Chapter 9 The Cytoskeleton and Cell Motility 354 and Developmentand lmnswt imtro 01 m Theywerename filamentsdiameterwitha 10–12 ofnm. termediatefilaments inner lining of in the Section nuclear 12. envelope, present whichareas lamins), (theIFs Vsider type a filaments, cytoplasmicconstructionof the in found We will restrict the present discussion to classes immunologic and genetic, biochemical, wellas9.2)as major five into classes based on divided the type of cell in be which they are can IFs of subunits polypeptide areencoded by approximately humans, 70 different ge IFsarechemicallya heterogeneous group structurof microt and microfilaments Unlike cavities. body’s the physica to subjected are and muscleincludingthe that epithelialcells, neurons, c cells to strength cal ropelike fibersments flexible, Intermedia thatare providstrong, cells. animal in identified been only have tein called called tein d elongated an of consist cross-bridges these cells, 9.41). (Figure cross-bridges wispy thin, by filaments cy other to interconnected often are and cells imal o aohr nemdae lmn,mcolmn,o mi tubule at the other end. or microfilament, filament, intermediate another for a bind depending ment on at the one isoform, end and, intermed an for site binding a has molecule plectin hm o om iia-okn fiaet.Ms notably, rod-shape Most polypeptides of IFs all contain a central, filaments. similar-looking form to them organizationstructuralsimilar a share all quences, Mammalian Intermediate Filament Proteins Table 9.2 9.4 etnI Neuroepithelia types cell All IV Peripheral neurons Muscle Neurons of central III cells Mesenchymal III Astrocytes proteins Lamin III EpitheliaNestin III Epithelia II Neurofilament proteins Peripherin I distribution acidic Sequence type Glial fibrillary Desmin Vimentin (basic) Keratin (acidic) Keratin protein IF More be found in detailed tables can Lamin A V (Nuclear envelopes) (Nuclear V nerves V and peripheral IV IV IV B Lamin A Lamin NF-H NF-M NF-L protein (GFAP) different(26 polypeptides) different(28 polypeptides) ai V C Lamin IFs radiate through the cytoplasm of a wide variety lhuh F oyetds ae ies aio cd se acid amino diverse have polypeptides IF Although | Intermediate Filaments plectin Properties and Distribution of the MajorProperties 118,20,and 2007, 21:1582, htcneiti ueosioom.Eachisoforms. numerous in exist can that lcrnmcocp ssld unbranchedsolid, electronmicroscopeas thein seen discussedwas be toelements The second of the three major cytoskeletal (or IFs Trends Cell Biol rnsBohmSciTrends Biochem .T ae intermediate Tofilaments date, ). 82,2008. 18:29, . 134 2006, 31:384, . Primary tissue -V which I-IV, are found (Table 2. ells that line that allowsthat d, imeric pro- imeric e mechani- part of theofpart sta,inthat, es toskeletal ␣ ae fila-iate In manyIn e.Thenes. criteria. d con-nd stress, l -helical Genes ing site ubules, e fila-te of an- d cro- the in- - polypeptides spontaneously interact as their their as Two interact spontaneously 9.42). Figure polypeptides 1, (step sequence and size variable fibrousdomain isflanked oneach side byglobular do actin subunits of microtubules Th and microfilaments. mediate filaments different very from the globular t acid This long amino fibrous domainquence. makes the subunits homologous o and length similar of domain the polypeptides and the opposite end by their N-te C-ter the by defined end one with polarity, has dimer are aligned parallel to one another in the same ori app dimer mately Because 45 the nm ropelike intwo lengthpol (step 2). a form to other each around wrap te iprat etr ta dsigihs F from elements. IFs cytoskeletal distinguishes is that which feature important filament, other assembled the does too so l blocks polarity, building tetrameric the Because GTP. or ATP involvement direct the require to thought is steps a these of None 5). (step filamentintermediate gated one another in an end-to-end fashion to form the hi filamentsaccomplishedlengthsassounitofthese as pol the of growth Subsequent 4). (step nm) 60 (about eral)arrangement to form a filament that is one uni side-by a in another one with associatetetramers 8 Recent studies of the self-assembly of IFs in vitro la itselftetramer the directions, opposite in point olia odprils(elw.(C (yellow). gold particles colloidal by ant is localized Plectin protein plectin (green). microtubules (red) by long wispy conscross-bridges filaments Intermediate (blue) are seen visualization. Individual components have been digitally filaments. selec of a fibroblast after tion of the cytoskeleton protein cross-bridges. Figure 9.41 Figure 9.42, step 3 and in Figure 9.42Figure in s and 3 step as 9.42, Figure directions, (antiparallel) opposite in pointing C- and N- their with fashion staggered a in side by alig become that dimers two by formedtetramer like The basic building block of IF assembly is thought Intermediate Filament Assembly and Disassembly AND G ARY Intermediate Intermediate filament filament B ORISY Cytoskeletal elements are connected to one another byCytoskeletal .) Electron micrograph of a replica por- of a small Microtubule Plectin UTS OFOURTESY Gold-labeled Gold-labeled anti-plectin anti-plectin antibodies antibodies b . Because the dimersthe Because . tive removal of actin T ibodies linked to ATYANA to be connected to isting of the fibrous colorized to assistcolorized ␣ hlcl rods -helical nain theentation, suggest that cks polarity.cks to be a rod- S ubulin and tin length rmini. ghly elon- -side (lat--side ciate withciate ITKINAVI ypeptides hown in in hown f eitherof mainsof ned sidened e central ssembly mini ofmini termini ymer isymer f inter- other such roxi- an- ack se- 1 2 3 355

C N N N N

N

N

C C C C

Monomer Dimer Tetramer (a)

6 5 4

~60 nm

N

(b) Figure 9.43 Experimental demonstration of the dynamic character of intermediate filaments. These photographs show the results of an

5 nm experiment in which biotin-labeled type I keratin was microinjected Intermediate Intermediate Unit length into cultured epithelial cells and localized 20 minutes later using filament filament of filament immunofluorescence. The photograph in a shows the localization of (a) (b) the injected biotinylated keratin (as revealed by fluorescent anti-biotin antibodies) that had become incorporated into filaments during the Figure 9.42 A model of intermediate filament assembly and architec- 20-minute period following injection. The photograph in b shows the ture. Each monomer has a pair of globular terminal domains (red or yel- distribution of intermediate filaments in the cell as revealed by anti- ␣ low) separated by a long -helical region (step 1). Pairs of monomers keratin antibodies. The dotlike pattern of fluorescence in a indicates associate in parallel orientation with their ends aligned to form dimers that the injected subunits are incorporated into the existing filaments at (step 2). Depending on the type of intermediate filament, the dimers may sites throughout their length, rather than at their ends. (Compare with be composed of identical monomers (homodimers) or nonidentical a similar experiment with labeled tubulin in Figure 9.26.) Bar, 10 ␮m. monomers (heterodimers). Dimers in turn associate in an antiparallel, (F ROM RITA K. M ILLER , K AREN VIKSTROM , AND ROBERT D. staggered fashion to form tetramers (step 3), which are thought to be the GOLDMAN , J. C ELL BIOL . 113:848, 1991, F IG . 4. R EPRODUCED basic subunit in the assembly of intermediate filaments. In the model WITH PERMISSION OF THE ROCKEFELLER UNIVERSITY PRESS .) shown here, 8 tetramers associate laterally to form a unit length of the in- termediate filament (step 4). Highly elongated intermediate filaments are then formed from the end-to-end association of these unit lengths (step 5). Once formed, intermediate filaments undergo a process of dynamic re- tergents extracts just about everything except the cell’s inter- modeling that is thought to involve the intercalation of unit lengths of fil- mediate filaments. Because of their insolubility, IFs were ini- 9.4

ament into the body of an existing filament (step 6). ( b) A model of a tially thought to be permanent, unchanging structures, so it FilamentsIntermediate tetramer of the IF protein vimentin. ( B: F ROM ANNA V. S OKOLOVA , ET came as a surprise to find that they behave dynamically in AL ., COURTESY OF SERGEI V. S TRELKOV , PNAS 103; 16209, 2006, F IG . vivo. When labeled keratin subunits are injected into cultured 3A. © 2006 N ATIONAL ACADEMY OF SCIENCES , U.S.A.) skin cells, they are rapidly incorporated into existing IFs. Sur- prisingly, the subunits are not incorporated at the ends of the filament, as might have been expected by analogy with micro- Intermediate filaments tend to be less sensitive to chemi- tubule and microfilament assembly, but rather into the fila- cal agents than other types of cytoskeletal elements and more ment’s interior (Figure 9.43). The results depicted in Figure difficult to solubilize. In fact, treatment of a cell with ionic de- 9.43 might reflect the exchange of unit lengths of filament Chapter 9 The Cytoskeleton and Cell Motility 356 f h si o tnu.Ti peoye er srn r humans, strong in disease skin-blistering rare bears a to blance phenotype This tongue. or skin the of b severe cause can newborn, the by nursing during or b the through passage during mild occurring that as such even that pressure mechanical to sensitive so These health problems. have serious layer, epidermal o cells by synthesized a normally polypeptide keratin K14, encoding gene the in deletions carrying mice For types. cell particular in filaments of intermediate importance altered the revealed an have studies produce These or polypeptide. knockout) gene parti (a a produce polypeptide to fail that mice engineered ically lc xnltasot leading to the death of neur axonalblock transport, aggregates NF These Parkinson’sdisease. and ALS ing disorder neurodegenerative human several in seen is Aggregation diameter. in dramatically increases axon becomes filled with neurofilaments that provide suppo ext fully become has cell nerve the Once crotubules. mi- supporting of numbers large but neurofilaments few very tion when the axon is growing toward a target cell, generalized epithelial cell, and Figure 9.44 Figure and cell, epithelial generalized filam keratin the of arrangement spatial the of view 249). Figure 9.44 Figure 249). cytopl the to connections by plaques of desmosomes and hemidesmosomes (pages 257 cell the of edge outer anch and cell the of center the in envelope nuclear tether cytoplasm, throughthe radiate IFs containing arrangement within a pair of cultured epidermal cel bobn mcaia srse apid y h extrace the by environment. applied and stresses architecture mechanical absorbing cellular scaff a maintaining as serve and to able organizing is network IF the physic nections, various these of Because cytoskeleton. grated a into elements separate w otherwise these microfilaments, transforms and microtubules cell’s the and IFs and pancreatic acinar cells). Figure 9.44 Figure cells). acinar pancreatic h and liver cells, epidermal (including cells epithelial Keratin filaments constitute the structural primary Types and Functions of Intermediate Filaments 9.42ure s in shown (as network IF existing an into directly nase A leads to their disassembly. phosphorylation of vimentin filaments by example, pro subuni the of dephosphorylation and phosphorylation prim controlled are IFs of disassembly and assembly the axon (see Figure 9.13 Figure axon(see the tain the proper spacing between the parallel neurofi outwar These sidearms are thought to project from the neurofilament. that sidearms have NF-M and NF-H IFs, polypeptides t the of Unlike all of Tablegroup 9.2. IV type NF-M, and NF-H, NF-L, proteins: distinct three or IFs, parallel parallel to that of the o cell nerve axon (see are Figure axes long whose filaments intermediate of dles Efforts to probe IF functionEfforts have relied on largely The cytoplasm of neurons contains loosely packed bu packed loosely contains neurons of cytoplasm The neurofilaments a ). Unlike the other two major cytoskeletal elements, cytoskeletal major two other the Unlike ). a also depicts the interconnections betweeninterconnections the depicts also , as they are called, are composed ofcomposed are called, are they as , b ). In the early stages of differentia-of stages early the In ). a shows a schematica shows b shows the actualthe shows 9.13 s Keratin-ls. epatocytes, ored at theat ored e ,Fig-6, tep proteins of it contains laments of called called f the basalthe f ons. irth canal irth ,includ-s, b ed to theto ed example, ne,itended, ents of aof ents ua IF cular mice are listering of otherof of NFsof .These). rt rt as the trauma, arily by arily al con- al tein ki- inte- n l for old genet- riented main- s Forts. type I type esem- asmic llular llular may hich and epi- IF for for he n- d rm hs suis ht F hv tsu-pcfi fun tissue-specific arewhich than in othe in more some cells important have IFs that studies these i It from IFs. cytoplasmic lack cells affected the a though abnormaliti minor macrophages, relatively show fibroblasts, cells, blood in white expressed su is which have gene, polypeptides IF the lack that mice all Forexample, Not functions. essential failure. heart gestive eventua and arrhythmias, cardiac weakness, muscle tal Personsfr suffer disorder this with desmin. encodes myopathy related named disease, human inherited An fragile. tremely the makes IFs these of absence the and cell, muscle myofi the of alignment the maintaining in role tural ke a plays Desmin abnormalities. muscle skeletal and seri exhibit producepolypeptide desminto thefail knock Similarly, cells situated in K5epithelial layers. stre mechanical the imparting in stud IFs These of role (or the confirm K14). with polypeptide dimers forms which K14 polypeptide, homologous the encodes mutationsthe in carry shownthey has that patients dermolysis bullosa simplex simplex bullosa dermolysis epidermis to the basement membrane.epidermis h which in proteinsby defects of the hemidesmosome, 4 microfilament-b toward bias This organelles. and cles cytopl of transport long-distance the for tracks as serve to microtubules, than rather microfilaments, on marily plant cel In fact, and cytokinesis. phagocytosis, cles, vesi-of movement the as such processes, motile intracellular invo also are Microfilaments element. cytoskeletal of maj third the microfilaments, on depend all they nent: of these various examples of motility share at leas the substratum and guide the cell toward a synaptic of a growing axon sends out microscopic processes that surveyleadin the Similarly, wound. the sealing area, damaged wound act the as motile devices at that pull the cells sheet of epithelial of projections broad motile; ca cells of parts Certain microorganisms. and debris se Leg body the of tissues the patrol cells 7.11). blood white Figure (see jaws the of cartilage the and s the of cells pigment the as products diverse such f embryo, the of width entire the across migrate and nervous developing the leave embryo vertebrate a in Cells The areneural capable of remarkable motility. 9.5 As mentioned in Chapter 7, similar types of blisteri similar types As mentioned in Chapter 7, .Give some examples that reinforce the suggestion 1. .Compare and contrast microtubule assembly and 2. W E I V E R intermediate filament assembly. common to all cells. specific functions rather than in basic activities t intermediate filaments are important primarily in tissue- | Microfilaments s asd y uain i te ee that gene the in mutations by caused is , ( EBS ). 4 Subsequent analysis of EBSof analysis Subsequent ng diseases can be causedng diseases can olds the basal layer of the t one compo- out mice that cells cells over the asmic vesi-asmic arching forarching ous cardiacous tre.All target. edge of aof edge i,teeth, kin, ls rely pri- crest cells gene thatgene om skele-om hat are evidents n also bealso n vimentin rs. brils of aof brils cells ex- cells s evenes, y struc-y desmin- system gh tongth orming os of ions vd in lved or typeor ctions, that g edgeg con-l ased nd ies ch 357

Cytoplasm +

ER

Chromatin

Nucleus MTOC NPC Adherens junction ONM INM Interchromatin space +

+ Desmosome

Hemidesmosome

ECM Focal adhesion

Protein of outer nuclear membrane Ribosome Nesprin Protein of inner nuclear membrane MFs Integrins IF-anchoring plaques IFs Plakin-type cross-bridging molecule (e.g., plectin) Actin-anchoring plaques MTs Lamins

(a) Figure 9.44 The organization of intermediate filaments (IFs) within an epithelial cell. (a) In this schematic drawing, IFs are seen to radiate throughout the cell, being anchored at both the outer surface of the nucleus and the inner surface of the plasma membrane. Connections to the nucleus are made via proteins that span both membranes of the nuclear envelope and to the plasma membrane via specialized sites of adhesion such as desmosomes and hemidesmosomes. IFs are also seen to be interconnected to both of the other types of cytoskeletal fibers. Connections to microtubules (MTs) and microfilaments (MFs) are made primarily by members of the plakin family of proteins, such as the dimeric plectin molecule shown in Figure 9.41. ( b) Distribution of keratin-containing intermediate filaments in cultured skin cells (keratinocytes). The filaments are seen to form a cagelike network around the nucleus and also extend to the cell periphery ( A: R EPRINTED WITH PERMISSION FROM H. H ERRMANN ET AL ., N ATURE REVS . MOL . C ELL BIOL . 8:564, 2007; COPYRIGHT 2007, MACMILLAN MAGAZINES LTD . N ATURE REVIEWS MOLECULAR CELL BIOLOGY BY NATURE PUBLISHING GROUP . R EPRODUCED WITH PERMISSION OF

(b) NATURE PUBLISHING GROUP IN THE FORMAT JOURNAL VIA COPYRIGHT CLEARANCE CENTER . 9.5 B: FROM PIERRE A. C OULOMBE AND M. B ISHR OMARY , C URR . O PIN . C ELL BIOL . 14:111, 2002, WITH Microfilaments PERMISSION FROM ELSEVIER .) Chapter 9 The Cytoskeleton and Cell Motility 358 ture with two helical grooves along running its len composed of globular subunits of the protein the of subunits globular of composed projections 9.66). (as in Figure and can provide structural support for various type shap the determining in role important an play also Micro 9.12). Figure (see cells plant many in tubules distribution restricted rather the reflects motility 95 R 1965. R CJ. Figure 9.45 EMSINOFPERMISSION lmn.A a eut f t sbnt raiain (Fi organization subunit its of 9.45 result a As hel filament. flexible, a form to polymerize monomers actin ATP, pres the In cells. most in protein abundant most the speeta h iu n ftefiaet (is present at the minus end of the filament. (in th The cleft is denoted as a plus and minus end. Actin filaments subunit is evident. in each ing cleft and 4 a 3, 2, one of the actin subunits are labeled 1, Th distinguish the consecutive subunits more easily. The actin subunits are represented in three filament. ( a replica of an actin filament showing its double-he A OBERT F: (a) ELL Microfilaments ROM a ), an actin filament is essentially a two-stranded st two-stranded a essentially is filament actin an ), POUE IHPRISO OF PERMISSION WITH EPRODUCED B .DH. IOL M ICHAE L 2:4,19,F 1994, 124:346, . Actin filament structure. filament Actin EPUE T HE J, .SF. R R . OCKEFELLER are approximately 8 nm in diameter anddiameter in nm 8 approximatelyare AND HI TAL ET CHMID R OBERT IG (b) .R 4. . U .RV. POUE WITHEPRODUCED ., NIVERSITY ( a OREYOFCOURTESY ) A model of an actin ICE b E ) Electron micrograph of LSEVIER .M J. , hv oaiy which have polarity, nd the ATP-bind- lical architecture.lical e upper red subunit) e subdomains in P colors to OL RESS c inact .) B. W ; gth (Figure s of cellular f micro- of IOL AH B , which is which , es of cellsof es : filaments FROM 12:302,. 100 nm ne ofence C HIU gure ruc- ical , cently labeled phalloidin (Figure 9.74 (Figure phalloidin labeled cently pro motor usi microscopy light by myosin localized be also can Actin a by moved be to likely are aments provides information as to the direction in which t c S1–actin the by formed arrowheads the of entation Figure of cells microvilliepithelial intestinal of i shown is “decoration” arrowhead this of example An pointedapp microfilament the of end one bound, are fragments Wh polarity. filament’s the reveal fragments S1 bound as filaments the identifying to addition In filament. S1 called binds to the actin molecules all along Figure 9.49), fragments, these of One fragment into enzyme. proteolytic cleaved is tissue) muscle from tained purified m To tein facilitate myosin. the interaction, wit manner specific highly regardless a in interact will source, filaments, actin that fact the of vantage that test cytochemical a using certainty with made ras os l-endntok,or tightly looseanchore ill-defined networks, arrays, actin filaments be can into organized highly engaged, w in activity the and cell of prope type the on Depending dynamic and a structures an of different ends have two filament the Consequently, polarity. has ment m entire the direction, same the in pointed are ment Because fila-actin an of subunits the all and polarity has subunit actin filament. of type this for synonyms basically rtis specifically those of the myosin superfamilproteins, activity the require actin involving processes most (pa own their on forces generate can filaments actin d from molecules actin Al filaments. hybridform to copolymerize sourcescan fact, In identical. percent 88 molecules from a yeast cell and from rabbit skeleta sequencesaminoacid the Forexample, eukaryotes. of ev the during conserved remarkably been have Actins products are specialized for different types of mot species possess a number of actin-coding genes whos Higher plantkaryotic cella that has been examined. been identified as a major protein in virtuallyth ever Since cells. muscle of proteins contractile major labeled or by fluorescently anti-actin anti filaments, 9.45 tg o filament of stage formation filament in nucleation stage initial the microtubules, c the in As ATP-actincontaining monomers. solutions of ADP-actin subunits. the bulk of an actin filament As a consequence, ment. growing the into incorporated is it after time some to hydrolyzed is monomer actin the with associated The 342). (page assembly microtubule in GTP of that ATProleof the and s is assembly actin in GTPase, a m binds a molecule actin of ATP. Actin an is just an as ATPase, filament, a into incorporated is it Before Microfilament Assembly and Disassembly The identification of actin filaments in a given cell can be Actinwasidentified more thanyears50 agooneaso Actin polymerization is readily demonstrated in vit in demonstrated readily is polymerization Actin b .The terms ). like an arrowhead, while the other end looks end other the while arrowhead, an like ocr sol i vto hra te subsequent the whereas vitro, in slowly occurs ) actin actin filament elongation cus uh oe ail.The rapidly. more much occurs , F-actin a ), which binds to actinto binds which ), and , microfilament 9.46. The ori- The 9.46. ile processes. y type of eu- he microfil- l muscle are the micro- ng fluores-ng y . actin fila-actin h the pro-the h d bundles. bodies. f motor of yosin (ob- nd animal e encoded en, it has it en, ci,theactin, tubulin is takes ad-takes hich it isit hich imilar toimilar ge 374),ge ordered ADP atADP icrofila- f their of onomer consists of actinof omplex though olution barbed b a by s n S1en ase ofase iverse (i.e., each then rties. ATP o inro (see f thef tein. ears ctin are . 359

Minus end

S1 – decorated actin filament Plus end

Pointed (minus) end of filament

(a) IF – +

Seed 1 Add to high concentration + 200 nm – Figure 9.46 Determining the location and polarity of actin filaments using the S1 subunit of myosin. Electron micrograph of a 2 Concentration replica showing the microfilament bundles in the core of the microvilli drops of an intestinal epithelial cell. The cell had been fixed, treated with S1 – + myosin fragments, freeze fractured, and deep etched to expose the fila- mentous components of the cytoplasm. The intermediate filaments 3 (IFs) at the bottom of the micrograph do not contain actin and there- Concentration fore do not bind the S1 myosin fragments. These intermediate drops filaments originate at the desmosomes of the lateral surfaces of the cell. – + (F ROM N. H IROKAWA , L. G. T ILNEY , K. F UJIWARA , AND J. E. HEUSER , J. C ELL BIOL . 94:430, 1982, F IG . 3. R EPRODUCED WITH PERMISSION OF THE ROCKEFELLER UNIVERSITY PRESS .) 4 Concentration remains constant – + nucleation stage of filament formation can be bypassed by in- cluding preformed actin filaments in the reaction mixture. 5 When preformed actin filaments are incubated with a high (b) concentration of labeled ATP-actin monomers, both ends of Figure 9.47 Actin assembly in vitro. (a) Electron micrograph of a the microfilament become labeled, but one end has a higher short actin filament that was labeled with S1 myosin and then used to affinity for monomers and incorporates them at a rate approx- nucleate actin polymerization. The addition of actin subunits occurs imately 10 times that of the other end. Decoration with the S1 much more rapidly at the barbed (plus) end than at the pointed myosin fragment reveals that the barbed (or plus) end of the (minus) end of the existing filament. ( b) Schematic diagram of the microfilament is the fast-growing end, while the pointed (or kinetics of actin-filament assembly in vitro. All of the orange subunits minus) end is the slow-growing tip (Figure 9.47 a). are part of the original seed; red subunits were present in solution at 9.5 Figure 9.47 b illustrates how the events that occur during the beginning of the incubation. The steps are described in the text. Microfilaments actin assembly/disassembly in vitro depend on the concentra- Once a steady-state concentration of monomers is reached, subunits are tion of actin monomers. Suppose we were to begin by adding added to the plus end at the same rate they are released from the minus preformed actin filaments (seeds) to a solution of actin in the end. As a result, subunits treadmill through the filament in vitro. Note: No attempt is made to distinguish between subunits with a bound ATP presence of ATP (step 1). As long as the concentration of versus ADP.( A: C OURTESY OF M. S. R UNGE AND T. D. P OLLARD .) ATP-actin monomers remains high, subunits will continue to Chapter 9 The Cytoskeleton and Cell Motility 360 ciiis s n xml of example an is activities two between balance of type This 4). (step constant the filaments and the concentration of free monomers the both that so balanced are filaments the of ends at reactions two the where reached is point a falls, concent monomer free the As end. minus their at curs but a net loss of to the plus ends of the filaments, be to continue monomers point, this At further. drops concentfreemonomer the continues, elongation ament ATP-actinfor lowera affinity has which 3 (step the at end, stops but ATP-actin, for affinity higher a has end, plus the at continues monomers of addition net reache is point a until drop to continues ATP-actin concentratio the filaments, the of ends the to dition As the monomers in the reaction mixture are consume cells, including protists, plants, nonmuscle cells of of cells nonmuscle plants, protists, including cells, of variety wide a from subsequently and tissue cle eadda ohed ftefiaet(tp2 Figure 2, (step filament the of ends both at added be ih h mjr xeto o moi I hc i disc is which below—move toward the plus end of an actin filament. VI, myosin of exception major Myosins the with superfamily. myosin the of members are ments act with conjunction in operate to known motors the da To microtubules. of tracks over directions posite in operate dynein—that and motors—kinesin molecular We have previously examined the structure and actio Myosin: The Molecular Motor of Actin Filaments to a halt contain when cells one of these compounds Microfilament-mediatedprocesses rapidly whi polymer. the sponge, incorporati their a blocks and from monomers free obtained to binds latrunculin, and turnover; which binds to intact actin filaments and preve room, poisonous a from obtained phalloidin, end; minus the which allowing depolymeri the plus ends of actin filaments, mold, a from obtained cytochalasin, activities: microfilament-b dynamic disrupt that drugs following most readily demonstrated by treating the cells wit The involvement of a these cell’s motile processes. locomot cell as and cytokinesis. such phagocytosis, shape, changes in cell processes dynamic for required reorganiz Such cytoskeleton. microfilament its ganize By the controlling cell this dynamic behavior, ments. mi of disassembly the or assembly the either toward conditions in a particular part of the cell can pus Changes in proteins. number of different “accessory” influenc be can cell the in filaments actin of sembly when the ATP-actinthe when approximately0 is concentration iln”(tp 45.Suis n iig el contain cells living rence 9.54 of treadmilling in vivo (see Figure on Studies fluorescently labeled actin subunits 4–5). have supported “trea (steps as known process milling” moving—a continually is ment within subunits individual of position relative the steady at filament each of ends minus a the from ends moved plus the to added being are subunits Because Myosin was first isolated from mammalian skeletal mu skeletal mammalian from isolated first was Myosin sntdaoe actin filaments play a role in nearly As noted above, sdsusdo ae32 the rate of and assembly d As discussed on page 372, tay statesteady pg 9) n occurs and 94) (page c ). h events there subunits oc- h one of the filaments is ah fila- each eukaryotic the occur- lengths oflengths can reor- opposite . ns of two opposing zation atzation n of freeof n te, all of all te, the local ). As fil- As ). nts their animals, whered d by ad- 9.47 d y a by ed remain n intoon ation isation .3 crofila- minus n fila-in added d re-nd mush- blocks which grind state, ration ration all of all ussed ␮ isas- ased op- ion, ing M. b — ch d- s- ). ie notobodgop:the groups: broad two general into vided are Myosins chains. (light) molecular-weight variety a contain also Myosins divergent. highly are tai the myosinsaresimilar, various of domains head Wherea ATP motor. myosinhydrolyzes the and drive to site a and filament actin an binds that site a tains head The domain. (head) motor characteristic a share and Al vertebrate cardiac and smooth muscle tissues. h olwn eto,the myosin IIthe filaments following that a section, desc As cell. muscle a in occurs as another, one ward of a myosin filament have the ability to pull actin Because they are the myosinbipolar, heads at the op ures 9.51 and 9.57). As a result, the filament is desc is bipolarfilament the result, a As 9.57). and 9.51 ures mentand the globular heads point away from the cen center the toward point tails the of ends the that the protein to Myosin form II filaments. molecules as portion of a myosin II molecule plays a structural key the The fib are discussed and on page 369. played by the neck, head, myosin the of action of mechanism singl a in contained is activity motor requiredfor m the of all Thus, 9.50. Figureshownthatin as such in an filamentsin attachedactinsliding of capable cov glass a of surface the on immobilizedbeen have terrupted terrupted each consisting of a s (2) a pair of necks, molecule; catalytic the contain that heads globular of pair a ige og o-hpd al omd y h intertwin the by long formed tail rod-shaped long, single, tion of the molecule in Figure 9.49 Figure in molecule the of tion Of the various myosins, type type II Of molecules the are various best myosins, u each presumed to have its own specialized f classes, l at from myosins different 40 about contain in Humans only present are XI and VIII myosins and brates, i only found is example, for X, Myosin restricted. are whereclasses are expressed widely among eukaryotes, Some III–XVIII). types and I (type classes different subdivided on the basis of amino acid sequence into myosins unconventional myosins duce a highly asymmetric protein (Figure 9.49 (Figure protein asymmetric highly a duce a way a such in chains—organized light of pairs two posed of six polypeptide chains—one pair of heavy c is shown in Figure 9.49 Figure in shown is vancing growth 9.48. cone in is Figure demonstrated The of effect inhibiting myosin II activi ure 9.76). and the turning behavior of growth cones migration, generating tension at focal ad during cell division, splitting requiredarefor myosins II type tivities, (barbed) Among end their of nonmus an actin filament. toward move IIs myosin All cells. nonmuscle in tion 3 of wh encodes 16 different myosin II heavy chains, also found in a The variety huma of nonmuscle cells. contract muscle formotors are primary the class II Conventional (Type II) Myosins II) (Type Conventional Isolated myosin heads (S1 fragments of Figure 9.49 An electron micrograph of a pair of myosin II molec II myosin of pair a of micrograph electron An ␣ indicating a reversal of polarity, at the filament’s -helical sections heavy chains. of the two -helical , which were first identified in muscle tissue, and the and tissue, muscle in identified first were which , ␣ helix and two associated light chains; and (3) a (3) and chains; light associated two and helix a . Each myosin II molecule is com-is molecule II myosin Each . h ucnetoa mois are myosins unconventional The . conventional b shows it to consist of (1)of consist showsto it Proteins of the myosinthe of Proteins a oe allowingrole, a cell in twoin cell a filaments to- .Examina-). ein,cellhesions, ed Thehead. e (or nl,unin-ingle, ty in an ad-ty site of theof site that bindsthat vitro assayvitro posite ends of the fila-the of unction(s). ion but arebut ion nderstood. at least 17 ssemble in l domainsl n genome hains and semble so l myosins erslip areerslip ich ich func- (see Fig- s to pro-to s as others of theseof ter(Fig- achinery f low- of type II type the plusthe n verte-n ie asribed rous tail ie inribed center. east 12east plants. b cle cle ac- n of ing y di- ly con- ) that s thes ules role ) 361

(a) (b)

Figure 9.48 Experimental demonstration of a role for myosin II in and continue to grow over the laminin-coated surface. Bar, 500 ␮m. the directional movement of growth cones. (a) Fluorescence (b) The tissue in this micrograph was obtained from a mouse embryo micrograph showing fine processes (neurites) growing out from a lacking myosin IIB. The growth cones no longer turn when they reach microscopic fragment of mouse embryonic nervous tissue. The neurites the edge of the laminin-coated surface, causing the neurites to grow (stained green) are growing outward on a glass coverslip coated with forward onto a surface (black) lacking laminin. Bar, 80 ␮m. (F ROM strips of laminin (stained red). Laminin is a common component of the STEPHEN G. T URNEY AND PAUL C. B RIDGMAN , N ATURE extracellular matrix (page 243). The tip of each nerve process contains a NEUROSCI . 8:717, 2005; © 2005, REPRINTED BY PERMISSION OF motile growth cone. When the growth cones reach the border of the MACMILLAN PUBLISHERS LTD .) laminin strip (indicated by the line with arrowheads), they turn sharply

Heads

S1 fragment

Tail Head ATP

Essential light chain

Neck Regulatory light chain

Tail 150 nm

(a) 20 nm (b) Figure 9.49 Structure of a myosin II molecule. (a) Electron micro- around one another to form a coiled coil and a pair of globular heads.

graph of negatively stained myosin molecules. The two heads and tail When treated with a protease under mild conditions, the molecule is 9.5 of each molecule are clearly visible. ( b) A highly schematic drawing of a cleaved at the junction between the neck and tail, which generates the myosin II molecule (molecular mass of 520,000 daltons). The molecule S1 fragment. ( A: F ROM S. A. B URGESS , M. L. W ALKER , H. D. Microfilaments consists of one pair of heavy chains (purple) and two pairs of light chains, WHITE , AND J. T RINICK , J. C ELL BIOL . 139:676, 1997, F IG . 1. which are named as indicated. The paired heavy chains consist of a REPRODUCED WITH PERMISSION OF THE ROCKEFELLER rod-shaped tail in which portions of the two polypeptide chains wrap UNIVERSITY PRESS .) Chapter 9 The Cytoskeleton and Cell Motility 362 Figure 9.50 ( is then incubated withwhich a preparation of actin myosining in which heads are bound to a silicone-c et ta fr i ms nnuce el otn displ often cells nonmuscle most in form that ments mi II myosin smaller the However, apparatus. tractile componentsstablehighlyarecellsskeletal muscle ( Figure 9.51 ue namoi Ifiaet (cules in a myosin II filament. of the staggereddiagram of the individ arrangement ohed,leaving a smooth section in the center of both ends, The heads of the fila myosin in vitro. filament formed B b ) Results of the experiment depicted in ) Results of the experiment C: (b) UTS OFOURTESY - (a) (a) In vitro motility assay for myosin.In vitro motility Structure of a bipolar myosin II filament.Structure H UGH Actin H Bipolar filament Heavy chains UXLEY b ) Electron micrograph of a bipolar .) a Two images were taken . + + ( a filaments. ) Schematic draw-) Schematic the filament. oated coverslip, ual myosin mole- ment are seen at ( a Myosin - of the con-the of ) Schematic ni-fila- ay re eidbtenepsrs (B exposures. between period brief movement of the actin filaments over the myosin head The dashed lines with arrowheads show of film. frame and as photographed a double expo 1.5 seconds apart ht ysnV omly a a iebe al segment, tail sizeable a has normally V myosin that (Keep cycle. a the singlemolecule mechanical during 9.52 slowed the rate at which the protein was able to tr in ular the obstacles path of the moving motor prot the these protein’sresearchers pl mechanical cycle, needed and then disassembling after they have acted th where and when assembling construction, transient vitro (Figure 9.52 (Figure vitro actin an along rapidly moves it as molecule single that micrographs force atomic of series remarkable by one of these myosins—myosin V—have been revealed steps The microtubules. along move dynein toplasmic ments in a manner analogous to the way that kinesin ac along processively moving of capable are myosins unconve best-studied The molecules. protein dividual ment formation and instead appear to operate primar membrane. pl role in could processes that require movement which or deformat membrane, plasma the on tension exert I myosin that suggested been bilayer has It lipid the membrane. plasma and cytoskeleton the of filaments betw cross-link a as serves often I myosin 9.66, ure known became protein microvillu a of drawing the in the reflected As I. myosin vitro; in filaments into ble tional myosin had only a single head and was unable Acanthamoeba unique myosin-like protein that was extracted from Edward Korn of the National Institutes of Health de novninl Myosins Unconventional (b) None of the unconventional myosins are capable of fi of capable are myosins unconventional the of None b shows a series of images that reveal the movements ofmovements the reveal that images of series a shows . Unlike muscle myosin, this smaller unconven- smaller this myosin, muscle Unlike . a ). In order to visualize the various steps insteps various the visualize to order In ). SDO OKBY WORK ON ASED n 93 hms olr and Pollard Thomas 1973, In sure on the same s during thes during the sliding .YT. aced molec- vl Figureavel. filament infilament AN A GID A i,whichein, the protist to assem- ion of the . capture acapture een actineen s and cy- scribed scribed a s in Fig-in s in mind ily ily as in- i fila-tin ntional f the of s de-as y areey taken can in a y aay as la- .) 363

Moving direction

Streptavidin

Vesicle

Melanophilin Rab27a Tail Myosin V Light chain Neck Head Minus Plus end end Filament (a) (b) (b ’) (c) Figure 9.52 Myosin V—a two-headed unconventional myosin corresponding HS-AFM images. A continuous movie of the protein’s involved in organelle transport. (a) Direct visualization of a single movement can be seen in the supplement to this article. ( c) Schematic myosin V molecule (one that is lacking its normal tail domain) as it drawing of a complete dimeric myosin V molecule, including its moves along an actin filament in vitro in the presence of ATP. Using numerous light chains, with both heads bound to an actin filament and high-speed atomic force microscopy (HS-AFM), this series of images its tail domain bound to a vesicle. Rab27a and melanophilin serve as shows the movement of the molecule over a period of about one second. adaptors that link the globular ends of the tail to the vesicle membrane. (b) Succesive HS-AFM images showing the hand-over-hand The long neck of myosin V binds 6 light chains. ( A-B: F ROM movement of a single myosin V molecule as it passes through a cluster NORIYUKI KODERA , ET AL ., FROM NATURE 468: 73, 2010; © 2010, of obstacles (consisting of streptavidin protein molecules). The swing- REPRINTED BY PERMISSION OF MACMILLAN PUBLISHERS LTD . ing neck (or lever arm) is highlighted with a thin white line. Interpre- COURTESY TOSHIO ANDO .) tive drawings ( bЈ) depict the motor protein as seen in several of the

picted in Figure 9.52 c, that has been removed for these partic- sively along an actin filament made up of helical strands of ular experiments.) subunits. The actin helix repeats itself about every 13 subunits In the first frame of Figure 9.52 b, both heads of the (36 nm), which is just about the step size of a myosin V mol- dimeric protein are seen to be attached to the underlying ecule (Figure 9.52 b). To accomplish this type of movement, filament, and a cluster of obstacles are seen in the path of the each myosin head must move a distance of 72 nm, twice the motor. In the second frame, the trailing head of the myosin V distance between two successive binding sites on the actin fil- molecule has detached from the filament and is in the process ament (Figure 9.52 b). As a result of its giant strides, myosin V of moving forward through the roadblocks in its path. By the can walk along the filament in a straight path even though the fourth frame, this swinging head has made contact with the underlying “roadway” spirals 360 Њ between its “feet.” filament at a forward position along the filament and thus has Several unconventional myosins (including myosin I, V, become the new leading head. These images provide visual and VI) are associated with various types of cytoplasmic vesi- confirmation that myosin V moves in a hand-over-hand fash- cles and organelles. In some cases these myosins may act pri- ion similar to that of the kinesin molecule in the drawing of marily as organelle tethers and in other cases as organelle Figure 9.15 b. To accomplish this type of movement, at least transporters. Some vesicles have been shown to contain mi- one of the two heads must be bound to its polarized track at crotubule-based motors (kinesins and/or cytoplasmic dynein) all times. Figure 9.52 c shows an illustration of an intact and microfilament-based motors (unconventional myosins) myosin V molecule. A seen in this illustration, myosin V is and, in fact, the two types of motors may be physically linked 9.5 noteworthy for the length of each neck, which at 23 nm is to one another. The movement of vesicles and other membra- Microfilaments about three times that of myosin II. nous carriers over long distances within animal cells occurs on Because of its long necks, which act as swinging arms (or microtubules, as previously described. However, once they ap- levers) during movement, myosin V can take very large steps. proach the end of the microtubule, these membranous vesicles This is very important for a motor protein that moves proces- are often thought to switch over to microfilament tracks for Chapter 9 The Cytoskeleton and Cell Motility 364 (and kinesin) motorsof to membraneattachment surfaces (Figure 9 the in involved also are Rabs ficking. ve regulatethat molecules as 302 page on discussed prote of family Rab The Rab27a. calledprotein brane my but lacked normal a gene, functional gene a encoding a had periph patients Griscelli of subset a that wa it 2000 In transport. vesicle defectsin tolated thoughtsymptoms other suffer coloration) and skin albinism partial exhibit individuals these syndrome; called disorder rare a from Vasuffermyosin coding Humanslacking normala g havemucha lighter color. th causing follicles, hair intomelanosomes transfer Micelacking myosin developingVahair. activity are incorporbecomethey where follicles hair toferred tr then are Melanosomes Va. called isoforms V one myosin by cell pigment the of processes peripheral fine transported are (melanosomes) granules pigment mals, hasbeen best studied inpigment cells (Figure 9.53 9.53). is mediated by myosins which (Figure cell, periphery actin-rich the through movement local the INPUBLISHED COPYRIGHT transpor microfilament-based motors in intracellular Figure 9.53 n ot ihnetne ellrpoess (Aand forthwithin extended cellular processes. pigment ofgr a frog in which pigment cell the case as of motors manner, may act in a cooperative types regions those present (cortical) in the peripheral over microfila their cargo transport which myosin Va, myosin also carry It is thought that some vesicles over their relati cargo carry which families, dynein is thought to be mediated by members of the kinesin andtransport et f h seecla y ehncl tml leads Ca of stimuli opening mechanical by stereocilia the of ment of the cell into the fluid-filled cavity of the inner stereocilia projectthat from hairlike the stiff, of the for named are cells Hair myosins. unconventional fu the studying for system good particularly a been oprto bten irtbls n microfilaments and microtubules between Cooperation Hair cells, whose structure is shown in Figure 9.54 shownis Figure structure whosein cells, Hair 98 R 1998, The contrasting roles microtubule- of contrasting andThe T HE 2 ϩ J OCKEFELLER channels in the plasma membrane and the and membrane plasma the in channels UNLOFOURNAL Myosin Va Pigment granule Actin filaments Kinesin Microtubules C U ELL NIVERSITY B IOLO GY FTER ftecl.The twoof the cell. P motors, such as such motors, vely long distances.vely RESS anules move back 143:1915.) illustrated hereillustrated in ,WX, et,includingments, t. er Displace- ear. O. apical surface apical Most vesicleMost s discovereds RIGINALLY U .Inmam- ). ated into ainto ated eir coat tocoat eir eral mem- nctions ofnctions unable to , sicle traf-sicle Griscelli .52 TALET lc of (lack to be re-be to sn Vaosin bundle eneen- o the to ins wasins f the of myosin f the of a c into ). have ans- ., hc,oe 10 m og n cnan hnrd o nuc of hundreds contains and long, mm 100 over thick, gle, cylindrically shaped muscle cell is typically1 cylindricallyiscell shapedmuscle gle, Skeletal muscle cells have a highly unorthodox stru untarycontrol andconsciouslycanbe commanded to them are anchored to bones that they They move. are th factthe from Skeletalnametheirmusclesderive 9.6 fluorescence micrograph 9.54of Figure captur is process This end. pointed the from sociate a treadmillfilament, the throughof body the filament, o end barbed the to bind continually monomers Actin const in are bundles actin the structures, permanent the stereocilia While the of the actin cytoskeleton. vided some of the most striking images of the dynam Stereocilia base. the at ends pointed and projection 9.54 filaments actin parallel of bundle a by supported is ste each microtubules, containing of Instead earlier. cili true the to relation no have Stereocilia sound. per we that impulses nerve of generation subsequent and cells of the inner ear; two of these are shown in Fi in shown are these of two ear; inner the of cells withi sites various at are localized XV) and VII, I VI, (I, myosins unconventional Several filament. each of ba the at subunits actin GFP-labeled of corporation teins interact to mediateteins interact a activi complex cellular we can see how these myosin structure and function, tions in myosin VI are the cause of several inherit and the maintenanceendosomes, M of Golgi morphology. themovement ofplasmauncoated membrane, vesicles t to be involved in the over formation of the clathrin-coated v to cytoskeleton Inplasma other membrane. myosintypes the of VIcells, is connect might it where actin Myosin filament. VI is located at the base of t towardpointedthe(minus) is, that versedirection,” isdistinguished byitsmovemenplasm ofmany cells, for the mutant encoding this motor allele protein a in Figure 9.54 Figure in my the in aremice shownof ear inner the of cells hair the on mutations gene VIIa of effects morphologic The ness. which by is both characterized deafness an syndrome, .Compare the structure of a fully assembled microt 2. Compare and contrast the characteristics of micro- 1. .How is it possible for the same vesicle to be tra 5. Contrast thestructureandfunctionofconventional 4. Describe three functions of actin filaments. 3. W E I V E R tubule assembly versus actin filament assembly. myosin II and the unconventional myosin V.myosin II actin filament,andintermediatefilament. along both microtubules and microfilaments?along both o ta w hv dsrbd h bsc o atn and actin of basics the described have we that Now processive a organelle transporter theinMysoin VI, b e . Mutations in myosin VIIa are the cause of Usher 1B Usher of cause the are VIIa myosin in Mutations . ) whose barbed ends are located at the outer tip of tip outer the at located are ends barbed whose ) | Muscle Contractility f , g . As in humans, mice that are homozygousare that mice humans, in As . c , which shows which the in-, ed diseases. 0 to 100 to 0 ty. he stereocilia tr.A sin-cture. esicles at the mselves aremselves a discusseda gure 9.54gure re deaf. at most ofmostat have pro-have end of anofend nsported under vol- n the hairthe n reocilium t inat “re- ed in thein ed rbed end rbed two two pro- ic nature contract. thought ant flux.ant (Figure d blind- ceive asceive ubule, nd dis-nd o earlyo f eachf cyto- I V,II, lying osin uta- the ␮ lei. m d 365

Tight junction

Stereocilium Actin filaments within stereocilium Stereocilia (b) (c)

Hair cell of cochlea

Supporting cell

Nerve (a) (d) (e)

(f ) (g) Figure 9.54 Hair cells, actin bundles, and unconventional myosins. Scanning electron micrograph of the hair cells of the cochlea of a con- (a) Drawing of a hair cell of the cochlea. The inset shows a portion of trol mouse. The stereocilia are arranged in V-shaped rows. ( g) A corre- several of the stereocilia, which are composed of a tightly grouped bun- sponding micrograph of the hair cells of a mouse with mutations in the dle of actin filaments. ( b) Transmission electron micrograph of a cross gene encoding myosin VIIa, which causes deafness. The stereocilia of section of a stereocilium showing it is composed of a dense bundle of the hair cells exhibit a disorganized arrangement. (A: T. H ASSON , C URR actin filaments. ( c) Fluorescence micrograph of a hair cell from the BIOLOGY 9:R839, 1999; WITH PERMISSION FROM ELSEVIER SCIENCE . vestibule of a rat inner ear. The tips of the stereocilia are labeled green B: COURTESY OF A. J. H UDSPETH , R. A. J ACOBS , AND P.G. G ILLESPIE ; due to the incorporation of GFP-actin monomers at their barbed ends. C,E: FROM A. K. R ZADZINSKA , ET AL ., COURTESY OF B. K ACHAR , J. Taller stereocilia contain a longer column of GFP-labeled subunits, CELL BIOL . VOL . 164, 891, 892, 2004, F IGS . 4, 5; D: FROM PETER 9.6 which reflects a more rapid incorporation of actin monomers. The GILLESPIE AND TAMA HASSON , J. C ELL BIOL . VOL . 137, C OVER #6, Muscle ContractilityMuscle stereocilia appear red due to labeling by rhodamine-labeled phalloidin, 1997; C–E, REPRODUCED WITH PERMISSION OF THE ROCKEFELLER which binds to actin filaments. ( d ) A hair cell from the bullfrog inner UNIVERSITY PRESS ; F–G: F ROM TIM SELF , ET AL ., COURTESY OF ear. The localization of myosin VIIa is indicated in green. The orange KAREN P. S TEEL , D EVELOP . 125:560, 1998; REPRODUCED BY bands near the bases of the stereocilia (due to red and green overlap) PERMISSION OF THE COMPANY OF BIOLOGISTS , L TD . indicate a concentration of myosin VIIa. ( e) Myosin XVa (green) is http://dev.biologists.org/content/125/4/557.full.pdf+html?sid=1524ec24- localized at the tips of the stereocilia of a rat auditory hair cell. ( f ) e6c0-413f-9c05-ac2111083a85) Chapter 9 The Cytoskeleton and Cell Motility 366 mononucleated because each fiber is a product of the fusion of lar priately called a Because of these a properties, skeletal muscle cell Figure 9.55 ino kltlmsl.(tion of a skeletal muscle. ucefie.( fiber. muscle aments staining staining of the partial overlap of two distinct types of fila electronmicroscope shows the banding pattern to be at ed ing staining o pair a sarcomerehas A zones. light and bandsdark extendsfrom one adn atr,whichgives themuscle fiberbandingstrip a pattern, sarcomeres ril consists of a repeating linear array of contrac ftinr yidia tad,called cylindricalstrands, ofthinner, a muscle fiber (Figure 9.55) reveals a cable made up ternal structure of any cell in the A body. longitud the region of overlap and contains both types of fil that part of the A band on either side of the H zon thezoneHonly thick filam tainsonlythin filaments, band (a) fiber (cell) Muscle I band bandA Skeletal muscle cells may have the most highly orde A appearance. Examination of stained muscle fibers in t in fibers musclestained of Examination appearance. Muscle bundle band I M line I bands and H zoneH zone H ah acmr i tr ehbt a characteristic a exhibits turn in sarcomere Each . The structure of skeletal muscle. structure The c oae bten h otr bns n a lightly a and bands, I outer the between located ) Electron micrograph of a sarcomere with the bands hc filamentsthick lies in the center of the TheH Izone. band con- muscle fiber myoblasts located at a itsmore outer densely edges, stain- located in the center of the A band. A densely A locatedinthe center ofthe band. A Nuclei Zline b Body muscle ) Light micrograph of a multinucleated tothe next Z line and contains several (premuscle cells) in the embryo. Muscle fibers. have multiple nuclei Z-line Fgr 9.56(Figure H-zone Z-line myofibrils Sheath Sarcomere Myofibril ( a a .Ec sarcomere Each ). ) Levels organiza-of ) Levels ieuis calledtile units, ments, Eachmyofib- . is more appro- ge numbers of inal section of aments. of hundreds e represents edor the result ns andents, lightlyf thin fil- red in- stri- he band (Figure 9.57).band (Figure o edges outer the contacted they until inward moved sa the of ends both on lines Z the progressed, ening decreased in width and then disappeared altogether. and band H the while length, in constant essentially rem sarcomere each in band A the shortened, fiber cle proces contractile the in stages different at meres th of pattern banding the of contra observations from came muscle underlying mechanism the to clue tant mos The muscle. entire the of length in decrease the accoun length in decrease combined whose sarcomeres, a shortening of units The work. perform can they way is there shortening; by operate muscles skeletal All The Sliding Filament Model of Muscle Contraction F etrd (lettered. ewe to hc fiaet (iue 9.56 (Figure filaments thick si two is filament between thin each that and filament, thick each arra hexagonal a in organized are filaments thin the ing thin filaments. n with attachments forming of capable cross-bridges projections The intervals. spaced regularly at ments the from projections of presence the show sections (b) (c) AWCETT Cross sections through the region of overlap show t show overlap of region the through sections Cross B /P E: HOTO RIC G R RAVE ESEARCHERS /P HOTO R I, ESEARCHERS NC .) I, b NC ). Longitudinal Longitudinal ). .; s. As a mus-a As s. C D: thick fila- thick represent no otherno Nuclei As short- y aroundy eighbor- t impor-t I bandsI ON rcomere e sarco-e te A the f tuated e there ained ts forts ction W. hat 367

I band ---- endA band ---- end I band H zone

Thick filament Thin filament

+end +end

Z line Z line

(a) (b) Figure 9.56 The contractile machinery of a sarcomere. (a) Diagram (cross-bridges). ( b) Electron micrograph of a cross section through an of a sarcomere showing the overlapping array of thin actin-containing insect flight muscle showing the hexagonal arrangement of the thin (orange) and thick myosin-containing (purple) filaments. The small filaments around each thick filament. ( J. A UBER /P HOTO transverse projections on the myosin fiber represent the myosin heads RESEARCHERS , I NC .)

H I zone band

H zone I band I band A-band RELAXED

Actin movement Cross-bridge Actin movement

H zone

I band A band I band CONTRACTED 9.6 (a) (b) Figure 9.57 The shortening of the sarcomere during muscle only a fraction are active at any given instant. ( b) Electron micrographs ContractilityMuscle contraction. (a) Schematic diagram showing the difference in the of longitudinal sections through a relaxed ( top ) and contracted ( bottom ) structure of the sarcomere in a relaxed and contracted muscle. During sarcomere. The micrographs show the disappearance of the H zone as contraction, the myosin cross-bridges make contact with the surround- the result of the sliding of the thin filaments toward the center of the ing thin filaments, and the thin filaments are forced to slide toward the sarcomere. ( B: T OP AND BOTTOM IMAGES FROM JAMES E. D ENNIS / center of the sarcomere. Cross-bridges work asynchronously, so that PHOTOTAKE .) Chapter 9 The Cytoskeleton and Cell Motility 368 ment is composed of the opposing tail regions of th of regions tail opposing the of composed is ment of center The sarcomere. the of center the at versed (see muscle of filaments thick the vitro of polarity the in 9.51), form that filaments the Like of proteins. amounts small with together molecules II myosin line. linked ends barbed their with aligned are sarcomere eac of filaments actin The tropomy and filament. the of components actin the both contact and filament thin alon apart nm 40 approximately spaced are molecules important molecu the of function overall the rolein distinct an having each subunits, three of composed protein globular a Troponinis 9.58). (Figure ament ecule is associated with seven actin subunits along tropomyosinrod-shaped Each filament. thin the within (approximately 40 nm long) that fits securely into t troponin The nebulin molecules (which are not discussed in the text) are o ecule domains contains and is spring-like capable The I-band portion the sarcomere contraction. during molecules are filament thought to maintain the thick mere at the Z line to the M band in the sarcomere c mere. Figure 9.59 was rapidly accepted, and evidence in its favor cont accumulate.favor its in evidence and accepted, rapidly was The 9.57). (Figure bands H and I the w decreased the and filaments the between overlap in observe the in result would sarcomere the of center The sliding of the thin overfilaments t one another. thei from rather but filaments, the of shortening the res not did sarcomeres individual of shortening the propose They contraction. muscle for account to 1954 mode far-reaching a proposed Hug Hanson, and Jean and Huxley Niedergerke, Rolf and Huxley Andrew tigators, kltl uce oti to te proteins, other two contain muscle skeletal FilamentsThinand Thick of Organization and Composition The ah hc fiaet s opsd f eea hundred several of composed is filament thick Each ae nteeosrain,two groups of i British Based on these observations, These huge elastic molecules stretch from the end o Z Fgr .8.Tropomyosin is an elongated molecule(Figure 9.58). The arrangement of titin molecules within the sarco the within molecules titin of arrangement The n diin o ci,te hn lmns f a of filaments thin the actin, to addition In I Nebulin sliding-filament modelsliding-filament f great elasticity. ne.Titinenter. s in the center of tropomyosin of the titin mol- Myosin e Troponinle. the thin fil- f the sarco- he grooves cells is re-is cells d increased oward the to the Zthe to e myosine the fila-the l fromult complex r slidingr inues to inues idth ofidth Figure d thatd h halfh mol- the g nves- other and osin and inl - Sarcomere h M A up the body of the filament (Figure 9.51).up the body of the filament (Figure t molecules myosin the of position staggered the to from each thick filament along the p remainder of heads its myosin The heads. of devoid is and molecules proper position within the center of the sarcomere sarcomere contraction.muscle the of center in the within filaments position myosin proper maintains also Titin stretching. vent the sarcomere from becoming pulled apart durin Titin is within though the molecule become uncoiled. cert as spring molecular a like stretches that tein nating at Titin is the a Z highl line (Figure 9.59). t and band A the past continuing filament, myosin the at the M line in the center of each sarcomere and e or molecules Titin acids. amino 38,000 than more ing pol a more than 3.5 million encodes daltons in molecular mass and length) different of isoforms to rise c (which gene titin entire The organism. any in ered is muscles T .S K S. C. (T. actin monomers that are allowed to assemble into a by regulatin thought to act like a “molecular ruler” C OYIH LAAC CENTER CLEARANCE COPYRIGHT in Figure 9.63.in Figure positional in these changes proteins c that trigger i as described molecules spaced at defined intervals, shaped tropomyosin molecules situated in the groove of actin s array thin filament consists of a helical Figure 9.58 J PNO NCL ILG BY BIOLOGY CELL IN OPINION OURNALS URRE NT h tid ot bnat rti o vrert skele vertebrate of protein abundant most third The Actin R. ELLER O titin PINION POUE IHPRISO OF PERMISSION WITH EPRODUCED The molecular organization of the thin filaments. molecular organization The C, , which is the largest protein yet to be discov-be to yet protein largest the is which , Actin URR 10 nm J UNL NTEFRA ORA VIA JOURNAL FORMAT THE IN OURNALS O. PIN rpnnTropomyosin Troponin Titin C E SVE LTDLSEVIER ELL .) B I IOL :3 95 C 1995. 7:33, . ,C., ubunits with rod- ontraction areontraction shown g the number of E URRE NT h et The n the text. thin filament. SVE LTDLSEVIER s and troponin Z lines y elastic pro- xtend along i regionsain URRE NT length due O hat makehat contain- g muscle ypeptide t to pre- PINION n givean iginate during ermi- roject ., their Each tal 369 The Molecular Basis of Contraction Following the for- Support for the “lever-arm” proposal was initially ob- mulation of the sliding-filament hypothesis, attention turned tained through a series of experiments by James Spudich and to the heads of the myosin molecules as the force-generating colleagues at Stanford University. These researchers con- components of the muscle fiber. During a contraction, each structed genes that encoded altered versions of the myosin II myosin head extends outward and binds tightly to a thin fila- molecule, which contained necks of different length. The ge- ment, forming the cross-bridges seen between the two types netically engineered myosin molecules were then tested in an of filaments (Figure 9.57). The heads from a single myosin fil- in vitro motility assay with actin filaments, similar to that de- ament interact with six surrounding actin filaments. While it picted in Figure 9.50. As predicted by the lever-arm hypothe- is bound tightly to the actin filament, the myosin head under- sis, the apparent length of the power stroke of the myosin goes a conformational change (described below) that moves molecules was directly proportional to the length of their the thin actin filament approximately 10 nm toward the cen- necks. Myosin molecules with shorter necks generated smaller ter of the sarcomere. Unlike myosin V (depicted in Figure displacements, whereas those with longer necks generated 9.52), muscle myosin (i.e., myosin II) is a nonprocessive motor . greater displacements. Not all studies have supported the cor- Muscle myosin remains in contact with its track, in this case a relation between step size and neck length, so the role of the thin filament, for only a small fraction (less than 5 percent) of lever-arm remains a matter of controversy. the overall cycle. However, each thin filament is contacted by a team of a hundred or so myosin heads that beat out of syn- The Energetics of Filament Sliding Like the other mo- chrony with one another (Figure 9.57 a). Consequently, the tor proteins kinesin and dynein, myosin converts the chemical thin filament undergoes continuous motion during each con- energy of ATP into the mechanical energy of filament sliding. tractile cycle. It is estimated that a single thin filament in a Each cycle of mechanical activity of the myosin cross-bridge muscle cell can be moved several hundred nanometers during takes about 50 msec and is accompanied by a cycle of ATPase a period as short as 50 milliseconds. activity, as illustrated in the model shown in Figure 9.61. Ac- Muscle physiologists have long sought to understand how cording to this model, the cycle begins as a molecule of ATP a single myosin molecule can move an actin filament approxi- binds to the myosin head, an event that induces the dissocia- mately 10 nm in a single power stroke. The publication of the tion of the cross-bridge from the actin filament (Figure 9.61, first atomic structure of the S1 myosin II fragment in 1993 by step 1). Binding of ATP is followed by its hydrolysis, which Ivan Rayment, Hazel Holden, and their colleagues at the occurs before the myosin head makes contact with the actin University of Wisconsin focused attention on a proposal to explain its mechanism of action. In this proposal, the energy released by ATP hydrolysis induces a small (0.5 nm) confor- Actin mational change within the head while the head is tightly filament bound to the actin filament. The small movement within the head is then amplified approximately 20-fold by the swinging Regulatory light chain movement of an adjoining ␣-helical neck (Figure 9.60). Ac- Neck (swinging lever) cording to this hypothesis, the elongated myosin II neck acts Essential light chain as a rigid “lever arm” causing the attached actin filament to slide a much greater distance than would otherwise be possi- Motor domain ble. The two light chains, which are wrapped around the neck, are thought to provide rigidity for the lever.

10 nm

Head Actin Myosin filament Neck filament

(a) (b)

Figure 9.60 Model of the swinging lever arm of a myosin II (depicted as the upper dark blue and lower light blue necks, 9.6 molecule. (a) During the power stroke, the neck of the myosin mole- respectively). It is this displacement of the neck region that is thought cule moves through a rotation of approximately 70 Њ, which would pro- to power muscle movement. The essential and regulatory light chains, ContractilityMuscle duce a movement of the actin filament of approximately 10 nm. ( b) A which wrap around the neck, are shown in yellow and magenta, respec- model of the power stroke of the myosin motor domain consisting of tively. ( B: F ROM MALCOLM IRVING ET AL ., N ATURE STRUCT . B IOL . the motor domain (or head) and adjoining neck (or lever arm). An at- 7:482, 2000; REPRINTED BY PERMISSION FROM MACMILLAN tached actin filament is shown in gray/brown. The long helical neck is PUBLISHERS LTD . C OURTESY OF MALCOLM IRVING , I VAN shown in two positions, both before and after the power stroke RAYMENT , AND CAROLYN COHEN .) Chapter 9 The Cytoskeleton and Cell Motility 370 idwal oteatnfiaet(tp3.The relea to the actin filament (step 3). bind weakly hydrolysis of the bound ATP the (step 2) energizes of the head fromthe detachment the ac causing head, begin in step 1 with the binding of ATPcycles to a of ATP is the basis for the condition of condition ATPthe of for basis the is The inability of myosin cross-bridges to detach in actin the to bound tightly remains head myosin the molecule so that In a the new absen cycle can of begin. release The 9.60. bound ADP (step 5) is followed by the Figure binding of a new ATP in shown as head myosin of stroke power the represents movement This comere. o center the toward filament actin the shifts change cha conformational large This conf a by the driven stored(step 4). free energy triggers which phosphate, boundits releases and 3) (step molecule actin a the to then taches myosin energized The movement. spontaneous of spri stretched a to analogous state, energized an in cross- the point, this At 2). step 9.61, (Figure whole pr the by absorbed is hydrolysis by released energy ening of muscles that ensues followingening of muscles death. P a ADP namely, ATPof productshydrolysis, The filament. 1994.) xiainCnrcin Coupling Excitation–Contraction tor unit are jointly innervated by branches from a ganized ganized into groups termed nte yl.(.Y J Y. (M. another cycle. The release of the ADP (step 5) sets the sarcomere. power stroke (step 4) that moves the thin filament t of the myosintighter attachment head to the thin fi yrlss n ees fAP D,and P and release ADP, of ATP, hydrolysis, involvi cycle of the head and adetachment chemical movemen the attachment, involving cycle a mechanical force-generating myosin head occurs as the result o i eanbudt h ciest fteezm,whil enzyme, the of site active the to bound remain , contractile cycle. contractile Figure 9.61 Figure ADP ATP A schematic model of the actinomyosin the of model schematic A IANG The movement of the thin filament by the 5 1 .P S P. & M. ADP Power stroke motor units ATP HEETZ i . In this model, the two the two In this model, . . All All the fibers of a . mo- B, 4 2 uce br ae or- are fibers Muscle rigor mortisrigor P IOESSAYS i ADP•P cleft in the myosin cleft f a linkage between oward the center of ADP•P head, causing it to causing head, se of P lament and the the stage for ng the binding, i lmn.Thetin filament. ,andt, 3 i single motor i the absence i 16:532, ormational ng capableng ce of ATP, causes acauses , the stiff- the , otein as aas otein f the sar-the f filament. bridge is bridge thee the nge the nd t- 10 ATPase molecules whose function is to Catransport atri ucecnrcin nterlxdsae t state, relaxed the In contraction. muscle in factor mined that calcium ions present in tap water were a fail but Ri contract in a solution made water, with distilled water. tap London with made solution saline cont would heart frog isolated an that found Ringer phys English an Ringer, Sydney by 1882 in shown first until needed.i it where SR, the of lumen the into and cytosol the the cell along membranous folds called called folds membranous tubules along cell the atedis propagatedincell a skeletal muscle into t action the impotential remains at the cell surface, coupling excitation–contraction deep within the muscle fiber constitute a process re muscle plasma membrane to the shortening of the sar an action potential. fiber muscle co of capable and excitable also the is membrane plasma to cleft synaptic a across axon the lar junction is a site of transmission of the nerve The ne of view the closer structure of the synapse). junctionmuscular up the that membranes cytoplasmic of system a to proximity of the integral protein of the SR membrane consists pe 80 Approximately myofibril. the around sleeve nous of a terminus of an axon with a muscle fiber is call of point The neuron. that along transmitted impulse stimulated when simultaneously contract and neuron rils. As a result, the intracellular the Caintracellular As a result, rils. m the to distance short the over and compartment SR and calcium diffuses out the SR membrane are opened, a of calcium potential by way of arrival the transverse tubules, the With contraction. for required tion proximately 2 proximately levels within of the a cytoplasm muscle fiber loware (ap-very tached troponin molecule. When Ca When molecule. troponin tached o position The control the under grooveis the tropomyosin within molecules. actin the on sites binding cules of the thin filaments (see Figure 9.58) block sider the protein makeup of the thin filaments. necessa is it fiber, muscle skeletal a in contraction units in the thin filament. which in turn controls the binding capacity of seve m tropomyosin one of position the controls molecule troponin Each filaments. thin the to attach allow to heads myosin molecules, actin my adjacent the the on exposes sites binding tropomyosin the of position in shift 9. Figure in a to b position (from groove filament’s which moves approximately 1.5 nm to closer the cent tro adjacent the to transmitted is troponin of ment the dominoes, of row a of collapse the Like molecule. t the of subunit another in change conformational a (troponinC troponin of subunits the of one to bind Ϫ 5 h iprac o clim n uce otato was contraction muscle in calcium of importance The The steps that link the arrival of a nerve impulse nerve a of arrival the link that steps The When the sarcomere is relaxed, the tropomyosin mole- tropomyosin the relaxed, is sarcomere the When M. To understand how elevated calcium levels trigger levels calcium elevated how understand To M. sarcoplasmic reticulum (SR) (Figure 9.62). The T tubules terminate in very close very in terminate tubules T The 9.62). (Figure ϫ 10 Fgr 96;se lo iue .6 o a for 4.56 Figure also see 9.62; (Figure Ϫ 7 M)—below the threshold concentra-threshold the M)—below . Unlike a neuron, where an where neuron, a Unlike . 2 ϩ , which which forms a membra-, 2 ϩ levels rise to rise about levels 5 levels rise, these ions these rise, levels rnvre (T) transverse impulse from he interior ofhe interior pulse gener- the myosin- ry to recon-to ry ed a n actin sub- channels channels in nger deter- ferred to as uromuscu- pomyosin, n essential of Ca ,causing), of the at-the of 3.This63). 2 nducting e Cahe actionn ract in ain ract er of the comeres ϩ roponin whose, storeds olecule, n the ing contact move- neuro- of the make yofib- out of t theat y anby d to ed ician. osin- the f rcent 2 ϩ 2 ϫ ϩ - 371

Neuromuscular Transverse Motor junction tubule neuron

Mitochondrion

Myofibril

Sarcoplasmic reticulum

Nucleus Z line

Figure 9.62 The functional anatomy of a muscle fiber. Calcium is membrane of the transverse tubule to the SR. The calcium gates of the housed in the elaborate network of internal membranes that make up SR open, releasing calcium into the cytosol. The binding of calcium the sarcoplasmic reticulum (SR). When an impulse arrives by means of ions to troponin molecules of the thin filaments leads to the events a motor neuron, it is carried into the interior of the fiber along the described in the following figure and the contraction of the fiber.

Once stimulation from the innervating motor nerve fiber to a position where they block the actin–myosin interaction. ϩ ceases, the Ca 2 channels in the SR membrane close, and the The process of relaxation can be thought of as a competition ϩ Ca 2 -ATPase molecules in that membrane remove excess for calcium between the transport protein of the SR mem- ϩ calcium from the cytosol. As the Ca 2 concentration de- brane and troponin. The transport protein has a greater affin- creases, these ions dissociate from their binding sites on tro- ity for the ion, so it preferentially removes it from the cytosol, ponin, which causes the tropomyosin molecules to move back leaving the troponin molecules without bound calcium.

R E V I E W S1 1. Describe the structure of the sarcomere of a skeletal S1 muscle myofibril and the changes that occur during its Tropomyosin contraction. b a 2. Describe the steps that occur between the time that a Actin Actin Actin Actin nerve impulse is transmitted across a neuromuscular b a junction to the time that the muscle fiber begins to shorten. What is the role of calcium ions in this process? S1 S1 9.7 Figure 9.63 The role of tropomyosin in muscle contraction. 9.7 Schematic diagram of the steric hindrance model in which the myosin- | Nonmuscle Motility MotilityNonmuscle binding site on the thin actin filaments is controlled by the position of Skeletal muscle cells are an ideal system the tropomyosin molecule. When calcium levels rise, the interaction for the study of contractility and move- between calcium and troponin (not shown) leads to a movement of the ment because the interacting contractile tropomyosin from position b to position a, which exposes the myosin- proteins are present in high concentration and are part binding site on the thin filament to the myosin head. of defined cellular structures. The study of nonmuscle Chapter 9 The Cytoskeleton and Cell Motility 372 another (arrow) and as a cross-linked meshwork in w the filaments in which are arrange as bundles arrays: in These filaments are seen to be organized filaments. the leading edge of a motile fibroblast shows the hi This electr of processes. types by extending various cell. are determined by interaction of actin with a remar ins filaments actin of behavior and organization The (Figure gels three-dimensional complex and (two-dimens thin networks, bundles, of types various including variety a act into organized are cells living contrast, in In ments straw. by covered barn a of re floor they the microscope, the Under activities. useful form another one with interact cannot filaments such but in vitro to form actin polymerize actin can Purified Actin-Binding Proteins tran cortex labile, more restricted ordered, they are typically Moreover, less arrangements. in present be to tend c critical the because challenging more is motility r ragdi aiu ietos (Care directions. in various arranged Figure 9.64 dependent on the assembly of microfilaments in the c cell into two cells Allduring of cell the division. sing a of constriction the and movement, cell during of extension the materials, extracellular of gestion responsible for such proces tive region of the cell, spatial patterns of actin filaments.spatial patterns leng numbers, assembly, of rates the govern that tors survey to important is it however, First, superfamily. members of the in some cases, on actin filaments and, that motility and contractility nonmuscle of amples In the following pages, we will consider a number of number a consider will we pages, following the In As described later in the chapter, cells move cells across later in the chapter, As described utbnahtepam ebae The cortex is an a just beneath the plasma membrane. Two within filaments actin of arrangements different UTS OFOURTESY on micrograph of .VJ. gh density of actingh density d parallel to oned parallel hich the filamentshich to two distinct to two se processes are ICTOR a substratum ses as the in- of patterns,of omponents kable vari- filaments, processes le animalle to a thin the fac- the ortex. S depend h,andths, ide cellside MALL myosin r per-or n fila-in semble 9.64). ional) sient ex- a .) c- ety ety of idn rti n h rviigcniin eg,the co binding protein and the prevailing conditions (e.g., depending on the con of activities listed, the types 2. 1. and H 5 difficult to extend the results to activities within e iie it svrl aeois ae o ter p their 9.65).function (Figure in the cell on based categories several into divided be pr Actin-binding types. cell different from isolated hav families numerous to belonging proteins binding differen 100 than More organelles. cellular with and one with interactions their and filaments, actin properties, the physical of disassembly or assembly ized It should be noted that some of these proteins can rtis the proteins, of nucleatin Another family actin filaments. branched complex generates networks of short, the Arp2/3 378, mosin proteins.Monomer-sequestering rapid elongation of the filaments helped c that they promote ve can they actin filaments, nucleate formins Thus not subunits at thatare site. as new inserted end ev with the barbed track formins filament, formed remains which at the pointed end of the newl Arp2/3, Unl 14.2). (Section of dividing cells rings tractile adhesions (pageas 248)those and found th at focal crotubule nucleation (Figure 9.20 (Figure crotubule nucleation way that analogous to t be added, actinto monomers which can that provides adopt a conformation two Arps a templ Once the complex is activated, actins. sidered “true” with actins but are not sequencesiderable homology proteins that shar that is, “actin-related proteins,” best studied is the of actin filaments identified that promote nucleation added (as in Figure 9.47added (as in Figure monomers can to which a preexisting seed or nucleus tion of an actin filament by the is accelerated pres the fo As noted earlier, on their own. molecules left process unfavorable for This is a very the polymer. together in the proper to begin formati orientation requires that at or least three two actin monomers which nucleation, an actin filament is the first step, Nucleating proteins. the time. is favor or depolymerization whether polymerization region and deter in a of certain a cell equilibrium the monomer–polymer proteins shift sequestering can of monomer changes in the concentration or activity the monomer to p bind G-actin and stabilize ability of Because of soluble actin monomers into filaments. would favor the near complete polymerizat cytoplasm conditions within the monomer-sequestering proteins, Without (50–200 mM). cells G-actin in nonmuscle responsiblelieved for the relatively high concentr These proteins are be proteins. monomer-sequestering Proteins are as ac described with this activity ing. called (often ϩ actin-binding actin-binding proteins. .Most studies of these proteins are conducted in v ). ␤ 4 ␥ ) are proteins that bind to actin-ATP monomers -tubulin is proposed a template for mi- to form G-actin formins Arp2/3 complexArp2/3 eeaeubace lmns such filaments, unbranched generate , The slowest step of in the formation ) and prevent them) and from prevent polymeriz- a ). Several proteins beenhave Several ). These proteins affect the local- 5 hmsn eg,thy-Thymosins (e.g., the cell. c , which contains which two, .As discussed on page). centration of the actin-centration carry out more than one ofcarry ncentration of Cancentration to and it is oftenitro, oteins canoteins ike mine tin ation of e con- ence of another resumed only doonly come actin-t on of actin e con- reate. rma- beene ool, the con- The. - ed at be ate their their ry ion g y 2 - en he ϩ 373

3 End-blocking (capping) 6 Cross-linking

2 Monomer- sequestering

Monomer Bundling Monomers Monomer polymerizing nucleating 6 4 1

5 7 Depolymerizing 8 Filament-severing Membrane-binding Figure 9.65 The roles of actin-binding proteins.

3. End-blocking (capping ) proteins. Proteins of this group body and at the pointed end of actin filaments (see Figure regulate the length of actin filaments by binding to one or 18.33). Cofilin has two apparent activities: it can frag- the other end of the filaments, forming a cap that blocks ment actin filaments, and it can promote their depoly- both loss and gain of subunits. If the fast-growing, barbed merization at the pointed end. These proteins play a role end of a filament is capped, depolymerization may pro- in the rapid turnover of actin filaments at sites of dynamic ceed at the opposite end, resulting in the disassembly of changes in cytoskeletal structure. They are essential for the filament. If the pointed end is also capped, depoly- cell locomotion, phagocytosis, and cytokinesis. merization is blocked. The thin filaments of striated mus- 6. Cross-linking proteins. Proteins of this group are able to cle are capped at their barbed end at the Z line by a alter the three-dimensional organization of a population protein called capZ and at their pointed end by the pro- of actin filaments. Each of these proteins has two or more tein tropomodulin. If the tropomodulin cap is disturbed actin-binding sites and therefore can cross-link two or by microinjection of antibodies into a muscle cell, the more separate actin filaments. Certain of these proteins thin filaments add more actin subunits at their newly (e.g., filamin) have the shape of a long, flexible rod and exposed pointed end and undergo dramatic elongation promote the formation of loose networks of filaments in- into the middle of the sarcomere. terconnected at near right angles to one another (as in 4. Monomer-polymerizing proteins. Profilin is a small pro- Figure 9.64). Regions of the cytoplasm containing such tein that binds to the same site on an actin monomer as networks have the properties of a three-dimensional elas- does thymosin. However, rather than inhibiting polymer- tic gel that resists local mechanical pressures. Other cross- ization, profilin promotes the growth of actin filaments. linking proteins (e.g., villin and fimbrin) have a more Profilin does this by attaching to an actin monomer and globular shape and promote the bundling of actin fila- catalyzing the dissociation of its bound ADP, which is ments into tightly knit, parallel arrays. Such arrays are rapidly replaced with ATP.The profilin-ATP-actin found in the microvilli that project from certain epithelial 9.7 monomer can then assemble onto the free barbed end of a cells (Figure 9.66) and in the hairlike stereocilia (Figure

growing actin filament, which leads to the release of pro- 9.54) that project from receptor cells of the inner ear. MotilityNonmuscle filin. Filament elongation at the barbed end is further Bundling filaments together adds to their rigidity, allow- promoted by members of the Ena/VASP protein family. ing them to act as a supportive internal skeleton for these 5. Actin filament-depolymerizing proteins. Members of the cytoplasmic projections. cofilin family of proteins (including cofilin, ADF, and de- 7. Filament-severing proteins. Proteins of this class have the pactin) bind to actin-ADP subunits present within the ability to bind to the side of an existing filament and Chapter 9 The Cytoskeleton and Cell Motility 374 membrane of the microvillus actimembrane and the peripheral is present which betwee The role of myosin I, fimbrin. proteins ordered by the bundling highly arrangement Actin filaments are ma tubule. of the kidney and wall as the such of solutes, that function in absorption 8. remains unclear. microvillus. Figure 9.66 skeleton of erythrocytes (see Figure 4.32 (see Figure skeleton of erythrocytes into the membrane actin polymers of short inclusion th Two examples were in previous chapters: described protei membrane means to aof peripheral attachment by indirectly, actin filaments to the plasma membrane facilitated by These activities are generally sis). or cytok phagocytosis during for example, (as occurs, locomotion) cell or to during invaginate in example, for (as occurs, outward it to protrude causing brane, ated by proteins the contractile act on the plasma the forces gene numerous activities, During membrane. lies just beneath the pl cells of nonmuscle chinery Membrane-binding proteins. filaments. of severing is also capable cofi 9.71, in Figure As indicated generate. ments they the frag- may cap or they additional free ends, barbed of actinpromote monomers by crea the incorporation gelsolin) m proteins (e.g., Severing break it in two. dystrophy). the protein responsible for muscular dystrophin, (includi family and members of the spectrin moesin), and radixin, members of the ERM (ezrin, family vinculin, Proteins that link membranes to actin include 7.26). adhesions and adherens 7.17 junctions (see Figures at foc of actin filaments to the membrane attachment Microfilament of filament Microvilli are of epi present surface on the apical Plus end Myosin I Actin filaments and actin-binding proteins actin-binding and filaments Actin ain Microvillus Much of the ma- contractile Much lining of the intestine d n filaments, ,and the ), intained in a villin and villin n the plasma Villin Fimbrin End cap linking the asma mem- thelia ay also and ward ine- ng al e ting lin n. r- food poisoning.food terium that infects macrophages and can cause encep ci plmrzto ad o o ivle h activit the involve not Consider the example of do myosin. and polymerization actin Mecha-nism Force-Generating a as Polymerization Actin f the by illustrated examples. are contractility and motility Non and changes in cell shape. axonal outgrowth, tion, p integral cell of cell–substratum interactions, within movements membranes, lateral activation, platelet traffic vesicle cells), plant large certain in occurs cytopla of flow bulk directed phagocytosis, (the streaming plasmic cytokinesis, including cells, nonmuscle act dynamic of variety a for responsible are my motors, with conjunction in working often filaments, Actin Examples of Nonmuscle Motility and Contractility ettepoeso ci oyeiain The proces rect the process of actin polymerization. together work that below) discussed complex, Arp2/3 recruits and activates a number of host proteins (i cytopla host the within exposed is ActA When terium. protein called ActA that is present only at one end Listeria part a and site particular a at machinery necessary as to able being cell a on depends motility because moti of type any of study the in important are tion ments at a particular site on its of surface? Question formation the induce to able cell bacterial the Ho 9.67). (Figure bacterium the behind just monomers polymerization the by cell infected an of cytoplasm taa nldn vros ye o three-dimensional of types may which various 18.22), Figure (see matrices extracellular including strata, sub-complex more develop to begun recently have searchers terr complicated more traversing cells to apply not ing evidence that some of the findings from these st thein cells and that there body don’tmind flat substrates, move in over bare, Keep research. of field this inated because these are the experimental conditions that two-dimensional tured cells moving over a flat studies (i.e., on concentrate will we discussion, following tion also contributes to the spread of cancerous tu ax of Cell and development protection against wound infection. healing, vessels, blood of formation opment, including tissue and tivities organ in devel-higher vertebrates, Cell Locomotion ria f el tesle,wih s h sbet f h fol the of subject the is section. which themselves, cells of the to organelles and vesicles cytoplasmic of sion utilized ranging for fro normal cellular activities, motility. req force the providing of capable is motors, myosin partic the without is, that itself, by tha polymerization conclusively demonstrate to researchers allowed rplin a be rcntttd n ir,wih ha which vitro, in reconstituted been has propulsion The same events that occur during Some Some types of cell motility occur solely as the res can accomplish this feat because it contains a surf a contains it because feat this accomplish can Listeria Cell Cell locomotion is required for many ac- is propelled like a rocket through thethrough rocket a like propelled is Listeria monocytogenes Listeria propulsion are m the propul- s about loca- ncluding ncluding the os In themors. ig bloodking, icular time.icular of the bac- semble thesemble have dom- movement ) substrate is increas- udies may ci fila-actin ipation ofipation s of le processle locomo- ollowing ollowing halitis or uired foruired vte inivities f actin of i.Re-ain. locomo- f cul- of ed to lead m thatsm a bac-, roteins muscle lowing lowing actin t o di-to Liste- m itsm, cyto- ult of of y isw osin ons, ace s 375

(a) Figure 9.67 Cell motility can be driven by actin polymerization. (a) Fluorescence micrograph of a portion of a cell infected with the bacterium L. monocytogenes . The bacteria appear as red-stained objects just in front of the green-stained filamentous actin tails. ( b) Electron micrograph of a cell infected with the same bacterium as in a, showing the actin filaments that form behind the bacterial cell and push it through the cytoplasm. The actin filaments have a bristly appearance because they have been decorated with myosin heads. Bar at the upper left, 0.1 ␮m. ( A: C OURTESY OF PASCALE COSSART ; B: FROM LEWIS G. T ILNEY ET AL ., J. C ELL BIOL . 118:77, 1992, F IG . 8. R EPRODUCED WITH PERMISSION OF THE ROCKEFELLER UNIVERSITY PRESS .)

Tail

Leading edge (b)

Figure 9.68 Scanning electron micrograph of a mouse fibro- blast crawling over the surface of a culture dish. The leading hibited by the fibroblast in Figure 9.68, shares properties with edge of the cell is spread into a flattened lamellipodium whose other types of locomotion, for example, walking. As you walk, structure and function are discussed later in the chapter. (F ROM GUENTER ALBRECHT -B UEHLER , I NT . R EV . C YTOL . 120:194, 1990.) your body performs a series of repetitive activities: first, a leg is extended in the direction of locomotion; second, the bottom of your foot makes contact with the ground, which acts as a point of temporary adhesion; third, force is generated by the 9.7

muscles of your legs that moves your entire body forward past MotilityNonmuscle revision of some aspects of the mechanism of cell locomotion the stationary foot, all the while generating traction against discussed below. the point of adhesion; fourth, your foot—which is now behind Figure 9.68 shows a single fibroblast that was in the your body rather than in front of it—is lifted from the ground process of moving toward the lower right corner of the field in anticipation of your next step. Even though motile cells when it was prepared for microscopy. Cell locomotion, as ex- may assume very different shapes as they crawl over a substra- Chapter 9 The Cytoskeleton and Cell Motility 376 been pulled forward.been pulled porti have been severed and the trailing substratum the rear 4 shows att after the cell Step substratum. accomplished byforce a(traction) contractile exer This remains which stationary. relatively attachment, shows the movement forward of the bulk of the cell the to grip uses this attachment The cell membrane. res that is mediated by integrins an attachment tum, the adhesion of the lower of the lamellipod surface of a lamellipo in leadingthe form edge of the cell substratum. the cell crawls over Figure 9.69 edd u fo te el s bod atnd veil-l flattened, broad, a a as called cell protrusion, the from out tended whic edge, leading its examining by seen is comotion m fibroblast a day, The key moveto the a fibrobldistance good of about 1 mm. a On withdrawing. times other advanci sometimes jerky, and erratic is movement Its Fig in (as narrowa and “tail” end frontal broadened flattensfibroblast fan-shapbecomes and substratum the to close itself a moves, it As 7.1). Figure (see tissue tive presenti predominant arecells the which broblasts, these cells under the microscope showstypically th the specimen and onto Exami the surface of the dish. individual cells migr an appropriate culture medium, is placed in a culture dish in such as skin or liver, living tissue, Substratum the over Crawl that Cells or “tail.” edge, of the trailing traction c substratum, the with contacts rear its breaks cell cel the of rear the of part become eventually which adhesiv the over forward pulled (3) is cell the of bulk anchorage. of sites temporary forming substratum, atta protrusion the of surface lower the of portion cell surface in the direction in which the cell is pa a of protrusion the by initiated is Movement (1) they display a similar sequence tum, of activities ( lipodia are typically devoid of cytoplasmic vesicle cytoplasmic of devoid typically are lipodia Nucleus The repetitive sequence of activities that occurs a sequence of activities repetitive The Cell body lamellipodium Integrin Step 1 shows of theStep the protrusion Lamellipodium Substratum Fgr 9.70 (Figure When a small piece ofpiece small a When dium. Step 2 shows Step dium. ted against the achments with theachments ium to the substra- on of the cell hason of the cell iding in the plasma over the site of movement is substratum. Step 3 Step substratum. omv.(2) Ato move. Figure Figure 9.69). 4 3 2 1 s and otherand s em to be fi- a ches to theto ches e contacts,e ausing re-ausing n connec-n .Lamel-). ed, with a with ed, ure 9.68).ure l. (4) The (4) l. ate out of nation of rt of theof rt ast’s lo- h is ex-is h g andng s a The ike ay n pedotit ellk aelpdu.(and spread out into a veil-like lamellipodium. membranes (of the lamellipodium. s micrograph of the leading edge of a cultured cell, monomers can provide the force that propels a propels that force the provide can monomers eim hog te yols.Ti tp o intracell of type of involvement the This without accomplished is movement cytoplasm. the through terium itself forw to pull for the cell sites of anchorage providing t substratum at underlying specific points, itAs adha lamellipodium is extended from the cell, uaigmto,giving it a ruffled appearance (Figu dulating motion, and the outer edge particulate often structures, exh B : FROM e a o pg 34 o te oyeiain f actin of polymerization the how 374 page on saw We (b) (a) edge of this motile fibroblast is flattened against t Figure 9.70 J EAN P AUL The leading edge of a motile cell. leading edge of a motile The R EVEL S, YMP A S. C: OC UTS OFOURTESY E. XP B. b ) Scanning electron) Scanning IOL howing the ruffled .VJ. 847 1974.) 28:447, . ard. ( he substratum a ICTOR Listeria ) Theleading ibits an un- eres to the emporary re 9.70 S MALL mo- bac- ular b ). ; 377

(a)

1 Capping protein

WASP Actin - Barbed end ATP

2 3 4 5 6 Cofilin Activated Profilin Arp2/3 complex Pointed end

Actin-ADP

Actin-ATP Profilin (b) Figure 9.71 Directed cell motility. (a) Micrograph of a white blood lamellipodium. Meanwhile, the barbed end of previously formed cell (a neutrophil) that has responded to a chemoattractant being deliv- filaments are bound by a capping protein, which prevents further ered by a pipette (seen on the right). The cell has become polarized and growth of these filaments, keeping them short and rigid. Eventually, is moving toward the source of the stimulus. ( b) A proposed the pointed ends of the older, preexisting actin filaments undergo mechanism for the movement of a cell in a directed manner. A stimulus depolymerization, releasing ADP-actin subunits (step 6). Depolymer- is received at the cell surface (step 1) that leads to the activation of ization is promoted by cofilin, which binds to ADP-actin subunits Arp2/3 complex by a member of the WASP/WAVE family (step 2). within the filament and stimulates dissociation of the subunits from the Activated Arp2/3 complexes serve as nucleating sites for the formation pointed end of the filament. Released subunits bind profilin and of new actin filaments (step 3). Once filaments have formed, Arp2/3 become recharged by ATP/ADP exchange, which makes them ready to complexes attach to the sides of the filaments (step 4), which stimulates engage in actin polymerization (as in step 3). (A discussion of the their nucleating activity. As a result, the bound Arp2/3 complexes initi- validity of this model can be found in Trends Cell Biol. 21:2, 2011 and ate side branches that extend outward (step 5) at an angle of about 70 Њ Nature Cell Biol, 13: 1012, 2011.) (A: F ROM CAROLE A. P ARENT , relative to the existing filaments to which they are anchored. As these CURR . O PIN . C ELL BIOL . 16:5, 2004; © 2004, WITH PERMISSION filaments polymerize, they are thought to push the plasma membrane FROM ELSEVIER .) outward, resulting in extension of the leading edge of the

lecular motors. A similar type of actin-polymerization mech- have a family of proteins (the WASP/WAVE family) that ac- anism is thought to provide the motile force required for the tivates the Arp2/3 complex at the site of stimulation near the protrusion of the leading edge of a lamellipodium. This type plasma membrane. WASP, the founding member of the fam- of nonmuscle motility also demonstrates the importance of ily, was discovered as the product of a gene responsible for actin-binding proteins (depicted in Figure 9.65) in orchestrat- Wiskott-Aldrich syndrome. Patients with this disorder have a ing the assembly and disassembly of actin-filament networks crippled immune system because their white blood cells lack a at a particular site within the cell at a particular time. functional WASP protein and consequently fail to respond to Suppose we begin with a rounded white blood cell that chemotactic signals. receives a chemical signal coming from one particular direc- Figure 9.71 b depicts a model for the major steps in for- tion where the body has been wounded. Once the stimulus is mation of a lamellipodium that would guide a cell in a partic- received at the plasma membrane, it triggers the localized ular direction. A stimulus is received at one end of the cell

polymerization of actin, which leads to the polarization of the (step 1, Figure 9.71 b), which leads to the activation of Arp2/3 9.7 cell and its movement toward the source of the stimulus (Fig- protein complexes by activated WASP proteins (step 2). In its ure 9.71 a). 6 Just as Listeria has a protein (ActA) that activates activated state, the Arp2/3 complex adopts a conformation MotilityNonmuscle polymerization at the bacterial cell surface, mammalian cells that resembles the free surface at the barbed end of an actin filament. As a result, free ATP-actin monomers bind to the 6This type of response can be seen in a remarkable film on the Web showing a Arp2/3 template, leading to the nucleation of actin filaments neutrophil chasing a bacterium. It can be found using the search words: (step 3). Polymerization of ATP-bound actin monomers onto “neutrophil crawling.” the free barbed ends of the growing filaments is promoted by Chapter 9 The Cytoskeleton and Cell Motility 378 have branched off of preexistinghave branched filaments. filaments polymerized newly the where junctions Y-shaped actin-filamen short resito seenare complexes Arp2/3 The immun labeling. gold of by highlighted complexes succession Arp2/3 with a branches, show 9.72 Figure in circular The lamellipodium. advancing an of brane tous actin network that resides just beneath the pl the of nature cross-linked branched, the shows 9.72 of micrograph electron The locomotion. cell of tures atce ylo) (F (yellow). particles by antibodies li branch at the base of each calized co Arp2/3 actin filaments. junctionsbranched between The circular insets show a succe individual “trees.” has been color which network, in a branched arranged The actin filaments are of a motile mouse fibroblast. Electron micrograph of a replica of the cytoskeleto the leading edge. fil actin of assembly the in reutilized be can which profilin-ATP-actinmono into conversion by recharged filamen disassembling the from released A subunits ADP 6). (step filaments the along subunits actin-ADP to poin their whi Disassembly is promoted ends by (step from cofilin, 6). disassembly undergo filaments capped growing by addition of subunits to their barbed end fil As newer stimulusthe attractive (steps 5 and 6). direct the in outward lamellipodium the of pushes membrane network the of filaments formed recently more barbe the to subunits actin of addition contrast, In protein capping of addition the by blocked is older ments of ends barbed the of growth Meanwhile, points. th at situatedare which ends, pointed the remainat com Arp2/3 The 5). (step branches as form that ments a additional of formation the nucleate and 4) (step Arp2/3 complexes bind to the sides formed, of these filament actin new Once 373). (page molecules profilin IHPRISO OF PERMISSION WITH B Figure 9.72 ORISY Figure 9.72 illustrates some of the major structura major the of some illustrates 9.72 Figure , FROM The structural basis of lamellipodial extension. basis of lamellipodial structural The .CJ. ELL ROM T B HE IOL T ATYANA R ., OCKEFELLER VOL 4,#,1999; #5, 145, . .SM. IKN ANDVITKINA U NIVERSITY nked to colloidal goldnked to colloidal ssion of Y-shaped n at the leading edge REPRODUCED seen to be ized to indicateized mplexes are lo- G asma mem- ARY P ,the olders, aments are RESS aments ataments e branch-e d ends ofends d filaments (step 5).(step tn fila-ctin de at theat de filamen- ch ch binds Figure G. es insets s haves ion ofion plexes .) s arets mers, l fea-l fila- ctin- the ted o- t substratum. Figure 9.73 Figure substratum. v un specifically the with contact to makes cell the investigators where structures allows This cell. ing w vinculin labeled fluorescently of localization the revealed best is attachment of sites these of ence substratum. underlying the to strongly adheres cell forces are exerted just behind the cell’s leading e the puterized greateimage of a migrating fibroblast, etdpnso h omto favr ra,thin broad, very a of formation the on depends ment gliding rapid their because locomotion studying for covers Keratocytes have the been fish’sa favored system epiderscales. the from derived cells are which keratocytes, cont These 9.69). roles of actin and myosin are bestin s illustrated Figure 3, (step forward cell the of the conjun pulling for (inresponsible is filaments) myosin actin with whereas 9.69), Figure 1, (step ward of edge leading the pushing for responsible is tion on these adhesion sites. tensio by stimulated probably is complexes focal of M adhesions. focal contractile more larger, into ture motile cell either disassemble as the cell moves fo complexes The focal that nearform the leading cell. outsid and inside the connect that molecules tegrin to the extracellular substrate by way of the transm t then and sites adhesion these with associated ton tion forces are thought to be generated in the acti cultured cells and arereferred often to as st spread, highly in seen adhesions focal mature the migrat adheres vin to the substratum tend to be a smaller and si The of edge leading the 7.17. where sites Figure containing in illustrated sites migrating containing a of th edge adhesions, focal of component major leading a is Vinculin the behind just trated ing the presence of red fluorescent vinculin molecul because they occur at thsites where they grips because the cell They are often described 3 as of Figure “trac 9.69). the main body of the cell quiredfo to pull or “tow” tha adhesion of sites at generated those are motion i involved forcesmajor The cell. the of bulk the of followedis by edge leading the of protrusion 9.69, pointed ends of the array toward the rear. added to barbed subu ends of the actin array at its which front end in 360) (page treadmilling of type und array actin-filament entire 9.71). the Figure whole, a as taken 6, (step lamellipodium the of back the t depolymerize then and direction rearward a fila in actin flow assembled lamellipodium, the of edge front th at continue branching and polymerization filament shown in Figure 9.73 Figure in shown portr and deformation substrate of patterns dynamic calculate be can cell migrating live a within tions var at exerted forces traction the Figure of magnitude The (see substratum underlying the of formation movements of the elastic cell material, are accompan A large body of evidence indicates that actin polym A indicates large body of evidence According to the sequence of events depicted in Fig in depicted events of sequence the to According actin As process. dynamic a is movement Lamellipodial When cells are allowed to migrate over a thin sheet thin a over migrate to allowed are cells When a . As seen by examination of this com-this of examination by seen As . b is a fluorescence micrograph show-micrograph fluorescence a is focal focal complexes dge where the tudies on fish and lost from rward rward or ma- e substrate. by followingby embrane embrane in- n cytoskele- rward (steprward tion forces” n cell loco- cell n d from thefrom d mpler than remainder a cell out- cell a ransmitted ithin a liv-a ithin movement es concen- st traction The pres-The ious loca-ious ied by de- edge of a exertedn aturation ae re- are t i thatmis derlying ationary o the of e ergoes aergoes e actin-e ing cell cell ing is arenits isualize move- yd asayed rasting Trac-. lamel- oward e verye ments culin- 7.18). eriza- Thus ction cell. ure of 379

(a) (b) Figure 9.73 Distribution of traction forces within a migrating that is adhering to the underlying substratum at numerous sites (red). fibroblast. (a) As a cell migrates it generates traction (pulling) This cell is expressing GFP-actin (green) and had been injected with forces against its substrate. The present image shows the traction forces rhodamine-tagged vinculin (red). The fluorescently labeled vinculin is generated per unit area by the surface of a migrating fibroblast. incorporated into dot-like focal complexes near the leading edge of the Traction forces were calculated at different sites on the surface based on cell. Some of these focal complexes disassemble, whereas others mature the degree of substrate deformation (see Figure 7.18). The magnitude into focal adhesions, which are situated farther from the advancing of the traction forces are expressed by varying colors with red edge. ( A: F ROM KAREN A. B ENINGO , M ICAH DEMBO , AND YU-L I representing the strongest forces. The largest forces are generated at WANG , J. C ELL BIOL . 153:885, 2001, F IG . 3. R EPRODUCED WITH sites of small focal complexes that form transiently behind the leading PERMISSION OF THE ROCKEFELLER UNIVERSITY PRESS ; B: F ROM J. edge of the cell where the lamellipodium is being extended (arrow). VICTOR SMALL , ET AL ., IMAGE COURTESY OF OLGA KRYLYSHKINA , Deformation at the rear of the cell (shown in red) occurs as the front NATURE REVS . M OL . C ELL BIOL . 3:957, 2002; © 2002, REPRINTED end actively pulls against the tail, which is passively anchored. ( b) A WITH PERMISSION FROM MACMILIAN PUBLISHERS LTD .) living, migrating fibroblast exhibiting a well-developed lamellipodium

lipodium. Figure 9.74 shows a moving keratocyte that has nerve cells not only remained healthy, but many of them been fixed and stained for actin (Figure 9.74 a) and myosin II sprouted processes that grew out into the surrounding (Figure 9.74 b). As expected from the previous discussion, the medium. Not only was this the first time that cells had been advancing edge of the lamellipodium is filled with actin. kept alive in tissue culture, the experiment provided strong ev- Myosin, on the other hand, is concentrated in a band where idence that axons develop by a process of active outgrowth and the rear of the lamellipodium joins the remainder of the cell. elongation. Electron micrographs of this region show the presence of The tip of an elongating axon is very different from the clusters of small, bipolar myosin II filaments bound to the remainder of the cell (see Figure 9.1 b). Although the bulk of actin network (Figure 9.74 c). Contractile forces generated by the axon shows little outward evidence of motile activity, the these myosin molecules are presumed to pull the bulk of the tip, or growth cone , resembles a highly motile, crawling fi- cell along behind the advancing lamellipodium. Myosin I and broblast. Close examination of a living growth cone reveals other unconventional myosins are also thought to generate several types of locomotor protrusions: a broad, flattened

forces for cell locomotion in some organisms. lamellipodium that creeps outward over the substratum; short, 9.7 stiff microspikes (Figure 9.75 a) that point outward to the edge Axonal Outgrowth In 1907, Ross Harrison of Yale Uni- of the lamellipodium; and highly elongated filopodia that ex- MotilityNonmuscle versity performed one of the classic experiments in biology. tend and retract in a continuous display of motile activity. Flu- Harrison removed a small piece of tissue from the developing orescence microscopy shows all of these structures in the nervous system of a frog embryo and placed the fragment into peripheral domain of the growth cone to be filled with actin a tiny drop of lymphatic fluid. Harrison watched the tissue filaments (shown in green, Figure 9.75 b). These actin fila- under a microscope over the next few days and found that the ments are presumed to be responsible for the motile activities Chapter 9 The Cytoskeleton and Cell Motility 380 aelpdu.Br 5 Bar, lamellipodium. rowheads) be seen projecting can ahead of the leadi and fine processes parent veil of the lamellipodium, microspikes (arrows) Rodlike be seen can substratum. is spread into that a flattened creeps lamellipodium growing axon.growing cone of a neuron showing the actin filaments (green) concentr Figure 9.75 the direction of movement, which can occur at rates occur at rates can which the direction of movement, The flattened lamellipodium. dish by means of a broad, moving micrographs of a fish keratocyte Fluorescence The distribution of filamentous actin is revealed in The distribution bodies. It is evident that the body of the lamellipo It is evident bodies. in part in part of myosin in the sa The distribution actin filaments. of fluorescently labeled phalloidin the localization lamellipodial-based movement of fish keratocytes.lamellipodial-based Figure 9.74 b , which shows the localization of fluorescent shows which antimyo the localization , (a) The structure of a growth cone: the motile tip of a tip the motile cone: of a growth structure The ( a ie mg falv rwhcn.The terminus) A video image of a live growth cone. (a) The roles of actin and myosin in theThe ␮ .(m. b ) Fluorescence micrograph of the growth) Fluorescence ci Myosin Actin (b) , which binds only to binds only which , dium contains actin part part forward over the forward ng edge of the called filopodia (ar-called me cell is revealedme cell of 10 within the trans- over a culture arrow shows a , which shows which , ␮ ( a,b m/min. sin anti- ated in ) (A: where they interact withthe acti peripheral domain, A number of microtubules be can domain. the central domain and the microtubules concenthe peripheral (orange) V OF (b) VOL R hw nrd ysnmlclsi le (B myosin molecules in blue. shown in red, T towardinteractions the rear of the lamellipodium. filamentous and t actin network of the lamellipodium egswt h oyo h el (merges with the body of the cell. in a band that lies just behind the lamelli instead, Myosin i of myosin. devoid filaments but is virtually W 3:9,19,F 1997, 139:397, 0:58 98 F 1988, 107:1508, OCKEFELLER ERKHOVSKY A TE RMAN T FROM 5,#5, 157, . HE R OCKEFELLER P AUL (c) -S COVER , TORER FROM U F IG NIVERSITY IG RCE ANDORSCHER .R 1. . 2;. 02 B 2002. , T , AND ATYANA B U POUE IHPRISO OF PERMISSION WITH EPRODUCED : FROM NIVERSITY C P T ERDCDWT PERMISSION WITH REPRODUCED OTH HRISTOPHER RESS .SM. F S c ENG ) A schematic drawing depicting the drawing ) A schematic TEPHEN .) IKN TAL ET VITKINA P -Q RESS UAN Y .) .SJ. .CS. A LEXANDER Z MITH OHAN HOU oim where itpodium, n-filament bundles. he actin network is s concentrated, ,J C J. ., he actin–myosin .C J. , C, seen to invade .C J. , LARE ELL B. ELL ELL trated intrated T M. B B HE IOL B IOL IOL . . . 381

Growth cone

(a) (b) Figure 9.76 The directed movements of a growth cone. (a) A video cell that is expressing the neuronal guidance factor ephrin (red). image of a live growth cone of a Xenopus neuron that has turned toward (A: F ROM ELKE STEIN AND MARC TESSIER -L AVIGNE , S CIENCE a diffusible protein (netrin-1) released from a pipette whose position is 291:1929, 2001. REPRINTED WITH PERMISSION FROM AAAS; indicated by the arrow. ( b) The growth cone (green) at the tip of a B: COURTESY OF IRINA DUDANOVA .) motor axon has made contact by means of its filopodia with a target

of the growth cone. Microtubules, on the other hand, fill the internal architecture that arises during embryonic develop- axon and the central domain of the growth cone, providing ment: the spinal cord is basically a hollow tube, the kidney support for the thin, elongating axon. A number of individual consists of microscopic tubules, each lung is composed of mi- microtubules are seen to penetrate into the actin-rich periph- croscopic air spaces, and so forth. Numerous cellular activities ery (shown in orange, Figure 9.75 b). These penetrating mi- are necessary for the development of the characteristic mor- crotubules are highly dynamic and are thought to play an phology of an organ, including programmed changes in cell important role in steering the growth cone in the appropriate shape. Changes in cell shape are brought about largely by direction. changes in the orientation of cytoskeletal elements within the The growth cone is a highly motile region of the cell that cells. One of the best examples of this phenomenon is seen in explores its environment and elongates the axon. Within the the early stages of the development of the nervous system. embryo, the axons of developing neurons grow along defined Toward the end of gastrulation in vertebrates, the outer paths, following certain topographical features of the substra- (ectodermal) cells situated along the embryo’s dorsal surface tum or responding to the presence of certain chemicals that elongate and form a tall epithelial layer called the neural plate diffuse into their path. The lamellipodia and filopodia from (Figure 9.77 a,b). The cells of the neural plate elongate as mi- the growth cone respond to the presence of these physical and crotubules become oriented with their long axes parallel to chemical stimuli, causing the pathfinding axons to turn to- that of the cell (inset, Figure 9.77 b). Following elongation, the ward attractive factors and away from repulsive factors. Figure cells of the neural epithelium become constricted at one end, 9.76a shows a cultured neuron whose advancing tip has made causing them to become wedge shaped and the entire layer of a direct turn toward a diffusible protein called netrin, which cells to curve inward (Figure 9.77 c). This latter change in cell acts as an attractant for axons growing within the early em- shape is brought about by the contraction of a band of micro- bryo. Figure 9.76 b shows a growth cone (green) making con- filaments that assemble in the cortical region of the cells just tact with a cell that is expressing another protein called ephrin beneath the apical cell membrane (inset, Figure 9.77c). Even- (red) that also acts as a neuronal guidance factor. Unlike tually, the curvature of the neural tube causes the outer edges netrin, ephrin is a non-diffusible, integral protein of the to contact one another, forming a cylindrical, hollow tube plasma membrane that binds to an ephrin receptor on the (Figure 9.77 d,e) that will give rise to the animal’s entire nerv- surface of the growth cone. The growth cone is making con- ous system. tact with the ephrin-expressing cell by means of its long R E V I E W filopodia, which serve a sensory function. Ultimately, the cor- 9.7 rect wiring of the entire nervous system depends on the un- 1. List the various types of actin-binding proteins and one canny ability of embryonic growth cones to make the proper function of each type. MotilityNonmuscle steering “decisions” that lead them to the target organ they 2. Describe the steps taken by a mammalian cell crawling must innervate. over a substratum. 3. Describe the role of actin filaments in the activities of Changes in Cell Shape during Embryonic Develop- the growth cone of a neuron. ment Each part of the body has a characteristic shape and Chapter 9 The Cytoskeleton and Cell Motility 382 lc oaohr p 324) (p. place to another. ce of movement the for responsible elements erating r machinery the movement a of materials and of organelles within cells; part as interior; cell the within internal framework responsible for positioning the shape the maintain helps that support structural a activities. cellularof number a in participate which m filaments), (actin ments and filaments, intermediate microtubules, structures: type distinct three of composed is cytoskeleton The | (e) (d) (c) (b) (a) Synopsis Neural tube Ventral Dorsal olciey the elements of the funct cytoskeleton Collectively, Ectoderm Neural plate various various organelles of the cell; as an as cell; the of nd as force-gen- qie fr the for equired l fo one from lls s of fibrous of s icrofila- ion as filaments Micro- ofcilia and flagella. form toskeleton, part of the mitotic centrio spindle, addit in proteinassembledtubulintheareand, from Microtubules are tubular hollow, structures 25 nm in steelgirders T provide supporttall building. fora microtubules often act in a supportive capacity not t of Because microtubule-associated (MAPs). proteins membeinfluencedby are capabilitiesinteraction, for includingth propertiesmicrotubules, theofof Many p or rows, in arranged are that heterodimers tubulin Microtubules Microtubulesare polymers assembled from C lt sbigfle ofr ue ( a tube. plate is being folded to form as its neural embryo of a chick sal surface ( ends of the cells.actin filaments at the apical forces by contractile bybe driven generated rolling of the plate into a tube is thought to whereas theelongation of microtubules, and by the orientation is thought to be driven in height The of initial the cells change tube. to roll into a neuralregion of the embryo at the mid-dorsal cells flattened ectodermal a layer of shape that cause changes in cell system. e ) Scanning electron) Scanning micrograph of the dor- UTS OFOURTESY Figure 9.77 development of the vertebrate nervous nervous vertebrate the of development ( a–d ) Schematic drawings of the drawings ) Schematic K ATHRYN Early stages in theEarly he structuralhe roleof unlike the way that .TW. e,and the les, core rs of a group ofgroup a of rs eir stabilityeirand diameter that ion to the cy-the to ion rotofilaments. OSNEY er stiffness,heir E : .) ␣␤ -