Download 34 Chemotaxis

The Dynamic Cell
Chemotaxis
S.K. Maciver Jan '03
Chemotaxis
Cells developed the capacity to move in order to feed and to avoid local harmful situations. They are attracted to all
sorts of stimuli and are repulsed by others. The term "Chemotaxis" was first coined by a W. Pfeffer in 1884 to describe
the attraction of fern sperm to the ova, but since then the phenomenon has been described in bacteria and many
eukaryotic cells in many different situations. Specialised cells within metazoans have retained the ability to crawl
toward bacteria in order to eliminate them from the body and the machinery is very similar to that used by primitive
eukaryotes to find bacteria for food. Much of what we know about chemotaxis has been learned from studying the
slime mould Dictyostelium discoideum, and comparing this to our own neutrophils, the white blood cells that detect and
consume invading bacteria in our bodies. Neutrophils are end differentiated and largely non-biosynthetic cells which
means that we cannot use the usual molecular biological tools that we would other wise use, such as gene knockout,
transient transfection, or expression of GFP-labelled proteins. Fortunately, Dictyostelium can be used for all such
studies. An understanding of neutrophils chemotaxis is of obvious importance for the treatment of human disease and
therapeutic intervention of these processes has resulted. Whereas chemotaxis is the sensing of a chemoattractant
gradient and climbing it cells can also accumulate by chemokinesis. Chemokinesis is an activity that increases the
overall speed of locomotion. In principle a cell be perceive gradients by a number of distinct mechanisms (Figure 14).
These hypothesis have been offered in various guises by a number of authors.
t2
C
Temporal
t2
t1
mC
e
Spatial
h
R2
R1
P1
oh
P2
t o h
tm C
ae
Pseudospatial
R2
R1
ho a t Cm r a e
Temporo-spatial
Figure 14. How cells may perceive chemo attractant gradients (after Lackie, 1986). The
temporal model assumes that the amoeba
measures and "remembers" the chemo attractant concentration at time zero and
compares this to a second reading at some time
point later as the cell crawls. In this way the
amoeba gets clues like that particularly
annoying party game "getting warmer" / "
getting colder" (Arghhh!!). The spatial model
assumes that the cell is able to read the
concentration across the span of the cell so that
it can compare the number of chemoattractant
molecules.
A related model is the
pseudospatial model, where the cell tests
concentrations by sending out pseudopods (P1
and P2 ) at various points to sense where the
highest concentrations lie. The temporo-spatial
model, detects waves of chemo -attractants
coming toward the cell from a particular
direction. Note that this last mechanism could
not read a static gradient. A huge amount of
work has been expended trying to work out
which (if any) of these hypotheses is correct.
to a a
mt t r
ea c t
The distribution of cAMP receptors on Dictyostelium has been determined by replacing the gene with a gene consisting
of the receptor fused to green fluorescent protein (GFP –see below). These studies (Xiao et al, 1997) indicate that there
is no particular concentration of receptors within pseudopods as expected from the spatial, pseudospatial or temporospatial models. However we cannot discount these models on this basis since they could all function in principle
without concentrated receptors (it would just work better). The temporo-spatial model cannot be valid for all cases as it
is well established that cells can chemotax in stable gradients of attractions.
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The Boyden chamber. Two chambers are separated by a filter through which cells migrate (page 7, figure15).
Chemotactic gradients can be set up by placing different concentrations of the putative chemo -attractant in the upper
and lower chambers. The advantage of the Boyden chamber is that is can discriminate between chemo -kinetic and
chemotactic influences. The use of this chamber requires that the cells under test have to move in three dimensions
(most do) and to squeeze between the pore size of the particular filter. Filters are available in different average pore
sizes, so this latter point is seldom a problem. The Boyden chamber is reproducible and the chemokinetic, chemotactic
response easy to quantify.
The Dynamic Cell
Chemotaxis
S.K. Maciver Jan '03
Upper chamber
Top
Bottom
Filter
Lower chamber
Media
1/1000
1/100
1/10
Media
0.2
0.2
0.3
0.5
1/1000
0.8
0.6
0.4
0.4
1/100
3.4
3.5
0.7
0.6
1/10
2.7
2.3
0.8
0.2
% of Cells in Bottom Chamber
Figure 15. The Boyden Chamber. The main advantage is that it can detect chemokinetic effects as well as
chemotactic effects. The Checkerboard assay (right) is a way to organise the Boyden chamber experiment.
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Figure 16. The checkerboard assay can be used to differential
chemokinesis from chemokinesis. If attractants are added to
both chambers at various concentrations, the relationship of
migration to attractant can be plotted. In this example, the
attractant is both a chemokinetic and a chemoattractant.
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.4
.2
2
0
0
0.05
0.1
0.15
Dilution of Chemo-attractant
Control
Attractant
Attractant
Cells
Control
Cells
Figure 17. The under agarose method (left) versus the over agarose method. The under agarose method (Laevsky &
Knecht, 2001 ) is simple and can be used for a variety of cell types. Chemo -attractant gradients are set up as the
attractant diffuses from the trough into the agarose. The presence of the agarose stabilizes the gradient that might
otherwise be dispersed or changed by thermal motion or minor agitations. An advantage of the various under agarose
methods is that it is possible to set up multiple gradients of various chemo -attractants to study their effect on cell
behaviour simultaneously (Heit et al, 2002).
Cell chamber
Figure 18. A stable gradient can
be created by placing the putative
attractant in one well and the cells
are then placed in a second well.
Chemo-attractant chamber
Agarose
Cells
Petri dish
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The Dynamic Cell
Chemotaxis
S.K. Maciver Jan '03
Chemotaxis in Development
Mammalian development begins with the meeting and fusion of the gametes, the female egg gives signals and the male
sperm comes swimming (setting the pattern for life?). Chemotaxis helps the sperm find the egg in humans (Eisenbach
& Tur-Kaspa, 1994), and in algae where a chemo -attractant increases the turning angle of the sperm cell so that it
spirals in toward the egg like a captured moon. Development involves mass movements of cells.
Attractant
fMLP
C5a
IL-8
LTP4
Type
End target
End target
intermediate
intermediate
Transduction pathway
p38 MAPK & PI(3) Kinase
p38 MAPK & PI(3) Kinase
PI(3) Kinase
PI(3) Kinase
Function
Liberated by bacteria, allows neutrophils to follow them.
Produced from inflammatory lesions via complement
Liberated from other cells to attract neutrophils
Liberated from other cells to attract neutrophils
Table 1 Chemoatrractants for Neutrophils
Figure 19.
Neutrophil migration into
inflammatory regions. (1). Neutrophils in the
microvascular vessels circulate passively in the
blood. At sites close to inflammation (2), the
endothelial cells alter their charge in response to
interleukins such as IL-8, so that now neutrophils
can stick. (3). Neutrophils now crawl along the
"-dimensional surface of the vessel and then
squeeze between the endothelial cells.
(4)
Neutrophils can now invade the 3-dimensional
space and begin to detect the fMLP and/or C5a
gradient at the same time as a negative IL-8,
LTP4 gradient. It has been suggested (Heit et al,
2002), that such an arrangement is more
stimulatory. (5) Neutrophils arrive at the site of
infection drawn by the gradient and now
consume the bacteria by phagocytosis.
Eventually the neutrophils die full of now dead
bacteria and accumulate as pus. The top line in
the graph represents adhesive forces which peak
fMLP
IL-8
LTP4
C5a
Adhesion
Chemoattractant
at the endothelial surface, drop after that as the neutrophils crawl in the 3-dimension matrix. Adhesion then increases
with fMLP/C5a gradient. The lower graph represents the expected gradient of the various classes of chemo -attractants.
Transduction of Chemo-attractant Signals
Most of what is known about the signalling cascade has been gleaned from Dictyostelium, but recent contributions are
available from knock out mouse models. From the top, chemotaxis requires the chemotactic receptor, heterotrimeric G-
Ligand
GPCR
γ
γ
Signal
P
L
C
β
Gα
GTP
PIP2
PI(3)K
IP3
PIP2
PI(3,4,5)P3
+
DAG
Superoxide production
Ca2+
PKC
Activation of cytoskeleton
for chemotaxis
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Figure 20. G-protein linked chemo attractant
receptor
dissociates
trimeric G-protein and the b,g
subunit activates both PLC and
PI(3)-kinase. The products of PI(3)kinase are short-lived messengers
that bind a number of targets
leading to their activation. The
activation of PLC is important for
activation of the superoxide burst
after phagocytosis in neutrophils but
not important in chemotaxis its self.
Mice bred without PI(3)-kinase
however have a reduced capacity to
chemotax.
The Dynamic Cell
Chemotaxis
S.K. Maciver Jan '03
proteins, Whereas the distribution of chemotactic receptors is uniform across the cell (Xiao et al, 1997), other signalling
molecules further down the cascade are found to have a polarized distribution. Mammalian PLCγ become localised to
the leading edge in a PI3-Kinase dependent manner (Piccola et al, 2002). PLCγ contains a PH domain that binds
PtdIns-3,4,5-P3, a product of PI3-Kinase, and so its probable that the PLCγ localization is downstream of PI3-Kinase
activity. The βγ subunit of the activated hetero-trimeric G-proteins are weakly polarised fashion with concentrations at
the leading edge (Jin et al, 2000). Another protein involved in chemotactic pathway AKT (protein kinase B), also
contain PH domains and are also localized to the leading edge of cells (neutrophils)(Servant et al, 2000). Surprisingly,
it has recently been reported that in mouse neutrophils, PLCs are not required for chemotaxis but are involved in
priming the superoxide burst (Li et al, 2000). Dictyostelium too seems to chemotax in the absence of PLC (Drayer et al,
1994).
The role of PI (3) kinase and p38/MAPK in chemotaxis
A number of studies have demonstrated the importance of the PI3-kinases in chemotaxis of both neutrophils and
Dictyostelium (and a number of other cell types). Most convincing are experiments where the genes for these enzymes
have been deleted (Hirsch et al, 2000). These experiments indicate that in the absence of PI3-kinase most cells do not
RAC
P21-activated kinase
PAK1
LIM Kinase
CDC42
PI(3)K
P
RAS
p21 G-proteins
PIP2 PI(3,4,5)P3
p38
MK2
Hsp27
MK2
Hsp27
AKT
PKB
Figure 21. The p21-activated kinase
(PAK1) is as its name suggests
normally activated by members of the
p21 G-protein family, CDC42 and
Rac. In addition to this route it has
been found that activated PI(3)kinase
also activates PAK1 even in the
absence of either p21 (Papakonstanti
& Stournaras, 2002). PAK1 activates
LIMK by phosphorylation Activated
enzymes are indicated with a flash
symbol.
chemotact, however some cells still did. It is suspected (Heit et al, 2002) that the remain cells respond to chemo attractants through the p38-MAPK pathway. Full activation of chemotaxis involves the phoshorylation of AFT/PKB
and although this is partially achieved through PI3-kinase is seems that additional phosphorylation by p38 is require at a
distinct site.
Name
ENTH
FYVE
PH
PX
Derivation
Epsin N-terminal Homology
Binds to
PI (4,5) P2 & PI (3,4,5)P3
Proteins containing
Epsin,
Fab1p, YotB,Vac1p, EEA1
Pleckstrin Homology
PI (3)P
PI 4,5 P2
Early endosome antigen
PLC, Unc104 Kinesin, Spectrin
Phox Homology
PI (3)P
Table 2 Protein domains that bind phosphatidylinositol lipids
Receptor
PI(3)K
PAK
PI3P
PTEN
Chemo-attractant gradient
10
phox
p40
Figure 22 The distribution of the
molecules primarily involved in
chemotactic signalling.
The Dynamic Cell
Chemotaxis
S.K. Maciver Jan '03
Human diseases resulting from failed chemotaxis
Chemotaxis is such an important defence mechanism that it is not a surprise that pathogens have developed toxins that
inhibit the process. These toxins are of course bad news for the infected, but they have provided researchers with a
valuable set of tools for the study of chemo taxis. Staphylococcus aureus inhibits neutrophil chemotaxis by producing
CHIP a small 14kDa protein that binds to and inactivates the receptor for FMLP and C5a. Bordetella pertussis is a gram
bacteria that is responsible for whooping cough. It produces a toxin that ADP-ribosylates the G-protein that transduces
signals from the receptor and many bacteria e.g. Clostridium produce toxins that target the actin cytoskeleton so that the
cells can no longer crawl.
Staphylococcus aureus
Bordetella pertussis
Clostridium difficile
Clostridium botulinum
CHIP
Pertussis toxin
Toxin B
C3 toxin
These toxins can be used as very specific
tools for research.
For example
Clostridium toxin B, can ADP-ribosylate
and so inactivate Rho type monomeric Gproteins thereby inactivating them.
Neutrophils can be loaded up with PI3P
lipids if histones (basic proteins) are used
as carriers (Weiner et al, 2002). When
the cells are loaded up they
spontaneously polarise and more PI3P is
produced in a positive feed back
mechanism. The absence of Rho proteins
(through toxin B treatment) prevents this
occurring so that the feedback
mechanism involves Rho proteins. The
details of this important mechanism are
not yet certain.
Receptor
G-protein
Rho G-protein
Actin
Ligand
Activation by
Ligand binding
Staphylococcus
aureus toxin
γ
γ
Gα
GDP
γ
β
β
Gα
β
GTP
Bordetella pertussis
toxin
Signal
Figure 23
Many others target the cytoskeleton
Ex1
Em1
Ligand
Activation by
Ligand binding
Ex2
Em2
γ
Gα
γ
β
CFP
EFP
Figure 24
γ
β
Gα
EFP
β
CFP
Wave length
Emitted yellow
light
Another use for green fluorescence protein (GFP) technology allows us to monitor the interactions of specific proteins
in living cells by using fluorescent energy transfer (FRET) combined with digital imaging. Two variants of the standard
GFP are made by the introduction of mutations around the site involved in the fluorophore so that GFP now emits at
cyan (cyan fluorescence protein) while another set of mutations changes the absorption spectra of another protein
(yellow fluorescence protein) matches the emission of the CFP. When the molecules (CFP and YFP) are close light
emitted by CFP is absorbed by the YFP which then emits this at a longer wavelength (yellow-green range). If as in the
case above, the CFP is fused with Gα, while YFP is fused with Gβ subunit, then the state of the complex can be
monitored by fluorescence microscopy. When cAMP is added to the Dictyostelium transfected with the two constructs,
a reduction in fluorescence is measured indicating that the complex has dissociated. This experiment now tells us
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The Dynamic Cell
Chemotaxis
S.K. Maciver Jan '03
where the G-protein is activated, and the kinetics of the dissociation. Thus information about the complex can be
measured in living cells – completely unthinkable even a few years back!! The GFP revolution only really started in
1990(ish) when it was introduced to cell biology. GFP forms also promise to reveal intracellular pH transients and even
molecular clocks as there are forms (mutants) of the protein that age within the time scale of hours changing their
fluorescent spectra!( Terskikh et al, 2000).
The take home message of all of the above is that the cell signalling molecules are arranged at the plasma-membrane by
the phosphatidylinositol lipids especially the more phosphorylated forms such as PIP2 and PI3Ps. In the next lecture we
will see that these lipid messengers also organise the cell body by the effects of the cytoskeleton. Myosins are crucially
stimulated to form multi-meric complexes and further stimulated to contract.
References:Drayer, A. L., Van der Kaay, J., Mayr, G. W. & Van Haastert, P. J. M. (1994) Role of phospholipase C in Dictyostelium: formation
of inositol 1,4,5-trisphosphate and normal development in cells lacking phospholipase C activity. EMBO J. 13, 1601-1609.
Eisenbach, M. and Tur-Kaspa, I. (1994) Human sperm chemotaxis is not enigmatic anymore. Fertil. Steril. 62: 233-235.
Funamoto, S., Meili, R., Lee, S., Parry, L. & Firtel, R. A. (2002) Spatial and temporal regulation of 3-phosphoinositides by PI 3kinase and PTEN mediates chemotaxis. Cell. 109, 611-623.
Heit, B., Tavener, S., Raharjo, E. & Kubes, P. (2002) An intracellular signaling hierarchy determines direction of migration in
opposing chemotactic gradients. J. Cell Biol. 159, 91-102.
Jin, T., Zhang, N., Long, Y., Parent, C. A. & Devreotes, P. N. (2000) Localization of the G protein βγ complex in living cells during
chemotaxis. Science. 287, 1034-1036.
Lackie, J. M. (1986) Cell Movement and Cell Behaviour, 1 eds, Allen & Unwin, London, Boston, Sidney.
Laevsky, G. & Knecht, D. A. (2001) Under-agarose folate chemotaxis of Dictyostelium discoideum amoebae in permissive and
mechanically inhibited conditions. BioTechniques. 31, 1140-1149.
Papakonstanti, E. A. & Stournaras, C. (2002) Association of PI-3 Kinase with PAK1 Leads to Actin Phosphorylation and
Cytoskeletal Reorganization. Mol. Biol. Cell. 13, 2946-2962.
Piccolo, E., Innominato, P. F., Mariggio, M. A., Maffucci, T., Iacobelli, S. & Falasca, M. (2002) The mechanism involved in the
regulation of phospholipase Cγ1 activity in cell migration., Oncogene. 21, 6520-6529.
Pollack, E. D. and Muhlach, W. L. 1981. Stage-dependency in eliciting target-dependent enhanced neurite outgrowth from spinal
cord explants in vitro. Dev. Biol. 86: 259 - 263.
Niggli, V. & Keller, H. (1997) The phosphatidylinositol 3-kinase inhibitor wortmannin markedly reduces chemotactic peptideinduced locomotion and increases in cytoskeletal actin in human neutrophils. Eur. J. Pharmacol. 335, 43-52.
Ralt, D., Manor, M., Cohen-Dayag, A., Tur-Kaspa, I., Ben-Shlomo, I., Makler, A., Yuli, I., Dor, J., Blumberg, S., Mashiach, S. and
Eisenbach, M. (1994) Chemotaxis and chemokinesis of human spermatozoa to follicular factors. Biol. Reprod. 50: 774-785.
Rane, M. J., Coxon, P. Y., Powell, D. W., Webster, R., Klein, J. B., Pierce, W., Ping, P. & McLeish, K. R. (2001) p38 kinasedependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils.
J.Biol.Chem. 276, 3517-3523.
Servant, G., Weiner, O. D., Hertzmark, P., Balla, T., Sedat, J. W. & Bourne, H. R. (2000) Polarization of chemo attractant receptor
signalling during neutrophil chemotaxis. Science. 287, 1037-1040.
Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao, X., Lukyanov, S., Matz, M., Kim, S.,
Weissman, I. & Siebert, P. (2000) "Fluorescent timer": Protein that changes color with time., Science. 290, 1585-1588.
Wang, F., Herzmark, P., Weiner, O. D., Srinivasan, S., Servant, G. & Bourne, H. R. (2002) Lipid products of PI(3)Ks maintain
persistent cell polarity and directed motility in neutrophils. Nature Cell Biol. 4, 513-518.
Weiner, O. D., Neilsen, P. O., Prestwich, G. D., Kirschner, M. W., Cantley, L. C. & Bourne, H. R. (2002) A PtdInsP3- and Rho
GTPase-mediated positive feedback loop regulates neutrophil polarity. Nature Cell Biol. 4 , 509-512.
Xiao, Z., Zhang, N., Murphy, D. B. & Devreotes, P. N. (1997) Dynamic distribution of chemoattractant receptors in living cells
during chemotaxis and persistent stimulation. J. Cell Biol. 139, 365-374.
http://dictybase.org/tutorial/spatial_gradients.htm
Queries to S. Maciver Room 444, Hugh Robson Building, George Square. E-mail [email protected].
Fax/Tel 0131 650 3714.
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