Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany
Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones. Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous cross talk between the cell surface and underlying cytoskeleton. The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed. Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast with the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin dependent. The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface. With their high proportion of binucleated cells, the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells.
THE surface of a motile cell receives signals upon
contact with another surface, and transmits these
signals through the plasma membrane to accommodate activities of the underlying actin system. The array of
actin filaments, which acts as a target, is furnished with
myosin motors of various types, as well as with other proteins that regulate actin polymerization or the cross-linkage and connection of actin filaments with the plasma
membrane. The actin system is not only a target of signals
arising in the environment of a cell; it modulates in turn
the interactions of a cell with an extracellular matrix or
with another cell. One of the proteins that link the plasma
membrane to the actin skeleton is talin (Burridge and
Connell, 1983 Human platelet and chicken gizzard talins are considered to be antiparallel homodimers (Goldmann et al.,
1994 Evidence that talin plays a major role in the interplay
between membrane proteins and the submembraneous
cytoskeleton has been accumulated. Talin is enriched in
phagocytic cups during FC receptor-mediated particle uptake (Greenberg et al., 1990 Cells of Dictyostelium discoideum interact during their
life cycle with three types of surfaces: with bacteria or
other microorganisms that are phagocytosed as a food,
with substrates on which the cells are ready to move, and
with cells of the same species to build a multicellular organism by aggregation. The highly motile cells of this microorganism are distinguished from fibroblasts by the absence of stress fibers and adhesion plaques that would stabilize cell shape and substrate interaction. D. discoideum is the first nonmetazoan known to contain a full-length talin homologue. In undifferentiated cells, this 269-kD
talin homologue is strongly accumulated at the tips of filopods, and in aggregating cells it accumulates at the leading
edge in a chemoattractant-controlled manner (Kreitmeier
et al., 1995 With the possibility in mind that Dictyostelium cells
make less specialized and less complicated adhesion complexes than fibroblasts, we have eliminated talin in D. discoideum by gene replacement. The talin-null cells moved
with small patches of their surface loosely adhering to a
substrate, indicating that cell motility is essentially independent in Dictyostelium of traction forces or of a gradient
in substrate adhesiveness established between the front
and tail of a cell. The absence of talin markedly affected the first steps of phagocytosis in cell suspensions where
particle attachment to the cell surface was critical for uptake. A slight impairment of cytokinesis observed in talin-null cells indicated that talin assists in the changes of cell
shape that lead to the separation of daughter cells.
Growth and Development of D. discoideum Cells
For axenic growth, D. discoideum wild-type strain AX2 and talin-null mutants were cultivated in shaken suspension at 150 rpm in liquid nutrient
medium at 23°C as described by Claviez et al. (1982) For growth in bacterial suspensions, Escherichia coli B/r or Salmonella
minnesota R595 were cultivated overnight in standard I nutrient broth
(Merck, Darmstadt, Germany), washed, and adjusted to a density corresponding to 1010 E. coli B/r cells per ml. The suspensions of washed bacteria were inoculated with 104-105 cells of D. discoideum per ml. For cultivation on agar surfaces, cells of E. coli B/r were spread on agar plates
containing 0.1% peptone and 0.1% glucose in non-nutrient buffer. AX2
or talin-null cells were picked onto the surface for measuring radial
growth.
Experiments shown in Fig. 3 were performed with the talin-null mutant
strain HG1664 and repeated with HG1666. Experiments in Figs. 2, 4, 5,
and 6 were performed with the talin-null strain HG1666 and repeated with
HG1663 (see Fig. 2), HG1664 (see Fig. 4, B/r suspension), or HG1665 (see
Figs. 5 and 6). The micrographs of cytokinesis and immunofluorescence
images of Fig. 7 were obtained with mutant HG1665.
Gene Replacement in D. discoideum
The targeting vector was constructed by inserting the Bsr cassette from
plasmid pBsr2 (Sutoh, 1993 For Southern blotting, genomic DNA was prepared according to Noegel et al. (1985) Monoclonal Antibodies
mAb 477 against D. discoideum talin was produced by Kreitmeier et al.
(1995) SDS-PAGE and immunoblotting with anti-talin antibodies was performed in 3-20% gradient polyacrylamide gels. mAb 477 was directly 125I-labeled, and mAb 341 was detected with iodinated sheep anti-mouse IgG
(Amersham Intl.). For detection of the csA glycoprotein on Western
blots, mAb 294 was used (Bertholdt et al., 1985 Reflection Interference Contrast
Microscopy/Bright-Field Double-View Microscopy
and Chemotactic Stimulation
Aggregation-competent Dictyostelium cells were monitored while migrating on the surface of a glass coverslip preincubated with 0.2% BSA solution (Serva, Heidelberg, Germany). The topography of the ventral cell
surface, including cell-to-substratum contacts, was imaged by reflection
interference contrast microscopy (RICM)1 using a double-view microscope (Weber et al., 1995 Cell Agglutination and Endocytosis Assays
Agglutination of wild-type and talin-null cells was assayed by measuring
light scattering according to Beug and Gerisch (1972) Analysis of Cytokinesis
For synchronization, cells from suspension cultures were washed and
shaken in non-nutrient buffer for 3 h, before they were allowed to adhere
for 30 min on glass coverslips. Subsequently, the buffer was replaced by
nutrient medium and the culture continued for 4 h (Neujahr et al., 1997 Knockout of a Talin Homologue in Dictyostelium
To inactivate the gene that encodes the Dictyostelium talin
previously described by Kreitmeier et al. (1995)
In all of the eight mutants obtained, the vector was selectively inserted into the talin gene; no second insertion
into other regions of the genome had occurred. In Fig. 1, B
and C, data for four of these mutants are shown. EcoRV
and BclI cut the gene at both sides of the inserted bsr cassette. When genomic DNA of the mutants was digested
with either one of these two enzymes and probed with a
sequence stretch of the talin gene downstream of the vector sequence, single bands were labeled. These bands indicated an increase in size by 1.4 kb relative to the wild-type
fragments, an upshift that corresponds to the size of the
bsr cassette (Fig. 1 B, upper panels). Using as a probe the
bsr coding region of the vector, the same single bands
showed up in the mutants. As expected, no hybridization
signal was seen with wild-type DNA (Fig. 1 B, lower panels).
Western blots of total cellular proteins separated by
SDS-PAGE were probed with two antibodies, one binding
COOH-terminally to the predicted site of disruption of
the talin polypeptide, the other recognizing an epitope at
the NH2-terminal side of the disruption (Fig. 1 A). The two
antibodies no longer detected the talin band in the eight
independent mutants, nor did the latter antibody recognize in the mutants any NH2-terminal fragment of this protein that might originate from a truncated message (Fig. 1 C).
Impairment of Cell-to-Substrate Adhesion and the
Chemotactic Response of Talin-Null Cells
A phenotypic alteration immediately obvious in the mutant cells was their weak adhesion to the polystyrol surfaces of plastic petri dishes, as revealed by their detachment upon gentle pipetting. To study this adhesion defect,
alterations in the interaction of talin-null cells with a substrate surface were analyzed by RICM. Areas of close cell-to-substrate contact are visualized by RICM as dark areas
(Gingell and Todd, 1979 Glass coated with BSA proved to be of optimal adhesiveness for the motility of D. discoideum cells. This substrate allows wild-type cells to attach and detach in a regular pattern (Weber et al., 1995 Talin-null cells had almost completely lost adhesiveness
to BSA-coated glass. This is seen in RICM images by the
absence of dark areas of contact in the center of interference patterns (Fig. 2 B). The finding that the area of closest approximation between the cell and substrate surfaces
was regularly bright means that the distance was in the order of the first maximum of Aggregation-competent wild-type cells are highly responsive to cAMP as a chemoattractant, and cell-to-substrate interactions are highly dynamic during reorientation. The cells turn into a new direction by bending their
established front or by extending a new front into the direction of the gradient. At the beginning of a chemotactic
response, the front does not need to be in contact with the
substrate, and sometimes two fronts compete with each other for becoming the leading edge (Weber et al., 1995 Talin-null cells efficiently responded to cAMP by turning toward the source. Stimulation with the chemoattractant also slightly enhanced interaction with the substrate,
so that grayish foci of attachment were seen in RICM images. However, the mutant cells were impaired in spreading and fusion of the contact areas (Fig. 2 D). Foci of attachment increased only marginally in size; often they
were fixed in a position essentially stationary on the substrate, with the cells gliding ahead of these local sites of
loose attachment. Most of the foci of cell-to-substrate contact in talin-null cells vanished when they had passed the
middle of a cell. Some of the patches existed for up to 2 min, whereas others were short-lived and never reached
the middle region of the cell.
A Conditional Defect in Phagocytosis Due to Reduced
Cell-to-Particle Adhesion
Consequences of the adhesion defect in talin-null cells for
particle uptake were assayed using heat-killed, fluorescently labeled yeast particles in shaken suspension. At a
shaking frequency of 150 rpm, the rate of phagocytosis in
talin-null cells was ~10% the rate in wild-type AX2 cells
(Fig. 3 A). Assuming that this reduction in the rate of particle uptake is a consequence of impaired adhesion, one
would predict a higher rate of uptake at conditions of low
shear. Fig. 3 B shows that reducing the frequency of shaking from 150 to 100 rpm increased substantially the rate of
particle uptake in talin-null cells and only moderately increased it in wild-type cells.
A particle adhering to a Dictyostelium cell triggers an
extension of the cell surface that spreads as a cup-shaped
lamella over the particle and finally engulfs it (Maniak et
al., 1995 Surface Properties of Bacteria Critical for their
Adhesion to Talin-Null Cells
Bacteria that differ in the carbohydrate moieties of their
surface lipopolysaccharides are convenient probes to evaluate the role of adhesion in phagocytosis. When fixed in a
lawn on an agar plate, a large variety of E. coli or Salmonella strains can support growth of Dictyostelium cells, independently of the chemical nature or physical properties
of the bacterial surfaces. If applied in shaken suspension,
adhesion of the bacteria to the phagocyte surface becomes
crucial for uptake, since a bacterium needs to adhere until
the phagocyte has produced a cup to envelop and eventually to engulf the bacterium. As far as wild-type cells of D. discoideum are concerned, the uptake of bacteria in suspension is primarily determined by surface lipopolysaccharides. Long polysaccharide chains prevent uptake by
the lack of adhesion. It suffices to coat these carbohydrate
chains with antibodies to render the bacteria appropriate
for uptake in suspension (Gerisch et al., 1967 E. coli B/r is commonly used for the efficient growth of
D. discoideum wild-type cells in shaken suspension. The
carbohydrate chains on the surface of this E. coli strain are
reduced to a glucose-containing core oligosaccharide (Malchow et al., 1967 To provide evidence that the core oligosaccharide present
on the surface of E. coli B/r interfered with adhesion to
talin-null cells, we used Salmonella minnesota R595, a
strain appropriate for D. discoideum suspension cultures
(Malchow et al., 1967 The EDTA-sensitive Type of Cell-to-Cell Adhesion Is
Talin Dependent
In suspension, undeveloped wild-type cells of D. discoideum aggregate into loose clusters. The cohesion of these
cells is distinguished from that of aggregating cells by its
EDTA sensitivity (Beug et al., 1973 Fig. 5 A shows the behavior of undeveloped cells: when
an equilibrium was reached after <1 h, light scattering in
suspensions of wild-type cells was low in the absence and
high in the presence of EDTA, whereas in suspensions of
talin-null cells it was high both in the presence and absence of EDTA. The photographs of Fig. 5 B illustrate
these quantitative data, indicating that the EDTA-sensitive cohesion of undeveloped cells is talin dependent.
In the aggregation-competent stage, talin-null cells adhered to each other in an EDTA-stable manner (Fig. 5 C),
and the aggregates formed in suspension were only slightly
smaller than those formed by wild-type cells (Fig. 5 D).
Cytokinesis Is Conditionally Impaired in
Talin-Null Cells
The initial observation that talin-null cells become larger
than wild-type cells when cultivated in suspension suggested a deficiency in cytokinesis. To establish this by
quantitative data, numbers of nuclei were counted in cells
that were cultivated either in suspension or on a glass surface. In suspension cultures, the majority of talin-null cells
contained two to four nuclei, whereas wild-type cells were
primarily mononucleated (Fig. 6, top). Under the same
conditions, the average volume of talin-null cells was 1.5-fold that of wild-type cells. These results indicate that, in
talin-null cells undergoing mitosis in suspension, cytokinesis fails to be reliably coupled to nuclear division. This deficiency was less distinct when cells grew on a solid surface
(Fig. 6, middle), and it was not at all evident when the liquid medium was replaced as a source of nutrients by a
lawn of bacteria on an agar surface (Fig. 6, bottom).
The finding that only a few of the talin-null cells acquired more than four nuclei in suspension culture with
nutrient medium suggests a process that counterbalances
the increase in nuclear number caused by the slight impairment of cytokinesis. To characterize this process, cytokinesis was recorded in bi- or multinucleated talin-null
cells. Fig. 7 A illustrates mitosis in a binucleated cell that
divided symmetrically into four daughter cells. In fixed preparations, mitotic stages of binucleated cells were characterized by the presence of two spindles and four asters
of microtubules, which held variable positions relative to
each other in the three-dimensional space of the cell body.
In the three cells shown in Fig. 7 B, the two mitotic complexes, each consisting of a pair of asters connected to a
spindle, were arranged in the same plane. These images illustrate most clearly that only two of the four cleavage furrows that are indistinguishable in Fig. 7 A are directed toward the middle of the spindles, the other pair incising the
cell body in between the mitotic complexes.
In addition to binucleated cells, cells that contained
three or four nuclei were found to form multiple spindles.
It appears therefore that the increase in nuclear number is
limited in talin-null cells by mitotic division cycles that
give rise to more than two daughter cells. This type of
cleavage is based on the shaping of cleavage furrows
around the asters of microtubules.
Differences in the Talin Dependency of Cell-to-Cell
Adhesion Systems
Dictyostelium has been the first developmental system in
which two types of cell-to-cell adhesion, an EDTA-stable
and a sensitive one, could be distinguished (Gerisch, 1961 In vertebrate cells talin interacts with integrins, which
link the cell surface to receptors on the extracellular matrix (Beckerle and Yeh, 1990 Whether or not Dictyostelium has a "protointegrin" on
its surface is not only of phylogenetic interest but is also
relevant for functional reasons. Dictyostelium cells manage in their natural habitat to move on soil particles of
varying chemical nature and physical surface properties.
The question is whether Dictyostelium cells have a chance
in their natural environment to use specific molecular interactions in regulating their adhesiveness to soil particles
or to a variety of bacterial surfaces. There are several possibilities of how coupling of talin to a membrane protein
regulates cell adhesion. (a) Adhesion might be regulated
by clustering of the membrane protein, as it is known for
integrins in other cells. (b) Some selectivity might be involved in the interaction of the putative adhesion protein
with the surface of soil particles, analogous to the stereospecificity that determines attachment of Xenopus kidney cells to the surfaces of (R, R) and (S, S) enantiomorphous tartrate crystals (Hanein et al., 1994 Cell Motility with a Minimum of Substrate Contact
The motility of fibroblasts or keratocytes depends on traction forces that these cells exert on their substrate (Oliver
et al., 1994 For the movement of talin-null cells on small patches of
contact with a substrate, the following possibilities can be
considered. First, loose adhesion might induce a local response, e.g., coupling of a membrane patch immobilized
on the substrate to one of the myosins, so that this patch is
driven in the membrane in a rearward direction (Jay and
Elson, 1992 Mutants to Dissect Phagocytosis
Phagocytosis mutants may be classified into four major
groups: (a) those deficient in the adhesion of particles to
the phagocyte surface, (b) mutants altered in signal transduction from the cell surface to the cortical cytoskeleton,
(c) cytoskeletal mutants impaired in the mechanism of
particle uptake, and (d) mutants in proteins that play a
role in later steps of the phagocytic pathway. To begin
with the third category, a series of gene disruption mutants
has been generated in Dictyostelium to eliminate proteins associated with the actin system. These proteins include
coronin, which accumulates together with actin early at
the phagocytic cup (Maniak et al., 1995 On the basis of the following results, we propose that
the talin-null cells are impaired in the initial adhesion to a
particle and in subsequent spreading on its surface. The
rate at which yeast particles are taken up by talin-null cells
in shaken suspension can be markedly increased by reducing the frequency of shaking (Fig. 3 B). This inverse dependence of uptake on the strength of shear indicates that
the average persistence time of a particle at the phagocyte
surface is shorter than the time a cell would need to irreversibly entrap the particle. Extreme differences in the uptake rate of bacteria that differ in their surface properties provide a second argument for an impairment of adhesiveness in talin-null cells (Fig. 4). Fluid-phase uptake shows
that talin-null cells can form surface extensions and convert them into macropinosomes large enough to include a
bacterium (Fig. 3 D). In suspensions of 1010 bacteria per
ml, as used in our experiments, accidental uptake will supply the cells with not more than one bacterium per 100 macropinosomes filled only with buffer. This explains why
talin-null cells are almost unable to grow on E. coli B/r in
suspension, whereas they are perfectly capable of growing
in a dense lawn of the same bacteria on an agar surface. By
the same argument, it is the strong adhesiveness of the carbohydrate-deficient R595 strain of Salmonella minnesota
that allows the talin-null cells to recruit these bacteria efficiently from a suspension.
Wild-type cells of D. discoideum extend a lamella with a
speed of ~10 µm/min around a particle. This process is
controlled by a zipper mechanism; it can be interrupted at
any stage before closure of a phagocytic vesicle (Maniak et
al., 1995 In several of the previously isolated phagocytosis mutants of Dictyostelium, the protein affected is unknown,
and inactivation of talin appears to be one possibility. Mutants characterized by Cohen et al. (1994) Cytokinesis in Talin-Null Cells
As in other eukaryotic cells, cytokinesis in D. discoideum
involves the formation of a cleavage furrow that initiates
separation of the daughter cells. Our data indicate that
talin, as an actin-associated protein, supports formation of
the cleavage furrow. Impairment of cytokinesis in talin-null cells that grow in shaken suspension indicates a role
for talin in cytoskeletal functions distinct from its involvement in making the cells adhesive. In the suspension cultures, a high proportion of bi- to tetranucleated cells is observed (Fig. 6).
Changes in cell shape during cytokinesis are brought
about by the actin-rich cell cortex in conjunction with the
microtubule-based spindle and asters. The synchronous division of multiple nuclei in a talin-null cell clearly reveals
that a cleavage furrow is induced wherever two asters of
microtubules are placed adjacent to each other (Fig. 7).
This finding extends observations made on a Dictyostelium mutant lacking myosin II (Neujahr et al., 1997 The cleavage stages in talin-null cells resemble experimentally disordered stages in dividing eggs of the sand
dollar, Arbacia lixula (Rappaport, 1986). In fibroblasts, talin acts in concert with other adhesion plaque proteins in anchoring stress fibers
to integrin clusters in the plasma membrane (Horwitz et
al., 1986
; Lewis and Schwartz, 1995
). On the outside, these
clusters are connected to fibronectin or laminin, thus associating the cells with the extracellular matrix (for review
see Geiger et al., 1995
; Jockusch et al., 1995
). In motile fibroblasts, talin accumulates also in membrane ruffles at
the leading edge (Hock et al., 1989
; DePasquale and Izzard, 1991
).
) or flexible monomers that partially associate into
parallel dimers (Winkler et al., 1997
). Talin binds directly
to actin (Muguruma et al., 1990
), vinculin (Burridge and
Mangeat, 1984
), and
integrins (Horwitz et al., 1986
;
Knezevic et al., 1996
). In the subunits of chicken talin,
three actin-binding sites linked to three vinculin-binding sites can be distinguished (Hemmings et al., 1996
). Talin
nucleates actin polymerization (Kaufmann et al., 1991
;
Isenberg et al., 1996
), cross-links actin filaments (Zhang et
al., 1996
), and anchors these filaments at membranes
(Kaufmann et al., 1992
). The
integrin, but not vinculin, is
essential for the assembly of talin at focal adhesions
(Moulder et al., 1996
). With its NH2-terminal domain, comprising a region typical of ezrin-radixin-moesin family proteins (Takeuchi et al., 1994
), talin can bind to membrane lipids (Niggli et al., 1994
).
), and is also enriched in contact areas between T-helper and antigen-presenting cells
(Kupfer et al., 1987
). Downregulation of talin by antisense RNA reduces the speed of cell spreading, the size of focal
contacts, and the formation of stress fibers (Albigès-Rizo
et al., 1995
). Similarly, microinjection of anti-talin antibodies inhibits the spreading of fibroblasts on a fibronectin
substrate (Nuckolls et al., 1992
). In neuronal growth
cones, chromophore-assisted laser inactivation of talin results in the cessation of both extension and retraction of
filopods, suggesting that talin couples signals generated in
substrate-attached filopods to actin-filament dynamics (Sydor et al., 1996
).
).
Materials and Methods
. For starvation, cells
were washed twice in 17 mM K-Na phosphate buffer, pH 6.0 (non-nutrient buffer) and were shaken at a density of 107 cells per ml in the buffer.
To achieve aggregation competence, AX2 cells were starved for 6 h, and
talin-null cells were starved for 8 h.
Fig. 3.
Reduced uptake rate of yeast particles contrasts to the
normal rate of fluid-phase uptake in talin-null cells. Wild-type
cells (closed symbols) and talin-null cells (open symbols) were incubated under shaking with heat-killed, TRITC-labeled yeast
particles for phagocytosis, or with TRITC-labeled dextran for pinocytosis. (A) Time course of particle uptake by wild-type (,
)
and talin-null cells (
,
), determined in two parallel experiments. Cells were shaken at 150 rpm with an excess of at least six
yeast particles per wild-type or mutant cell. (B) Dependence of
particle uptake on shearing stress. Wild-type (
,
) and talin-null (
,
) cells were incubated with yeast particles and shaken
either at 150 rpm (
,
), as in A, or at 100 rpm (
,
). (C) Time
course of fluid-phase uptake determined in three parallel experiments (
,
,
and
,
,
). (D) A talin-null cell taking up liquid medium by macropinocytosis. To visualize uptake, TRITC-dextran was added to the medium, and the cell was scanned by
confocal fluorescence microscopy at the times indicated. The
nonfluorescent cytoplasm is seen in black, the fluorescent medium in light grey, and endocytic vacuoles at grey-to-white values
depending on the degree of concentration of the internalized
TRITC-dextran. During the time period shown, the cell formed
three macropinosomes in the confocal plane (asterisks). Bar, 5 µm.
[View Larger Versions of these Images (14 + 116K GIF file)]
Fig. 2.
Cell-to-substrate
adhesion, as visualized by
RICM, and chemotactic responses in wild-type (A and
C) and talin-null cells (B and
D). Cells were starved for
6-8 h to induce development
up to the aggregation-competent stage, and then allowed to settle on a BSA-coated glass surface. (A and
B) Differences in cell-to-substrate adhesion exemplified
by one wild-type and one
talin-null cell, both freely
moving on the glass surface
in a fluid layer. The RICM
images show black areas at
sites of close contact of the wild-type cell with the substrate surface. Such areas are
not seen in the RICM images
of the talin-null cell, demonstrating the lack of close contact. The long lateral distances between the maxima
of the interference patterns
in the mutant cell indicate that major portions of the
bottom surface of this cell
formed small angles with the
substrate surface. The rapid
changes of these patterns reflect the instability of interactions with the substrate. (C
and D) A wild-type and a
talin-null cell exposed to
changing gradients of chemoattractant. These cells were
stimulated with cAMP through
a micropipette, the tip of
which was moved into different positions relative to the
cells. Actual directions of the
gradients are indicated by arrows. Despite its weak adhesion to the substrate, the
talin-null cell was not slower
than the wild-type cell: during straight movement toward the attractant, the front
of the wild-type cell propagated with an average speed of 13 µm/min, and that of the
talin-null cell with 17 µm/
min. To make allowance for
movement of the cells out of
frame, white and black asterisks were introduced as stationary markers to denote points on the substrate. The cells were imaged using a
double-view microscope (Weber et al., 1995). Contours of cell body projections were copied from bright-field images as white outlines
into the RICM images. Intervals between the frames: 10 s. Bars, 10 µm.
[View Larger Version of this Image (115K GIF file)]
Fig. 4.
Growth of wild-type (closed symbols) and talin-null
cells (open symbols) on bacteria. Two bacterial strains with different defects in lipopolysaccharide synthesis were used: Salmonella minnesota R595 synthesizes only 2-keto-3-deoxyoctonate
(3-deoxy-D-manno-octulosonic acid; KDO) linked to lipid A. E. coli B/r adds a core oligosaccharide consisting of heptose and glucose residues to this basal structure (Malchow et al., 1967). (A)
Growth with E. coli B/r on agar plates. On lawns of bacteria, colony diameters increase linearly with time. Since the bacteria are
completely used up by both wild-type and mutant cells at the border of the colonies, this increase is an indirect measure of uptake
rates. Under these conditions, the growth rates of wild-type (
)
and talin-null (
) cells were indistinguishable. Data are averages
of 20 colonies measured for each strain. (B) Growth on E. coli B/r
in suspension. Wild-type cells grew with a generation time of 3.4 h
and entered the stationary phase upon consumption of the bacteria. Growth of talin-null cells became negligible after uptake of
~10% of the bacteria, as indicated by generation times >20 h.
,
,
, and
, cultures with inoculates of 9, 7, 5, and 3 × 104 cells per ml, respectively. (C) Growth on Salmonella minnesota R595 in suspension under the same conditions as in B. On
this bacterial strain, talin-null cells grew efficiently with only marginally longer generation times (tgen) than wild-type cells. Symbols represent cultures inoculated with 8, 6, 4, and 2 × 104 cells
per ml, in the same order as in B.
[View Larger Version of this Image (19K GIF file)]
Fig. 5.
Cell-to-cell adhesion of undeveloped and aggregation-competent wild-type AX2 and talin-null cells in the absence and presence of EDTA. (A) Cell adhesion of undeveloped wild-type and talin-null cells, assayed in an agglutinometer to expose the cells to standardized shear forces. Within 1 h in the agglutinometer, equilibria of aggregated and single cells were obtained, as determined by light scattering measurements (Beug and Gerisch, 1972). Light scattering in arbitrary units indicates agglutination (low values) or dissociation into single cells (high values). Comparison of wild-type cells in the absence (
) and presence of 10 mM EDTA (
) shows that cohesion of the undeveloped cells was sensitive to EDTA, in accordance with previous reports (Beug et al., 1973
). Talin-null cells reached almost identical, high equilibrium values without (
) or with (
) EDTA, indicating strong reduction of cohesiveness in these mutant
cells. (B) Photographs of cell suspensions taken after 1 h of agitation in the agglutinometer. In wild-type AX2, irregularly shaped clusters of cells are recognizable in the absence of EDTA, and almost complete dissociation into single cells is seen with 10 mM EDTA (left
panels). Nearly all of the talin-null cells remained single in the absence as in the presence of EDTA (right panels). (C) Cell adhesion of
aggregation-competent wild-type (closed symbols) and talin-null cells (open symbols) in the absence (
,
) or presence (
,
) of 10 mM EDTA. (D) Illustration of EDTA-stable cell adhesion in wild-type AX2 and talin-null cells in the same experiment as shown in C. The developmentally regulated contact site A cell adhesion protein was expressed in the wild-type and mutant cells to the same level
within a tolerance of 7% (data not shown). Bar (B and D), 100 µm.
[View Larger Versions of these Images (89 + 85K GIF file)]
Fig. 6.
Histograms showing the probability of nuclei residing
in mononucleated or multinucleated cells under various growth
conditions. In wild-type AX2 (left panels), the majority of nuclei
was located in mononucleated cells under all conditions tested. In
talin-null strains (right panels) cultivated in shaken suspension
culture, most of the nuclei were found in multinucleated cells carrying up to 12 nuclei. This tendency of talin-null cells to become
multinucleated was less pronounced during growth in nutrient
medium on a solid surface, and was undetectable during growth
on agar plates in a lawn of Klebsiella aerogenes. For each panel,
between 400 and 500 nuclei were counted in DAPI-stained cells.
[View Larger Version of this Image (24K GIF file)]
Fig. 7.
Mitotic division of binucleated talin-null cells. (A) Sequence of
shape changes of one cell dividing into
four. Numbers on the video record
show that for progression of the cleavage furrow from the second to the seventh frame ~2 min was required. (B) Anaphase and telophase stages of
three binucleated cells shown in phase
contrast (top), stained with DAPI for
DNA (middle), and labeled with anti-
-tubulin antibody to depict spindles
and microtubule asters (bottom). Bar,
10 µm.
[View Larger Version of this Image (91K GIF file)]
) into the single blunted PstI site of a genomic
DNA fragment comprising base pairs 58-3,031 of the talin coding region.
This fragment was obtained by PCR using primers 5
-GCGGATCCTTTGCACCAGATATGTGTATTC and 3
-CGGCAATTCAACTTAGC, and was cloned into pUC19. The construct was excised using
BamHI and HindIII, and the linearized and dephosphorylated fragment was used to transfect AX2 wild-type cells of D. discoideum by electroporation. Talin-null mutants were selected with 7.5 µg/ml blasticidin S (ICN
Biomedicals, Inc., Costa Mesa, CA) in nutrient medium.
. 15 µg of either AX2 or mutant DNA was run on 1% agarose gels and blotted onto Hybond N nylon membrane (Amersham Intl.,
Little Chalfont, UK). The blots were hybridized under high stringency for
15 h at 65°C in RapidHyb buffer (Amersham Intl.) with a PCR-generated
probe comprising base pairs 3,086-3,442 of the talin coding region.
. The antibody recognized an epitope between amino acid residues
2,053 and 2,290, as deduced from bacterially expressed talin polypeptide
fragments. mAb 227-341-4 (here designated as mAb 341) was obtained
from a Balb/c mouse immunized with a His-tagged NH2-terminal fragment comprising residues 20-124 of the talin sequence. For the production of this fragment, a genomic DNA fragment was generated by PCR
and cloned into pQE30 (QIAGEN Inc., Chatsworth, CA). The sequence
was verified and the fragment was expressed in E. coli M15. The protein
was purified on a Ni2+-agarose column under denaturing conditions using
6 M guanidinium chloride followed by 8 M urea. The fragment was injected together with Freund's adjuvant, and spleen cells were fused with
PAIB3Ag81 myeloma cells.
).
). The wavelength of the green light used for
RICM was
= 546 nm. Contours of two-dimensional projections of the
cell body, as seen in bright-field images, were extracted by an image processing routine (Weber and Albrecht, 1997
) and superimposed onto the
RICM images. Chemotactic responses were recorded while the cells were
stimulated from a micropipette filled with a solution of 10
4 M cAMP and
placed with its orifice at a distance of 10-20 µm from the cell surface
(Gerisch and Keller, 1981
).
in a microprocessor-controlled agglutinometer (Bozzaro et al., 1987
). Phagocytosis assays using TRITC-labeled heat-killed yeast in shaken suspension were carried
out essentially as described by Maniak et al. (1995)
, and quantitative and
microscopic assays of fluid-phase uptake were performed using TRITC-labeled dextran according to Hacker et al. (1997)
. For the quantitative assays of Fig. 3, A-C, and Fig. 5, A and C, suspensions of wild-type and talin-null cells were adjusted to the same total cell volume by determining the
volume of densely packed cells (Maniak et al., 1995
). The volume of the
mutant cells was, on the average, 1.5-fold the volume of wild-type cells.
).
Cytokinesis in the adherent cells was monitored by video recording. For
fluorescence labeling, the cells were fixed for 15 min in a solution of 15%
saturated picric acid, 2% paraformaldehyde, pH 6.0, postfixed with 70%
ethanol, and processed according to Humbel and Biegelmann (1992)
.
-Tubulin was labeled with rat mAb YL 1/2 (Kilmartin et al., 1982
) and
TRITC-conjugated goat anti-rat antibodies (Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA). DNA was stained with 4
,6-diamidino-2-phenylindole (DAPI) (Sigma, Deisenhofen, Germany). Micrographs were taken with a Phaco ×100 oil Neofluar objective using a Zeiss
Axiophot microscope (Oberkochen, Germany).
Results
, cells of D. discoideum were transfected with a vector causing gene replacement by homologous recombination. The vector was
designed to interrupt the gene by a blasticidin resistance
(bsr) cassette at the codon for T516 of the talin sequence
(Fig. 1 A). Probing 40 blasticidin-resistant clones by
Southern blotting revealed eight independent transformants in which the gene was disrupted. These mutant lines
were characterized by two phenotypic alterations: (a)
weak adhesion to the polystyrene surfaces of petri dishes,
and (b) the tendency to form larger cells in suspension. All
transformants completed development on agar with the
formation of normal fruiting bodies.
Fig. 1.
Generation of
talin-null mutants by gene replacement. (A) Map of the
talin gene of D. discoideum,
the construct used for the talin knockout (hatched), the
probe used to identify knockout mutants by Southern
blotting, and location of two
mAb binding sites at the protein. To produce the vector
construct used for transfection, the blasticidin resistance (bsr) cassette was inserted into the PstI site of a
genomic fragment comprising nucleotides 58-2,999 of
the coding region. The PstI
site resides in the codon for
T516 of the talin sequence.
The antibody binding regions
were mapped by the use of
bacterially expressed fragments. (B) Southern blots
probed either with a fragment of the talin gene that
flanks the 3-end of the vector as indicated in A (upper
panels), or with the bsr coding region (lower panels).
Genomic DNA was digested with EcoRV (left panels) or
BclI (right panels), according
to the map in A. Lane numbers indicate wild-type AX2 (0), and four independent transformants HG1663-1666 (1-4). (C) Western blots of total cellular proteins of the same strains as in B. Proteins were separated by SDS-PAGE and probed with anti-talin mAb 477 (left
panel) or mAb 341 (right panel). Talin in the wild-type runs at an apparent molecular mass of ~220 kD, although its calculated mass is
269 kD. Neither intact talin nor any fragment is detectable in the mutants. Since the promoter and the coding region up to amino acid
515 should not be affected by homologous recombination of the vector into the talin gene, the lack of an NH2-terminal fragment appears to be due to instability of the transcript, inefficient translation, or degradation of the incomplete polypeptide.
[View Larger Versions of these Images (32 + 10K GIF file)]
), which means that the reflecting
cell surface approaches the glass surface with a distance
<50 nm close to the zero order intensity minimum of the
interference pattern (Rädler and Sackmann, 1993
). The
first interference maximum corresponding to a distance of
/4 appears then as a bright area.
) and to move persistently
with negligible loss of material retained on the substrate
surface (Schindl et al., 1995
). In the aggregation-competent stage, which is reached after several hours of starvation, wild-type cells adhere to the substrate only at portions of their basal surface, and the areas of contact rapidly change in size and shape with movement of the cells (Weber
et al., 1995
). An example is given in Fig. 2 A.
/4 = 136 nm for the green
light used. This distance made firm attachment of the
talin-null cells impossible, so that they easily drifted with
slight convections in the fluid.
).
In the example shown in Fig. 2 C, the wild-type cell initially moved toward the top of the frames, then turned
with its front toward the right, and shortly later produced a
second front, which subsequently established itself as the
leading edge. New sites of attachment at the fronts increased in size and fused with each other, followed by detachment at the tail of the cell.
). Uptake of liquid medium occurs in D. discoideum by macropinocytosis which, like phagocytosis, requires a response in the actin system to internalize a vesicle
(Hacker et al., 1997
). Macropinocytosis differs, however,
from phagocytosis in being independent of the stimulus of
a particle that impinges on the cell surface. To distinguish between a defect in adhesion and the process of uptake,
internalization of TRITC-dextran, a fluid-phase marker,
was measured. The rate of fluid-phase uptake proved to be
indistinguishable in talin-null cells from that in wild-type
cells (Fig. 3 C). Microscopic observations revealed that the
mechanism of this uptake in talin-null cells is similar as recently described for wild-type cells (Hacker et al., 1997
).
Protrusions extending
2 µm from the cell body eventually closed at their rim, thus entrapping an aliquot of medium (Fig. 3 D).
).
). On a lawn of E. coli B/r on an agar
plate, talin-null cells grew as fast as wild-type cells, which
confirmed that engulfment of the bacteria is not impaired
in the mutant cells (Fig. 4 A). In a suspension, however,
the growth of talin-null cells on E. coli B/r was dramatically slowed down (Fig. 4 B).
; Gerisch et al., 1985
; Cohen et al.,
1994
), which lacks the entire carbohydrate moiety apart
from two residues of 3-deoxy-D-manno-octulosonic acid
linked to lipid A (Lüderitz et al., 1966
). In suspensions of
this bacterial strain, the generation times of wild-type and talin-null mutant cells differed only slightly, indicating that bacteria of proper adhesiveness can be taken up by the
mutant cells even under the restrictive conditions of a
shaken suspension (Fig. 4 C).
). Cell cohesion can be
quantitatively assayed by an automated light scattering assay. Suspended cells are rotated in an agglutinometer in
cuvettes that are designed to apply standardized shear to
dissociate the cells (Beug and Gerisch, 1972
). The strength of cell cohesion is then reflected in the size of aggregates
formed against the dissociating forces, and the decrease of
particle number accompanying aggregation results in reduced light scattering.
Discussion
)
and shown to be blocked separately by antibody Fab
(Beug et al., 1973
). According to the data presented here,
the EDTA-sensitive "type B contacts" and also cell-to-substrate contacts represent talin-dependent types of cell adhesion (Fig. 5, A and B). Quantitative measurements confirmed that the energy of cell-to-substrate adhesion is
strongly reduced in the talin-null cells of Dictyostelium
(Simson, R., personal communication). A glycoprotein anchored to the membrane by a ceramide-based lipid (Stadler et al., 1989
) is the major adhesion protein involved in
the EDTA-stable cohesion of aggregating cells (Müller
and Gerisch, 1978
). Cell-to-cell adhesion mediated by this
"contact site A" glycoprotein turned out to be rather independent of talin (Fig. 5, C and D).
). The distinction of two adhesion systems in Dictyostelium on the basis of their linkage to talin suggests that also in this microorganism talin
interacts as part of a specific complex with one type of adhesion protein in the plasma membrane. Integrins have
not been identified in Dictyostelium; a candidate for a
talin-regulated adhesion protein is a 126-kD (Chadwick et
al., 1984
) or 130-kD glycoprotein (Chia, 1996
).
). (c) Thermally
induced out-of-plane fluctuations of soft membranes lead
to a repulsive force, which has the same distance dependence as the Van der Waals attractive forces (Sackmann,
1994
). Coupling of the cell membrane to the actin cortex
via talin will suppress the undulations, thereby increasing
cell-to-substrate adhesion (Zeman et al., 1990
). (d) Single
cells of Dictyostelium might coat a surface with a secreted protein and then adhere on this layer in an analogous fashion to an integrin-fibronectin type of cell interaction with
an extracellular matrix. In fact, a matrix is produced in the
multicellular slug stage of Dictyostelium and is used as a
surface for migration (Freeze and Loomis, 1977
; Abe et
al., 1994
).
; Lauffenburger and Horwitz, 1996
). The traction is thought to convert intracellularly generated contractile forces into net movement. Stress fibers and focal
contacts appear to be optimized for efficient and directed
pulling on the surrounding extracellular matrix. In contrast, aggregating cells of D. discoideum efficiently move
while adhering only with part of their basal surface to the substrate. These cells lack static structures like stress fibers or focal adhesion complexes and are fast moving with
speeds of up to 30 µm/min. In talin-null cells, substrate adhesion is even further reduced to an absolute minimum
(Fig. 2). These almost nonadhesive cells exemplify a case
of cell motility largely uncoupled from traction forces.
). As a result, the cell will move ahead while
the patch remains stationary on the substrate. Second, cell
locomotion might be effected by a continuous membrane
flux, either in the form of a rolling movement (Anderson
et al., 1996
) or a lipid flow generated by exocytosis at the
front and by endocytosis along the lateral cell surface
(Bretscher, 1996
). Talin-null cells offer themselves for a
detailed analysis of membrane events essential for cell locomotion because of the absence of spreading, which in
wild-type cells masks the local interactions at small areas of cell-to-substrate contact.
) and later reassociates with postlysosomal endosomes (Rauchenberger et al.,
1997
), and two F-actin cross-linking proteins, 120-kD gelation factor (ABP120) and
-actinin (Cox et al., 1996
; Rivero et al., 1996
). Since integrity of the actin system is essential for phagocytosis to occur (Maniak et al., 1995
), the
lack of proteins that cross-link actin filaments or otherwise
contribute to the viscoelastic properties of the actin cortex
most likely affects particle uptake directly.
). If the assumption is correct that the initial stages
of phagocytosis resemble the spreading of a motile cell on
a planar substrate (Grinnell, 1984
), one can deduce from
the dotlike contacts that talin-null cells form on a substrate
that phagocytosis is similarly altered by weak initial adhesion and insufficient spreading of the mutant cells on a
particle surface. Along the same line, it has been inferred
from the enrichment of talin in phagocytic cups of mouse
macrophages that talin plays a role during Fc receptor- mediated phagocytosis in linking transmembrane receptors to the force-generating cytoskeleton (Greenberg et
al., 1990
), similar to its implication in focal adhesion assembly.
do not grow in
suspensions of S. minnesota R595, which distinguishes the
defect in these mutants from the impaired adhesion of
talin-null cells. Mutants selected earlier by Vogel et al.
(1980)
differ from the talin-null mutants by their ability to
grow in suspensions of E. coli B/r. Two separate adhesion mechanisms are involved in the binding of these bacteria
to Dictyostelium cells, one unspecific and a sugar-specific
one. If the unspecific type of adhesion is eliminated by mutagenesis of D. discoideum cells, bacteria of the E. coli B/r
type can still stick to the mutant cells by virtue of terminal
glucose residues on their lipopolysaccharides (Vogel et al.,
1980
). These and other sugar residues are recognized by
lectinlike receptors exposed on Dictyostelium cell surfaces (Bozzaro and Roseman, 1983
). The fact that talin-null
cells are almost incapable of growing on E. coli B/r in suspension implies that talin is linked to both adhesion systems, the sugar-specific and the unspecific one.
). Mitosis in multinucleated cells of this mutant is accompanied by folding of the cell surface around each aster of microtubules.
). If in these eggs
the formation of a cleavage furrow is prevented during the
first nuclear division by dislocating the mitotic apparatus,
four furrows are simultaneously induced during the second division at spaces between the asters, giving rise to
four blastomers. In Arbacia blastomers as well as Dictyostelium cells, cruciform stages are diagnostic of single cells
dividing into four. The spindle is dispensable in these cases
for a cleavage furrow to be formed, and it is the apposition of two asters closely associated with the cell cortex that induces a furrow at the midline between them. Since this effect is easily observed in talin-null cells, these cells may
provide a tool to identify the proteins or molecular configurations that specify a cleavage furrow.
Received for publication 14 April 1997 and in revised form 16 May 1997.
Please address all correspondence to Günther Gerisch, Max-Planck-Institut für Biochemie, Abteilung Zellbiologie, D-82152 Martinsried, Germany. Tel.: (49) 89-8578-2326. Fax: (49) 89-8578-3885. e-mail: gerisch{at}biochem.mpg.deWe thank Alicija Baskaya for mAb production, Gerhard Rahn for iodination, and Jean-Marc Schwartz for digitalization of the video images.
DAPI, 4,6-diamidino-2-phenylindole;
RICM, reflection interference contrast microscopy.
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