Download Mechanics of the Cvtoskeleton

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Peter Nick
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Abstract This chapter summarizesevidencefor a cytoskeletalfunction in tensegral integrationon both the organismaland the cellular levels.The plant cytoskeleton consistsof two major elements,microtubulesand actin filaments.The spatial
organization of these elements is highly dynamic and changes fundamentally
during the cell cycle, with conspicuouseffects on the predicted stress-strain
patterns.In interphasecells, microtubule bundlesare thought to control the direction of cellulose depositionand thus to reinforce the axiality of cell growth. By
microtubule-actinlinkers such as the novel classof plant-specifickinesins with a
calponin-homologydomain, the rigid microtubulesand the flexible actin bundles
can be integratedinto a systemendowedwith mechanicaltensegrity.Becausethe
plant cytoskeletonis relieved of the load-bearingtask it fulfils in the non-walled
animal cells, it has adoptedsensoryfunctions.Stretch-inducedchangesof protein
conformation and stretch-activatedion channelsseem to act in concert with the
cytoskeleton,which acts either as a stress-focussing
susceptorof mechanicalforce
uponmechanosensitive
ion channelsor as a primary sensorthat transducesmechanical force into differential growth of microtubule pl,.:sends. This cytoskeletal
tensegrity sensor is used both to integrate the growth of individual cells with
mechanicalload oftissues and organsand as an intracelluliu sensorusedto control
holistic propertiesof a cell such as organellepositioning.The distinct nonlinearity
of microtubulesin particular rendersthem an ideal tool for self-organizationin
responseto mechanicalinput from the exterior.
P. Nick
Botanical Institute and Center of Functional Nanostructures, Karlsruhe Institute of Technolosv.
Kaiserstr. 2,'7 6128 Karlsruhe, Germany
e-mail: [email protected]
P. Wojtaszek(ed.1,MechanicalIntegrationof Plant Cellsand Plants,
Signalingand Communicationin Planrs9, DOI 10.1007/918-3-642-19091-9*3
Berlin Heidelberg201I
e Springer-Verlag
5,1
P. Nick
Prologue:The SensitiveCytoskeletonor the Hidden
Face of Plant Tensegrity
Even before Ledbetterand Porter (1963) described"microtubules" in plant cells,
which they hadobservedby transmissionelectronmicroscopy.the plantcytoskeleton
was predictedto exist on the basisof biomechanicalconsiderations.
It was Paul
Greenwho concludedfrom their geometrythat growing plantcellsrepafiitiongrowth
by an unknown"reinforcementmechanism"from the spontaneously
preferredlateral
expansionin favour of elongation.and he predictedthat the cell can establishand
maintainthe mechanicalanisotropyof cell walls throughan as-yet-unknownlattice
of tubularelementsthat are orientedin an orderedfashion(Green 1962).Thus.ever
sinceits discovery,the plant cytoskeletonhas beenintimately linked with mechanical aspectsof morphogenesis,and this chapterwill thereforenot follow the usual
approachto describethe plant cytoskeletontiom its molecularbasisand then derive
structuresand functions.It will ratherassumea view of the cytoskeletonthat derives
from its function in mechanicalintegrationof cell and organmorphogenesis.
As a consequence
of their photosynthetic
lif'estyle,plantsincreasetheir surface
by folding outwards,producinga considerabledegreeof mechanicalload. As long
as they remainedaquatic,this load was partiallyrelievedby buoyancy,allowing
considerable
body sizesevenfor fäirly simplearchitectures.
However,when plants
began to move into terrestrial habitats"they had to develop flexible yet robust
mechanicalsupports.The inventionof vasculature-based
modules.the so-called
telontes(Zimmermann1965),was the decisivefactorfbr the evolutionarysuccess
of the cormophyticlandplants.
Mechanicalload shapedplantarchitecture
down to the cellularlevel.Plantcells
areendowedwith a rigid cell wall, and this afl'eclscell divisionand cell expansion
both specilicallyand fundamentally(see also chapter"Micromechanicsof Cell
Walls"). The depositionof a new crosswall will definethe pattemsof mechanical
strain that, during subsequentcell expansion,will guide the complex inteplay
betweenthe expandingprotoplastand the yielding cell wall. It is even possible
to describethe shapeof individual cells in a plant tissueas a manifestationof
minimalmechanicaltension(Thompson1959),emphasizing
the stronginfluenceof
mechanicalload on plantdevelopment.
Whenplantsarechallengedby mechanicalstimuli.theyrespondby changingthe
architecture,
which will allocateload-bearingelements(vesselsand fibreson the
organ level, cellulosemicrofibrilsand lignin incrustationson the cellular level)
guided by the imposedfield of forces.An impressiveexampleis the formation
of tensionand compressionwood (for a recenlreview, see Funada2008). This
architecturalresponseensuresthat mechanicalstrainsare balancedin an optimal
fashionfor minimal investmentof energyand biomatter.Moreover,this mechanical
balanceis continuouslyadjustedto the curent environment- a must in a system
endowedwith open morphogenesis.
where the Bauplon is continually extendedby
additionof newly formedorgans(seealsochapter"MechanicalForceResponses
ol
P l a n tC e l l sa n dP l a n t s " ) .
P. Nick
or the Hidden
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Mechrnics ol lhe C) tO\keleton
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It was only'in the 1920swhen such self-supporting,
flexible structureswere
deliberatelyconstructedby mankind (Robby 1996).Startingfrom the Equilibrist
Studies (consistingof three solid sticks interconnectedby ropes) of the Soviet
artist Karl Joganson,it was mainly the American architect and engineerRichard
BuckminsterFuller (1895-1983)who systematically
adoptedbiologicalprinciples
and developedself-supportivestructuresthat all consistedof a continuousnetwork of tensileelements(which can transmitforcesby pulling) linked to a discontinuoussystemof stiff elements(which can transmitforcesby pushing).lt was also
Buckminster Fulier who later coined the term "tensegrity" as a combination of
"tension"and "integrity".
A few yearslater it becameclear that animal cells are shapedby tensegrit)'(for
reviews see Ingber 2003a, b; chapter"Introduction: Tense-eralWorld of Plants").
The part of the tensile elementsis played by actin microfilaments,which are not
only contractile,but are also mechanicallycomparableto silk fibres (Gittes et al.
1993).The part of the stitT elementsis played by the microtubules,which are
not only hollow cylinders, but are also mechanicallymuch more rigid than actin
filamentsand can be approximated
as very delicateglasslibres(Gitteset al. 1993).
The actinfilamentsareconnectedthroughthe membranewith a suppofiingscaffbld
the extracellularmatrix.
What is the secretthat rendersbiological tensegrityso successful?Biological
tensegrityis not constructeda priori, but emergesa posteriorifrom reorganization
in responseto ever-changing
patterns.To usea term ofJacob (1977).
stress-strain
biological shapeis not producedby intelligentdesignbut ratherfrom bricolctge,and
therefbreit oscillatesaround the stateminimal energy without every reachingil.
Plantcellsareendowedwith a cell wall that is built asa compositestructurewith
elongateload-absorbingelements(cellulosemicrofibers)that are embeddedin an
amorphousmatrix (hemicelluloses,
pectins,proteins).It hasbeenshownfor technical applicationsthat suchcompositematerialsoptimallycombinebendingflexibility with mechanicalstabiiity(Niklas 1992).This meansthatthe tensegrityfunction
fulfilled by the interphase
cytoskeletonin animalcells is replacedby tensegrityof
the cell wail. The plant cytoskeletonis thereforenot directly required to support
cellular architectureand is thereforefree to adopt other functions (Fig. I ).
Cellular architectureusesthe tensegralprinciple to achievemaximal mechanical
stability and, simultaneously,flexibility on the basis of parsimonioususe of
resourcesand load-bearingelements.In addition,it adaptscontinuouslyto the
ever-changing
conditionsof growing and developingcells.This requiresefficient
sensingofforces and strainsfbllowed by appropriatereorganizationofthe tensegral
elements.Thus,the tensegralcytoskeleton
is not only a deviceto providemechanical stability.it mustalsoparticipatein the sensingof stressand strainpatterns.This
mechanicalstimulationf'eedsback to the organizationof the cytoskeletonin sucha
way that a stableminimum of mechanicalenergy is reachedand continuously
adjusted.It is this hidden face of tensegritythat becomesespecially important in
the walled plant cells that are undercontinuousturgor pressureand usethis pressure
fbr regulatedexpansion.The evolutionof the interphasicplant cytoskeletonwas
thereforeshapedby selectivepressurestowardsoptimized sensingand integration
P. Nick
56
Animal cell - architectural tensegrity
b
Plant cell - dual tensegrity
Misotubules:compression
sensory tensegrity
cytoskeleton:
cell wall: architecturaltensegrity
Fig. 1 Functional shift of the cytoskeleton from architectural tensegrity in animal cells towards
sensory tensegrity in plant cells. In animal cells (a), architectureresults from the interplay between
stiff microtubules absorbing compressive forces and flexible actin filaments elastically tethering
the cells through focal adhesionsto the substrate.This set-up is used both to maintain cell shapeand
to senseand to adjust to mechanical force. In plant cells (b), cellulosic microfibers in combination
with a flexible cell wall matrix maintain cell shape,such that the tensegralcytoskeleton is released
from its architectural function and can be optimized for sensory integration ofmechanical stimuli
of mechanicalstimuli. The aim of this review is to give a survey of the role of the
cytoskeletonin mechanicalintegrationof plant cells, tissuesand organs.
2 Cellular Players:The Microtubule-Actin TensegritySystem
The plant cytoskeletonconsistsof two major elements,microtubules and actin
filaments - intermediatefilaments have remained elusive in plants. The spatial
organization of these elements is highly dynamic and changes fundamentally
P. Nick
tectural tensegrity
Mechanics of the Cytoskeleton
51
during the cell cycle, with conspicuouseffects on the predicted stress-strain
patterns.Since microtubules and actin filaments are functionally, but probably
also structurally,interconnected(Wasteneysand Galway 2003; Collings 2008), it
seemsmore appropriateto describe their dynamics as a functional entity. The
molecular players have been extensivelyreviewed elsewhere(for recent reviews
seeHamada2007; Sedbrookand Kaloriti 2008),and thus the focus of this chapreris
on cellular function, ratherthan on molecular composition.
2.1
Cell Expansion
2.1.1 Microtubules
rll - dual tensegrity
. ' c h , t e c t ur a l t e n s e g r i t y
'- -rrrr in animal cells towards
u
t-
..
: . , i l ! ' n t \ e l a s t i c a l l yt e t h e r i n g
. ,.. I
t : . t ( Jn t a i n t a i nc e l l s h a p ea n d
i - . . . ; r r er t r f i b e r si n c o m b i n a t i o n
-.
. . : : . i l i r t o s k e l e t o ni s r e l e a s e d
- -:.rrrt)lo
t l m e c h a n i c a ls t i m u l i
:. . i .: .ur\ e1 of the role of the
- : , . . . - , \u n do r g a n S .
-Actin TensegritySystem
'-..:: -':.:. ::l!rotubules and actin
s d - - - . . , ' r n p l a n t s .T h e s p a t i a l
trrr- .:. : ,h.rnges fundamentally
In interphasecells, microtubulesare organizedin arraysofparallel bundlesperpendicular to the axis of preferentialcell expansion(Fig. 2a). Thesebundlesarethought
to control the direction of cellulosedepositionand thus to reinforce the axiality of
cell growth. Corlical microtubules can change their orientation in responseto
variousexternaland internalstimuli, and this reorientationwill shift the preferential
direction of cellulosedepositionand thus the mechanicalanisotropyof the yielding
cell wall suchthat the proportionalityofcell expansioncan be alteredin responseto
the stimulus(for a recentreview seeNick 2008a).The cell wall in cells that are not
endowedwith tip growth is formed by appositionof celluloseon the inner surfaceof
the cell wall. Cellulose is synthesizedby specializedenzyme complexesthat, in
freeze-fracturepreparations,appearas rosettesof six subunitsof about 25-30-nm
diameterarounda centralpore (Kimura et al. 1999)and are thereforedesignatedas
terminal rosettes.The terminal rosettesare integratedinto the membraneby fusion
of exocytotic vesicles.UDP-glucoseis transportedtowards the central pore and
polymerizedin a B-1,4 configuration.Each subunit of the cellulose synthasewill
producesix cellulosechainsthat will be integratedby hydrogenbonds into a long
and fairly stiff cellulosemicrofibril. Theseenzymecomplexesare thoughtto move
within the fluid membraneand leave a "trace" of crystallizing cellulose behind
them. This movementwill thereforedeterminethe orientationof cellulosemicrofibrils and thus the anisotropyof the cell wall. It is at this point that the microtubules
come into play. ln fact, it was cell-wall anisotropythat led Green (1962) to predict
that microtubulesmust exist even beforethey were actuallydiscoveredmicroscopically by Ledbetterand Porter (1963).
The intimate link between cortical microtubules and the preferential axis of
growth is supportedby the following main arguments(for a review seeNick 2008a)
(l) upon plasmolysis, direct contact between cortical microtubules and newly
formed cellulose microfibrils can be demonstratedby electron microscopy; (2)
when the axis of cell expansion changes in responseto a stimulus or during
development,this is accompaniedby a correspondingswitch in the preferential
axis of cellulosedeposition,precededby a correspondingreorientationof cortical
microtubules; (3) elimination of cortical microtubules by inhibitors produces a
58
a
P. Nick
c
F i g . 2 M o l e c u l a r p l a y e r s o f m e c h a n o i n t e g r a t i o ni n e x p a n d i n g ( a . c ) a n d d i v i d i n g ( b . d ) p l a n t
cells. Microtubules and actin filaments cooperate to fbrm tensegral structures together with other
proteins. (a) Organization of microtubules (M7s) and actin filaments in an expanding cell;
ntolecular details o1'the cell wall-plasma menrbrane*cytoskeleton(WMC) continuum are shou'n
in (c). (b) Organization ofthe microtubular preprophaseband (PP^B),and the actin-based phrag,
mosome in a cell preparing for mitosis; molecular details of the nuclear envelope are shown in (d).
(c) Molecular details of the cell wall-plasma membrane{ytoskeleton continuum. PLD phosphoIipase D anchor for microiubules. CE,Scellulose synthasecomplexes that are pulled by kinesins of
the KIFzI family along microtubules towards the plus-end complexes (+I1P) that are linked via
c r o s s - w i r i n g p r o t e i n s ( C r W i P s ) s u c h a s k i n e s i n s c o n t a i n i n g a c a l p o n i n - h o m o l o g yd o m a i n w i t h
actin, putative plant homologues of integrins (lntH) and mechanosensitive ion channels (MSC).
The functional integrin homologues are linked with the exiracellular matrix (ECM). e.g. arabinogalactan proteins, and cellulose n-ricrofibrils.(d) Molecular details ofthe link berween the nuclear
envelope and the cytoskeleton. Through cross-wiring proteius, a tensegral link between aetin
lilaments and microtubules is generated, whereby microtubules might confer compression forces
between the periphery and the nucleus. whereas the flexible actin filaments transfer tension fbrces.
NPC nuclear pore complex
progressiveloss of orderedcellulosetexture and the axiality of cell expansion,
leading, in extreme cases,to lateral swelling and bulbous growth. The mode of
action of severalherbicide classes,such as the phenyl carbamatesor the dinitroanilines.is basedon the eliminationof cortical microtubulesand the subsequent
inhibitionof elongationgrowth.
The striking parallelismbetweencofiical microtubulesand newly deposited
cellulosemicrofibrilsstimulatedtwo alternativemodels:The original "monorail"
model postulatedthat cofiical microtubulesadjacentto the plasmamembraneguide
the movementof the cellulose-synthesizing
enzymecomplexesandthusgeneratea
patternof microflbrilsthat parallelsthe orientationof microtubules(Heath 1974).
The driving force for the movementof cellulosesynthases
in the monorailmodel
P. Nick
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c and dividing (b. d) plant
. i l u . t u r e s t o g e t h e rw i t h o t h e r
.i:r.'nt\ in an expanding cell;
ri l/C) continuumare showrr
.', . untl the actin-basedphrag. . u c n v e l o p ea r e s h o w n i n ( d ) .
' ,n
e o n t i n u u m .P I D p h o s p h o - - : h , r ta r c p u l l e d b y k i n e s i n so f
' . T l P t t h r t a r e l i n k e dv i a
: ' , : r r n - h o n t o l o g yd o m a i n w i t h
- . ' r \ 1 l i \ e i o n c h a n n e l s( M S C ' ) .
:rirtri\ (ECM), e.g.arabinot.'
. r : . ,, r n k b e t w e e nt h e n u c l e a r
. : r : r l l i n k b c l u e e na c l r n
- , L r n t e rc o m p r e s s i o nl b r c e s
- r r \ l r a n \ f e r t e n s i o nf o r c e s .
,litr of cell expansion.
{ r ( ) \ \ t h .T h e m o d e o f
i..rnr.rtes
or the dinitror. .rnclthe subsequent
rnrl newly deposited
, r r i g r n a l" m o n o r a i l "
.ir;.i rnembraneguide
' unil thus generatea
r i . L r l e (sH e a t h 1 9 7 4 ) .
tir.' nronorail model
Mechanics of the Cytoskeleton
-59
would be an activetransportthroughmicrotubulemotors.Alternatively.the interaction between microtubulesand cellulose synthasescould be more indirect.
whereby the microtubulesact as "guardrails" inducing small fblds of the plasma
membranethat confine the movement of the enzyme complexes (Giddings and
Staehelin199I ). The driving force for the movementwouid be the crystallizationof
cellulose.The solidifyingmicrofibrilwould thuspushthe enzymecomplexthrough
the fluid plasma membrane,and the role of microtubuleswould be limited to
delineatingthe directionof this movement.
The practical discriminationbetweenthesetwo models is far tiom straightforward becauseexperimentalevidencewas mostly basedon electronmicroscopy
observationand thus prone to fixation artefacts.Moreover, great luck was required
cellulosemicrofibrils
to locatethe right section.For instance.the newly synthesized
fbrmed afier treatmentwith taxol were sometimesfound to be directly adjacentto
individualmicrotubules.
fbr instancein tobaccoBY-2 cells(Hasezawaand Nozaki
1999),supportingthe monorail model. On the other hand,the cellulosesynthase
complexeswere observed"in gap" betweenadjacentmicrotubulesin the alga
Closterium(Giddingsand Staehelin1988),favouring the guardrailmodel. The
situation was further complicatedby situationswhere the orientationsof microtubulesand cellulosemicrofibrilsdiff'er(for a review seeBaskin2001;Wasteneys
2004),leadingto a debateon therole of microtubulesin guidingcellulosesynthesis.
This debate stimulateda key experimentexploiting the potential of live-cell
imaging in Arabidopsisthaliana (Paredezet al. 2006).A componentof the terminal
rosette,cellulosesynthasesubunit A6 (CESA6), was expressedas fusion with
yellow fluorescentprotein under the native promoter in the background of a
cesa6-null mutant, such that overexpressionaftefacts could be excluded. The
resultingpunctuatesignal was observedto be localizedadjacentto the plasma
membraneand to move along parallel pathwaysthat resembledcortical microtubules.By crossingthis line into a background.v'hereone of the s-tubulinswas
protein,it becamepossibleto fbllow this
expressed
as lusionwith a blue fluorescent
movementundersimultaneous
visualizationof CESA6andmicrotubuies.
This dual
visualizationdemonstrated
very clearlythat CESA6 was moving along individual
microtubule bundles.Moreover. in a recent publication. a central problem of the
monorail model, i.e. the existenceof polylamellate walls with layers of differing
microfibril orientation, could be plausibly explained by a rotary movement of
groups of microtubules(Chan et al. 2007; for a recent review see Lucas and
Shaw2008).
The original monorail model postulateda microtubule motor that pulls the
cellulose svnthasecomplex along the microtubules.lf this motor were defective.
cellulosemicrofibrilswould deviatefiom the orientationof microtubules.
A screen
for reducedmechanicalresistance
in A. thalianayieldeda seriesof so-calledliagrle
fiber mutants(Burk et aI.2001: Burk and Ye 2002) that were shown to be
completelynormal in tems of cell wall thicknessor cell wall composition,but
were afl-ectedin terms of wall texture.One of thesemutants,.fi'agilefiber 2, allelic
to the mutanthotero(Bichetet al. 2001).was affectedin the microtubule-severing
proteinkatanin.leadingto swollencellsand increasedlateralexpansion.A second
60
P. Nick
mütant,fragilefiber -1,was mutatedinto a kinesin-relatedprotein belongingto the
KlF4 family of microtubule motors (Ftg. 2c). As expected,the array of cortical
microtubules was completely normal; however, the helicoidal arrangementof
cellulose microfibrils was messed up in these mutants. This suggeststhat this
KlF4 motor is involved in guiding cellulose synthesisand might be a component
of the monorail complex. Thus, the original monorail model for microtubule
guidanceof the terminal rosettes(Heath 1974) was rehabilitatedafter more than
three decadesof dispute.
However, the microtubule-microfibril model is still far from complete. ln
addition to occasionally discordant orientations of microtubules, there are cell
wall textures that are difficult to reconcile with a simple monorail model. For
instance. cellulose microfibrils are often observed to be intertwined (Preston
1988).This has stimulatedviews that claim that microtubulesare dispensablefor
the correct texture of microfibrils. The self-organizationof cellulose synthesis
would be sufficient to perpetuatethe pattern becausethe geometricalconstraints
from microfibrils that are alreadylaid down would act as templatesfor the synthesis
of new microfibrils (for a review seeMulder et aL.2004').There are two problems
with this model. First, it ignores that microtubules and microfibrils are actually
parallel in most cases,at least if cells in a tissuecontext are analysed.Second,it
ignoresthat disruption of microtubuleseither by inhibitors (for a review seeNick
2008a,b) or by mutationsthat impair the formation of orderedmicrotubule arrays
(Burk et al. 2001; Bichet et al. 2001 for katanin;Whittington et al. 2001 for mor l) \s
accompaniedby a progressiveloss of orderedcell wall texture and a loss of growth
axiality. Nevertheless,the issue of cellulose self-organizationhighlights that the
original microtubule-microfibril model hasto be extendedby a feedbackcontrol of
microfibrils upon cortical microtubules.
2.1.2 Actin Filaments
Similar to microtubules,actin is organizedinto severaldistinct arraysthat presumably exert different functions.For cells with pronouncedtip growth, suchas pollen
tubesor root hairs, actin functionsas a track for the transpofi of vesicleswith cellwall material that are insertedinto the tip by intussusception(reviewed in Hepler
Mechanicsof Tip Growth")
et al. 2001;chapter"Generatinga CellularProtuberance.
and are probably conveyedby actin polymerization (Gossotand Geitmann200l;
Cärdenaset al. 2008). However, cells growing in a tissuecontext grow by apposition to the stretchedcell wall rather than by intussusception.The role of actin
therefore must be different and is not as obvious as for tip growth. During the
diffuse elongationof tissue cells, longitudinal actin bundlesprevail, especiallyin
vacuolated cells (Parthasarathyet al. 1985; Sonobe and Shibaoka 1989). The
rigidity of thesetransvacuolarstrandsand the degreeof their bundling is regulated
by signalssuch as plant hormones(Grabski and Schindler 1996),kinase cascades
(Grabski et al. 1998) or light (Waller and Nick 1997'1.\naddition to the transvacuolar bundles,a fine network of highly dynamic microfilamentscan be detectedin
P. Nick
rn-:: .,iJli irrr)teinbelonging to the
{. ;'.;.,'.ted. the array of cortical
: . : : . J h . l l e ( ) i d a l a r r a n g e m e n to f
r',-i-:i'.i. Thrs suggests that this
r!h.... .,n!l nticht be a component
:::.'r' ,:.rrLnrodel for microtubule
^r- :rit.rbilitated after more than
:. .. .trll t'al fiom complete. In
. . : :lt!r()lLlbules, there are cell
lh r .i:lpic ntonorail model. For
':-.r-: r,, he intertwined (Preston
I :r:. :,'rubules are dispensablefor
jii-.:./.ilr()n of cellulose synthesis
'.:-.- tlri rcometrical constraints
rl ;. i .:. tlnrplates for the synthesis
. . _ rrr_,. There are two problems
u.i. .,irti tntcrofibrils are actually
. -,,:'.1.'\tale analysed. Second, it
:n:..ill\\r\ (lbr a review seeNick
r..':'.': ,Jrlleredmicrotubule anays
\ i:::.r!t()n et al. 2001 for morl) is
^ .r .:. . i r'\ t ure and a IOSSOf growth
.:-,':i.rnrllrion highlights that the
:r:::r.i.'.i br a f'eedbackcontrol of
< r r : . : . . : t . t l n c l a r T a y st h a t p r e s U m t" r*: , r.- llI rrowth, such as pollen
t:< ::-:: .p1r11
llf veSiCleSwith Cell- \ . - . , , . ; r i r ( ) l tl r e v i e w e d i n H e p l e r
::::.^-: \lrihanics of Tip Growth")
!),.:.. r : ..rrt .rnd Geitmann 2007;
' - : i . . . . r . ( ) n t e x tg r o w b y a p p o s i ni-.....-:|tl()ll. The role Of actin
r-. ":. r : trp growth. During the
: . : : : - . . . . . : . J \ p r e v a i l , e s p e c i a l l yi n
rf,r .-.: .,r .i Shibaoka 1989). The
::.:rr bundling is regulated
F:.i
: : - . : i 9 9 6 t . k i n a s ec a s c a d e s
i'
.+, : . r J . l i t i o nt o t h e t r a n s v a :1r- - ..::]int\ can be detected in
Mechanics of the Cytoskeleton
6l
the corlical cytoplasm of elongating cells often accompanyingcortical microtubules (for a review see Collings 2008). This corlical network can be rendered
visible after pretreatmentwith protein cross-linkers(Sonobeand Shibaoka 1989),
upon very mild fixation (Waller and Nick 1991)or by fusing binding domains of
actin-bindingproteinsto fluorescentproteins(Voigt et al. 2005; Nick et al. 2009).
The transvacuolaractin cableswere suggestedto limit cell expansionby their
rigidity and auxin was thought to stimulate growth by releasing this rigidity
(Grabski and Schindler 1996). This mechanical model for the growrh control
through actin was supported by experimentswhere growth was modulated by
light (Waller and Nick 1997).ln the dark, when cells underwentrapid elongation,
actin was organized into fine strandsthat became bundled in responseto lightinduced inhibition of growth. This actin reorganizationwas rapid and preceded
the changesin growth rate (Waller and Nick 1997).Moreover, this responsewas
confined to the epidermis, the target tissue for the signal control of growth.
However, the mechanicalmodel of actin function was shatteredby experiments
with actin inhibitors. The mechanicalmodel predicted that elimination of actin
cables should releasethe rigidity that limits growth. However, experimentswith
cytochalasinD (Thimann et al. 1992:-Wang and Nick 1998) and latrunculin B
(Baluika et al. 2001) revealedthat even mild elimination of actin inhibited. rather
than promoted, cell elongation.Thus, actin, possibly in combination with directional vesicletransport(Baskin and Bivens 1995),is a positiveregulatorof growth.
Subsequently,two actin populationscould be separatedowing to differencesin
sedimentability(Waller et al. 2002). Whereasthe fine actin filaments correlated
with a cytosolic fraction of actin, actin became progressively trapped on the
endomembranesystemand partitionedinto the microsomalfraction when bundling
was inducedby light (perceivedby phytochrome),by fluctuationsof auxin contenr
or by brefeldin A. This bundling of actin was accompaniedby a reduced auxin
sensitivityof cell elongation.This led to a model where auxin-signallingtriggered
the reorganizationof actin bundlesinto finer filamentsthat more efficiently transported auxin-signalling/transport
componentstowards the cell pole. The debundling in responseto auxin predictedby this model was later demonstratedin intact
rice coleoptilesin vivo usingthe actin-bindingdomainof mousetalin in fusion with
yellow fluorescentprotein first upon transientexpression(Holweg et al. 2004) and
later after stableexpression(Nick et aI.2009).
To understandthe link betweenactin and the auxin responseof growth, excessive actin bundling was inducedby overexpressionof the actin-bindingdomain of
talin in tobaccoBY-2 cells (Maisch and Nick 2007) and in rice plants (Nick et al.
2009). In both systems,the reversion of a normal actin configuration can be
restored by addition of exogenousauxin and this fully restoresthe respective
auxin-dependentfunctions.These findings led to a model of a self-referringregulatory circuit betweenpolar auxin transportand actin organization.Thus, although
actin can stimulate growth by virtue of its mechanicalpropertiesin tip-growing
cells (Gossotand Geitmann2007), within a tissuecontext it does not act through
mechanics,but acts by controlling the proper localization and thus activity of the
signallingmachinerythat regulatescell expansion.
62
P. Nick
2.1.3 Actin-Microtubule Interaction
For cell growth, coordinationand cross talk betweenmicrotubulesand actin fila(for
mentshavebeeninf'erredliom their closecoalignmentand structuralinteraction
ubiquitous
to
be
seem
that
reviewsseeWasleneysandGalway2003;Collings2008)
andhavebeenobservedin differentspeciesandcell types.This conclusionhasbeen
manipulation,where both
supporledby experimentsinvolving pharmacological
cell elongation'otten
reduced
agents
microhlament-and microtubule-eliminating
andBivens 1995:
Baskin
(Arabidopsis:
by a lossof growthanisotropy
accompanied
et al. 2004:
Wang
1998;
et
al.
Gianf
collings et al. 2006; Gramineanseecllings:
proposed
been
even
it
has
Blancaflor2000;cottonfibres:Seagull1990).Recently,
memfrom
microtubules
of
that microtubulereorientationis causedby cletachment
D:
phospholipase
of
contactpoints(controlledby specificisoforms
brane-associated
streaming
of
actin-based
Fig. 2c) followeclby their realignmentwith the direction
(Sainsburyet al. 2008).Despiteextensivestudieson microtubularassociationof
actin filaments,the proteinsthat mediatetheseinteractionshave remainedelusive.
Interactionsbetweenmicrotubulesand actin filamentscould be mediatedeither by
bifunctionalproteinsthat can bind to both cytoskeletalelementsor, alternatively.by
a connectingcomplex of two or more monofunctionalproteinsharbouringa microIn animalandfungalcells.
or an actin-bindingdomain,respectively.
tubule-binding
a numberof proteinshave beenidentiliedthat mediatesuchinteractions.either in a
(Goode
bifunctional way or as complexesconsistingof monofunctionalproteins
suchas
candidates
a
few
et al. 2000;Rodriguezet al. 2003).ln plants,however.only
MAPl90 havebeenproposedso fär (Igarashiet al. 2000)'
In animal cells. the microtubularminus-endmotor dynein is connectedwith and
activateclthrough the clynactincomplex (for a review see Karki and Holzbaur
1999).The dynactin complex is further linked to the microtubule tip component
EBI and thusregulatesthe stabilityof microtubules(fbr a review seeTirnauerand
Bierer 2000).Dyneins as centralelementsof the dynactincomplex are not fbund in
of the lossof flageliatecellsandcentrioles
plants.This is probablythe consequence
(for a review see Schmit and Nick 2008). Plantsmust have evolveda functional
compensationfor the loss of dyneins in the fbrm of other minus-end-directed
motors that are able to interact,either directly or indirectly. with actin filaments.
domain(KCH kinesins)were identiRecently,kinesinswith a calponin-homology
lied as plant-specificsubsetof the kinesin-14fämily (Tamuraet al. 1999;Preuss
et al. 2004; Frey et al. 2009; Xu et al. 2009). In addition to the characteristic
microtubule-bindingkinesin motor domain, these proteinspossessa conserved
domain.well known as an actin-bindingdomainfiom a variety
calponin-homology
proteinssuchas q.-actinin,spectrinand fimbrin. Thus. KCHs are
of actin-associated
for a bifunctional mediationbetweenboth cytoskeletalelements.
strongcandiclates
and several studies confirmed that they can bind, in fact, both elementsof the
cytoskeleton(cotton:Preusset al. 2004: Xu et al. 2009; rice: Frey et al. 2009).
These cross-linkingmicrotubule motors are presentin higher plants, but also in
Pltvslornitrella ytatens(Richardsonet al. 2006; Frey et al. 2009)' Their cellular
function is not really understood,but for one of the cotton KCHs, a role in cell
T_
P. Nick
,\ --'r
, : ( ) t L l b u l e \a n d a c t i n f i l a :..: .. .: .ll.ueturalinteraction (for
- - . . irt,it \eerr to be ubiquitous
r . : . . ' - ' . l h i . c o n c l u s i o nh a s b e e n
, i . - . . . r : r . r n i p u l a t i o nw, h e r e b o t h
r - - r . . - i ' i i . c l l e l o n g a t i o n .o f i e n
. -it
r:: -
Il.r:kinand Bivens 1995:
lei)S: Wang et al. 2004:
ir h.t\ even been proposed
, nrrenrtubulesfiom memr : - .,:,)rrll\ofphospholipaseD:
! . : - ' - . , r , , 1u c t i n - b a s e ds t r e a m i n g
: - " : . : - : .' n . h i r r e r e m a i n e de l u s i v e .
r - - :: . - , L r , ,bi c ' m e d i a t e de i t h e r b y
l:... - . r:)ent\ ol'. alternatively, by
- . : i ' 1 . ' r r lh: r r r b o u r i nag m i c r ( ) : . . - . . l r r i n i n r a la n d f u n g a l c e l l s .
. i . . : r - . . r . h i n t e r a c t i o n se, i t h e r i n a
::.
' . J'
: . . n ! t i ( ) l r a lp r o t e i n s ( G o o d e
: .. \ .r lL-\\candidatesSuchas
-,
. ,
, : . : r n i : c o n n e c t e dw i t h a n d
, :'
, : . ( ) t t l b L l ltei p c o m p o n e n t
.'.
.. ::r ieu see Tirnauer and
:.- . - ' J i n p l e xa r e n o t f o u n d i n
' ..
- - ' . , . r 1 c! 'e l l s a n d c e n t r i o l e s
. ": ... ....' i\olved a functional
:-:irlr nrinus-end-directed
r
. - -r.,.. *ith actin ltlaments.
::
r
.'\i .
t- -
r r l 1 r . i n e s i n sw
) ere identi. . r . r l i .cr t a l . 1 9 9 9 ; P r e u s s
':, I,\ the characterislic
-
. Ir()\\eSSa COnSefVed
- t,'rrrrinfromavariety
'r::l.r'irT
r .h u s .K C H s a r e
' . r 1 ',. l s l s 1 1el l e m e n l s .
L
r ::
- : i . , , t he l e m e n t so f t h e
' -. Fler el aI.2009).
. u t a l s oi n
-irrr plj115b
1lr 191.Their cellular
' K ( l l \ . a r r l l ei n c e l l
t
Mechanics of the Cytoskeleton
63
elongation through cross-linking of microtubules and microfilaments has been
proposed,howeverwithout experimentalevidence(Xu et al. 2009). For the rice
homologueOsKCHl, the phenotypeof insertionmutantsand a KCH overexpressionline generated
in tobaccoBY-2 cells(Freyet aI.2010)suggestthatthis kinesin
phenomstimulatescell elongation,althoughthis stimulationmight be a secondary
enoncausedby changesin the timing of cell division.
2.1.4 The Cell-Wall CvtoskeletalContinuum
A continuum betweenthe cytoskeletonand the extracellularmatrix is central fbr
mechanosensing
in animalcells(1bra review seeGeigerand Bershadsky
2001)and
involves interactionbetweenintegrinsand extracellularmatrix proteinsthat contain
Arg-Gly-Asp (RGD) motifs (for a review seeGiancottiand Ruoslahti 1999).Plants
seemto lack integrin homologues,but there is evidencefor cytoskeletalreorganizationin responseto treatmentwith RGD peptides(Canutet al. 1998;Wang et al.
2007). As a molecular basis for the cytoskeleton-plasmamembrane-cell wall
procontinuumin plants (Fig. 2c), cell-wall-associated
kinases.arabinogalactan
(for a revrew
teins,pectinsand cellulosesynthases
themselves
havebeendiscussed
see Baluika et al. 2003; chapter "lntroduction: TensegralWorld of Plants").
Recently,the rich but circumstantial
evidencefor suchtransmembrane
interactions
was assembledinto a model for a so-calledplasmalemmalreticulum as a third
elementof the plant cytoskeleton(Pickard2008).This plasmalemmalreticulumis
consideredto be a tensile structureand to participatein the control of cellulose
deposition.It can be visualizedas a reticulatestructureby antibodiesraisedagainst
componentsof the mammalian extracellularmatrix and also contains arabinogalactanproteins.The reticulumis reorganizedin the contextof cell elongationin
a mannersimilarto cellulosedepositionand is suqgested
to representa morphological manifestationof lipid rafts.
2.2
Cell Division
2.2.1 Microtubules
ln dividingcells,the ensuingmitosisis heraldedby a displacement
of the nucleusto
thecell centre,wheretheprospective
cell-platewill be fbrmed(fbr a reviewseeNick
2008a).Simultaneously,radial microtubulesemanatefiom the nuclearsudaceand
merge with the cortical cytoskeleton,tetheringthe nucleusto its new position
(Fig. 2b). In the next step,the preprophaseband is organizedby the nucleusas a
broad band of microtubulesaround the cell equator,marking the site where after
completedmitosisthe new cell plate will be formed.In fern protonemata.wherethe
formation of the preprophaseband can be manipulateclby centrifugationof the
nucleusto a new location (Murata and Wada 1991).a causalrelationshipbetween
P. Nick
the preprophaseband and cell-plateorientationwas elegantlydemonstrated'Moreover, in cells where the axis or symmetry of cell division changes,this changeis
always predicted by a correspondinglocalization of the preprophaseband. The
division spindle is always laid down perpendicularto the preprophaseband, with
the spindle equatorlocated in the plane of the preprophaseband' As soon as the
chromosomeshave separated,a new array of microtubules,the phragmoplast'
appears at the site that had already been marked by the preprophaseband' This
microtubularstructureconsistsof a double ring of interdigitatingmicrotubulesthat
increasesin diameter with increasingsize of the cell plate. New microtubulesare
organizedalong the edge of the growing phragmoplast(Vantard et al. 1990).The
phragmoplasttargetsvesicle transportto the peripheryof the expandingcell plate.
Microtubules seem to pull at tubular-vesicularprotrusionsemanating from the
endoplasmicreticulum(Samuelset al. 1995).The guidingfunctionof the preprophase
band is supportedby evidence from Arabidop.sismutants (tonneaulJass),where the
preprophaseband is absentowing to a mutation in a phosphatasePP2A regulatory
subunit(Camilleri et a|.2002).In thesemutants,the orderedpatternof cell divisions
that characterizesthe development of the wild type is replaced by a completely
randomizedpattem of crosswalls (Traaset al. 1995;McClinton and Sung 1991)'It
should be mentioned,however,that during meiosisthe division plane can be controlled in the absenceof a preprophaseband (for a recent review see Brown and
Lemmon 2007),suggestingthat thereexist additionalmechanismsof spatialcontrol.
2.2.2 Actin Filaments
In contrastto the obvious and dramatic reorganizationof the microtubule during
mitosis, actin filaments seem to be more persistent.Two decadesago, actin filamenls were shownto accompanymitotic mic:otubulearrayssuchas preprophase
band, spindle and phragmoplast(Kakimoto and Shibaoka 1987; Lloyd and Traas
1988).However, there is still some controversyas to the exact behaviour,persistence and orientation of actin filamentsduring M phase (for a recent review see
Panteris 2008). The microtubular preprophaseband that disappearswith the breakdown of the nuclear envelopeleavesa so-calledactin-depletedzone as a negative
imprint that later, upon reestablishmentof the daughter nuclei, will be the site
where the new cell plate is formed. Despite some debateregardingto what extent
this zone is completely void of actin or whether it just contains fewer actin
filaments,there is a clear correlationbetweenthe actin-depletedzone and the site
of the prospectivecell plate.When the actin filamentslining this depletionzone are
eliminatedby inhibitor treatment,this will affect the subsequentcell division when
the treatmentoccurs during the presenceof the microtubular preprophaseband'
However. actin inhibitors will have only a marginal effect once the preprophase
band has disappeared(Sanoet al. 2005). What is the actin-dependentfunction that
definesthe cell plate? It might be linked to a belt composedof endosomesthat is
laid down adjacentto the preprophaseband by joint action of microtubule-driven
and actin-driven transport (Dhonukshe et al. 2005). This belt persists during
P. Nick
r :. :.- :.!nti) demonstrated.
More^ - J . . . r { } nc h a n c e st,h i s c h a n g ei s
\\.. : ihr preprophaseband. The
r..: : ' rhj preprophase
band,with
p:.t-:,,rh.r\eband.As soon as the
: : . - : , , t L r b u l etsh. e p h r a g m o p l a s t ,
i.t :. ihr preprophase
band. This
.: :::l::Jrgrtatingmicrotubulesthat
: -.. l.irtc. \ew microtubulesare
n , ' r . . : . i V a n t a r de t a l . 1 9 9 0 ) T
. he
rrn.:\ \)1the expandingcell plate.
' l:,,l:u\l()ns emanatingfrom the
J!r.r:irLIunctionof thepreprophase
:r -:.1:rl\\ t ()tlleauffass),where the
,.. ., ;.ilir:phatasePP2A regulatory
:ht ,,:.irrerlpattemof cell divisions
:\t\i r. rcplacedby a completely
r.r: \1.('linton and Sung 1997).It
hr. ::'..'Jir isionplanecan be conr. r :J.Jltt review seeBrown and
-{:r- ::Jjhanisms of spatialcontrol.
r:1i: ,ir ,,1 the microtubule during
!...: i.',,, clecadesago, actin fila(Ll,..: ,rr-rJ\s SuChaS preprophase
\:..^.,,'x.rl9lt7: Lloyd and Traas
: , I : : . r ' r ' \ u L Ib e h a v i O u rp. e r s i s \ t , . - . 1 ( ) ra r e c e n t r e v i e w s e e
r:L l:..r: .ir.rtppsalt with the break:-:
: - 1 r 1 . ' 1 .Z' io n e a s a n e g a l i v e
. : r - : : ' : r : n L r c l e i .w i l l b e t h e s i t e
F -:- ^..:-' :e-larding to what extent
-,i.
...1 r'onlaifls fewer actin
E j-: : --lrl)lcteclzone and the site
rr::.
: :nL rhis depletion zone are
!.:.: .---','quent cell division when
!:
- - : . . h u l . r rp r e p r o p h a s eb a n d .
E:i-; - r:--. i r)ltce the preprophase
t ::^:i . -: : -Jtpendent function that
:-1 - : :\ ,.:.1 Lrf endosomes that is
s : : : . - - r . , n L r ln t i c r o t u b u l e - d r i v e n
. nr' belt persists during
--\r <
Mechanics of the Cvtoskeleton
mitosis,and is, upon completedseparationof the chromosome,"read out" by a new
set of microtubulesemergingfrom the spindlepolesthat "explore" the cell periphery in different directions. The lifetime of these exploratory microtubules is
increasedwhen they hit the endosomalbelt, whereas microtubules that fail to
interact with the endosomesare prone to undergocatastrophicdecay (Dhonukshe
et al. 2005). Thus, the actin-depletedzone is rather a manifestationof and not the
causefor the correct positioningof the cell plate.
2.2.3 NuclearMigration
In cells that preparefor mitosis, the nucleusis tetheredby the so-calledphragmosome forming the characteristic"Maltese cross" to its new position in the cell
centre, and persists during the subsequentmitosis (Fig. 2b). The cytoskeletal
reorganizations
accompanyingmitosis assigna centralrole to nuclearmigration
in the control of division symmetry,and to the preprophaseband for the control of
division axis. Nuclear migration can be blocked by actin inhibitors such as cytochalasinB (Katsutaand Shibaoka 1988). However, microtubulesalso seem to be
involved in nuclearpositioning,since antimicrotubularcompoundssuch as colchicine (Thomaset al. 1977) and pronamide(Katsutaand Shibaoka 1988)have been
found to loosen the nucleus such that it can be displacedby mild centrifugation.
This indicates again that proteins mediating actin-microtubular interaction are
relevant for nuclear positioning. In fact, the plant-specific KCHs (Frey et al.
2009) were found to be dynamically repartitionedduring the cell cycle: in premitotic cells, KCHI was clearly aligned in a punctuatepattern along filamentous,
mesh-like structureson both sides of the nucleus and on perinuclear filaments
spanningover and surroundingthe nucleus.At the onsetof mitosis,KCH I retracted
to both sides of the nucleus, but not in preprophasebands nor in the spindle
apparatusnor at the division plate. During late telophaseand the beginning of
cytokinesis,KCHI was shifted towards the newly forming nuclei and lined the
filamentsthat tetheredthesenuclei to the peripheryand the new cell wall (Frey et al.
2010). Tosl7 ftcl21knockout in rice showedincreasedcell numbersin the seedling
shoot, whereasoverexpressionof KCHI in tobacco BY-2 cells reducedthe cell
number. This effect was assignedto a delay in the premitotic nuclear migration
towardsthe cell centre,suggestingthat KCH regulatesnuclearpositioningand thus
the progressionof mitosis (Fig. 2d).
3 MolecularPlayers:Protein ConformationVersus
Stretch-ActivatedChannels
Mechanicalintegrationrequiresthe perceptionof stress-strainpatterns.In turgescent plant cells, the expandingprotoplastexertsconsiderableturgor pressureupon
the yielding cell wall. This cell-autonomouscomponent of mechanical load is
6t)
P. Nick
complementedby mechanicaltensionacrossthe tissuethat is causedby the limiting
extensibility of the epidermis(for a recent review seeKutschera2008). Thus, any
has to cope with this strong, but tonic
mechanismfor plant mechanosensing
backgroundstimulus.
Essentially,there are two basic models for the molecularbasis of mechanoperceptlon:
l. Stretchingof proteinswill changetheir conformationand createnew binding
sitesfbr the recruitmento1 associatedproteins(for reviews see Janmeyand
weirz 2004: Orr et al. 2006).
channelsare ableto directly detectand respondto forcesfrom
2. Mechanosensitive
the lipid bilayer. Such channelswill open when the plasma membraneis
deformedor whenthe channelis pulledby a tether(for a reviewseeKung 2005).
Mechanosensingby stretch-inducedconfbrmationalchangesis well supported
for the adhesionof mammalian cells (fbr a review see Geiger and Bershadsky
2001). Here. the growth of focal contacts"where the actin cytoskeletonis tethered
to the extracellularmatrix througha complex of associatedproteinsand integrins,is
promotedby local mechanicalforce (Rivelineet al. 2001).A similarmechanosensing network was proposedfor plant cells, whereanaloguesof integrinslink the
cytoskeletonat the inner face with the cell wall at the outer face of the plasma
membrane(Jaff'eet a|.20021.However"the transferof this model from mammalian
cells to plants is not straightforwardbecausethe molecular componentsdiffer
considerably(fbr a review seeBaluika et al. 2003).lt is the cell wall with a completely difTerentset of moleculesthat replacesthe extracellularmatrix of mammalian cells. Furthermore,canonicalintegrins are obviously absentfrom plants.
suggesting
that the link betweenactin filamentsand the extracellularbindingsites
must usedifferentgroupsof molecules.Further.althoughplantsand animalsshare
severalactin-bindingproteins,importantcomponentsof focal conlacts,such as
talin. do not exist in plants. On the other hand, there seem to exist integrin
analoguesthal can bind to RGD tripeptidesin a way similar to the way integrins
do in animalcells.It seemsthat classVIII myosinsand fbmins mi-ehtact as linkers
betweenthe actin cytoskeletonand the plant analoguesof the extracellularmatrix.
proteins(Baluika and
kinasesand arabinogalactan
such as cell-wall-associated
that move along microtubules
Hlavaöka2005). Additionally.cellulosesynthases
(Paredezet al. 2006)tetherthe cytoskeletonto the cell wall, and severalmicrotuproteins.fbr instancephospholipase
D (Gardineret al. 2001)and
bule-associated
(Wang
microtubulesand the
act
as
linkers
between
corlical
MAPIS
et aI.2007),
plasma membraneThus, although the molecular componentsdiffer considerably
from their animal counterpart,a contiguouslink betweenthe cytoskeletonand the
cell wall doesexist and is commonly ref'enedto as the cell wall plasmamembranecytoskeletoninterface(Telewski2006).
channelsin plantswas originallydiscovered
The existenceof mechanosensitive
in specializedcells,wherea touch stimulusinducedan actionpotential(Shibaoka
leavesof Mimosapudic'a(Toriyamaand Jaffe
1966)such as in the seismonastic
1912),or internodalcells of Charo (Kishimoto 1968).From electrophysiological
P. Nick
'r.
- . r r . r r r hr t1l t h e l i m i t i n g
\..t.ihc'rrr 2008).Thus, any
. . thr. \l|ong. but tonic
, ' , L r i r i rb a s i s o f m e c h a n o -
:r .Lnrlcreate new binding
:.'\ i!'\\ s see Janmey ancl
' .irrrlrc\pond
to forces from
':r. yrl;1;1n1
membraneis
; .i rcr ierv see Kung 2005).
i.::: .: -lr,rnges is well supported
, . : . , . i ' . ' C i e i g e ra n d B e r s h a d s k y
::i ..-:in irtoskeleton is tethered
'. l
I \ l \ ) r c i n a\ n d i n t e g r i n si.s
., ; .\ :imilar mechanosenr-' .: .. '-.Lrc:of integrins link the
. .:i r , ,\uter filce of the plasma
t--:
rr. nr()(lelfrom mammalian
t: J
.' . LrI ur components differ
: I . the ccll wall with a com: - ' , " . . .- . l L r l l rm a t r i x o f m a m m a u '- ,,r.lr absent from plants,
:.: .: ' - ,'.,rr.acellular
binding sites
. - . I r l i l n t \a n d a n i m a l s s h a r e
:.ii
-r- '
l o r i l l c o n t a c t s ,S u c h a S
::
. . , i . r . n lt o e x i s t i n t e g r i n
l .rl lt) the waY integrins
r. .:- . :':trnrmight act as linkers
t
. r r . i \ t r i . l c e l l u l am
r alrix,
r -. - .irr It'()tcilt\ (Baluika and
'ri alongmicrotubules
{\'
h.: - .
.,.,. .rnti several microtu. " : , - , i , . , r ' t l i r l e t ' eatl . 2 0 0 l ) a n d
.,-:
r 'r'
.. :.' .
-.r lric|otubules and the
-':rt' tlift'er considerably
r : t J . \ t o s k e l e t o na n d t h e
.. .t,1 plasma membrane-
: '
si i
r,
+r
. . . , , r ' i g i n a l l yd i s c o v e r e d
- : , r r| ( ) t c n t i a l ( S h i b a o k a
, I Lrr.irama and Jaffe
:
: :icitrophysiological
M e c h a n i ts o f t h e C y t o . k e l e t o n
6l
and phamacological evidence, a model emerged where these touch-sensitive
channelsmediatean influx of calcium (for a review seeJaffeet al. 2002).In lact.
with useof aequorin-transformed
plants,mechanicalstimulationwas demonstrated
to triggercalciuminflux with stimulus-specific
signatures(Knight et al. 199l). A
causativerole of calciumfluxeswas supportedby the isolationof touch-inscnsitive
ArahidopsismutantsafTectedin calmodulingenes(Braam and Davis 1990),and
inhibition of touch responsesby inhibitors of calmodulin (Jonesand Mitchell
1989). A mechanosensitive
calcium channel was demonstratedfbr epidermal
cellsof onion (Ding and Pickard1993).but the molecularidentityof mechanosensitive calcium channelshas remainedelusive. Molecular identificationis also
hamperedby the highly artilicial conditionsrequired to identify stretch-activated
ion fluxesby patch-clamp
techniques.
Removalof the cell wall, isotonicconditions,
and suction by the holding electrodecreate conditions where most ion channels
(Gustinet al. l99l).
would be definedas mechanosensilive
Although both mechanisms,stretch-inducedconformationalchanges and
stretch-activatedion channels, are often discussed separately,they mi-eht. in
fäct. act in concert,as componentsof a so-calledpiasmalemmalreticulum (for a
review seePickard2008).This integrativestructurecomprisesadhesiveeomponents (among others arabinogalactan
proteinsand wall-associated
kinases)that
link the plasma membranewith the cell wall, and are also connectedwith
mechanosensorycalcium channeis. This structure has been demonstratedand
describedfor tobacco BY-2 as a cell biological model system (Gens et al. 2000:
Pickard and Fujiki 2005). The plant integrin analogueshave been reported to
connect microtubules. plasma membrane, actin filaments and stretch-activated
membranechannels(Telewski2006).It is highiy conceivablethat sucha network
could act, on the one hand, as a tensegral entity that can convey and focus
mechanicalfbrce upon stretch-activating
membranechannelsand,simultaneously.
transduceforces into conformationalchangestl'at can result in diff'erentialdecoration with associatedproteins that can act as a trigger for signallin-g.The
necessityfor stress-focussing
is supportedby estimationsof the activation energiesfor mechanosensitive
channels(aroundI mN m l. SachsanclMorris 1998)in
a range not fär below the lytic tension of plant membranes(around 4 mN m I,
Kell and Glaser 1993).
If the tensegralcytoskeletonis linked to the cell wall through such integrarive
linkers,this shouldbecomemanifestas organizinginfluencesof the cell wall upon
the cytoskeleton.This has been observed.For instance,removal o1 the cell wall
rendersmicrotubulescold-sensitive
in tobaccocells (Akashiet al. 1990).When in
the samecellsthe incorporationof UDP-glucoseinto the cell wall was blockedby
the herbicideisoxaben(Fisherand Cyr 1993).this impairedthe axialiry of cell
expansion,resultingin isodiametriccells and disorderedcorticalarraysol'microtubules.Thus. the mechanicalstrainsproducedby cellulosemicrofibrils ali-sn
cortical microtubules,closing a regulatorycircuit betweenthe cell wall and the
cytoskeleton.Since expansionis reinforcedin a direction perpendicularto the
orientation of microtubulesand microfibrils, fbrces will be generatedparallel to
the major strain axis. These forces are then relayed back through the plasma
68
P. Nick
membraneupon cortical microtubulesthat are aligned in relation to these strains.
Since individual microtubules mutually compete for tubulin heterodimers,and
since the number of microfibrils is limited by the quantity of cellulose synthase
rosettes,this regulatory circuit should follow the rules of a reaction*diffusion
system (Turing 1952) and should therefore be capable of self-organizationand
patterning.
How could mechanicalstrainsfrom the cell wall causea correspondingalignment of cortical microtubules?The so-calledmicrotubule plus-end tracking proteins (+TIP proteins)seemto play an importantrole in this context.Theseproteins
associatewith growing plus endsof microtubulesand form complexesthat control
microtubule dynamics, organization and the interaction of microtubules with
membranes,organellesand proteins (for a review see Akhmanova and Steinmetz
2008). EB I, a central componentof this complex, is important in searchingfor socalled exploratorymicrotubulesfor intracellularcapturesitesthat are often marked
by specific actin structures.For instance, such capture sites are laid down by
the preprophaseband and the phragmosomeprior to mitosis and are recognized
by exploratory microtubules emanating from the cell poles during telophase
(Dhonukshe et al. 2005). Because of this function, several members of +TIP
proteins interact with the actin cytoskeleton.Some +TIP proteins,such as adenomatouspolyposiscoli (Moseleyet al. 2007) and CllP-associatedprotein (Tsvetkov
et a|.2007), interactwith actin filamentsdirectly, othersinteractthrough the actinbinding formins. A third group, including EB l, interactwith the dynacrincomplex
linking the minus-endmicrotubulemotor dynein with actin filaments(for a review
seeTimauer and Bierer 2000). EB I binds to microtubuleplus endsat the seamthat
joins the tubulin protofilaments(Sandbladet al. 2006) and is therefore a good
candidatefor a conformationalmechanosensor.During microtubule catastrophe,
the protofilamentsbend outwards,which meansthat they have to be actively tied
togetherto sustainmicrotubulegrowth. The +TIP complex,in general,and EB l, in
particular, are thereforesubjectto mechanicaltension and must be consideredas
primary targets for mechanical strains on microtubules. ln fact, Arabidopsis
mutants in members of the EBI family have been found to be touch-insensitive
(Bisgroveet al. 2008).
Summarizing,in plant cells both stretch-inducedchangesof protein conformation and stretch-activated
ion channelsseemto act in concertduring the perception
of mechanicalstimuli. The cytoskeletoncan participatein both pathways,either as
a stress-focussing
susceptorof mechanicalforce upon mechanosensitive
ion channels or as a primary sensorthat transducesmechanicalforce into differentialgrowth
of microtubule plus ends. Microtubules are endowed with nonlinear dynamics,
leadingto phasetransitionsbetweengrowth and catastrophicshrinkage.ln addition,
they have to compete for a limited pool of free heterodimers.Microtubules are
thereforeideal devicesto amplify the minute inputs from mechanicalstimulation
(small deformationsof the perceptivemembranes,changesin the dynamic equilibrium betweenassemblyand disassemblyof microtubulesat the microtubule plus
end) into clear and nearly qualitative outputs that can then be processedby
downstreamsignalling cascades(for a review seeNick 2008b).
P.Nick
L:::-.J.rrrrrelationto thesestrains.
:r. : ,: rubultn heterodimers,and
u'fc --,.rntll\ clf cellulosesynthase
l.:iii :.,.J. Lrf a reaction-diffusion
.:1.:r.t Lrl self-organization
and
*.1.. ,-,u\c a corresponding
align: i - : : . r h u l ep l u s - e n dt r a c k i n gp r o : ' . : . : .l l l . e o l l t e x tT. h e s ep r o t e i n s
'. ::..i :\)r'nteomplexesthat control
: n l : : . r , l l \ ) l to f m i C r o t u b U l ews i t h
:.r .:: .\khmanovaand Steinmetz
\. :. ::r)porll-rflt
in searchingfor so. j!-:il:. .rtesthatareOftenmarked
-. -.,r:Lrr. sites are laid down by
:,,: :., ntitosisand are recognized
:hr.rll poles during telophase
1 . 1 1 , ' l r\ c. \ e r a l m e m b e r so f + T I P
,:rr * I IP proteins,suchas adeno1'l-I l'-.r.'ociatedprotein(Tsvetkov
. L\tli:s rnteractthroughthe actinn:j:,:- r ri ith the dynactincomplex
r * : i r .. r .r i r rl i l a m e n t s( f o r a r e v i e w
:,'r-ru,r' plusendsat the seamthat
'
1. x ,rr, und is therefore a good
D*: :r: nricrotubulecatastrophe,
:r:r ilij\ haveto be activelytied
P - :r.:..r ',. in general,and EB I , in
e:..: :. .lnrl ntust be consideredas
r,r r-.^:,.i:. ln fäct, Arabidopsis
ct:. : ..nJ ro be touch-insensitive
'-t:
c \ .
rnLd:of proteinConforma: ,:'11Juringthe perception
.:r hothpathways,eitheras
:.:. hanosensitive
ion chan:- i rnto differentialgrowth
,..rth nonlineardynamics,
:-:rr..hrinkage.In addition,
.ilin.r\. Microtubulesare
. : r r e h a n i c aslt i m u l a t i o n
- r r rl h ed l n a m i ce q u i l i b . : t r h c r n i c r o t u b u lpel u s
tlr;rr he processedby
.,rh
Mechanics of the Cytoskeleton
4
69
The Cytoskeleton as a Sensor: Intercellular Sensing
It was the loss of buoyancy as a supporting force that drove the evolution of
mechanicalintegrationin terrestrialplants.Signalling through a mechanicalsignal
is much faster than any diffusion-basedprocessand its velocity equals or even
exceedsthat of electric signalling.Moreover, mechanicalintegrationis holistic in
nature,sinceit allows the sensingalmostsimultaneouslyof the presenceor absence
of building elementseven if they are remote from the sensingcell (as long as they
are mechanicallycoupled).The stiffer this mechanicalcoupling, the lessenergy is
dissipatedduring signalling. Microtubules are endowed with a high degree of
rigidity (Gitteset al. 1993)and thereforerepresentideal transducersfor mechanical
integrationeven acrossthe bordersof individual cells.
Such microtubule orientations that transcendthe borders of individual cells
have been reportedduring phyllotaxis (Hardhamet al. 1980; Hamant et al. 2008).
In wounded pea roots, a supracellularalignment of microtubulesheraldscorresponding changesof cell axis and cell divisions such that the wound is efficiently
closed (Hush et al. 1990). A curious case of microtubule patterning was discovered in the Arahidopsis mutants spiral, lefty and tortifolia (Furutani et al.
2000; Thitamadeeet aI. 2002; Buschmannet al. 2004).In thesemurants,microtubulesare alignedover many cells in the distal elongationzone of the root (spiral
and lefty) or the petiole (tortifolia), accompaniedby twisted growth.
The twisted growth phenotypesof thesemutantsareconventionallyexplainedon
the basis of uniformly oblique arays of microtubules (and consequentlymicrofibrils). In the spiral, lefty and tortifolia mutants, it is either tubulin itself or
microtubule-associated
proteins that are affected by these mutations. Moreover,
spiral growth can be phenocopiedin the wild type by inhibitors of microtubule
assembly(Furutaniet al. 2000). As pointedout earlier,the microtubule-microfibril
circuit is endowed with self-amplificationlinked to mutual inhibition. A typical
systemicproperty of sucha self-organizingmorphogeneticsystemis an oscillating
output (Gierer 1981).Any factorthat altersthe lifetime of microtubuleswill alter
the relay times within this feedbackcircuit. Since neighbouringcells are mechanically coupled by tissue tension, even a weak coupling will result in a partial
synchronizationof the individual circuits (Campanoniet al. 2003), and the degree
of synchronywill dependon the velocity of the feedbackcircuit. Thus, mutationsin
an associatedprotein such as the tortifolia gene product (Buschmannet al. 2004),
mutationsin tubulin itself, as in caseof lefty (Thitamadeeet al. 2002), or treatment
with microtubuleinhibitors (for a review seeHashimotoand Kato 2006)is expected
to enhancesynchrony,leading to the observedoscillationsof growth. In contrast,
the synchronyshould be reducedwhen microtubule lifetimes are increased,which
seemsto be the casefor the mutant radially sw,ollen6 (Banniganet al. 2006), where
microtubulearraysare orderedwithin individual cells,but deviatestronglybetween
neighbouringcells, suggestingthat supracellularalignmentis affected.
The impact of microtubules for mechanointegrationcan be exemplarily
studied in the context of gravity responses(see chapter "Mechanical Aspects of
P. Nick
10
To compensate
Gravity-ControlledGrowth, Developmentand Morphogenesis").
of fbrcethe
arrangement
optimize
plants
have
to
gravity,
load
by
for mechanical
mechanical
provide
optimal
they
that
such
in
a
manner
in
space
bearingelements
consumeminimal biomassand are as light as possible.
support,but simultaneously
only
be achievedwhen the arrangementof supportive
can
This optimization task
pattern
of mechanicalstrain.Thus, gravity has to be
guided
by
the
structuresis
has.
in addition. to be linked to morphogenesis.
it
and
perceiveclvery efficiently
with respectto gravity. the plant will
plant
is
changed
a
of
When the orientation
restore
the original orientationand thus to
way
as
to
a
in
such
respondby bencling
(gravitropism).
When
new organsdevelop,they areofien
stress
minimizemechanical
role
(gravimorphosis).
When
the mechanointegrative
gravity
to
adjustedwith respect
separate
imponant
to
gravity,
it
is
to
respect
with
is
of the cytoskeleton discussed
so-calledsusceptionfrom perceptionin the strict sense."Susception"meanstransformationof physicalenergyinto a differenttype of energythat can be perceivedby
the perceptivesystem(Björkman 1988).For instance,the differencein gravitational
field strengthbetweenthe two flanksof a misorientedplant would be cefiainly far too
small to be sensedby any biochemicalprocess.lt is generallyacceptedthat gravity is
first transfomed into mechanicalforce by acting on heavy panicles.the so-called
statoliths.Thesestatoliths(as well as their accessorystructures)themselvesare not
gravisensitive,but they assistsensingby acting as susceptors.
4.1
Microtubules and Gravitropism
For the rhizoid of Chara, experimentsby JohannesBuder ( 1961) demonstratedthat
vesiclesfilled with barium sulfate, the Glanzkirperchen,are necessaryand suffitheory
For higher plant::,the classicalstarch-statolith
cienf for gravisusception.
perceptive
in
the
postulated
amyloplasts
(Nemec 1900; Haberland 1900)
that
tissues(e.g. root cap or bundle sheathcells) are responsiblefbr the susception
of the gravitropic stimulus. A long tradition of experimentationdemonstrated
that amyloplastsare necessaryfbr efficient gravitropism.For instance,gravitropic
mutants.However, it took almost a
sensitivitywas reducedin starch-deficient
of
centuryuntil it was shown that susception energyby amyloplastsis sufflcient
to trigger a curvatureresponse.By using high-gradientmagneticfields, Kuznetsov
in inducingbendingin verticallyorientedroots
and Hasenstein(1996)succeeded
and thus were able to prove very elegantlythat the generationof mechanicalfbrce
by statolithsis sufficientfor gravisusception.lt is an irony of sciencehistory that
this breakthroughwas not achievedby the elaborateand expensivemicrogravity
experimentsin the contextof spaceresearch.but insteadthrough a very cheap.but
well-designedground experiment.Thus, in higher plants as well. the primary
althoughthe actual
by statolithicparticles(the amyloplasts),
stimulusis procluced
perceptionevent has remainedelusive so far.
For the rhizoid o1 Cltara the sedimentationof lhe Glanzlörperchento the lower
flank ofthe rhizoid was found to divert vesicleflow towardsthe upperside suchthat
p. Nick
\ 1 :'
-. nL'\l:" ). To compensate
. ::rJ ln'angement of force. . . 'r rrlc optimal mechanical
.- .:r!i .ircas light as possible.
: - . . : r ' , r n s e m e notf s u p p o r t i v e
. ; I-hLrs.eravity has to be
' . .nk.,l lo morphogenesis.
: ' - , i r ! \ - u | a v i t y ,t h e p l a n t w i l l
- n.rl olicntation and thus to
: _.rn\ (lc\elop, they are often
-" :trr nrcchanointegrativerole
: . . 111\ mportant to separate
.-' \Ll\ception" means trans' -':i\ thrt can be perceived by
': ,' .lrllcrence gravitational
in
' t .i , 'rrlrlhe cerlainly far too
' ' . 1 , ,r t . r ' e p t e dt h a t g r a v i t l i s
r r.r\ \ pufiicles, the so-cailed
-:.'.1!lLlfcs) themselvesare not
--l\l\)l\.
r r . r 1 t ) 6l ) d e m o n s t r a t e d t h a t
,
.r'e necessary and suffi.. -.r, rturch-statolith theory
1 . 1 . 1 . 1r n. t h e p e r c e p t i v e
r . r h r c I ' o rt h e s u s c e p t i o n
.'. : .:).lrtation demonstrated
f ' r i r r ' l r n c e .g r a v i t r o p i c
i: ,icrer. it took almost a
. . : r \ l , r p l u s l si s s u f f i c i e n t
. : i n e t i c f i e l d s ,K u z n e t s o v
' . : r t i c l l l y o r i e n t e dr o o t s
' ' rrt
n l e c h a n i c a lf o r c e
:-.."
l'ri
-
l\
--
:-'
.
. Lrl \cience history that
. ' r ' i ' t r . i re m i c r o g r a ri t y
.
: , , 1 r - cah v e r y c h e a p ,b u t
'- .1: \\ell.
the primary
. i . : r l t h o u g ht h e a c t u a l
t , t l t t ' t tt o t h e l o w e r
L r p p 6 1 - s i 6s lusc ht h a t
M e c h a n i c ro l t h e C 1t o s k e l e t t ' n
1l
more materialis intussuscepted
into the upperflank. resultingin a growth dif'ferential driving downwardbending(Sieversand Schröter197l). This hypothesiswas
iaterextendedto negativegravitropismby combiningsedimentationwith a dilferent
mode of growth (Hodick 1994).Accordingto this model,the actualperceptionof
gravity would rely upon a proximity mechanism.It is doubtful that proximity is used
for graviperception
in higher plants becauseclassicalstudies(Rawitscher1932)
using intermittentstimulalion showedthat perceptioncan occur in the absenceof
amyloplastsedimentation.Moreover,dose-responsestudiesemploying centrifugaeven fbr
tion have shown that the output (gravitropiccurvature)is dose-dependent
stimuli that completely saturateamyloplastsedimentation.Even for the rhizoid of
Chara, fbr which the proximity mechanism was originally postulated, it was
demonstratedthat strong stimuli that saturatethe sedimentationof the Glan:kir(HenelandFriedrich1973).This suggests
perchencanstill be discriminated
thatthe
actual perceptionof gravity is not basedon proximity, but is basedon the force
exertedby the statolithson a mechanosensor
suchas stretch-activated
ion channels
and/orthe cell wall-plasma membrane-cytoskeletoninterfäce.
If gravity is perceivednot by proximity but by pressure.this poses a big
challengefor the sensingmechanism.Sincegravity is sensedby individualcells
(in contrastto the direction of light in phototropism;Buder 1920;Nick and Furuya
1996),the maximal energyavailablefor stimulationis the potentialenergyof the
sensingcell. This energybarely exceedsthermalnoise if it is not focussedupon
small areas.Theseconsiderations
stimulatedresearchon a potentialrole of microtubulesas amplifiersof gravitropicperception.In fäct. gravitropismcan be blocked
by antimicrotubulardrugs in the rhizoid of Chcn'o(Hertel and Friedrich 1973) as
(Schwuchowet al. 1990:Walker and Sack 1990)or in
well as in mossprotonemata
coleoptilesof maize(Nick et al. 1991) and rice (Godbol6et al. 2000: Gutjahrand
Nick 2006) at concentrationsthat leave the machinery fbr growth and bending
essentiallyuntouched.Conversely,when the dynamicsof microtubulesis reduced
as a consequence
of eithera mutation(Nick et al. 1991'lor treatmentwith taxol,this
results in a strong inhibition of gravitropic responses(Nick et al. 1991 Godbol6
et al. 2000: Gutjahr and Nick 2006). The gravitropically induced reorientationof
cortical microtubuleshas been observedfor both shoot gravitropism(Nick et al.
l99l) and root gravitropism(Blancaflorand Hasenstein1993).In maize coleoptiles, the microtubulesin the epidermal cells of the upper flank of the stimulated
organ assumeda longitudinalorientation.whereasthe microtubulesin the lower
flank remainedtransverse.By microinjectionof fluorescenttubulin into epidermal
cells of intact maize coleoptiles, it was later even possible to demonstratethe
gravitropicmicrotubulereorientationin vivo (Himmelspachet al. 1999). The
time courseof this responsewas consistentwith a model wheregravitropicstimulation induceda lateralshifi of auxin transporttowardsthe lower or-{anflank and,as
a consequence,
a depletionof auxin in the upperflank.The microtubularresponse
was thought to be primarily by this decreasein auxin concentrationrather than by
gravity itself. In maize roots,however,where a similar reorientationwas observed
in the cortex(BlancaflorandHasenstein| 993),the time courseof reorientation
was
found to be slowerthan the changesin erowthrate inducedbv gravitv.
i)
p. Nick
This leadsto the questionof whetherthe gravitropic
responseof microtubulesis
,.
direct or whethermicrotubulesmerely ."rpo-nd
to changesin growth rate.rn fact, it
is possibleto induce microtubulereorientation
by bendingcoi.optit", *ith manual
force (Zandomeniand Schopfer r9g4) - the
microtubuleswill then becomelongi_
tudinal in the concaveflank, but remain transverse
in the convex flank. To dissect
the gravitropic responseand a potentialresponse
to changedgrowth rate, microtu_
bule behaviour was followed in coleoptiles
that were]..änr"J
U], a surgicat
adhesivefrom elongating and were kept
either in a horizontal orientation (such
that a gravitropic stimulation occurreä) or
in a vertical orientation (such that
growth was inhibited in the absence
of a gravitropic stimulus). In this set-up,
microtubule reorientation from transverse
tJ longitudinal was obse.ved only in
the horizontalorientation(Himmerspachand
Nick 2001), demons,*iing unequivocally that microtubules,at least in this system,
respondedto gravity rather than to
t h e i n h i b i r i o no f g r o w r h .
4.2
Microtubules and Gravimorphosis
It seemstrivial that roots form at the lower
pole of a plant organ, but this is a
manifestationof gravimorphosis.Although
a considerabi.umouit of phenomeno_
logical work was dedicatedto this probi-em
at the tum of the nineteenthcentury
(vöchting 1878;Sachslgg0; Goebei
r90g), the underlyingmechanismsare still
fär
from being understood.This is partially'due
to the use of adult organs, where
polarity has arreadybeenfixed and is
haid to invert. However, in germinatingfem
spores' the first asymmetric,division that
separatesa larger, vacuoratedrhizoid
precursorfrom a r-"11:1.u1gdenser tha'us
precursorcan be oriented by graviry
(Edwardsand Roux 1994).This lirst
cell division is clearly of fomative character;
when it is rendered symmetric by treatment
with antimicrotubular herbicides
(vogelmannet al. 1981),the two
daughtercells both give rise to thalloid tissue.
when this sporeis tilted after the axis Jf
th. firrt divisiÄ has beenderermined,the
rhizoid will grow in the wrong direction
and cannot adjust for this error (Edwards
and Roux 1994).prior to division, at the
time when the spore is lornpetent to the
influence of gravity, a vivid migration of
the nucleus towards the lower
ltt.t:ilghalf of the sporeis observed.This movem"ent
is not a simple sedimentationprocess
becauseit is oscillatory and interrupted
by short periods of active sign reversal,
indicating that the nucleusis tetheredto
a Äotive force (Edwardsand Roux r997).
The action of antimicrotubular compounds
strongly suggeststhat this guiding
mechanismis basedon microtubulesthat probabty
aügn-,irrr, tne gravity vector,
resemblingthe determinationof the grey
crescentin amphibian
.t ut.
1981), where the dorsoventralaxis is determined
"ggiC".nurt
by an interplay
among gravltydependentsedimentationof yolk particles,
spem-induced nucleation of micro_
tubules and self-amprifying alignment
of ne*ry formed microtubules that drive
cortical rotation (Elinson and Rowning lggg).
P. Nick
1 rl: :'. - - j:pfrilS€ of microtubules is
r,, - i..:t.rJr ln growth rate. In fact, it
t r r;;1.1,',. colr-optileswith manual
r-: ':..r...c\ rrill then becomelongi.:\' :. tilr' c()nvex flank. To dissect
< ' : - r . . r r r j c tsl r o w t h r a t e . m i c r o t u i:: .1j:t pre\ented by a surgical
: ::'. -: it')rirontal orientation (such
i : .:rlrctl orientation (such that
:!.::,.i.
\ttnlulus). In this Set-up,
l . ' r : . : . r J r n u i w a s o b s e r v e do n l y i n
-r I
l'.,lemonstratingunequivo:.\l'. \n!l.rl to -eravity rather than to
:s
r'.:' ,: .i plant organ, but this is a
rn.r.1i:.rblr-amount of phenomeno::.j :-.:'n o1 the nineteenth century
LnltJ:.\ iltg mechanismsare still far
l,' ::lr u\c o1 adult organs, where
r :::i llr rr\ c\ er. in germinating fern
i:r:. .r lrtrser. vacuolated rhizoid
,'- -:. ,: .,1n be oriented by gravity
r :. -.-..:;.1\ttf fbrmative character;
- ::: .:nrinticrotubular herbicides
. :\ :: ir\c'rise to thalloid tissue.
I . : . , . . . , : r h a : b e e n d e t e r m i n e d ,t h e
ßi, i .:.r .r.r tbr this error (Edwards
r:..3: ::.J .f ()re is competent to the
' : : - i : ' . u i l e u st o w a r d s t h e l o w e r
$.r: -: ..:--.rrla:cclimentationprocess
r: t.i: ,.1. ()t tctive sign reversal,
< : :, J [:.lri rLrdsand Roux 1997).
:.r.- . ..r-jL.:rs that this guiding
1 1 . . .. : t r . . \ l l h t h e g r a v i t y v e c l o r .
s -i .: .::rl.r.itreggs (Gerhart et al.
.r: - ,
.: rr.rpl.namong gravity:::.
: . _ - : - i n u c l e a t i o no f m i c r o r : - - - - . : : l l ! r ( ) t u b u l e st h a t d r i v e
tt
I
Mechanics of the Cytoskeleton
4.3
13
Microtubules and the Sensing of Gravity
Since microtubulesguide the anisotropicdepositionof cellulose in the cell wall,
it is not trivial to discriminatetheir function in gravity sensingfrom their role in
the responseto gravity during the development of tropistic curvature. When
gravitropic bending is inhibited by antimicrotubularagents,this might be caused
by a block of either the sensoryfunction or the effectorfunction of microtubules.To
discernthesemicrotubularfunctions,the lateral transportof auxin can be usedas a
responseupstreamof differential growth. With use of radioactivelylabelledauxin
in rice coleoptiles(Godbol6 et al. 2000), lateral auxin transportwas found to be
blocked by ethyl-1V-phenylcarbamate,
a herbicidethat binds to the carboxy terminus of ü-tubulin and inhibits assemblyof tubulin heterodimersto the growing ends
of microtubules(Wiesler etal.2002).Interestingly,Taxol inhibited lateraltransport
partially without any inhibition of longitudinal transportof auxin. This indicates
that sensorymicrotubuleshaveto be not only present,but, in addition,also dynamic
to fulfil their function. The high dynamicsof this sensorymicrotubule population
might also explain the extreme sensitivityof gravisensingto low temperaturethat
would be difficult to explain otherwise(Taylor and Leopold 1992).The necessityof
high microtubular turnover favours a model where microtubules sensegravityinducedforcesactively ratherthan merely acting as gravisusceptors.
The gravisensory function of microtubulescan be specificallyblocked by acrylamide (Gutjahr
and Nick 2006), a widely used inhibitor of intermediate-filamentfunction in
mammalian cells (Eckert and Yeagle 1988). Similar to ethyl-N-phenylcarbamate,
acrylamide interrupts a very early step in the gravitropic responsechain, clearly
upstreamof auxin redistributionand differential growth. No clear homologuesof
intermediate-filamentproteins are known in the plant kingdom, but acrylamide
treatmentspecificallydisruptsmicrotubules,leaving actin filaments,for instance,
untouched(Gutjahr and Nick 2006). The immediatetarget of acrylamidein mammalian cells seemsto be a kinase that phosphorylateskeratin (Eckert and Yeagle
1988).Since kinasesand phosphatases
have been shown to regulatethe organization of plant microtubules(Baskinand Wilson 1997),the inhibition of gravitropism
by acrylamidemight be causedby interferencewith the regulatorycircuits activein
the highly dynamic microtubule populationresponsiblefor gravisensing.
4.4
Microtubules and Mechanosensing
Gravity is not the only source of mechanicalstimulation used to integrateplant
architecture.In contrast to terrestrial animals, the cells of land plants are not
surroundedby an isotonic medium, but are surounded by a hypotonic medium,
with the consequencethat their plasma membrane is under continuous tension
from the expanding cytoplasm that is counterbalancedby the cell wall. On
the level of organs, considerabletissue tensions develop that can be used for
a1
P. Nick
when,fbr instance,new organsemergeand will thusgenerate
mechanointegration
local tension.This phenomenonhas been inlensivelystudiedand modelledfor
phyllotaxisby PaulGreenand co-workers.who showedthat modelsof stress-strain
patternscouldperfectlypredictthe positionsof incipientprimordia(fbr a revieu'see
Green 1980).In the growing meristem,the formation of new primordia is suppressedby the olderprimordia.The tissuetensionpresentin an expandingmeri\tem
producesmechanicalstresses
resultingfiom bucklingof the precedingprimordia.
One of the earliesteventsof primordial initiation is a reorientationof cortical
microtubulesthat are perpendicular
with respectto the microtubulesof their noncommittedneighbours.This differenceis sharp.but later it is smoothedby a
transitionalzoneof cellswith obliquemicrotubules,
suchthat eventuallya gradual.
progressive
chan-ee
in microtubularreorientation
emergesover severalrows of cells
(Hardhamet al. 1980).This phenomenon
hasbeenrevisitedrecentlymaking useof
microtubulemarkerlineslabelledwith greenfluorescent
proteinin the developing
shootapexof A. tlnliana (Hamantet al. 2008).wherea feedbackbetweenmicrotubule orientationand organgrowth was demonstrated.
Mechanicalmodellingof the
expandingshootmeristempredictedthe transcellular
patternof rnicrotubuleorientation that was predictedanclobservedto be alignedwith the directionsof maximal
slress.By ablationof specificcells in the outermeristemlayer of the meristem,a
redistributionof stresswas inducedand modellecl.As expected,this redistribution
causeda conespondingredistributionof microtubularorientation.
4.5
Candidatesfor the Underlying Mechanism
The sensingof gravity relies on the mechanicalforcessusceptedby the amyloplasts.This would, at first sight, suggestthat the sensingof gravity and the
sensingof mechanicalstimuli shouldrun in parallel.This can be testedby antagonisticapplicationof arlificialbendingstressin antagonism
to a gravitropicstimulus:
it is possibleto separatethe responseof gravity fiom the secondarymechanical
stimulus that is induced by the diff'erential growth during gravitropic bending
(lkushima and Shimmen 2005). When, under these conditions,the activity of
mechanosensitive
channelswas suppressed
by gadoliniumin hypccotylsof adzuki
beans,this suppressed
the (mechanicallyinduced)reorientation
of microtubulesin
the effectortissue,whereasgravitropiccurvatureproceededunaltered(indicating
that the microtubulepopulationresidentin the inner tissuesof the apical hook that
is responsiblefor gravisensing
remainedfunctional).Thus. at leastin this system,
mechanosensing
is sensitiveto gadolinium,whereasgravisensing
is not. Similarly.
when the gravitropicbending of maize coleoptileswas inhibited by a surgical
adhesive,
the gravity-induced
reorientation
of microtubuleswasnevefiheless
developed (Himmelspachand Nick 2001).suggestingdiff-erentsignalchainsfbr gravisensingand mechanosensing.
Althoughthe responses
of plant cellsto gravityand
mechanicalstimuli are generallydiscussedin the contextof stretch-activated
ion
channels(Ding and Pickard 1993),the proteinconformationparadigmof sensing
r
I
P. Nick
:i: . -. -':-r' und will thus generate
.iLr.lrctlancl modelled fbr
. : . . - . : t : , . r 1l l i r ) d e l so f s t r e s s - S t r a i n
- :
:-' . :
: -.
.t.
.
: l n l r , l ( l t i(rl b r a r e v i e ws e e
,,: nc\\ primordia is sup: r ' r. r nc \ p a n d i n gm e r i s l e m
: rhr preceding primordia.
-i
'..: .rr.'r'
it is smoothed by a
- ' . . . - : t l h l t e v e n t u a l l ya g r a d u a l ,
-": -. _-.. rr\ cr SeV€fäl rows of cells
- - : .: . . l J r l f c c e n t l ym a k i n g u s e o f
:- . - -. .1 lrr'()teinin the developing
. . . l h r r c kb e l w e e nm i c r o l u - : : : : \ 1 . ' . h u n i c a lm o d e l l i n g o f t h e
. -. .. :-.,11.'t'n
of rnicrotubule orienr,' :
tlt rhe directions of maximal
:' . '
. l . r rc r o f t h e m e r i s t e m ,a
.: i- -.\ |e cted. this redistribution
^ . . . . , :l i n l a t i o n .
'c hani snt
- . . u \ e c p t e db y t h e a m y l o .-:r.in! of gravity and the
. .iin Lretestedby antago. l\Jit sravitropicstimulus;
' :.-' .cr'ondary
mechanical
:..: n.r gravitropicbending
. , : r ( i r l i ( ) n tsh. e a c t i v i t yo f
rn hr pocotylsof adzuki
' . r r,' n , ' l 'n r i c r o l u b u l e s
in
. :-'.1LrnrLlrered
(indicating
. - . r r 1t h c a p i c a lh o o kt h a t
. . , 1l t u s [ i n t h i s s y s t e m .
r . r r i i . n o t .S i m i l u r l l .
. r h r h i r e cbl y a s u r g i c a l
. - . .. i . n . \ e r t h e l e sdse v e l ' -n,rl chlins for grav' .,'r.tll: to gravity
and
I : : ' 1l .l - u c t i v a t ei o
dn
: .,t.r,li!n
o tf s e n s i n g
M e c h a n i c so f t h e C y t o s k e l e t o n
75
should not be neglected.This is emphasizedby the phenotype ol Arabiclopsis
mutants,wherethe microtubuleplus-endprotein EBI is aff'ected(Bisgroveet al.
2008).ln thesemutants,both the initiationof gravitropiccurvatureand a speciltc
touch-inducedwaving of roots on inclined agar plateswere affected.Although our
knowled-ee
of the primary eventsof mechanosensing
and gravisensing
in plantsis
extremelylimited.it is clearevenat this stagethal the rolesof microtubulesmight
ditfer qualitatively.[n mechanosensin_e,
microtubulesseem to act as susceptor
structuresthat focus defbrmation stress towards ion channels. In contrast. in
gravisensing.
the necessityfbr high dynamicsand dimer turnoverfavoursa direct
sensoryrole of microtubules.
Thus,naturemight utilize both mechanisms
simultaneouslyto sense(andpossiblyto discriminate)differentstimuli.The challengefor
future researchin this field will be to design experimentalapproacheswith clear
outputsbasedon clear conceptsof the sensingmechanism.Only in a secondstep
will it becone possibleto defineand testmolecularand cellularcandidates.
5
The Cytoskeletonas a Sensor:Intracellular Sensing
The previoussectiondealtwith the mechanicalintegrationof individualcellsinto a
coordinatedresponseof the entire organ. However, mechanosensingis also used
to integratethe diff-erentcomponentsof a cell into an individual.This becomes
evident as redistributionof organellesin responseto mechanicalstimulation(thigmomorphogenesis;chapter "Mechanical Force Responsesof Plant Cells and
Plants"),but also involvesresponses
that are lessevident.such as the adaptation
(chapter"Osmosensing")
to cold, the inductionof plant defence.osmoregulation
and the regulationof division symmetry.The fundamentalrole of intracellular
mechanosensinghas emergedin recent years.but its full impact is still far fiom
beingrecognized.
5.1
Thigmomorphogenesis
Morphologicalresponses
to mechanicalstimulationhave been demonstrated
not
only on the supracellularlevel. but also on the sub-cellularlevel. When I'ern
protonemataare squeezedby a needle,chloroplastsavoid the site of contact (Sato
et al. 1999).In epidermalcells(Kennardand Cleary 1997)or in suspension
cellsof
parsley(Gus-Mayeret al. 1998)sucha localpressure
can inducenuclearmovement
towardsthe contactpoint. When regeneratingprotoplastsor cells are challengedby
eithermild centrifugation
or touch.the axisof cell divisionis alignedwith the lbrce
vector(Lintilhacand Vesecky1984:Wymer et al. 1996:Zhot et al. 2007).Using
tension-freeprotoplasts.Wymer et al. (1996) aligned microtubulesby a shorl
centrifugationand thus orientedthe axis of cell expansionin a direction perpendicularto the forcevector.They usedthis systemto demonstrate
a role of microtubules
P. Nick
76
To separatethis sensoryrole from the microtubularfunction in
in mechanosensing.
cell-wall synthesis,microtubuleswere eliminatedtransientlyduring the application
of force by amiprophos-methyland allowed to recover by washing out the herbicide, suchthat cellulosesynthesiscould occur and thus the cell axis could develop.
When this transient microtubule elimination was performed either immediately
before or immediately after the centrifugation,the alignment of cell division was
not observed.ln a control, microtubuleswere eliminated subsequentto the centrifugation, which did not impair the alignment of the cell axis by the mechanical
stimulus.This demonstratedclearly that microtubulesare necessaryfor the sensing
walled cells of
of this mechanicalstimulus. A recent study in agarose-embedded
Chrl-santhemum(Zhou et al. 2001) extendsthese findings by the interactionwith
the cell-wall cytoplasmiccontinuum.Here,cell divisionswere alignedby compression force (Lintilhac and Vesecky 1984). When microtubules were removed by
oryzalin prior to the treatmentor when the cell-wall cytoplasmic continuum was
impaired by treatmentwith RGD peptides,this alignmentresponsewas interrupted.
ln contrast.elimination of actin filamentsby cytochalasinB was not effective.
5.2
Cold Sensing
The sensing of temperature must occur cell-autonomously.This is generally
ascribed to a reduced fluidity of membranesthat will alter the activity of ion
channelsor the balanceof metabolites(Lyons 1973).For instance,overexpression
has been shown repeatedlyto modify chilling sensitivity in plants
of desaturases
(Murata et al. 1992). Since microtubulesdisassemblein the cold, they have long
been discussedas alternativesensorsfor low temperatures.ln fact, when microtubuleswere manipulatedpharmacologically,this was accompaniedby changesin
cold hardiness(Kerr and Carter 1990). Microtubule disassemblyof plants and of
animalsin the cold differ dependingon the type of organism.Whereasmammalian
microtubules disassembleat temperaturesbelow 20"C, the microtubules from
poikilothermic animals remain intact far below that temperature(Modig et al.
1994).ln plants, the cold stability of microtubulesis generally more pronounced
than in animals, reflecting the higher developmentalplasticity. However, the
critical temperaturewheremicrotubuledisassemblyoccursvariesbetweendifferent
plant species,which is correlatedwith differencesin chilling sensitivity(Jian et al.
1989). The close correlation between microtubular cold sensitivity and general
chilling sensitivity is supportedby the observationthat abscisicacid, a hormonal
inducer of cold hardiness(Holubowicz and Boe 1969; lrving 1969; Rikin et al.
1975; Rikin and Richmond 1916),can stabilize cortical microtubulesagainstlow
temperature (Sakiyama and Shibaoka 1990; Wang and Nick 2001). Tobacco
mutants, where microtubules are more cold-stable owing to expressionof an
activation tag, are endowedwith cold-resistantleaf expansion(Ahad et al. 2003).
Conversely,destabilizationof microtubulesby assemblyblockers such as colchicine and podophyllotoxinincreasedthe chilling sensitivityofcotton seedlings,and
t
I
P. Nick
. :: : . li"i nlcrotubularfunctionin
:.: ::.:: .i,'nrl\ duringthe application
:i- ,:: hr *ashing out the herbi,:rr if .... rhc ccll axis could develop.
s j\ -\r'''\rrnredeither immediately
: i : . , . : * n r r r conlt' c e l ld i v i s i o nw a s
r.i:'.:r.:lJrl\ubsequentto the centri,': :::: .tll axis by the mechanical
for the sensing
ua-.r. .:r. necessary
i i . : : , , . i - e n r b e d d ew
d a l l e dc e l l so f
:.. :rr!irngsby the interactionwith
j,,, .;1r11r
*ere alignedby compres:r'.r.lr)tubules
were removedby
.n
was
[ . - . - . , .. r t o p l a s m i c o n t i n u u m
was
interrupted.
:. : :::::r:nt fesponse
, : , \ : ' . . r . . r \ iB
n w a sn o t e f f e c t i v e .
[-r-: 'n()nror.lsly.
This is generally
r.rr-:l:rrll alter the activity of ion
. - - I I - ( , ri n s t a n c eo.v e r e x p r e s s i o n
r'..r:: . .hilling sensitivityin plants
{':.^.r rn the cold, they have long
rs:.:-L:.rture
s. ln fact, when micro.
.
.
,
.
Lr.
. r . eo m p a n i e db y c h a n g e isn
-^.. -. .ir.r\\ernblyof plantsand of
-' : : -.1ili\m.Whereasmammalian
." - ('. the microtubules from
r'niperature (Modig et al.
- :rne rally more pronounced
rf:-- -.: r.: plir\ticity. However, the
ria... - - !ri\ \ aries between different
- : . . i r n g s e n s i t i v i t y( J i a n e t a l .
!s.
-5-.-:r -,.J 'ensitivity and general
\-..-
il: : ::'..r:.rl,scisicacid, a hormonal
i.-lc " ' lrring 1969; Rikin et al.
- - . : : I r . r ( ) t u b u l easg a i n slto w
- . , : ' . 1\ i c k 2 0 0 1 ) . T o b a c c o
\f, .: r
1..3i:
.
i \ \ :
. . l n ! t o e x p r e s s i o no f a n
' : - . , n \ r ( ) rtlA h a d e t a l . 2 0 0 3 ) .
^
.. hirrckers such as colchi. : . L r r. o t t o n s e e d l i n g s a
, nd
I
Mechanics of the Cvtoskeleton
this effect could be rescued by addition of abscisic acid (Rikin et al. 1980).
Conversely,gibberellin,a hormonethat hasbeenshownin severalspeciesto reduce
cold hardiness(Rikin et al. l9l5; lrving and Lanphear 1968), renders corrical
microtubulesmore cold-susceptible(Akashi and Shibaoka1987).
It is possibleto increasethe cold resistanceof an otherwise chilling-sensitive
speciesby pre-cultivationat moderatelycool temperature.Cold hardeningcan be
detectedon the level of microtubules as well. Microtubules of cold-acclimated
spinachmesophyllcells copedbetterwith the consequences
of a freeze-thawcycle
(Bartolo and carter 1991a).Microtubulesare not only the targetof cold stress,they
seem,in addition, to participatein cold sensingitself, triggering a chain of events
that culminates in increasedcold hardiness.when microtubule disassemblyis
suppressedby Taxol, this can suppresscold hardening (Ken and Carter 1990;
Bartolo and Carter 1991b).This indicatesthat microtubuleshave to disassemble
to a certaindegreeto trigger cold hardening.To test this hypothesis,cold hardening
was followed in three cultivars of winter wheat that differed in freezing tolerance
(Abdrakhamanovaet al. 2003). During cultivation at 4"C, the growth rate of roots
recoveredprogressivelyas a manifestationof cold hardening.In parallel,the roots
acquiredprogressiveresistanceto a challengingfreezingshock(-7'C) which would
impair growth irreversiblyin non-acclimatedroots. when microtubuleswere monitored during cold hardening, a rapid, but transient partial disassembly was
observedin cultivars that were freezing-tolerant,but not in a cultivar that was
freezing-sensitive.However, when a transientdisassemblywas artificially generated by a pulse treatment with the antimicrotubularherbicide pronamide in the
sensitivecultivar, this induced freezing tolerance.This demonstratesthat a transient,prutial disassemblyof microtubulesis necessaryand sufficientto trigger cold
hardening,suggestingthat microtubulesact as "thermometers".
Similar to mechanosensingand gravisensing,this leads to the question of
whether microtubulesact as susceptorsor as true receptorsfor low temperature.
The primary signal for cold perceptionis thought be increasedmembranerigidity
(Los and Murata 2004; Sangwanet aI.2001). For insrance,the input of low
temperaturecan be mimicked by chemical compoundsthat increaserigidity, such
as dimethyl sulfoxide, whereasbenzyl alcohol, a compound that increasesmembrane fluidity, can block cold signalling (Sangwan et al. 2001). With use of
aequorinas a reporterin transgenicplants,rapid and transientincreasesof intracellular calcium levels in responseto a cold shock were demonstratedby monitoring
changesof bioluminescence(Knight et al. 1991). Pharmacologicaldata (Monroy
et al. 1993) confirmed that this calcium peak is nor only a by-product of the cold
response,but is also necessaryto trigger cold acclimation.This peak is generated
throughcalcium channelsin conjunctionwith calmodulin.Calcium and calmodulin
in tum are intimately linked to microtubule dynamics.Immunocytochemicaldata
show that microtubulesare decoratedwith calmodulindependingon the concentration of calcium (Fisherand Cyr 1993).Ir was further suggestedrharthe dynamicsof
microtubulesis regulatedvia a calmodulin-sensitiveinteractionbetweenmicrotubules and microtubule-associated
proteins such as the bundling protein EF-lr
(Durso and cyr 1994).However,the interactioncould be evenmore direct.because
78
P. Nick
cleavageof the carboxy terminusof maize tubulin was shown to renclermicrotubulesresistantto both low temperature
and calcium(Bokroset al. 1996).When
the releaseo1'calciumfiom intracellularpools was blocked by treatmentwith
lithium, an inhibitor of polyphosphoinositide
turnover,this resultedin increasecl
cold stabilityof microtubulesin spinachmesophyll(BartoloandCarter1992).wirh
use of a cold-responsive
reportersystemit was demonstrated
that disassembly
of
microtubulesby oryzalin or treatmentwith the calcium ionophoreA23 187 could
mimic the ef'tectof low temperature,whereasthe calcium channelinhibitor gadolinium or suppression
of microtubuledisassembly
by taxolpreventedthe activation
of this promoterby low temperature(Sangwanet al.200l). Thesedata fävour a
model wheremicrotubulesact as receptorsthat limit the permeabilityof calcium
channelsthat are triggered by membranerigidification. when microtubulesfunction as modulatorsof calcium channelactivity and when microtubuleintegrityis
regulatedthroughcalcium/calmodulin,
this would set-upa regulatorycircuitcapable of self-amplilication:
Stablemicrotubulesthat limit the activityof cold-inducecl
voltage-dependent
calcium channelswould. upon disassernbly,
releasethis constraintand this would elevatethe activityof the channels.resultingin an increased
influx of caicium.This calciuminflux,in tum, would resultin furtherclisintegration
of the microtubularcytoskeletonand thus trigger by this positive-f'eedback
the
influx of additionalcalcium. A very small initial calcium influx might thus be
amplifiedinto a strong signal that can be easily processedby the activationof
calcium-dependent
signallingcascades.
The resultingsignallingcascadewill activate colclhardeningas an adaptiveresponseto cold stress.Interestingly.microtubules will be renderedcold-stableas a consequence
of this coid hardening
(Pihakaski-Maunsbach
and Puhakainen1995; Abdrakhamanovaer al. 2003),
which. in turn, should result in reducedactivity of the calcium channelsthat
respondto membranerigidification.Thus, microtubuleswould not only enclow
cold sensingwith high sensitivity,but, in acdition.would also endow it with the
ability to downregulatesensitivityupon prolongedstimulation.a key requirement
lbr any biological sensoryprocess.
5.3 Plant Defence
The interactionof pathogensand their hostsis subjectto an evolutionaryarmsrace.
wherethe pathogensdevelopvariousstrategies
to circumventor suppress
det'ence
responsesof the host, whereasthe host developsvarious strategiesto senseand
attack the invading pathogenor its effector molecules.Cellular responsesto
elicitorsincludeformationol cell-wall papillaearoundsitesof pathogenpenetralion. The fomation of these papillae is preceded by a reorganizationof the
cytoskeletoncausinga redistributionof vesicletraffic and a cytoplasmicaggregation towards the penetralionsite (for reviews see Takemoto and Hardham 2004:
Kobayashiand Kobayashi2008),and a somewhatslowermigrationof rhe nucleus
(fbr a review seeSchmelzer2002). By localizedmechanicalstimulationof parsley
P. Nick
f-.
. . . . . . h r r u n t o r e n d e rm t c r o l J , ' k r o se t a l ' 1 9 9 6 ) W
. hen
- ' . , r t k c d b y t r e a t m e nw
t ith
. -.: rhi: resultedin increased
i i . , : t L r iior n dC a r t e r1 9 9 2 )W
. ith
' r i . l | l t e d t h a t d i s a s s e m b loyf
- . . : : rr ( ) n o p h o rAe2 3 1 8 7c o u l d
.. - nrnt ehannelinhibitorgado-.
. ..,rrrl plsvsJlted
the activation
. . l r ) () l i . T h e s ed a t af ä v o u ra
r :ll. permeabilityof calcium
: r'L\\'hen microtubulesfunc: ,.hL'ltrtricrotubule
integrityis
r:\
.,i Lrp1 regulatory circuit caparl thc ilctivity of cold-induced
.: -.r..entbly, release this con-
L - .
. . : . : r , ' r \r.e s u l t i n gi n a n i n c r e a s e d
: . . r r l l i n I ' u r r h ed
r isintegration
'.,. rhrs positive-feedback
the
-., .iLun influx might thus be
' ,,.;'::erl by the activation of
' - . r - g n l l l i n - sc a s c a d e
will acti.t !.\\. Interestingll.micro-...Jr. j of this cold hardening
'..: .rt'hurnanova et al. 2003),
.
rhc calcium channelsthat
'.. ..
!.\ riould not only endow
r:.
l _ -
'
Lrl.lrrlso endow it with the
. , . . t t i o l la. k e r r e q u i r e m e n t
' - \ ( ) l u t i o n a rayr m sr a c c .
'.rnt of suppressdef-ence
\;
\-:
F.: i
. .t|ute-eies
to senseand
- C e l l u l a rr e s p o n s etso
l-. ()1'pathogenpenetra.. r'.()rganization
o1' the
: -rlrrplu:mia
c ggrega' ' ' . r n . lH a r d h a m2 0 0 4 ;
' : . r r i r t l o no f t h en u c l e u s
. . , . l r r u l a t i 0 no f p a r s l e y
Mechanics of the Cytoskeleton
19
cells, it was possibleto partially mimic an attack by Ph,-tophthoraso.jaeand to
induceseveralaspectsof a non-hostresistance,
includingnuclearmigration.cytoplasmic reorganization,formation of reactiveoxygen speciesand the induction of
(Gus-Mayeret al. 1998).In contrast.localizedappliseveraldefence-related
-qenes
cation of the corespondingelicitor (pep-13)failed to induce the morphologicai
changes,althoughit induceclthe full setof defence-related
genesand the formation
of reactiveoxygenspecies.Interestingly,
the elicitorcompletelyinhibitedcytoplasmic aggregationand nuclearmigration in responseto the mechanicalstimulus.
Since pep-13 inducesin this systemthe activity of a mechanosensitive
calcium
channel(Zimmermannet al. 1997).ftseemsthat chemicaland mechanicalsignalling converge during the cytoskeletalresponseto pathogen attack. Neither the
mechanicalstimulus nor the elicitor nor their combinationwas able to induce
hypersensitive
cell deathin theseexperiments,
leadingthe investigators
to conclude
that additional chemical signals are required to obtain the complete pathogen
response.
This suggests
an interactionbetweenmicrotubulesand mechanosensitive
ion channelsthat are imDortantlbr the induction of defence.
5.4
Osmoregulation
The ability to maintainionic balancerepresents
a basiccapacityof all living beings.
Prokaryoteshave already developed osmoregulation.In plants, the mechanical
tensionsproducedin the context of an expandingtissue have to be balanced
by osmoticpressure(chapter"Osmosensing").Microtubulesseemto be directly
involved in osmoadaptation.
By applicationof osmotic stressto root tips of
Tritit-um turgidLrm,microtubuleswere induced to disassembleand to reorganize
into massivebundles(Komis et al. 2002).when the fbrmationof theseso-called
macrotubuies
was suppressed
by treatmentwith oryzalin,the protoplastswere no
longer able to adapt to osmotic stressby controlled swelling and perished.A
pharmacological
study (Komis et al. 2006) revealedthat inhibitorsof phospholipaseD. suchas butan-1-oland N-acetylethanolamine.
suppressed
osmoticadaptation as well as the formationof the macrotubules.
In contrast,phosphatidicacid.a
product of the action of phospholipaseD, enhancedosmoadaptationand macrotubule lbrmation and was able to overcomethe inhibitory effect of butan-l-ol.
Theseobservationsdemonstratethat the microtubuleresponse(formationof macrotubules)is essentialfor osmoadaptation,
and that signallingthroughphospholipase
D actsupstreamof microtubulesin this response.
5.5 DivisionSymmetry
A homologueof the bacterialmechanosensitive
channelMscS.MSL3, localizedin
discretepatchesin the plastidenvelopeand co-localizedwith the plastiddivision
80
P. Nick
factor MinE, indicating an interaction with plastid division (Vitha et al. 2003).
When this bona fide channel was mutated,this resultedin chloroplaststhat were
irregular in size, shapeand partially number. Thus, these channelsregulatemorphogenesisand developmentof plastids,suggestinga functional shift from osmoregulationtowardsregulationof plastid morphogenesis.However, it is not only in
the symmetry of organelledivision where mechanosensingseemsto play a role.
Mechanosensingseemsto be the integratorthat allows the nucleusto determinethe
correct position prior to mitosis - this premitotic nuclear movement is an active
processand is driven by the cytoskeleton.It will detemine the symmetry of the
ensuing cell division and thus the basic morphology of the prospectivedaughter
cells. The discovery that overexpressionor knockout of the plant-specifickinesin
motor KCHI (Frey et al. 2010), which binds to actin filaments,retardsor acceleratespremitotic nuclearpositioning,indicatesthat cytoskeletaltensegrityis used
to determinethe correct position of the nucleus.Two principal modesare conceivable that are not necessarilymutually exclusive.Microtubules and actin filaments
might transmit forces that are generatedby the KCHI motor at the perinuclear
contact sitesto the cortex such that the nucleusis either pulled or pushed,or both
(Fig. 2c, d). Alternatively, KCHI might simply anchor the perinuclearnetwork at
the cell cortex and move the nucleus by mutual sliding of actin filaments and
microtubules in the cortical cytoplasm.From studiesin yeast, filamentousfungi
and a variety of animal cells,the molecularmechanismsthat orient and move nuclei
were found to be moderatelyconservedand involve as key playersdynein,dynactin
and otherproteinsat the plus endsof astralmicrotubules,mediatinginteractionwith
the cell cortex and actin filaments (Morris 2003; Yamamoto and Hiraoka 2003).
Both repulsiveand attractiveforcesare generatedby a combinationof microtubule
polymerizationand de-polymerizationevents,complementedby dynein-mediated
sliding of microtubulesalong the cell cortex (Adamesand Cooper2000). In plants,
which lack dynein and its associatedproteins (Lawrenceet al. 2001), the mechanisms for nuclear movement must involve fundamentallydifferent players that are
able to interact with both premitotic microtubulesand actin filaments.Could KCH
proteinsbe thesemissing links as functional homologuesof dyneinsby anchoring
minus-end-directedmotor activity to the cortex?
6 EvolutionaryPerspective:The Cytoskeleton
as a Central Integrator
This chaptersummarizedevidencefor a cytoskeletalfunction in tensegralintegration on both the organismaland the cellular level. We are still far from understanding the molecular set-up of tensegral sensing.But even at this stage, the first
differencesbetweenthe different stimulusqualitieshave emerged.
For mechanoperception,
microtubulesseemto interactwith stretch-activated
ion
channels,probably focussingminute deformationsof the membraneor changesin
P. Nick
.lrrr.ron (Vitha et al. 2003).
.:. :J....1r(ltn chloroplaststhat were
I i .-.. '.ttire channels regulate more . : l :': .1 :L[tcltonal shift from osmoi : ' . : ' . r . .H o w e v e r , i t i s n o t o n l y i n
i:'. ,.Jll\lllg seems to play a role.
! g-. .,.. rhe nucleus to determine the
r , : : , : - . r r i r f n l o v e m e n ti s a n a c l i v e
li i.. .tJl.ntrlne the symmetry of the
).,
_,. ()t the prospective daughter
Ir\.r ,.rl ,)1 the plant-Specifickinesin
: . ' , r , : - i t l t l a m e n t s ,r e t a r d s o r a c c e l r li..:i .\rLr:keletal tensegrity is used
. I . . ' n r r r r ei p a l m o d e s a r e c o n c e i v -
\ 1:.r,,tubulesand actin filaments
f'('llI motor at the perinuclear
. :. r.lltCf
pulledor
hcr puiled
pushed,or
or pushecl,
or both
both
ia
i r:1.:t(\r the perinuclear network at
r : - : . . . r r l r n go f a c t i n f i l a m e n t sa n d
.:-.:.ii. in veast,filamentousfungi
' : : : r r . : : t \ t h a to r i e n ta n d
m o v en u C l e i
,r.\ :' ..\ Nc\ playersdynein,dynactin
,,1-r,..r'. ntediatinginteractionwith
r 'i. \ .rnlulnotoand Hiraoka 2003).
3 J ' . . , ! ( ) n t b i n a t i oonf m i c r o t u b u l e
-.,:r.:-.Jrltented
by dynein-mediated
.i:: .'. .rntlCooper2000).In plants,
L: ",:-':',.i.'t al. 2001).the mechan:::'.::ri.,.lrditl'erentplayersthat are
.:r..r.:.tut filaments.Could KCH
:lJ\ ()t dyneinsby anchoring
rtoskeleton
. . . : . . . . : , , t l r ) l li n t e n s e g r a li n t e g r a 'ri.
3- -.:: .till far from understandI b- -..-'n ar this stage,the first
i:::.
'.:.:
l:llCfged.
. { ' : : 1 i - . - - : . r r t h : t r e t C h - a C t i v a t ei od n
r(.-.
::.r :llinlbrane or changes in
Mechanics of the Cytoskeleton
8l
membranefluidity towardsspecificmembraneareas.Becausethe demonstrationof
mechanosensitivityby patch-clampexperimentsis experimentallyvery problematic and prone to artefacts(Gustin et al. l99l), the molecular identity of stretchactivatedchannelshas remainedelusive in plants.When mechanosensitivityis not
an intrinsic propertyof suchchannels,but is conferredby accessorystructures(such
as cytoskeletaltensegrity), the identification of these channels will go beyond
simple expressionin heterologoussystems(such as frog oocytes),and will require
the reconstitutionof the entire structure,i.e. it will rely on syntheticbiology.
The situation might be different for the sensingof gravity. Here microtubules
themselvescould act as primary sensors.The findingsof the few experimentswhere
the involvementof ion channelshas been addressedexperimentally(Ikushima and
Shimmen 2005) suggest that these channels might be dispensablefor gravity
sensing.The necessityof microtubule tumover in the sensingof gravity indicates
a true perceptivefunction ratherthan stimulus susception.
The sensingof cold seemsto representa third mechanism,where the gating of
cold-sensitivechannels(which probably respondto membranerigidity as an input)
is limited by microtubules.when these microtubulesdisassemblein responseto
cold, the constraintsupon the activity ofthe ion channelswill be releasedsuchthat
calcium can enter, which will facilitate, through interaction with calmoclulin,
further disassemblyof microtubulesand thus trigger a positive-feedbackloop. In
this systemmicrotubuleswould play a dual function - first as a perceptivedevice
and later as accessorystructuresfor the perceptivechannels.
why is the cytoskeleton central for mechanical integration? The reason is
probably linked to the innate properties of cytoskeletal dynamics that render
microtubulesand actin filamentsideal for the sensingof minute and noisy inputs.
These dynamics are nonlinear and endowed with autocatalyticproperties.In the
cell, the abundanceof monomersis limited, which meansthat different polymers
compete for the incorporation of free monomcrs. For instance,in all organisms
investigatedso far, tubulin synthesisis tightly regulatedby an elaboratesystemof
transcriptionaland post-transcriptionalcontrols, probably to avoid the accumulation of (highly toxic) supemumerousfree heterodimers(for a review seeBreviario
2008). Although the term "cytoskeleton" was coined in the model of a rigid
framework that stabilizesthe structureof a cell, such associationsare far from
reality. The half+ime of a plant microtubule,for example,has beenestimatedto be
in the range of 30-60 s (Hush et al. 1990). Therefore, it is more appropriateto
conceive of microtubules and actin filaments as statesof dynamic equilibrium
between assemblyand disassemblyof monomers.It is this dynamic equilibrium
that provides the major source for the characteristicnonlinearity of cytoskeletal
dynamics.
Interestingly,the relationbetweenassemblyand disassemblyis practicallynever
balanced-one always dominatesover its antagonist.This statementis valid in both
spaceand time: in space,becausedimer addition and dispersaldefine a distinct
polarity of eachindividual cytoskeletalpolymer, with dimer addition dominatingat
the plus end and dimer dissociationdominating at the minus end; in time, because
eachpolymer can switch betweena growing state,when dimer addition at the plus
t32
P. Nick
end predominatesover dimer dissociationat the minus end, and a shrinking state.
when dimer dissociationat the minus end exceedsdimer additionat the plus end.
The switch between both statesis so swili and dramatic that it has been termed
"microtubule catastrophe".These conversionsclependon associatedproteins that
can increaseor decreasethe frequencyof transitionbetweengrowth and shrinkage.
Becauseof its nonlineargrowth, the cytoskeletonis an ideal tool for developmental patterning.This has been exemplarily shown fbr the induction of the grey
crescentin developingfrog eggs.This manifestationof dorsoventralityis produced
in an epigeneticprocess,where a gravity-dependentgradient of developmental
determinants
in the centralyolk interactswith a second.displaced.gradientin the
egg cortex(Gerhartet al. 198l). The displacement
of the egg coftex is driven by
microtubulesand is triggeredby the penetrationof the sperm.The sperm induces
the nucleationof microtubulesthat act as tracksfbr a kinesin-drivenmovement
(Elinsonand Rowning 1988).The movement,in turn, triggersshearfbrces that
align the nucleationof additionalmicrotubulesin a directionparallelto the movement, whereasdeviant microtubulesmore fiequently undergocatastrophictransitions. The resultingnet alignmentof tracksincreases
the efficiencyof ntovement
andthusthealigningforce.This culminatesin a rapidrotationof thecorticalplasma
in a direction fiom the sperm towards the more remote equatorof the egg. This
movementwill then causean overlapof uppercortex with a small region of the
lower core and eventuallytrigger inductiveeventsthat lay down the Spemann
organizer.
The combinationof nonlinear,autocatalyticdynamic statesof individual cytoskeletal polymers with the tight control of free monomers accentuatingmutual
competition generatessystempropertiesthat are highly relevant lbr sensoryprocesses.Microtubules and actin filaments fullil all formal criteria of a reactitlndifTusionsystem(Turing 1952).This meansthat they can be understoodas ideal
pattern generatorsthat are able to produce qrralitativelyclear, neat outputs liom
minute and highly noisecontaminatedinputs.One can model how owing to their
innate dynamic properties microtubules will spontaneouslyself-organizein
responseto even a weak external factor such as gravity or mechanical fields
(Tabonyet al. 2004).It thus seemsthat naturehasmadeampleuseof theseunique
molecular propertiesto builcl sensorysystemsthat are both sensitiveand robust
againststochastic
noiseandcanbe usedto integrateevenminutemechanicalstrains
on the level of individualceils as well as on the level of entireorsans.
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