1, arrow). Under this
condition, we observed an emission peak at 475 nm, which corresponded
to the CFP emission, and another peak at 527 nm, which corresponded to
YFP emission (Fig. 4). This emission at
YFP wavelength should represent a mixed emission of directly excited
YFP resulting from a slight overlapping of the YFP excitation
spectrum in 425 nm, emission of CFP at the YFP region from the
overlapped emission spectrum of CFP, and true FRET. If the interaction
between MacMARCKS and dynamitin is blocked, the FRET should
disappeared, which should translate to a decrease in the YFP emission
peak and an increase in the CFP emission peak. Therefore, we treated
the cells with 100 nM of PMA for 15 min before adding
Triton X-100, because PMA-promoted MacMARCKS phosphorylation decreases
the binding between these two proteins (12). Indeed, we observed an
~10% decrease in the YFP emission peak and a corresponding increase
in the CFP emission peak. These data indicate that the increase in YFP
emission observed using microscopic methods indeed resulted from
FRET.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Emission spectral analysis of FRET.
Cells cotransfected with CFP-dynamitin and MacMARCKS-YFP were treated
without (solid line) or with (dashed line) PMA.
After cells were lysed with Triton X-100, the lysates were
immediately subjected to emission spectral scanning using an SLM 8000 spectrofluorometer excited at 425 nm. PMA treatment caused an increase
in CFP emission; a decrease in YFP indicated a decreased FRET.
|
|
Microscopic Study of the Spatial and Temporal Characteristics of
FRET in Living Cells--
Next we examined the FRET of the transfected
cells under the microscope. The FRET and FRETN value of each cell were
represented by averaging the 30 areas at the size of 20 × 20 pixels with the highest FRETN value. Then the average FRET and FRETN
values of ~30 cells of each sample group were compared with those of
the negative control group (cells cotransfected with empty CFP and YFP
plasmid). In RAW cells, we found that the FRETN value of CFP-dynamitin and MacMARCKS-YFP-coexpressing cells was 67-fold greater than the
control group, which is a statistically significant difference (Table
I). To be sure that the positive FRET is
not limited only to RAW cells, we also examined 293 cells cotransfected
with wild type dynamitin and MacMARCKS. Similar data were obtained in
which the FRETN was 159-fold greater than its negative control (Table I).
View this table:
[in this window]
[in a new window]
|
Table I
FRET and FRETN comparison of different cells
The FRETN and FRET value in each row is an average of n cells. To
obtain the FRETN and FRET value of each cell, 30 areas (20 × 20 pixels) with the highest FRETN value within cell boundary were used.
The average FRETN and FRET of the 30 areas represents the FRETN and
FRET of this cell. For statistical analysis, each group of cell was
compared with the group transfected with CFP/YFP empty plasmids using
the standard t test provided in SigmaPlot software.
|
|
Because the directly observed image was a mixture of contributions from
FRET and from CFP and YFP, we calculated the FRET values pixel by pixel
and plotted them using in-house software. This process provided spatial
information about the interaction site in live cells. Non-PMA-treated
RAW cells maintained their spherical shape, and MacMARCKS-YFP
concentrated on the plasma membrane in these cells when compared with
the uniform distribution of CFP and YFP mock transfected cells (Fig.
5). Although CFP-dynamitin was primarily
distributed in the perinuclear region, the FRET was concentrated at the
cell periphery where MacMARCKS was located, thereby suggesting
that the portion of dynamitin interacting with MacMARCKS is that
located at the periphery (Fig. 5). Because the interaction between
MacMARCKS and dynamitin is regulated by PMA-stimulated phosphorylation
of MacMARCKS (12), we then tested whether PMA treatment also abolishes
the FRET observed in cells coexpressing CFP-dynamitin and
MacMARCKS-YFP. After being treated with 100 nm PMA for 15 min, the RAW
cells spread out. Most MacMARCKS-YFP was concentrated at the
perinuclear region with small residual amounts left on the plasma
membrane (Fig. 5). Although CFP-dynamitin was also concentrated at the
perinuclear region, no FRET was observed in the perinuclear region or
anywhere else in these cells. The FRETN values of these PMA-treated
cells were at the same level as the negative control groups (Table I).
This observation suggested that merely having two proteins colocalized
in the same subcellular localization was not sufficient to induce FRET.
Again, PMA also abolished the FRET between CFP-dynamitin and
MacMARCKS-YFP in 293 cells (Table I).

View larger version (71K):
[in this window]
[in a new window]
|
Fig. 5.
Localization of FRET in cells. RAW cells
transfected with CFP-dynamitin and MacMARCKS-YFP or with
CFP-MBD and MacMARCKS-YFP were treated with or without PMA.
Fluorescent images were taken with a CCD camera using CFP, YFP, and
FRET filter sets as described under "Experimental Procedures." The
net FRET values were calculated and replotted using software written
in-house.
|
|
To be sure that the FRET observed had indeed resulted from a true
protein-protein interaction and not from a random encounter between two
overexpressed proteins in a crowded space, we expressed the
MacMARCKS-binding domain deletion mutant of dynamitin (CFP-MBD) instead
of wild type dynamitin in RAW cells. Although MacMARCKS was still
concentrated at the plasma membrane in RAW cells and the mutant
dynamitin still at the perinuclear zone, the mutant dynamitin no longer
caused the FRET (Fig. 5), because the mutant dynamitin did not bind to
MacMARCKS (12). The FRETN values were extracted from these cells
coexpressing the mutant dynamitin and wild type MacMARCKS; the values
were close to those of the negative control (Table I). These data
provided convincing negative control to support the hypothesis that the
FRET between MacMARCKS and wild type dynamitin resulted from actual
protein-protein binding. Thus, we conclude that the interaction between
MacMARCKS and dynamitin indeed exists in cells and is subjected to the
regulation of MacMARCKS phosphorylation.
 |
DISCUSSION |
In this report, we have show that MacMARCKS and dynamitin occur in
proximity in the peripheral membrane in live macrophage cells and on
the perinuclear vesicles of live 293 cells. As required by the FRET
principle, these two molecules must be within 10 nm of each other for
FRET to occur. Considering in vitro binding data and
coimmunoprecipitation data supporting their binding (12), we conclude
that these two proteins indeed interact in cells. In addition, the
in vivo interaction is regulated by PMA-stimulated MacMARCKS
phosphorylation, because FRET no longer occurred after cells were
treated with PMA. These data correlate with previously reported data
that PMA regulates the direct binding of these two proteins in binding
assays with both purified proteins and coimmunoprecipitation.
Thus, this report provides the first evidence of in vivo
interaction of MacMARCKS and dynamitin in live cells. We can safely say
that these two proteins interact with each other in unstimulated macrophages near the cell periphery. During PMA-stimulated cell spreading, the localization of FRET at this site disappears. This observation also agrees with our finding that when the interaction of
these two proteins is inhibited, the cells spread (12). Thus, it seems
that the interaction is a mechanism of maintaining the cells in a
spherical shape. Because intact microtubule is required for the
maintenance of cell shape (32), microtubules must be anchored to
the plasma membrane or to the microfilaments beneath the plasma
membrane. We speculate that dynamitin may be such an anchor, because it
is linked to microtubules through a dynactin complex on one end and to
the plasma membrane through its interaction with MacMARCKS on the other
end. There are two possible ways for this anchor to mediate the
involvement of microtubules in cell spreading. One possibility is
integrin activation, which is the initial step of cell spreading (33).
Our recent work showed that the intact microtubule cytoskeleton is
indeed responsible for keeping integrin from being
activated.3 The intact
microtubule cytoskeleton could exert its influence by linking to
dynamitin through dynactin and dynein. Then interaction of dynamitin
with MacMARCKS translates the effect of the microtubule to the
integrin. This possibility is supported by reports that MacMARCKS is also required for integrin activation (2, 7-9). The
second possible mechanism for microtubules keeping cells in a spherical
shape is to interfere with focal adhesion. It is known that intact
microtubule cytoskeleton is required for the disassembly of focal
adhesion (34). Dynamitin-MacMARCKS may be responsible for anchoring the
microtubules to the focal adhesion. Because the assembly of focal
adhesion, which promotes cell spreading, and disassembly of focal
adhesion, which returns cells to spherical shape, are a balanced
process, when dynamitin mutation interferes with disassembly, cell
returns to a spherical shape.
All of these possible mechanisms are dynamic processes in living cells.
The FRET assay of the MacMARCKS and dynamitin interaction thus provides
us with a good tool for further studying these possible mechanisms of
the involvement of microtubules in cell spreading.