Cytoskeletal Mechanisms for Breaking Cellular Symmetry

Cytoskeletal Mechanisms for BreakingCellular Symmetry

R. Dyche Mullins

N312F Genentech Hall, UCSF School of Medicine, 600 16th Street, San Francisco, California 94158

Correspondence: [email protected]

Cytoskeletal systems are networks of polymers found in all eukaryotic and many prokaryoticcells. Their purpose is to transmit and integrate information across cellular dimensions andhelp turn a disorderly mob of macromolecules into a spatially organized, living cell.Information, in this context, includes physical and chemical properties relevant to cellularphysiology, including: the number and activity of macromolecules, cell shape, and mechan-ical force. Most animal cells are 10–50 microns in diameter, whereas the macromoleculesthat comprise them are 10,000-fold smaller (2–20 nm). To establish long-range order overcellular length scales, individual molecules must, therefore, self-assemble into larger poly-mers, with lengths (0.1–20 m) comparable to the size of a cell. These polymers must thenbe cross-linked into organized networks that fill the cytoplasm. Such cell-spanningpolymer networks enable different parts of the cytoplasm to communicate directly witheach other, either by transmitting forces or by carrying cargo from one spot to another.

Because they control the architecture andinternal organization of the cell, cytoskeletal

systems play uniquely important roles in break-ing symmetry and establishing cell polarity (Liand Gundersen, 2008). Broadly speaking, cyto-skeletal networks participate in symmetrybreaking in three ways: (1) by recording or“freezing” the results of symmetry-breakingevents performed by other signaling networks;(2) by providing feedback loops to associatedsignaling networks that enable them to breaksymmetry; and (3) by directly generating asym-metry via intrinsic cytoskeletal mechanisms.In this article, I concentrate on symmetry-breaking mechanisms intrinsic to cytoskeletalsystems. First, I briefly describe some

components and properties of the major eu-karyotic cytoskeletal systems. Recent reviewsprovide more detailed descriptions of theactin (Pollard and Borisy 2003), microtubule(Wittman and Desai 2005), and intermediatefilament (Goldman et al. 2008) cytoskeletons.Next, I discuss some basic principles usefulfor understanding how polymer networks gen-erate asymmetry. Finally, I outline specificexamples of cytoskeleton-mediated symmetrybreaking.

POLYMERS, PUSHING, AND PULLING

The three main cytoskeletal systems in eukary-otic cells are composed of actin filaments,

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microtubules, and intermediate filaments. Allthree systems play a role in determining theshape and mechanics of a cell by resisting andresponding to externally applied forces. Inaddition, networks of actin filaments andmicrotubules can: (1) generate pushing forcesby self-assembly or expansion; (2) transmitpulling forces caused by motor molecules; and(3) provide oriented tracks for directed move-ment of cargo (Fig. 1). Little is known abouthow intermediate filaments are assembled orhow they contribute to symmetry breakingand cell polarity, so this discussion focuses onactin filaments and microtubules.

Individual actin filaments and microtubulesare structurally and kinetically polarized.Structural polarity means that the polymersare not symmetrical. That is, the polymerlooks different from its reflection in a mirroror from itself rotated 1808. One consequenceof this is that motor molecules move preferen-tially in one direction or the other on an actinfilament or microtubule. In addition, someaccessory factors bind specifically to one end

of a polymer but not the other. Kinetic polaritymeans that one end of each polymer growsfaster than the other. The fast end of a micro-tubule is called the plus end and the slow, theminus end. For actin filaments, these arecalled the barbed (fast) and pointed (slow)ends (Woodrum et al. 1975). Both structuraland kinetic polarity are essential for the abilityof actin filaments and microtubules to formorganized and polarized networks.

The two most important differencesbetween microtubules and actin filaments aremechanical rigidity and assembly dynamics.Microtubules are far more rigid than actin fila-ments and they have more complex assemblydynamics. One measure of polymer rigidity isa parameter called persistence length, essentiallythe distance over which the polymer can trans-mit force. Actin filaments have a persistencelength of 10–20 mm, whereas the persistencelength of a microtubule is 300–500 timeslarger (Gittes 1993). Because of their rigidity,individual microtubules can form relativelystraight tracks that span the length of a cell.Many animal cells take advantage of this andconstruct radial arrays of microtubules thatfunction as hubs for intracellular trafficking.These arrays are polarized, with microtubuleminus ends collected together at the center andplus ends radiating out to the cell edge. To movetoward the center of the cell, cargo moleculesmust recruit motors that carry them towardminus ends of microtubules. To move to theplasma membrane, they recruit motors thatmove toward microtubule plus ends. Becausetheyare less rigid, actin filaments that participatein large scale cellular organization are generallycross-linked into ordered bundles or more disor-dered gels. Stress fibers, for example, are mixed-polarity bundles of actin filaments that connectsites at which cells make intimate contact withthe extracellular matrix (Ridley and Hall 1992).They generate contractile forces and enablecells to pull against their external environment.In addition, networks of more randomly cross-linked actin filaments adjacent to the plasmamembrane give most animal cells their shape.Filament assembly and disassembly withinthese networks produce changes in cell shape

A

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Figure 1. Mechanisms by which cytoskeletal networksintegrate information over long distances. (A)Pushing forces generated by polymer assembly ornetwork expansion, (B) pulling forces caused bymotor sliding filaments, and (C) oriented tracks fordelivery of cargo. Note that pulling forces can alsobe generated by polymer disassembly (Lombilloet al. 1995) and pushing forces can be generatedby motor-mediated filament sliding, but theseprocesses are less common.

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and amoeboid motility. Other forms of motility,such as swimming driven by the beating offlagella (Riedel-Kruse et al. 2007) or the prop-agation of kinks in helical spiroplasma(Shaevitz et al. 2005) do not involve such dra-matic, global reorganization of the cytoskeleton.

The assembly dynamics of actin filamentsand microtubules are adapted to their distinctcellular functions. To a good approximation,the assembly of an actin filament can be de-scribed by a single set of rate constants (Pollard1986) and, under most conditions, the rate offilament assembly depends directly on the con-centration of free monomeric subunits available.The steady elongation of actin filaments is wellsuited to producing sustained forces requiredto advance the leading edge of a migrating cell(Pollard and Borisy 2003). Microtubules, in con-trast, can exist in one of two states: steadilygrowing or rapidly shortening. In the first state,microtubules are similar to actin filaments andtheir growth rate depends on the concentrationof available tubulin subunits. In the secondstate, microtubules shorten rapidly, regardlessof the concentration of available subunits.Individual microtubules can switch betweenthese two states, growing for a time and thenrapidly shrinking. This behavior is known asdynamic instability (Mitchison and Kirschner1984) and is important for the ability of micro-tubules to find chromosomes during mitosis(Holy and Leibler 1994; Wollman et al. 2005)and for the ability of radial microtubule arraysto find the center of a cell (Holy et al. 1997).

In addition to polymer assembly and dis-assembly, motor-driven processes also playimportant roles in cellular organization andsymmetry breaking. Different classes of motormolecules are responsible for sliding filamentsand for moving cargo. Actin-based motors areall part of the large myosin family, whose mem-bers are adapted to carry out many different cel-lular tasks. Myosin II, for example, assemblesinto bipolar minifilaments and induces slid-ing of anti-parallel actin filaments past eachother (Reisler et al. 1980; Pollard 1982). Toprevent interference between the multiplemotor domains, each subunit of a myosin mini-filament interacts only briefly with an actin

filament, imparting a brief tug and then lettinggo (De la Cruz and Ostap 2009). Addedtogether, all the tugs produced by a myosin IIminifilament produce a sustained, contractileforce in the network. In contrast, other familymembers, such as myosins V and VI, remainbound through many cycles of motor activityand can move for a considerable distance alonga filament before falling off (Mehta et al. 1999;Rock et al. 2001). Single molecules of these“processive” motors can, therefore, transportcargo over long distances. Myosins can alsomove cargo in both directions along an actinfilament. Myosin V transports cargo towardthe fast-growing barbed end while myosin VImoves in the opposite direction (Wells et al.1999). Unlike actin-based motors, microtubulemotors appear to have arisen twice in eukaryoticevolution. The newer class of motors, which arenot found in primitive eukaryotes such asGiardia labmlia, are called dyneins (Gibbonset al. 1965) and they move from plus end tominus end. Dyneins help construct and powerflagella and mitotic spindles and they carry themajority of minus-end-directed cellular cargo(Vaisberg et al. 1996). The older class ofmotors is a large protein family, called the kine-sins (Lawrence et al. 2002; Vale et al. 1985).Some kinesins (e.g., kinesin-1) are processiveand move cargo long distances, whereas others(e.g., Eg-5 and other class-five kinesins) arespecially adapted to cross-link microtubules orcause them to slide past each other (Miki et al.2005; Vale and Milligan 2000). Similar to themyosins, the kinesin family contains bothplus-end- and minus-end-directed motors.

BASIC PRINCIPLES

I now focus on three principles that appear tobe important in the majority of cytoskeleton-mediated symmetry-breaking events: (1) posi-tive feedback, (2) architectural heterogeneity,and (3) network mechanics.

POSITIVE FEEDBACK

Nothing in nature is perfectly symmetricalor homogeneous, so a simple way to generateasymmetry from apparently symmetrical

Cytoskeletal Mechanisms for Breaking Cellular Symmetry

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starting conditions is to amplify very small,pre-existing defects or inhomogeneities. Auto-catalytic processes, in which the product of areaction feeds back positively to speed up itsown production, can dramatically amplifytiny, spatial fluctuations. This is such a generalprinciple that it is difficult to imagine anyform of symmetry breaking in which positivefeedback does not play some role. In fact, insome situations, a single positive feedbackloop appears to be sufficient to generate cellularasymmetry (Kozubowski et al. 2008; Altschuleret al. 2008). The best experimental evidence forthis comes from budding yeast, which deter-mine the position at which a daughter cellforms by localizing the machinery for exocyto-sis and cell wall synthesis to a single spot on themembrane (see Slaughter et al. 2009). This spotis usually determined by pre-existing cellularlandmarks but mutants that lack the landmarksor the ability to recognize them still (randomly)select a single, unique bud site (Chant andHerskowitz 1991). One signaling moleculethat is important for this process is a smallRho-family GTPase, Cdc42. Two positive feed-back loops have been shown to participate inlocalization of Cdc42: one involving actin-dependent transport of activated Cdc42 to themembrane (Wedlich-Soldner et al. 2003) andthe other involving autocatalytic activationof Cdc42 mediated by a scaffold protein,Bem1 (Kozubowski et al. 2008). Interruptionof both feedback loops abolishes polarizationbut removal of either one alone does not.Mathematical analysis of autocatalytic recruit-ment of molecules to a membrane poses impor-tant constraints on the ability of such a simplemechanism to generate polarity (Altschuleret al. 2008). A unique site on the membranecan be specified only when: (1) the totalnumber of signaling molecules is small and(2) the rate of autocatalytic recruitment ishigh compared to the rates of spontaneousmembrane binding and dissociation. The rela-tive lack of robustness of this simple mechanismmay explain why many cellular systems rely onmultiple, redundant feedback loops.

In cytoskeletal networks, positive feedbackcan include autocatalytic mechanisms for

assembling or disassembling component poly-mers as well as cooperative mechanisms forchanging the local architecture of the network.The Arp2/3 complex, for example, is an impor-tant nucleator of new actin filaments and itsactivity is stimulated by the presence of pre-existing filaments (Welch and Mullins 2002).To generate a new filament, the Arp2/3 com-plex must interact with a pre-existing (or“mother”) filament, an upstream activator,called a nucleation promoting factor (NPF),and an actin monomer bound to the NPF(Fig. 2). After nucleation, the newly formed“daughter” filament remains attached to themother filament, leading to formation of across-linked filament array. As the density offilaments increases, the rate at which thecomplex is recruited and activated increases.This feedback loop is important for buildingrobust and stable actin networks at the leadingedge of motile cells (Svitkina and Borisy 1999;Iwasa and Mullins 2007; Wang 2009) as well assites of endocytosis (Kaksonen et al. 2006;Orlando and Guo 2009). Obviously, this feed-back cycle cannot continue indefinitely, produ-cing an infinite density of actin polymer. Likeall positive feedback loops in biology, itis ultimately limited by some physical or bio-chemical constraint, in this case by the avail-ability of actin monomers required to activatethe complex. When the number of growing fil-aments becomes very large, they act as a sink,competing with the complex for NPF-boundactin (Akin and Mullins 2008). Assembly ofmicrotubules is not as well understood butthere does not appear to be an equivalent posi-tive feedback system controlling their nucle-ation. Instead, a motor-mediated feedback loopcontrols assembly and stability of the microtu-bule organizing centers that focus the minusends of microtubules to discrete spots (dis-cussed later).

ARCHITECTURAL HETEROGENEITY

In cell signaling, the most common type ofspatial heterogeneity is a fluctuation in thelocal concentration of an activated signalingmolecule, say a kinase or a G-protein. Because

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cytoskeletal networks are collections of cross-linked, linear polymers, they show additional,often subtle, classes of spatial heterogeneity.These include local differences in: (1) polymerdensity; (2) polymer alignment and orientation;(3) activity of motor molecules; or (4) numberof bound cross-linking proteins (Fig. 3). Eachof these architectural heterogeneities can beamplified by positive feedback to producelarge-scale variations in network properties.As noted above, local polymer densitycan affect the activity of the actin-nucleatingArp2/3 complex. In addition, polymer orien-tation and alignment can affect interactionwith motors and cross-linkers. Myosin II, forexample, prefers to interact with antiparallelbundles of actin filaments. By recruiting andaligning additional filaments, myosin II motoractivity can produce additional myosin IIbinding sites (Schaub et al. 2007). This positive

feedback loop participates in polarization ofmany motile cells (see the following discussion).Also, in addition to motors, the number ofbound cross-linking proteins can determinethe degree of filament orientation and thelocal mechanical properties of a network. Atlow concentrations, many actin filament cross-linking proteins, including, for example, a

actinin, will stabilize filament networks with avariety of geometries, from parallel bundles toorthogonal arrays. At higher concentrations,these cross-linkers cause filaments to zippertogether and preferentially form mixed-polaritybundles (Wachsstock et al. 1994). In vivo, theasymmetric localization of the actin-cross-linking protein cortexillin, for example,helps drive shape changes required for celldivision, even in the absence of myosin IImotor activity (Girard et al. 2004; Weber et al.1999).

Actinmonomer

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B

Arp2complex

Motheractin

filament

V C

V C AWASP-familyVCA domain

a. Requirements for nucleation

b. Assembled components

c. Activated state

d. Stable filament

Figure 2. Positive feedback in the activation of the Arp2/3 complex. (A) Molecular mechanism of Arp2/3activation. (a) Four factors must come together to produce a new filament: An Arp2/3 complex, an actinmonomer, a VCA domain, and a pre-existing actin filament. (b) All the components assemble into apreactivation complex. (c) A conformational change in the complex results in formation of a new stablefilament (d) attached to the side of the pre-existing (mother) filament. (B) The requirement for apre-existing filament makes the nucleation reaction auto-catalytic. The existence of a single filament near asurface covered with an Arp2/3 activator leads to nucleation of more filaments, which, in turn, stimulatefurther Arp2/3-dependent nucleation.





NETWORK MECHANICS

The mechanical properties of a material can bedescribed by how it responds to an appliedforce. Elastic solids are initially deformed butthen return to their original shape after forceis removed. Viscous liquids, by contrast, flowin response to force and do not regain their orig-inal shape when force is removed. Cytoskeletalnetworks can behave either like viscous liquidsor elastic solids, or combinations of both,depending on the architecture of the network

and how force is applied to it (Sato et al.1987; Wachsstock et al. 1993). Generally, thedensity and orientation of the polymers andthe number of cross-linkers connecting themdetermine whether a network resists defor-mation like an elastic solid or flows likeviscous liquid (Fig. 4). In addition to forcesapplied from the outside, cytoskeletal networkscan also respond to forces generated within thenetwork by motor molecules. One importantclass of symmetry-breaking processes makesuse of the asymmetrical flow of cortical actinnetworks caused by nonuniform distributionsof motors and cross-linkers. These flows cangather signaling molecules and polarity deter-minants that are bound to actin filaments andconvert a uniform distribution into a highlypolarized one. Mechanisms of this type areresponsible for polarization of oocytes and fer-tilized eggs in many species (see the followingdiscussion).

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Figure 3. Typical heterogeneities in the architecture ofpolymer networks. (A) Variations in polymer density.Arrow denotes region of low density. Lower panel:Electron micrograph of actin filaments in a sea ur-chin coelomocyte (Henson et al. 1999). (B) Variationsin orientation or alignment of polymers. Arrowdenotes region of filament alignment in an otherwiserandom gel. Lower panel: Variations in actin filamentalignment in the periphery of a fibroblast cell (Svit-kina et al. 2003). (C) Variations in density or activityof accessory factors such as motororcross-linker mol-ecules. Arrow denotes region of high concentration ofmotor or cross-linker. Lower panel: distribution ofmyosin motors (yellow) in the actin network of afish epidermal keratocyte (Svitkina and Borisy 1997).

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Figure 4. Elastic versus viscous behavior of cross-linked polymer networks. (A) Some cross-linkedcytoskeletal networks respond to some types ofapplied forces (arrows) like elastic materials. Theydeform and then return to their original shape afterthe force is removed. (B) Other networks flow likeviscous liquids in response to applied forces and donot return to their original shape after the force isremoved. For most networks, the response toapplied forces is best described by a combination ofthese viscous and elastic behaviors.

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In addition to deformation and flow, me-chanical failure of cytoskeletal networks can alsocontribute to symmetry breaking. To under-stand how this works, imagine a breakable,macroscopic object with spherical symmetry—say a balloon. A straightforward way to breakthe spherical symmetry of this particularobject would be to over-inflate it until the skinof the balloon is stretched beyond its elasticlimit and fails (i.e., pops). Failure occurs at aspecific site, probably determined by micro-heterogeneities in the material. In a similarway, external and internal forces acting on acytoskeletal network can produce symmetry-breaking ruptures.

The remainder of this article is devoted todiscussing specific examples of cytoskeleton-mediated symmetry breaking in light of thegeneral principles discussed previously.

ASTERS AND SPINDLES: THESPONTANEOUS ASSEMBLY OFMICROTUBULES INTO ORGANIZEDSTRUCTURES

In many cells, the bipolar mitotic spindle andmonopolar interphase microtubule array areorganized around well-defined microtubuleorganizing centers, called centrosomes. Thearchitecture and composition of centrosomesare not completely understood but they con-tain microtubule nucleating and binding fac-tors that collect and anchor microtubule minusends. Centrosomes, however, are not essentialto life. All plant cells and many meiotic animalcells lack them entirely. In addition, work fromseveral laboratories over the past two decadeshas shown that cells that normally containcentrosomes can carry out many essential,microtubule-based functions without them.Laser ablation of centrosomes in animal cells,for example, does not prevent assembly of func-tional mitotic spindles (Khojakov et al. 2000)and fission yeast lacking spindle pole bodies(functional equivalents of centrosomes) as-semble functional interphase microtubulearrays, indistinguishable from those normallyassembled around spindle pole bodies(Carazo-Salas and Nurse 2006). These results,

together with the observation that microtubulesand motor proteins can spontaneously self-assemble into radial arrays and bipolar spindles(see following discussion), suggest that centro-somes evolved, in part, to increase the robust-ness and fidelity of pre-existing pathways forspontaneous assembly of microtubule arrays(Marshall 2009).

Asters

Radially symmetrical arrays of microtubules,like those found in interphase animal cells, areoften called asters. It might seem odd todescribe assembly of such an array as symmetrybreaking, but, from the point of view of a mol-ecule in the cytoplasm, the broken symmetry isobvious. Rather than a linear, anterior–posterior or dorsal–ventral axis, the axis ofpolarity established by such an array is frominside to outside. Leibler and coworkersshowed that radial arrays of microtubules canbe spontaneously assembled from mixtures ofmicrotubules and motors (Nedelec et al.1997). These authors constructed artificial,four-headed kinesins and mixed them withpreformed, stabilized microtubules. At certainconcentrations, the motors convert the spatiallyuniform, random distribution of microtubulesinto a collection of well-separated, radial arraysof approximately the same size. Using plusend-directed kinesins, aster assembly beginswith the motors binding to microtubules andmoving toward their plus ends. Because theycan bind multiple microtubules simultan-eously, the motors can draw plus ends together.If a motor binds a pair of antiparallel micro-tubules, for example, it will force their minusends in opposite directions and bring theirplus ends to the same point. For microtubulesthat cross each other at an arbitrary angle, amotor that binds both will pull both plus endsto the crossover point. Because each microtu-bule can bind many motors and contact manyother microtubules, many plus ends wind upmoving to the same location, producing aradial array, or aster, centered on this point(Fig. 5). The remarkably uniform size ofthe asters produced in this experiment is





determined by the average lengths of the micro-tubules and the concentration of the motormolecules (Surrey et al. 2001).

What about the three principles outlinedabove? In this system, the architectural hetero-geneities required to break symmetry are therandomly occurring points of microtubuleoverlap, where motors can bind simultaneouslyto multiple microtubules. As more plus ends arecollected together in one spot, the microtubulesradiating from this spot will contact and reelin additional microtubules from a larger andlarger volume, resulting in positive feedback.Finally, the high mechanical rigidity of themicrotubules is required to prevent them fromsnarling and buckling as they are moved

around by motors. If the microtubules wereless rigid, they would form a tangled hairballrather than an organized radial array.

In this system, plus end-directed motorsproduce asters with microtubule minus endsfacing out while minus-end-directed motorsproduce asters of the opposite polarity. Asimilar mechanism can probably constructradial microtubule arrays in vivo. In cells,dynein motors carry cross-linking factors (e.g.,NuMA) to the minus ends of microtubules, pro-moting their assembly into radial arrays inwhich plus ends face out. When radial microtu-bule arrays are placed in a confined volume,they tend to find the center of the space. Thisself-centering is the result of polymerization-dependent pushing forces and requires dy-namic instability of the microtubules (Holyet al. 1997). A similar self-centering of radialarrays is observed in vivo and involves the addi-tional participation of motor proteins locatedat the cell periphery (Rodionov and Boirsy1997; Burakov et al. 2003).

Spindles

Microtubules assemble, during mitosis, into abipolar, DNA-segregating machine called themitotic spindle. In animal cells, the poles ofthe spindle are formed by centrosomes and, inaddition to aligning and segregating chromo-somes, the microtubules between the centro-somes determine the size and morphology ofthe spindle. Centrosomes, however, are notabsolutely required for spindle assembly orDNA segregation. As noted above, plant cellsand animal cells undergoing meiosis form spin-dles without centrosomes and laser ablation ofcentrosomes in animal cells does not preventthe assembly of functional spindles. Healdet al. (1996) used meiotic Xenopus egg extractsto investigate spindle assembly in vitro andfound that neither centrosomes nor intactchromosomes were required for bipolarspindle formation. Addition of micron-sizedpolystyrene particles coated with plasmid DNAwas sufficient to induce localized polymeri-zation of microtubules. These microtubulesthen spontaneously assembled into bipolar

–

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Figure 5. Assembly of polarized, radial arrays ofmicrotubules. (A) Multi-valent microtubule-basedmotors can cross-link microtubules and cause themto slide past each other. (B) The polarity of themotors (in this case, minus end-directed) causesthe minus ends of the microtubules to be pulledinto proximity. (C) Additional motor-dependentinteractions recruit additional microtubules, even-tually forming (D) a polarized radial array.

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spindles with plus ends facing the DNA-coatedparticles in the middle and minus endsfocused at the poles. The first step in thisprocess, DNA-induced microtubule assembly,involves activation of a small G-protein, Ran,by a DNA-associated GTP-exchange factor,RCC1. Activated Ran then releases microtubulenucleation factors sequestered by importin-family proteins (Zheng 2004). The cloud ofmicrotubules assembled around the DNA-coated beads is then cross-linked by multivalentmotors. Multiple cross-links between pairs ofmicrotubules drives their alignment into amixed-polarity bundle. Formation of a more-or-less linear bundle at this stage ensures thatthe final spindle has two poles and not, say,three or five. Next, plus end-directed kinesin-family motors bound to DNA push the minusends of the microtubules out, away from theDNA-coated beads. Finally, dynein motorscarry microtubule cross-linking factors to theminus ends and focus them into a tight pole(Fig. 6) (Heald et al. 1997).

ACTIN-BASED MOTILITY OF PATHOGENSAND PARTICLES: A ROLE FOR MECHANICALFAILURE

Many micron-sized objects, including somepathogens and newly endocytosed vesicles,harness the assembly of actin networks to gener-ate force and move through the cytoplasm ofeukaryotic cells. Motile pathogens, including:Listeria, Shigella, and Rickettsia species, as well

as Burkholderia pseudomallei, Mycobacteriamarinum, and vaccinia virus, hijack the hostcell actin cytoskeleton and use it to powerintracellular motility and cell-to-cell spread.Conversely, in many cells, newly endocytosedvesicles appear to be pushed away from themembrane and into the cytoplasm by polarizedactin assembly (Merrifield et al. 2002; Kaksonenet al. 2006). A particularly dramatic exampleof this type of motility is observed in Xenopuseggs, where fertilization produces a globalburst of actin-based vesicle motility (Tauntonet al. 2000).

Motile pathogens and vesicles expressdifferent molecules on their surfaces and inter-act with different host cell factors but, ulti-mately, they all recruit and activate the sameactin-nucleating Arp2/3 complex (Gouinet al. 2005). In fact, the only molecular require-ment for this type of actin-based motility is asurface-attached activator of the Arp2/3 com-plex. A remarkable feature of this type of motil-ity is that, even when the Arp2/3 activatoris uniformly distributed on the surface of theparticle, it can robustly form a polarized actinnetwork and move in a vectorial fashion(Cameron et al. 1999). In vitro, polystyrenemicro-spheres and lipid vesicles uniformlycoated with activators of the Arp2/3 complex,induce formation of spherically symmetricalactin networks which then break symmetry toform motile actin “comet tails.” Several modelshave been proposed to describe how these sym-metrical particles generate asymmetrical actin

Nucleation

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Figure 6. Formation of bipolar spindles in the absence of centrosomes. (A) In cell extracts, DNA-coated particlesinduce nucleation of microtubules whose polarities are random with respect to the DNA. (B) Microtubulecross-linkers and multivalent motors align the microtubules while minus-end directed motors begin tocluster their minus ends. (C) Plus end-directed microtubule motors associated with DNA push the clusteredminus ends away from the DNA-coated particles, forming a bipolar, spindle-shaped structure.





networks (van Oudenaarden and Theriot 1999;van der Gucht et al. 2005). The most successfulis based on treating the crosslinked actinnetwork as an elastic gel with a finite breakingstrength (Van der Gucht et al. 2005; Van derGucht and Sykes 2009). In this model, actinnucleation at the surface of the particle producesa symmetrical actin shell and then continuednucleation and polymerization at the surfaceof the particle pushes the original shelloutward. The network behaves like the skin ofa balloon. As more actin polymerizes at thesurface, it stretches the network until eventuallyit ruptures. The energy stored in the elasticdeformation causes the expanding network topull away from the site of rupture, producing adramatic asymmetry in the distribution ofactin around the particle. This asymmetry per-sists because, even if new actin assembles uni-formly over the particle, the new networkcovering the site of rupture will be thinner andweaker than that on the opposite side, whereno rupture has occurred. This asymmetricnetwork will expand and rupture again at thesame site (Fig. 7).

The role of mechanics in this system isobvious. The material properties of the actinnetwork will determine how much force isrequired to inflate the network and how muchstretching it can withstand before it ruptures.The important architectural heterogeneity inthe system is a (possibly slight) fluctuation innetwork density that determines the initial siteof rupture. Because the outer network isunder tension, the loss of a small number ofcross-links results in increased tension on theremaining cross-links. This increased tensionincreases the probability that some of thesecross-links will fail, and so on. This positive feed-back means that, once a small rupture occurs, itwill tend to propagate, leading to a large scalemechanical failure.

POLARIZATION AND MOTILITY OF CELLSAND CELL FRAGMENTS

To move in a given direction, a cell must firstestablish an axis of polarity, with functionallydistinct regions at the front and back (Xu 2003;

Wang 2009). At the front of the cell, the leadingmembrane is pushed forward by assembly ofactin networks similar to those that push patho-gens and vesicles through the cytoplasm. At therear of the cell, the trailing edge is pulledforward by myosin-dependent contraction ofthe actin network. Adhesion to a substrate isrequired for some types of amoeboid loco-motion but the most basic form of movementthrough a three-dimensional matrix appears todepend only on actin-dependent protrusionand expansion of the cell’s front and retractionof its rear (Lammermann et al. 2008).

A

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Figure 7. Polarization of actin networks because ofexpansion and mechanical rupture. (A) Sphericalsurfaces coated with activators of the Arp2/3complex (black circle) direct assembly of sphericallysymmetrical actin networks (gray rings). Continuousnucleation of new filaments at the surface causesoutward displacement and stretching of the initialactin shell (1). (B) Continued expansion eventuallystretches the outer portions of the actin network tothe point of mechanical failure. The elastic energystored in the stretched shells is released bycontraction. The initial rip propagates because ofpositive feedback. As the rip progresses, theremaining stress in the network is distributed over asmaller number of cross-links, increasing theprobability that that they will fail. (C) Recoil of theactin shell away from the rip results in a polarizeddistribution of actin on the surface of the particle.

R.D. Mullins




Cells that crawl toward or away from chemi-cal cues determine direction of motion basedon activity of signaling systems linked to cellsurface receptors (Parent and Devreotes 1999).In many motile cells, however, the cytoskeletonappears capable of breaking symmetry anddetermining direction of migration withoutexternal cues. Evidence for this comes fromthe fact that a population of chemotactic cellsplaced in a spatially uniform concentration ofchemoattractant, which activates receptorsuniformly over the cell surface, spontaneouslybreak symmetry and move in random directions(Tranquillo et al. 1988; Weiner et al. 1999; Xu2007). Also, some cells, including fish epider-mal keratocytes, move constitutively in theabsence of any known external cue. The mem-brane of these cells appears to be uniformlypermissive for assembly or contraction of actinnetworks and the direction of migrationappears to be determined by mechanismsintrinsic to the cytoskeleton (Verkhovsky et al.1999; Yam et al. 2007; Xu et al. 2007). Recentreviews (e.g., Weiner 2002) provide a detaileddiscussion of how cytoskeletal assembly isintegrated with cellular signaling systems con-trolling chemotaxis. For purposes of thisdiscussion, I focus on symmetry-breakingmechanisms that appear to be independent ofexternal chemical cues.

Important insight into cytoskeletal pro-cesses that establish and maintain polarity hascome from the study of motile cell fragments,called cytoplasts. Cytoplasts can be producedfrom many cell types by several techniques(Malawista et al. 1985). They can be generated,for example, by treatment of fish keratocyteswith nonspecific kinase inhibitors, which causemotile regions at the cell front to pull awayand detach from the cell body (Verkhovskyet al. 1999). Cytoplasts lack a nucleus, a micro-tubule organizing center, and contain only afraction of the cytoplasm of the original cell.Despite this, they maintain a shape similar tointact cells and move for hours in a directedfashion. The organization of cytoplast actin net-works is remarkably similar to that of intactcells. The network at the leading edge of bothis a gel of cross-linked filaments with a large

fraction of barbed ends pointing toward theplasma membrane (Svitkina et al. 1997).Continuous assembly of this network pushesforward the leading edge (Theriot et al. 1991).At the rear of both cells and cytoplasts, actin fila-ments are collected into large, mixed-polaritybundles. These bundles also contain a largenumber of myosin II minifilaments that causethem to contract (Anderson and Cross 2009)and pull forward the rear of the cell (Lammer-mann et al. 2008; Xu 2003). The combinationof polymerization and protrusion at theleading edge and contraction at the trailingedge gives both keratocytes and their cytoplastsa characteristic crescent or canoe shape.

Occasionally, cytoplasts lose polarity andstop moving. Such cytoplasts go from crescent-shaped to round and actin assembly occursuniformly around their entire circumference.It appears, in these cases, as if the cell fragmenthas lost its rear end and that the entire peripheryis now functionally equivalent to the front of amigrating fragment. These nonpolarized cyto-plasts can spontaneously break symmetry andbegin moving again. Symmetry can also bebroken by small, externally applied pertur-bations and this type of forced symmetry break-ing has provided insight into cytoskeletalmechanisms of generating and maintainingpolarity. Verkhovsky et al. (1999) showed thata stream of liquid strong enough to deformthe edge of a nonmotile cytoplast can inducepolarization and sustained motility. Theseauthors found that induced deformations oftenpropagate along the edge of the cytoplast,becoming a zone of sustained network contrac-tion and defining a new rear. The cytoplast thenbecomes motile and crawls away from the site ofinitial perturbation (Verkhovsky et al. 1999).This phenomenon appears to depend on thepreference of myosin II minifilaments forantiparallel actin filaments. When the Arp2/3complex is active all around the periphery, itgenerates actin networks in which filamentsintersect at a variety of angles, all with theirbarbed ends facing toward the membrane(Svitkina and Borisy 1999; Mullins et al.1998). This type of network does not containmany favorable binding sites for myosin





II. Mechanically deforming the network bycrushing (or stretching) it forces some of thefilaments into an antiparallel alignment, pro-ducing new binding sites for myosin II. Oncebound, myosin II pulls additional filamentsinto the bundle, creating more myosin IIbinding sites (Anderson et al. 2008). This posi-tive feedback causes an initially small architec-tural heterogeneity in the network to propagate(Fig. 8). Given the strong positive feedback,why does this architectural change in thenetwork fail to propagate around the entire cir-cumference of the cytoplast? The simplestanswer is that it is ultimately limited by celllocomotion. As the zone of contraction propa-gates, the forces acting on the cytoplastbecome unbalanced. The pushing force causedby actin assembly on the opposite side combineswith the contraction to cause the cell to migrate.Cell migration pushes the leading edge of thecell away from the original actin network. Seenfrom the perspective of the leading edge itself,the actin network appears to treadmill back-wards. When myosin II binds to the networkanywhere but the trailing edge, it is swept back-ward with the flow of actin filaments. This rear-ward flow continuously sweeps myosin II awayfrom the leading edge and further concentrates

it in the rear of the cytoplast (Anderson et al.2008). This type of flow-induced asymmetryof actin binding proteins also plays a key rolein establishing polarity of many types ofoocytes and fertilized eggs (see the followingdiscussion). In addition, the myosin-dependentcollection and contraction of actin filamentsappears to be limited by a poorly understood,myosin-dependent mechanism for disassem-bling actin filaments (Biron et al. 2005;Medeiros et al. 2006).

So much for cell fragments. What aboutintact cells? Yam et al. (2007) found that themechanism by which intact keratocytes breaksymmetry and establish polarity is remarkablysimilar to the mechanism described for cyto-plasts. These authors watched nonpolarizedcells break symmetry and initiate directed moti-lity and found that, in all cases, symmetrybreaking began as a contraction in the actinnetwork that propagated to establish a trailingedge for the cell.

POLARIZATION OF OOCYTES ANDFERTILIZED EGGS

In most metazoans, the major axes of embryo-nic development—anterior–posterior and

A B C D

Figure 8. Polarization and motility of cytoplasts. (A) In cytoplasts that have lost polarization, actin assemblyoccurs symmetrically around the periphery, producing uniform outward forces (arrows) that sum to zero.Myosin II minifilaments (red symbols) are randomly oriented and do not produce coherent, large-scale pull-ing forces. (B) Random fluctuations in myosin localization or activity or external mechanical perturbation (-large arrow) can produce local alignment of actin filaments. (C) Alignment of filaments produces morefavorable binding sites for myosin minifilaments and makes their pulling forces more coherent (arrows at bo-ttom of cell). As the contraction proceeds, more filaments are aligned and recruited into the contractilebundle. This bundle is a poor substrate for the Arp2/3 complex and inhibits production of polymerization-driven outward forces. This imbalance of outward forces enables polymerization on the opposite side of thecell to produce net protrusion. (D) Translocation of the cell in the direction of protrusion results in treadmillingof the actin network away from the leading edge. This treadmilling carries myosin II away from the leading edgeand concentrates it in the rear of the cell, further enhancing the polarization of the cell and the spatial separationof protrusive and contractile forces.

R.D. Mullins




dorsal–ventral—are established at the singlecell stage, either during oogenesis or soonafter fertilization. Different organisms usedifferent mechanisms to establish polarity butall rely heavily on the actin and microtubulecytoskeletons. One of the most completelyunderstood polarity generating mechanismsoccurs in the Caenorhabditis elegans zygote(Munro and Bowerman 2009). In this case,anterior–posterior polarity is determined bysperm entry. Both the actin and microtubulecytoskeletons work together to convert thisinitial asymmetry into a global cell polarity.

In C. elegans eggs, the determinants thatspecify posterior cell fates, including thePar-3/Par-6/PKC-3 complex, are uniformlydistributed throughout a cortical actin net-work, just below the plasma membrane.Determinants that specify the anterior fatesare found deeper in the cytoplasm. Fertilizationinduces a dramatic, global activation of myosin-dependent contraction of the actin network,probably via a wave of calcium influx. At thesite of fertilization, the sperm nucleus entersthe egg, bringing along its centrosome, whichnucleates formation of a radial array of microtu-bules. The microtubules of this sperm aster actas tracks to deliver factors that inhibit myosincontraction to the cortex (Jenkins et al. 2006).

The cortex adjacent to the site of sperm entryreceives the largest dose of these factors andbecomes the site of weakest contraction.Because of the reduced contraction, this zonecannot resist the pulling forces coming fromthe rest of the cortex and the cortical actinnetwork is, therefore, pulled away from thissite, dragging along the associated posteriordeterminants (Fig. 9).

SUMMARY AND PERSPECTIVE

Cytoskeletal systems are organized networksof polymers that control cell shape, organizeintracellular spaces, and integrate informationover cellular length scales. Establishing cell po-larity or otherwise breaking cellular symmetryrequires, at the very least, reorganizing thecytoskeleton and, in many cases, is actuallydriven by cytoskeletal mechanisms. Autocata-lytic assembly, disassembly, or reorganizationof cytoskeletal networks can amplify small per-turbations and help convert subtle externalcues into large-scale cellular responses. In theabsence of external cues, these feedback loopscan often amplify spontaneous fluctuations innetwork architecture and break symmetry ontheir own.

A B C

Figure 9. Contraction-induced polarization of C. elegans zygotes. (A) Before polarization, many posteriordeterminants (e.g., par6, yellow symbols) are uniformly distributed throughout the cortical actin network ofthe zygote. Soon after sperm entry, the activity of myosin II minifilaments (red symbols) causes contractionof the cortical actin network. The sperm centrosome (near the right side of the zygote) delivers factors to thecortex that locally inhibit myosin activity. (B) The asymmetric activity of myosin II pulls the cortical actinnetwork away from the sperm centrosome toward the opposite end of the cell. (C) Actin-associated polaritydeterminants pulled along with the cortical network define the posterior pole of the zygote and, ultimately,the embryo. Cortical actin removed from the future anterior pole is replaced by newly polymerized filamentsor filaments that well up to the surface from the underlying cytoplasm (green filaments).





In many examples of biological symmetrybreaking, the specific contributions of signalingand cytoskeletal systems are difficult to parse.Correct polarization of Drosophila oocytes, forexample, is known to require participation ofboth the actin and microtubule cytoskeletons,as well as multiple cellular signaling systems(Roth and Lynch 2009). At present, however,the specific molecular event that determinesthe positions of the anterior and posteriorpoles of the oocyte is unknown. In many cases,a complete understanding of a symmetry-breaking process will require its reconstitutionin vitro, either in cell extracts or from purifiedcomponents. A relatively small number ofcomplex cytoskeletal systems have already beenreconstituted in vitro and they have provideddeep insight into the pathways by which theyare assembled and the mechanisms by whichthey are regulated. They have also impressedon the field the fact that remarkably complex,and otherwise unpredictable, properties canemerge from the interaction of a small set ofmolecules. The next phase in cell biologicalresearch will be to use such reconstitutedsystems to bridge the gap between structuraland biochemical studies of individual moleculesand complex behavior of living cells andorganisms.

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published online September 30, 2009Cold Spring Harb Perspect Biol R. Dyche Mullins Cytoskeletal Mechanisms for Breaking Cellular Symmetry

Subject Collection Symmetry Breaking in Biology

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Symmetry Breaking in the Life Cycle of the

Brian D. Slaughter, Sarah E. Smith and Rong Li

Cell's CompassPlanar Cell Polarity Signaling: The Developing

AxelrodEszter K. Vladar, Dragana Antic and Jeffrey D.

Neuronal PolaritySabina Tahirovic and Frank Bradke

Cellular Polarity in Prokaryotic OrganismsJonathan Dworkin Polarity

Membrane Organization and Dynamics in Cell

Kelly Orlando and Wei Guo

ArabidopsisDivisions in Mechanisms Regulating Asymmetric Cell Symmetry Breaking in Plants: Molecular

Philip N. BenfeyJalean J. Petricka, Jaimie M. Van Norman and

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OogenesisDrosophilaSymmetry Breaking During Siegfried Roth and Jeremy A. Lynch

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Kenneth E. PrehodaEstablishment of Cell PolarityWidely Conserved Signaling Pathways in the

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Jasper van der Gucht and Cécile SykesShaping Fission Yeast with Microtubules

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Cytoskeletal Mechanisms for Breaking Cellular Symmetry