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Sackmann Keber Heinrich

Physics of Cellular Movements

Erich Sackmann1,2,*, Felix Keber2, and Doris Heinrich2

Physics Department, Institute for Biophysics E22, Technische Universitt Mnchen, D-85748 Garching, Germany; email:

Fakultt fr Physik and Center for NanoScience (CeNS), Ludwig-Maximilians-Universitt Mnchen, D-80539 Munich, Germany; email:, *Corresponding author.

Supplemental Material
Table of contents
A. B. C. D. E. F. G. Extended model of travelling force field motors, mediated by gradients of the signalling lipid PI-3,4,5-P3 1 Actin regulating proteins involved in cell motions 4 Regulation of actin network structures by molecular switches and actuators 7 Regulation of the actin cortex structure by signal molecules and scaffolding proteins 8 Adhesion induced membrane domain formation by receptor clustering 11 Magnetic bead microrheometry of Dictyostelium cells 12 14 Passive and active membrane bending excitations and biological functions

A. Extended model of travelling force field motors, mediated by gradients of the signalling lipid PI-3,4,5-P3
In the following we propose an extended model of the amoeboid cell movement during locomotion or the engulfment (phagocytosis) of large objects by the SAGW. The basic concept is that the nucleation and polar growth of actin gel clusters is mediated by the propagating gradient of the phosphoinositides PI-3,4,5-P3 and PI-4,5-P2, with the signalling lipid PI-3,4,5-P3 being enriched in adhesion domains. The domains serve as docking stations for cooperative binding of the scaffolding protein CARMIL, which triggers the actin growth by uncapping actin plus ends, thus enabling the attachment of G-actin or pieces of F-actin provided by the actin provider coronin. Branched gels are formed by subsequent activation of Arp2/3 resulting in the growth 1 of nascent filaments off the side of prolonged filaments. The gradient is generated by the interplay of two antagonistic enzymes: the PI-3-kinase (PI-3K) acting as PI-3,4,5-P3 generator and the PI3-phosphatase (PI-3PH, such as PTEN) acting as PI-3,4,5-P3-annihilator. Evidence for the initiation of phagocytosis by the local accumulation of the (PI-3,4,5P3) generator P3-K has been visualized by fluorescence labelled proteins with specific domains preferentially binding to PI-3K (1, 2). This model of polar growth of the actin gel in the direction of propagation and the subsequent branching implies several requirements which will be discussed below, to-

gether with experiments justifying the assumptions made: 1) Once a local excess concentration of PI3,4,5-P3 is generated, it has to propagate in one direction along the pillar. A possible answer to this question will be given below. 2) To generate a solitary wave the F-actin must be added at the front of the wave and dismantled at the opposite end. This process is attributed to the actin polymerization promoter coronin which plays a two-fold role (cf. Supplemental material B). It removes small pieces of F-actin at the trailing end and transfers them to the growing front. Here its unique capacity to bind preferentially fragments of F-actin with bound ADP and P (and not to monomeric ADP-actin) comes into play. Evidence for this role of coronin is provided by the observation that it is intimately associated with newly formed F-actin (as shown in Figure 5 of the main text). 3) The rate of actin polymerization at the front has to be triggered. This task is fulfilled by the scaffolding protein CARMIL (3, 4). Similar to the VASP complex in Figure 4b this supramolecular complex recruits the players involved in the polar actin growth (namely: Arp2/3, ATP-actin, the molecular GTPase switch Rac and the motor protein myosin-IB).The accelerated growth of the actin gel may be mediated by two mechanisms: by activated unbinding of the capping proteins at the plus-end and attachment of fragments of F-actin bound to coronin. by nucleation and growth of branched fragments through Arp 2/3, as shown in Figure 4b). Following Uruno et al. (3) and Miyoshi et al. (5) we consider the first mechanism more probable for several reasons. First, coronin (in its non-phosphorylated state) prevents the growth of the actin gels by inhibiting the binding of Arp2/3 to F-actin. Second, in the 2

resting state the capping protein CP binds strongly to F-actin preventing its growth. Third, the capping protein binds very strongly to CARMIL, with Kd 0.4x10-6M (similar to binding of myosin-IB to CARMIL with Kd 1x10-6M). Thus, after recruitment of activated CARMIL to the membrane the capping protein is removed and the actin filaments can elongate rapidly by attachment of fragments of F-actin provided by coronin. By the uncapping process by CARMIL only assemblies of linear actin filaments can be generated. Branched networks can be formed, however, in a second step by activation of Arp2/3. This can only occur after the coronin is phosphorylated which is mediated by a specific kinase (protein Kinase C). Evidence for the simultaneous recruitment of CARMIL and myosin IB and the delayed formation of branched networks is provided by the following observation. At the front of the solitary waves the newly synthesized actin (as indicated by LIM) and myosin IB appear simultaneously (e.g. within about 1 sec after a sudden generation of a gradient of chemo-attractants cAMP). In contrast, the recruitment of Arp2/3 to the front is delayed by 1-2 seconds (7). The key questions discussed now are: (i) How is the PI-3,4,5-P3PI-4,5-P2gradient maintained that determines the direction of growth? (ii) How is the protrusion force generated that drives the cell forward? (iii) Does the MT-aster play a role? The gradient of the phosphoinositides is generated by competition of the PI-3K and PI-3PH. In the case of lymphocyte locomotion, the PI-3K is switched-on by binding of proteins of the tissue to the chain of the integrins (the major CAM mediating cell tissue adhesion) which activates the kinase through the GTPase Ras (8). Very important recent studies showed that Dictyostelium cells exhibit an integral CAM which shares

some features with the chain of integrin (9). We can assume with some confidence that the same mechanism holds in our case. To drive the SAGW the PI-3,4,5-P3PI4,5-P2-gradient has to propagate. This could be achieved by the constant generation of active PI-3K at the front of the spreading pseudopod where the adhesion domains grow and new CAMs are recruited and activate the PI-3K. The adhesion domains enriched in PI-3,4,5-P3 can now serve as docking station for the CARMIL-myosin-IB complex. CARMIL could be recruited to the adhesion domains in two ways: First, by direct binding to PI-3,4,5-P3 and second with the help of myosin-IB. Myosin-IB can couple to the plasma membrane either directly, by electrostatic binding with its long basic tail to the negatively charged lipid moiety of the membrane, or indirectly by binding with its tail domain to the actin filaments already anchored to the adhesion domains via talin (cf. Figure 4b). Evidence for the recruitment of CARMIL by myosin-IB is provided by observations that at the front of the actin polymerization waves the newly synthesized actin (as indicated by LIM) and myosin-IB appear simultaneously (e.g. within about 1 sec after a sudden generation of a gradient of chemo-attractants cAMP). Since a large number of CARMILcomplexes can simultaneously assemble at the adhesion domains, the recruitment of these proteins at the front of the growing actin gel is a cooperative process. Some evidence for such a role of the adhesion domains is provided in the work of Weiner et al. (10), which shows that cells that are stimulated with chemo-attractants produce a random distribution of spots (~1m diameter) of membrane bound Hem-1 (reflecting the heterogeneous distribution of the scaffolding protein). These patches fuse at the leading front forming the solitary wave and disappear at the rear. The same punctuated 3

formation of scaffolding proteins precedes the re-establishment of actin networks decomposed by latrunculin, a poison strongly binding G-actin (11). In the present model the motion of the actin gel patches along the side of the elongated pillars is a natural consequence of the dismantling of F-actin at the trailing end by coronin. As shown previously (12) disruption of the actin fragments from the adhesion domains drastically weakens the adhesion strength of the receptors resulting in their unbinding (cf. Supplement Appendix E). This would also provide a new reservoir of CAMs for the growing front of the SAGW. The role of the MT network is poorly understood although it is well established that it plays a regulatory role for global cell movements and shape changes. Thus microtubules are required for effective phagocytosis since the particle uptake is much slower if the MTs are removed. One established role of the MTs is to recruit PI-3,4,5-P3 to membranes since its concentration in the membrane is lowered after decomposing the MTs. This enrichment of the signalling lipids is caused by the recruitment of the PI-3 kinase to membranes since concentration of the signalling lipid in the membrane is lowered after decomposing the MTs (13). The most important role of the MT-aster is to stabilize the global shape of the cell during locomotion as predicted by the tensegrity model. Reconstructions of the MT-aster by confocal microscopy of cells with GFPlabelled MTs show that they are tightly connected to the actin cortex over several m long segments. Moreover, the motion of the actin gel clusters and the MTs are correlated, strongly suggesting that the very large forces (of some 100 pN) on the MTs are generated by the propagation of the actin columns during cell locomotion.

References to Appendix A
1. Dormann D, Weijer G, Dowler S, Weijer J. 2004. J. Cell Sci. 117:6497509 2. Cai L, Holoweckyj N, Schaller M, Bear J. 2005. J. Biol. Chem. 280, 31913-23 3. Uruno T, Remmert K, Hammer JA. 2006. J. Biol. Chem. 281:1063550 4. Jung G, Remmert K, Wu X, Volosky JM, Hammer JA III. 2001. J. Cell Biol. 153:147997 5. Miyoshi T, Takahiro T, Higashida C, Hertzog M, Fujita A, Narumyia S. 2006. J. Cell Biol. 175:94755 6. Cai L, Holoweckyj N, Schaller M, Bear J. 2005. J. Biol. Chem. 280, 31913-23 7. Etzrodt M, Ishikawa H, Dalous J, Mueller-Taubenberger A, Bretschneider T, Gerisch G. 2006. FEBS Lett. 580:670713 8. Melikova S, Dylla S, Verfaillie C. 2004. Exp. Hematol. 32:105156 9. Cornillon S, Gebbie L, Benghezal M, Nair P, Keller S, et al. 2006. EMBO Rep. 7:61721 10. Weiner O, Marganski W, Wu L, Altschuler S, Koirschnert M, et al. 2007. PLoS Biol. 5: 2053-62 11. Gerisch G, Bretschneider T, Mller-Taubenberger A, Simmeth E, Ecke M, et al. 2004. Biophys. J. 87:3493509 12. Simson R, Wallraff E, Faix J, Niewoehner J, Gerisch G, Sackmann E. 1998. Biophys. J. 74:51422 13. Khandani A, Eng E, Jongstra-Bilen J, Schreiber D, Douda D, Samavarchi-Tehrani P, 2007. J. Leukocyte Biol. 82: 417-28

B. Actin regulating proteins involved in cell motions

Biochemical properties of G-actin: The biochemical behaviour of actin monomers is determined by the state of the nucleotide bound in the centre of the molecule. The binding constant of the G-actin to the fast growing plus end of filaments is highest when ATP (denoted as ATP-actin) is bound. It decreases after hydrolysis of ATP (denoted ADP-P-actin) and is changed further after P dissociates (yielding ADP-actin). After formation of F-actin the nucleotide is hydrolysed and a gradient of ADP-actin ADP-Pactin to ATP-actin is formed. Thus, one has to consider three species of actin monomers. This is expected to play a key role for the treadmilling process in pseudopods and the generation of solitary gelation waves as shown in Figures 4 and 5. Capping proteins: Proteins that bind to the fast growing plus ends of actin filaments and block their growth. Capping protein for Dictyostelia cells are severin and CP. The dissociation constant of severin is Kd 10-7 M. The CP-capper binds very strongly to the plus end (with dissociation constants of 4 Kd 10-9 M and a life time of 30 min). The CP-content of cells is 0.1-1 x 10-6 M. It is only two to three orders of magnitude smaller than the G-actin concentration and the actin filaments in cells are therefore short (~1m). Only activated CARMIL can remove CP from the plus end which is essential for the treadmilling process described in Figure 4 of the main text. Arp 2/3 can bind to the minus ends of actin monomers. Since this actin binding protein can also bind to the side of actin filaments, it can generate branched networks by forming F-actin budding off the side of existing filaments (as shown in Figure 1 of the main text. Thus, Arp2/3 can play a twofold role as capping and cross-linking protein. Cofilin and Profilin: Cofilin is a F-actin depolymerization factor that destabilizes actin filaments resulting in the dissociation of actin monomers (G-actin). It is most active for hydrolysed ADP-actin and is thus responsible for the dismantling of mature

actin monomers in cells. Cofilin can also sequester G-actin in the cell. Profilin provides activated ATP-actin by accelerating the exchange of ADP by ATP. It can take over ADP-actin from cofilin and activate it by ADP ATP exchange. Profilin loaded with ATP-actin can also bind to the scaffolding proteins WASP or CARMIL. Coronin (Coronin 1B): A promotor of Factin dismantling. In contrast to Cofilin it binds strongly to F-actin rods through electrostatic forces but not to monomers. Importantly, it binds much stronger to ADP-Pactin (dissociation constants Kd170 x 10-9M) than to ADP-actin (8 x 10-6M). It can therefore disrupt fragments of F-actin from matured actin filaments and deposit them at the plus-end after removal of the capping protein from this end. The concentration of coronin in cells is about 0.73 x 10-6 M (compared to actin with typical concentrations of 100 x 10-3 M). Coronin often works together with cofilin in dismantling actin filaments. It is important to note that in its nonphosphorylated state, coronin inhibits Arp2/3 binding to actin. However, this inhibition is abolished after phosphorylation of coronin. Formin (mDia-1): A group of proteins involved in the promotion of actin polymerization. It mediates preferentially the growth of actin bundles and is involved in the formation of thin cellular protrusions (named filipodia). The proteins exist in the cytoplasm in an auto-inhibited state. Activation is most efficiently mediated by GTPase switches of the Rho family (see Reference (1)). The proteins are also assumed to play a key role for binding of the microtubules to the actin cortex.

LIM protein domains, are cystein- and histamin-rich protein motifs. Dimers of LIM can act as non-specific linkers between proteins. Each LIM domain coordinates two Zn(II) ions (forming a so called zinc-finger). LIM binds to actin with dissociation constants of Kd 10-7M and stabilizes actin bundles at low Ca2+-concentration, while it unbinds at high Ca2+-content (>10-6M). It also protects actin filaments against depolymerization by cofilin. Most importantly, the LIM-proteins bind only transiently to newly polymerized actin. This can be used to visualize freshly polymerized actin by labelling LIM protein domains with GFP. Myosin IB: This motor exhibits only one motor head. It has a large positively charged tail which can mediate the direct binding to the inner leaflet of the plasma membrane (which contains about 10% negatively charged lipids). It exhibits a specific domain (SH3) of about 60 amino acids at the end of the tail that can bind the motor to all proteins exhibiting SH3 binding domains, such as CARMIL the scaffolding protein of Dictyostelium cells. Members of the myosin I family have some distinct features. They exhibit two actin binding sites (one at the motor head group) and the other at the tail and can thus crosslink actin filaments. The motor function is activated by phosphorylation of the motor domain by myosin light chain kinase, similar to muscle myosin. It has been reported that myosin I can exhibit some type of gear. Under load it can switch from a non-processive (tension generating) to a processive motor that can move towads the plus end of actin filaments. Myosin IB can thus act as force controlled actuator (see Reference (2)).

Talin (member of family of FERM proteins): Talin is a major constituent of the composite cell envelopes of most mammalian cells or amoebae. It mediates the direct coupling of actin to the intracellular domains of glycolproteins (such as glycophorin in the case of erythrocytes) and integrins, which is present in all animal cells. The binding is in general mediated by the head group of talin exhibiting a so called FERM domain (150 amino acids in length). The FERM domain is found in many other actin membrane couplers such as band IV.1 protein (the major actin-membrane coupler of erythrocytes), ezrin, radixin and moesin. It can exist in an inactive and an active state. In the dorming state the binding site for the membrane protein is shielded. Similar to the situation for band IV.1 (shown in Figure 1) the FERM domain of talin becomes activated by phosphorylation. Talin plays also a key role for the control of cell adhesion strength mediated by integrins. These CAMs exhibit a weakly binding and a high affinity conformation, whereby binding of talin triggers the transition to the latter state. Talin exhibits two binding sites for actin and can thus crosslink filaments (For a comprehensive review of the role of talin see Reference (3)). Microtubule-actin crosstalk: There are several candidates mediating the coupling of the microtubules to actin cortex. For the present work we consider two mechanisms: 1. Coupling via end binding proteins (EB1) with the help of the actin growth pomotor formin (and mDia-1). References to Appendix B

2. Coupling via Dynactin: This rod-like supramolecular complex (of ~1.1MDa) is about 37 nm long and 10 nm thick. Its major constituents are (i) the actin-like filament Arp1, (ii) capping proteins determining the length of the rod and (iii) a constituent mediating the binding of the motor protein dynein. The thickness of the complex (together with the dynein motor) is about 50 nm. The dynein motor moves towards the minus end of the MT (that is towards the centrosome) and thus generates tension in the filament and may be responsible for the prestress of the cells mentioned in the main text (cf. also Figure S.M.6) In many quiescent cells the microtubules are highly flexible and are not bound to the actin cortex. They grow and shrink continuously on the time scale of minutes. However, cross-linking of MTs to the actin network comes into play during adhesion and cell crawling on surfaces and it is essential for the growth of nerve axons. For this purpose a fraction of the MTs becomes fixed with their plus ends at the actin cortex (as shown in Figure 2 of the main text). The structure of the tubulin monomers of these stable MTs is modified post-translationally (see Reference (4)). Although EP1 is called end-bindingprotein, it can mediate the binding of several m long segments of the MTs to the actin cortex. It can also mediate the formation of MT bundles which can mutually slide over each other (for References see (5)).

1. Wallar BJ, Stropich BN, Schoenherr JA, Holman HA, Kitchen SM, Alberts AS. 2006. Biol.Chem. 281:4300-07 2. Laakso JM, Lewis JH. Shuman, Ostap EM. 2008. Science 321:133-36 3. Critchley DR, Gingras A. 2008. J. Cell Sci. 121:134547 4. Gundersen G, Bulinski J. 1988. Proc. Natl. Acad. Sci. USA 85:594650 5. Reilein A, Nelson WJ. 2005. Nat. Cell Biol. 7:463-73

C. Regulation of actin network structures by molecular switches and actuators

The rapid reorganisation of the actin cytoskeleton structure is often mediated by molecular switches of the GTPasesuperfamily (called Ras). They are further divided into the subfamilies consisting of Ras, Rho, Rab, Cdc42. These GTPases are small proteins of 20-30 kDa which bind guanine nucleotides (GTP or GDP). They can thus exist in an inactive state (with GDP bound) and an active state (with GTP bound). They are all actuated by external signals (mechanical forces, hormones, growth factors) through the exchange of GDP by GTP. This process is mediated by GDPGTP exchange factors (GEF, cf. Figure S.M.1). As shown in Figure S.M.1, the members of the three subgroups evoke distinct responses by activating specific actuator proteins (also called effectors or adaptors in the biological literature) which in turn activate different actin manipulation proteins, such as capping proteins (impeding the actin growth); crosslinkers (such as Arp2/3), actin-to-membrane linker, such as band IV.1 (shown in Figure 1b of the main text) and talin. The GTPase switches are regulated in a sophisticated way to trigger or switch off bursts of cellular activities. Two pathways of activation are known: 1) The GTPases exhibit lipid anchors mediating their membrane coupling. But they reside in the cytoplasm in a sleeping conformation, often mediated by binding of a specific protein: guanine nucleotide dissociation inhibitors (GDI). The GTPase is activated by exchange of GTP for GDP: a process mediated by a GDP GTP exchange factor (GEF) shown in Figure S.M.2 (left). The lipid chain of the GTPase is now exposed. It can bind to the membrane and interact with actuators of the cytoskeleton. 2) The intrinsic GTPase activity of the switches is rather weak resulting in a long lifetime of the activated switch. In order to accelerate this low rate of hydrolysis (which is about 0.01/min), and switch-off the GTPase fast, another regulatory proteins has to come into play. An example is the GTPase activating protein(GAP). It stimulates the Rho-GTPases to hydrolyse the GTP resulting in a rapid de-activation of the molecular switches. GEF and GAP together control their net activity. The GAP control factor could introduce a timer into GTPase controlled signalling pathways.

Figure S.M.1 Schematic view of the activation of effector proteins (actuators) involved in the control of the structure of the actin-based cytoskeleton.

Figure S.M.2 Activation of the effectors (actuators) by molecular switches of the Ras super-family. The super-family comprises over 100 members, including Ras, Rho and Rab. Left: activation and membrane binding of a GTPase by exchange of GTP for GDP. After membrane binding the switch controls the activity of a specific actuator. Right: control of the activity of the molecular switches by the tandem GEFGAP (described in the text). Activation of proteins by phosphorylation: a general principle. Numerous signal transmission processes in cells are controlled by signal cascades associated with phosphorylation-dephosphorylation reactions of proteins and lipids (such as phosphoinositides). These are mediated by tandems of antagonistic enzyme: the kinases (which add phosphate groups) and the phosphatases (which remove phosphate groups). The proteins consist of a universal catalytic domain mediating the reactions and a specific domain controlling the interaction with other proteins or with membranes (such as the PI3-kinase or PI-3-phosphatase). In general, the tandems are part of a whole cascade of phosphorylation-dephosphorylation reactions. It is important to note that the activation of the kinases and phosphatases requires the cooperation of the GTPase-switches with the associated helper proteins (GEF, GAP and possibly the inhibitor GDI). Therefore, the activation of a certain enzyme is controlled by a set of at least four proteins working together.

D. Regulation of the actin cortex structure by signal molecules and scaffolding proteins
Second messenger phosphatidylinositol3,4,5-triphosphate (PI-3,4,5-P3). This phosphoinositide exposing three phosphate groups is generated by phosphorylation of PI-4,5-P2 by phosphoinositol-3-kinase (PI3K). PI-3,4,5-P3 has a twofold function. It can first act as second messengers and second, serve the recruitment of proteins (which regulate the structure of the actin cortex) to the membrane. PI-3,4,5-P3 and PI-4,5-P2 are connected by the antagonistic pair of enzymes PI-3K and the PI-3phosphatase (an example is PTEN) removing the phosphate group at the 3-position of the inositol head group.

Figure S.M.3 Illustration of states of phosphorylation of head groups of the phospholipid phophatidytidylinoisitol.

It is important to note that both enzymes bind to PI-4,5-P2 and PI-3,4,5-P3 through the interaction with a specific protein domain (named C2 homology domain). The binding involves two to three Ca2+-ions. Note first: PI-4,5-P2 is much more abundant (about 1% of the total lipid content) than PI-3,4,5-P3 (~ 0.1%). Note second: the membrane binding of the proteins is enforced by electrostatic forces due to the binding of basic sequences of the proteins to other negatively charged lipids (mainly phosphatidylserine) as shown in Figure 1c. Scaffolding proteins: (CARMIL, VASP, WASP). The scaffolding proteins serve the recruitment of proteins and molecular switches required for the polar growth of actin gels at the front of pseudopods. They are all composed of five constituents with defined functions. At the N-terminal they exhibit a specific domain (WH1) which binds the molecule to proteins associated with focal adhesion domains, such as vinculin. In the middle they expose a prolin rich domain (PPP). It acts as docking pocket for proteins exhibiting so called SH3-domains. Important examples of such proteins are PI-3-kinase and myosin 1B. It exhibits also four binding sited for profilin (which recruits activated ATP-actin to the complex). Finally, the

VCA-domains bind both G-actin and Arp2/3. The scaffolding protein CARMIL (shown schematically in Figure S.M.4) mediates the SAGW generation in Dictyostelium cells. The supramolecular complex (molecular weight of 116 kDa) consists of a 66 nm long tail and a 19 nm large head. In the resting state of cells CARMIL is in a sleeping state with the tail assumed to be wrapped around the head. The CARMIL complexes consist also of five parts binding the different proteins required for the directed actin polymerization. A very important difference to the other scaffolding proteins is the strong binding of myosin IB (dissociation constant Kd = 100 x 10-9 M). Another essential feature is the very strong binding of the capping protein CP of Kd = 0.4 x 10-6 M. Finally CARMIL can also form dimers with Kd = 1 x 10-6 M. The concentration of CARMIL and myosin IB in cells are about 1 x 10-6 M and 2 x 10-6 M, respectively. The concentrations are thus adjusted in such a way that the association-dissociation equilibrium of the CARMIL-myosin IB and CARMIL-CP complexes can play a key role under physiological conditions. In particular CARMIL can remove the capping protein CP from the plus end of actin. This process is assumed to play a key role for the rapid prolongation of actin filaments.

Figure S.M.4 Schematic structure of the scaffolding proteins mediating the solitary actin gelation waves. Example of CARMIL. Integrins can serve as docking stations for scaffolding proteins which recruits kinases and activation of PI-3-kinase (on the local generation of PI-3,4,5-P3). Recently it was shown that clusters of integrin can play a key role as docking station for the recruitment of cytoplasmic proteins involved in cell signalling (1). The -tail of integrin exhibits a specific binding motif (domains) which binds proteins exposing phosphorylated tyrosine side chains. An important example mentioned above is phosphorylated talin which switches the transition of the integrin from a weakly binding to a high affinity state and simultaneously mediates the coupling of actin gel patches to the adhesion domains as mentioned above (Supplement Appendix B). Another imporReferences to Appendix D
1. Calderwood DA, Fujioka Y, Pereda JM, Garcia-Alvarez B, Nakamoto T, Margolis B, et al. 2003. Proc. Natl. Acad. Sci USA. 100: 2272-77 2. Melikova S, Dylla S, Verfaillie C. 2004. Exp. Hematol. 32:105156 3. Cornillon S, Gebbie L, Benghezal M, Nair P, Keller S, et al. 2006. EMBO Rep. 7:61721

tant role of the specific binding domain is the recruitment of scaffolding proteins to the integrin chains which serve as docking stations for actuators triggering the activity of other proteins of the signalling pathways. By this mechanism the PI-3-kinase could be recruited to the adhesion domains (see Figure 5 of original manuscript), resulting in the local generation of the signalling molecule PI-3,4,5-P3 as postulated in our model of SAGW generation. This mechanism of PI-3,4,5-P3 generation has indeed been verified for leucocytes (see Reference (2)). In the case of Dictyostelium cells, the role of the integrins could be played by the CAM named SibA which was discovered recently by Cornillon et al. (see Reference (3) and Supplement Appendix F).


E. Adhesion induced membrane domain formation by receptor clustering

The adhesion behaviour of the amoeba-like Dictyostelium cells depends on the state of the live cycle. In the presence of enough food (e.g. bacteria) the cells are in the vegetative state. They can survive as isolated quasi-spherical cells, search food by random walk and divide. After starving for about 6h the cells become elongated (worm-like). They associate and eventually generate slime molds. In this so-called adhesion-competent state the cells express a specific cell adhesion molecule (CAM) called contact site A. The outside domain is composed of three IgGlike domains to which N-linked oligosaccharides are coupled while the CAM are linked to the plasma membrane by lipid anchors. In the vegetative state the situation is less clear. First, the adhesion could occur by nonspecific forces. Second, it has been postulated that a modification of the contact side A protein (denoted as contact side B protein) acts as CAM. Third, an integral protein SibA (synonym for Similar to Integrin Beta) has recently been discovered as potential CAM of vegetative cells (see Reference (1)). This protein is supposed to penetrate the membrane with nine hydrophobic chains. The most interesting point is that this CAM shares some similarities with the -chain of integrins. In particular it mediates the direct binding of talin which then recruits F-actin to the membrane. This is important for the following reason. As noted above the activation of the PI3-kinase is triggered by binding to the chain of integrins. The same may hold for the SibA and therefore the adhesion of Dictyostelium cells could stimulate the activation of the PI-3,4,5-P3 generator similar to the situation in mammalian cells. The adhesion of Dictyostelium on glass substrates leads to the formation of adhesion domains (of about 1 m diameter) which are stabilized by coupling of talin to the inner 11 domains of the CAM (see Reference (2)). The primary event of adhesion is the formation of adhesion domains which can occur within seconds after contact formation between cells and surfaces. They form by lateral segregation of the receptor-ligand pairs interacting via lock-and-key forces (see Reference (3) for a summary of the physical basis of this process). In a second step the adhesion domains are stabilized by coupling of actin assemblies to the intracellular domains of the CAM. Model membrane studies strongly suggest that the adhesion induced formation of micro-domains is an inevitable consequence of the interplay between the strong short range specific interaction between the CAM and ligands of the tissue and long range repulsive forces induced by molecules of the glycocalix or by membrane bending excitations described below (in Supplement Appendix F). The distribution and size of the adhesion domains depends on the mechanical forces exerted on the cells. The adhesion strength of cells (measured in terms of the work of unbinding) depends sensitively on the membrane bending elasticity. Therefore, the coupling of F-actin to the membrane (e.g. via talin) increases the adhesion strength drastically. For instance, knock out of talin by mutations reduces both the bending modulus and the work of adhesion of Dictiostelium cells by a factor of five. The adhesion domains could play a key role for adhesion-induced cell signalling. They can form scaffolding entities for the local assembly of specific proteins involved in signalling. In particular, in the case of SAGW generation the adhesion domains could account for the cooperative binding of the scaffolding protein CARMIL to PI3,4,5-P3.

References to Appendix E
1. Cornillon S, Gebbie L, Benghezal M, Nair P, Keller S, et al. 2006. EMBO Rep. 7:61721 2. Tsujioka M, Joshida K, Inouye K. 2004. EMBO J. 23:221625 3. Sackmann E. 2006. J. Phys. Condens. Matter 18:R78595

F. Magnetic bead microrheometry of Dictyostelium cells

In the following some pertinent results of our previous microrheometry studies of the cytoplasmic space of Dictyostelium cells are summarized. Superparamagnetic beads were taken up by the cells through phagocytosis. They are therefore wrapped by the plasma membrane as other phagosomes (such as bacteria) and moved around in the cell for some time, before they are expelled again. Frequently the beads are bound to microtubules and transported along the filaments in both directions. Figure S.M.5 shows a micrograph of the cell with fluorescence labelled microtubules. Intracellular motions of 1.4 m diameter magnetosomes reflect movements of microtubules. To study the viscoelastic behaviour of the cytoplasm of Dictyostelium cells a large number (about 1400) of viscoelastic response curves (evoked by force pulses with force Figure S.M.5 Fluorescence micrograph of cell with two centrosomes showing the magnetic bead coupled to a microtubule (Adapted from Reference (1) with permission of the publisher) amplitudes ranging from 100 to 700 nN) were recorded. In Figure S.M.6 we show a frequently encountered scenario. The beads do not follow the applied force but move predominantly perpendicular to the applied external force Fex which is directed towards the left. The force amplitudes for pulses P1 to P4 are f1=450 pN (yellow line), f2=500 pN (orange line), f3=110 pN (red line), and f4=290 pN (purple line), respectively. The most remarkable finding is that the beads return close to the initial position after the pulses P1 to P3. After pulse P4 the bead does not return any more and is most likely detached from the MT. In summary, the repeated return of the bead close to the initial position strongly suggests that the cell exhibits some mechanical shape memory; which is most likely determined by the mechanical equilibrium of the MT-aster.


Figure S.M.6 Section of the bead trajectory shown in Fig 3a of the main text, The bead motion was observed for about 10 min while viscoelastic response curves were recorded at different times. The section shows a frequently occurring scenario where the bead moves predominantly perpendicular to the applied external force direction. Due to the (diffusive) reversibility of the microtubule deflection, the bead can revisit the same region within the cell several times. (Adapted from Reference (1) with permission of the publisher).

To relate the motion of the bead and the microtubules, cells with GFP-labelled tubulin were studied. The trajectory of the bead motion and the conformational change of the MT before, during and after application of a force pulses could be observed, since the

bead remained attached to the MT for the time of observation. A typical scenario is shown in Figure S.M.7b and the major findings are discussed in the Figure Caption. A detailed report and analysis of these experiments is given in Reference (1).

Figure S. M.7 (a) Motion of microtubule and attached bead in cell, induced by a force pulse of Fext= 40 pN. The thick green dashed line (#1) marks the contour of the MT immediately (56 ms) before application of the force and the thin black lines (numbered 2 to 5) during application of the force. Contour #6 indicates the MT position after switching off the force pulse. The thick red curve with the arrow indicates the motion of the MT. It is drawn to guide the eye. The time interval between contours is 0.5 s. 13

(b) The left side shows the trajectory of the bead centre (maximum initial velocity ~1m/sec). Note that the bead follows the force direction after an initial short deflection in the opposite direction (see small arrow). The right side shows the trajectory of the left centrosome #1 (closest to the bead) with the dots indicating the switching on and off of the external force. The maximum initial velocity of the centrosome is v~3 ms-1. It is seen that also the centrosome moves first fast in a direction perpendicular to the applied force before it returns close to the initial position again (Adapted Reference (1) with permission by the publisher). References to Appendix F
1, Heinrich D. and Sackmann E. (2006). Acta Biomaterialia 2:619-625

G. Passive and active membrane bending excitations and biological functions

A striking manifestation of motions in cells is the pronounced random bending excitation of cell envelopes exhibiting amplitudes of up to 30 nm (which is also called flickering). This dynamic membrane roughness has stimulated many physicists to enter the field of cell physics. Measurements of the bending excitation spectra (amplitude versus wave vector curves) provide insights into the molecular architecture of the composite cell envelopes and enables measurements of the bending moduli of membranes. Theoretical basis of membrane excitations (the Helfrich model) The mechanical properties of vesicles and erythrocytes are dominated by the bending elastic energy of the fluid membranes. The shape of free and adhering soft shells can therefore be described in terms of the simple model of flexible shells proposed first by Helfrich (and well documented in Chapters 8 and 9 of Reference (1)). The total elastic energy of the fluid plasma membrane is determined by the energy functional
i ela

where u is the deformation amplitude in the direction normal to the membrane. The first term accounts for the bending deformation ( is the bending modulus) the second for the lateral tension and the third for the interaction between both membranes or between a membrane and a wall (assuming that it can be described in terms of a harmonic potential) is a consequence of the closure of the shell. The state of adhesion and the adhesion energy of tension free shells are determined by the adhesion energy and the pronounced thermally excited bending undulations resulting in a dynamic roughness of the membrane of the order u 2 k B TL2 / . As shown by Peliti and Nelson (2), the energy functional (Equation S.M.1) can also be applied to tethered membranes exhibiting shear elasticity (such as the composite cell envelopes of erythrocytes) if the bending

2 u 2 u 2 u 2 u 2 = dA 2 + 2 + + + v (h h0 ) 2 (S.M.1); y 2 x y 2 x


modulus is replaced by a renormalized modulus c(q): 9 k BT C ( q) = c 0 + 16 c 0 q 2 The second term accounts for the coupling between the bending and the extensional deformations of the shell resulting in the apparent increase of the bending modulus at short wavelengths of excitation. Following Auth et al. (3), the dynamic roughness of the composite shells can be understood on the basis of a two shell model by considering the coupling between the sub-shells. Thus one has to distinguish between two situations: For large wave vectors q>>q* the amplitudes are small and the two sub-shells fluctuate independently. The mean square amplitudes of each shell (i) can be obtained from Equation (S.M.1) by application of the equipartition theorem and can be expressed in terms of the normalized bending modulus i (q) as follows: 2 k B T ui ( q ) 2 = 2 . L i ( q)q 4 For fluid plasma membranes (such as vesicles) it is i ( q ) = B + q 2 + v q 4 , (where B is the bending modulus) and for cells envelopes with tethered cytoskeleton i ( q ) = C ( q ) + q 2 + v q 4 . For small wave vectors the fluctuation of the two shells are synchronized and the amplitudes are k BT 2 2 < u >= 2 4 L Bq + C q4

The two-shell-model can well explain the experimental data at long wavelengths, with = 2/q large compared to the mesh size of the cytoskeleton ( ~0.1 m). The bending excitation is dominated by the plasma membrane and exhibits a bending modulus of about 50 kBT (B = 4 x 10 -19J) in agreement with measurements (4). However, the experiments show an anomalously large amplitude for = 2/q corresponding to an apparent bending modulus of 5 kBT (over an order of magnitude larger than the value of 50 kBT expected for a PM containing 50mole % cholesterol). This points to non-thermal random driving forces that could be explained in terms of a higher effective temperature Teff. One possible source of these chemical forces could be the random phosphorylation and dephosphorylation of the spectrinmembrane coupling protein band IV.1. (cf. Figure 1). This is expected to result in the dynamic binding and unbinding of the spectrin-membrane linker which is associated with the generation of defects in the hexagonal spectrin network (such as local pairs of +60 and -60 disclinations, corresponding to dissociated edge dislocations). As illustrated in the inset of Figure S.M.8 (at the upper left), this results in the formation of pairs of saddle like and conical caps and gives rise to an additional roughness of the tethered membranes.


Figure S.M.8 Mean square amplitude of erythrocyte flicker spectra as function of the excitation wave vector (bottom abscissa) and wavelength (top abscissa)): the dots indicate experimental data for discocytes shapes and the stars for echinocytes (see urchin like shapes). The dashed curve is calculated by the two shell model (3). Note that the bilayer exhibits an excess area of about 10% with respect to the average area of the cytoskeleton Inset at top left: Illustration of the Nelson mechanism of buckling of two-dimensional hexagonal tethered network by generation of +60 and -60 disclinations at a distance large compared to the mesh size of the tethered network (after Reference (5))

Flickering is a universal phenomenon. Membrane undulations are not unique for erythrocytes but have been observed also for numerous other cells, in particular white blood cells (6). This is remarkable, considering the fact that the bending and shear moduli of these composite shells are 1000kBT and ~4 10-4 Jm-2 are thus much larger than the values for erythrocytes (10 kBT and 10-6Jm-2). This can be explained in

two ways. First, the thermally excited plasma membrane and the actin cortex are locally decoupled (e.g. between two talin mediated anchoring sites in Figure S.M.8) and the lipid-protein bilayer exhibits some excess area (with respect to the actin cortex). Second, the bending excitations are driven by non-thermal noise generated by the intracellular motions (6).


The biological functions of flickering: Considering the ubiquitous presence of passive and actively driven membrane bending excitations the question concerning its possible biological function arises. One important consequence of membrane bending excitations is the generation of entropic disjoining pressures (7). It is a consequence of the freezing-in of long wavelength modes if the membrane approaches a solid surface (or the cytoskeleton) to a distance which is smaller than the root mean square amplitude <u> of the bending excitations. It is of the order (k T ) 2 pdisj B 3 (corresponding to pdisj.~ d 0.1N/m2 at d~100 nm, for ~ 25 kBT). Three possible biological functions of the dynamic surface roughness of cell envelopes are summarized below. 1) It generates a disjoining pressure between the two sub-shells of the erythrocyte cell envelope. Thus, their average distance (of d 30 nm) is controlled by the balance between pdisj and the attractive forces generated by the coupling of the spring like linkers band IV.1 and ankyrin to the membrane proteins

2) The thermal excitations generate attractive isotropic forces between two membrane-cytoskeleton linkers (typically 1mN/m). One consequence is the generation of excess area of the PM with respect to the cytoskeleton (about 5% in erythrocytes). The attractive forces between linkers are however balanced, resulting in an isotropic distribution of the anchoring sites. 3) The undulation forces play important roles for the control of cell adhesion. It controls the swelling of lipids in water and prevents the sticking of cells to solid surfaces through nonspecific forces. However, the entropic pressure also facilitates the formation of specific bonds between cell adhesion molecules (CAM) that are hidden within the glycocalix of the cells (8).

References to Supplemental Material G

1. Lipowsky R, Sackmann E. 1995. Handbook of Biological Physics, Vol. I. Elsevier: Amsterdam 2. Nelson DR, Peliti L. 1987. J. Phys. France 48:1085 3. Auth T, Safran SA, Gov N. 2007. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 76:0519109 4. Strey H, Peterson M, Sackmann E.1995. Biophys. J. 69:478-88 5. Gov N, Safran S. 2005. Biophys. J. 88:185974 6. Zidovska A, Sackmann E. 2006. Phys. Rev. Lett. 96:048103-7 7. Helfrich W. 1978. Z. Naturforschung. 33 a:30515 8. Pierres A, Benoliel AM, Touchard D, Bongrand P. 2008. Biophys. J. 94:4114-22