patterning and lifetime of plasma membrane-localized ...control seedlings (c), seedlings grown on...

Patterning and Lifetime of Plasma Membrane-LocalizedCellulose Synthase Is Dependent on Actin Organizationin Arabidopsis Interphase Cells1[W]

Arun Sampathkumar, Ryan Gutierrez, Heather E. McFarlane, Martin Bringmann2, Jelmer Lindeboom,Anne-Mie Emons, Lacey Samuels, Tijs Ketelaar, David W. Ehrhardt, and Staffan Persson*

Sainsbury Laboratory, University of Cambridge, Cambridge CB2 1LR, United Kingdom (A.S.); Max PlanckInstitute for Molecular Plant Physiology, 14476 Potsdam, Germany (A.S., M.B., S.P.); Department of PlantBiology, Carnegie Institution for Science, Stanford, California 94305 (R.G., J.L., D.W.E.); Botany Department,University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (H.E.M., L.S.); and Laboratoryof Plant Cell Biology, Wageningen University, 6708 PB Wageningen, The Netherlands (J.L., A.-M.E., T.K.)

The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cellwalls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organizationfor wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioningthe delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils.The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cellwall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA deliveryrate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analysesrevealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments wereassociated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected bythe actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulosedeposition, and organization of cell wall biogenesis.

Plant cells are surrounded by a flexible yet durableextracellular matrix that makes up the cell wall. Thisstructure offers mechanical strength that counters os-motically driven turgor pressure, is an important fac-tor for water movement in plants, acts as a physicalbarrier against pathogens (Somerville et al., 2004), andis a determining factor for plant cell morphogenesis.Hence, the cell wall plays a central role in plant biology.

Two main types of cell walls can typically be dis-tinguished: the primary and the secondary cell wall.The major load-bearing component in both of thesecell walls is the b-1,4-linked glucan polymer cellulose(Somerville et al., 2004). Cellulose polymers are synthe-sized by plasma membrane (PM)-localized cellulosesynthase (CesA) complexes (Mueller and Brown, 1980),which contain several CesA subunits with similar amino

acid sequences (Mutwil et al., 2008a). The primary wallCesA complexes are believed to be assembled in the Golgiand are subsequently delivered to the PM via vesiculartrafficking (Gutierrez et al., 2009), sometimes associatedwith Golgi pausing (Crowell et al., 2009). Furthermore,the primary wall CesA complexes are preferentiallyinserted into the PM at sites that coincide with corticalmicrotubules (MTs), which subsequently guide cellulosemicrofibril deposition (Gutierrez et al., 2009). Hence, thecortical MT array is a determinant for multiple aspects ofprimary wall cellulose production.

The actin cytoskeleton plays a crucial role in organizeddeposition of cell wall polymers in many cell types, in-cluding cellulose-related polymers and pectins in tip-growing cells, such as pollen tubes and root hairs (Huet al., 2003; Chen et al., 2007). Thus, actin-depolymerizingdrugs and genetic manipulation of ACTIN genes impairdirected expansion of tip-growing cells and long-distancetransport of Golgi bodies with vesicles to growing regions(Ketelaar et al., 2003; Szymanski, 2005). In diffuselygrowing cells in roots and hypocotyls, loss of anisotropicgrowth has also been observed in response to mutationsto vegetative ACTIN genes and to actin-depolymerizingand -stabilizing drugs (Baluska et al., 2001; Kandasamyet al., 2009). While actin is clearly important for cell wallassembly, it is less clear what precise roles it plays.

One well-known function of actin in higher plants isto support intracellular movement of cytoplasmic or-ganelles via actomyosin-based motility (Geisler et al.,

1 This work was supported by the Max Planck Society (to A.S.,M.B., and S.P.) and a Canadian Natural Sciences and EngineeringResearch Council Discovery Grant (to H.M. and L.S.).

2 Present address: Department of Biology, Stanford University, 371Serra Mall, Stanford, CA 94305.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Staffan Persson ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.113.215277

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2008; Szymanski, 2009). During primary wall synthesisin interphase cells, treatment with the actin assemblyinhibitor latrunculin B (LatB) led to inhibition of Golgimotility and pronounced inhomogenities in CesAdensity at the PM (Crowell et al., 2009; Gutierrez et al.,2009) that coincided with the density of underlyingand immobile Golgi bodies (Gutierrez et al., 2009).These results suggested that Golgi motility is impor-tant for CesA distribution (Gutierrez et al., 2009). Theactin cytoskeleton also appears to be important forsecondary wall cellulose microfibril deposition. Forexample, longitudinal actin filaments (AFs) define themovement of secondary wall CesA-containing Golgibodies in developing xylem vessels (Wightman andTurner, 2008). In addition, it has been proposed thatthe AFs also can regulate the delivery of the second-ary wall CesA complex to the PM via pausing of theGolgi (Wightman and Turner, 2008). It is thereforeclear that actin organization is important for CesAdistribution and for the pattern of cellulose microfi-bril deposition.

Despite the above findings, very few reports haveundertaken detailed studies to elucidate the role of theactin cytoskeleton in the distribution and trafficking ofspecific proteins in plant cells. Here, we have investi-gated the intracellular trafficking of CesA-containingvesicles and delivery of CesAs to the PM, in the con-text of the actin cytoskeleton. We quantitatively dem-onstrate that the organization of the actin cytoskeletonregulates CesA-containing Golgi distribution and theexocytic and endocytic rate of the CesAs. However,

actin organization has no effect on the localized in-sertion of CesAs at sites of MTs at the PM.

RESULTS

Motility of CesA-Containing Golgi Bodies Is Facilitated bythe Actin Cytoskeleton

CesA-containing Golgi motility depends on the or-ganization of the actin cytoskeleton (Crowell et al.,2009; Gutierrez et al., 2009). We confirmed this usinga yellow fluorescent protein (YFP):CesA6-expressing line(Paredez et al., 2006) in which we monitored interphaseepidermis cells in 3-d-old etiolated hypocotyls exposed toLatB (1 mM). In agreement with previous reports, we ob-served aggregation of the Golgi in response to LatB (Fig.1; Supplemental Fig. S1, A and B). While these effects aremost likely caused by impairment of the actin cytoskele-ton, our aim was to further characterize the role of theactin cytoskeleton in cellular CesA distribution. In addi-tion to actin-disrupting drugs, we therefore also took agenetic approach using mutant analyses. The Arabidopsis(Arabidopsis thaliana) genome contains 10 ACTIN genes, ofwhich three, ACT2, ACT7, and ACT8, are expressed invegetative tissues, two are potential pseudogenes, andthe rest, ACT1, ACT3, ACT4, ACT11, and ACT12, areexpressed in reproductive tissues (Supplemental Fig. S2,A and B; Gilliland et al., 2002). ACT2, ACT7, and ACT8are assumed to be functionally redundant, and singlemutants only result in weak phenotypes (Kandasamyet al., 2009). In agreement with these assumptions, act2act7

Figure 1. Defects in actin organization cause re-duced Golgi motility and accumulation of post-Golgivesicles. A and B, Actin organization in elongatinghypocotyl cells of 3-d-old etiolated control (A) andact2 act7mutant (B) seedlings expressing GFP:FABD.Red arrowheads indicate thin transverse AFs thattypically appear shorter in the mutant compared withcontrol; yellow arrowheads indicate free GFP. Bars =5 mm. C to E, Transmission electron micrographs ofcontrol seedlings (C), seedlings grown on 100 nMLatB (D), and act2 act7 mutant seedlings (E). Redarrowheads indicate Golgi; green arrowheads indi-cate regions with dense populations of post-Golgiaggregates in seedlings grown on 100 nM LatB and inthe act2 act7 mutant. Bars = 500 nm.

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displayed additive phenotypes, including severe dwarf-ism and organ twisting (Gilliland et al., 2002; Kandasamyet al., 2009). Using live-cell imaging of GFP fused to theF-actin-binding domain (GFP:FABD) lines, we confirmedthat the act2act7 mutant held disorganized AFs, withfewer AF bundles and fragmented cortical AFs withmore free or broken ends (Fig. 1, A and B; SupplementalMovie S1).To monitor CesA-containing Golgi in the act2act7 mu-

tant, we made reciprocal crosses between the mutant andYFP:CesA6-expressing plants. Similar to the LatB-treatedseedlings, we observed some cells showing aggregationof Golgi (Supplemental Fig. S1, A–C). Transmissionelectron microscopy (TEM) supported these observations,showing regions with high Golgi density as well as highdensities of vesicles adjacent to the Golgi (Fig. 1, C–E),presumably representing post-Golgi vesicles, in theact2act7mutant and LatB-treated seedlings. Furthermore,to quantify Golgi movement in the different conditions,we classified static Golgi as those that remained pausedfor periods up to 30 s. In the wild-type control, only 3%of the Golgi (n = 216 Golgi, three cells) were classified asstatic, whereas 98% (n = 81 Golgi, three cells) and 21%(n = 109 Golgi, three cells) were classified as static in theLatB-treated and act2act7 mutant seedlings, respectively(Supplemental Fig. S1, A–G). These results indicate thatthe organization of the actin cytoskeleton is important forCesA-containing Golgi distribution in Arabidopsis inter-phase cells and that the Golgi motility is only partiallyaffected in the act2act7 mutant.

Defects in the Organization of the Actin CytoskeletonResult in Cellulose Deficiencies

The reduced motility of the CesA-containing Golgiin the actin-deficient cells suggested the possibility thatthe deposition of cellulose might be affected. Severalcellulose-deficient mutants, including procuste1 (prc1-1,affecting CesA6) and cobra (required for cellulose de-position), display isotropic root cell growth and ectopiclignin deposits (Desprez et al., 2002). Close examinationof the act2act7 mutants revealed clear root cell swellingin light-grown seedlings, and both mutant and LatB-treated seedling roots and hypocotyls accumulated ec-topic lignin, which frequently accompanies diminishedcellulose deposition (Caño-Delgado et al., 2003), asassessed by phloroglucinol staining (Jensen, 1962; Fig. 2,A–F; Supplemental Fig. S1, I and J). To examine furtherthe impact on cellulose production by actin impairment,we measured the cellulose content in 5-d-old etiolatedact2act7 mutant seedlings using the Updegraff method(Updegraff, 1969) and in seedlings grown on low levelsof LatB (100 nM). Both the mutant and the LatB-treatedseedlings contained significantly less cellulose per unitdry mass compared with control seedlings (Fig. 2G). Incontrast with the LatB-treated seedlings, seedlingsgrown on media containing the MT-destabilizing drugoryzalin (500 nM) did not show reduced levels of cellu-lose (Fig. 2G). One possible reason for the reduction incellulose content in the actin-deficient seedlings could

arise from slower movement of CesA complexes at thePM. To investigate this, we measured the velocity ofCesA particles in the actin double mutant and LatB-treated seedling; however, there was no significant dif-ference observed between control and the treatment ormutant (P . 0.05, Student’s t test; n = 123, 126, and 108CesA particles for wild-type, act2act7, and LatB threeseedlings, respectively; Supplemental Fig. S1K).

Several cellulose-deficient mutants, including prc1-1,also develop incomplete cell walls (Fagard et al., 2000). Toassess whether this also is the case in the actin-impairedseedlings, we took two different approaches. First, weanalyzed time series projections of YFP:CesA6 at the PMin etiolated hypocotyl cells in the wild type (SupplementalFig. S3A) and act2act7 mutants (Supplemental Fig. S3B).Similar to what was reported in LatB-treated cells, weobserved uneven distribution of CesA complexes at thePM (Supplemental Fig. S3B; Supplemental Movie S2), aphenotype correlated with and explained by unevendistribution of Golgi (Gutierrez et al., 2009). Second, westained transverse sections of seedling roots of the act2act7double mutant with calcofluor, which stains cellulose,callose, and other simple b-linked glucans (Maeda andIshida, 1967). Consistent with the uneven CesA distribu-tion at the PM and the reduced cellulose levels in theact2act7mutants, the cell wall material appeared unevenlydistributed in the epidermal, cortical, and endodermalcells of the mutant seedling roots when compared withthe wild type (Fig. 2, H and I). To corroborate this ob-servation, we analyzed TEM sections of act2act7 mutantand LatB-treated seedling roots. These analyses confirmedthat both the mutant and LatB-treated seedlings had cellwalls with varying thickness compared with wild-typesections (Fig. 2, J to M). These results show that the un-even distribution of Golgi and CesA complexes in the PMobserved during impairment of actin are accompanied bydefects in cellulose deposition and cell wall thicknessdistribution. While the uneven cell wall distribution couldbe due to an uneven distribution of the CesAs at the PM,it is also plausible that these irregularities are caused bydefects in the distribution of other cell wall components.

Motility of Small CesA-Containing Compartments IsFacilitated Both by the Actin and MT Cytoskeleton

In addition to Golgi, CesA is also observed in smallmotile CesA compartments (SmaCCs; SupplementalFig. S4A; Crowell et al., 2009; Gutierrez et al., 2009). Aheterogeneous collection of compartments (Gutierrezet al., 2009), SmaCCs associate with cortical MTs underconditions of osmotic stress or inhibition of cellulosesynthesis and can be translocated by tracking depo-lymerizing MT ends in a polarity-independent fashion(Gutierrez et al., 2009). When associated with MTs, thesecompartments are also termed MT-associated CesAcompartments (MASCs; Crowell et al., 2009). Consideringthat some SmaCC compartments are observed associatedwith Golgi (Gutierrez et al., 2009) and that Golgi havebeen observed to be translocated along the actin cyto-skeleton (Akkerman et al., 2011), we investigated whether

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Figure 2. Actin deficiency mimics cellulose-related mutants and leads to aberrant cell wall deposition. A to C, Root phenotypesof 5-d-old light-grown control (A), prc1-1 (affecting CesA6; B), and act2 act7 mutant (C) seedlings. Inserts display magnifiedregions of the seedling roots. Bars = 100 mm. D to F, Phloroglucinol-HCl staining of control (D), prc1-1 (E), and act2 act7mutant(F) seedlings grown as described in A to C. Bars = 100 mm. G, Cellulose levels in etiolated 5-d-old seedlings (genotypes anddrugs as indicated in figure). Error bars indicate SD, and asterisk shows significance (P , 0.05, Student’s t test). Calcofluor-stained root sections of 5-d-old wild-type (H) and act2 act7 mutant (I) seedlings. Arrowheads indicate regions of uneven cellwall deposition. Bar = 50 mm. J to L, Transmission electron micrographs of control seedlings (J), act2 act7 mutant seedlings (K),and seedlings treated with 100 nM LatB (L). Uneven cell walls are indicated with red arrowheads. CW, Cell wall. Bars = 500 nm.M, Variability in cell wall thickness is indicated by the mean SD of the wall thickness arbitrary unit; error bars represent SE.Asterisk shows significance (P , 0.0001, Student’ t test; n = 70–80 cells per genotype).


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SmaCCs also may be associated with and perhaps betranslocated along the actin cytoskeleton. To investigatethe relationship between SmaCCs and the actin cyto-skeleton, we generated a dual-labeled GFP:FABD- andtdTomato red fluorescent protein (tdTomatoRFP):CesA6-expressing Arabidopsis line. Importantly, the phenotypesof the majority of the progeny from these crosses wereindistinguishable from wild-type plants (SupplementalFig. S3D), indicating that the fluorescently labeled pro-teins did not interfere with plant growth to any majorextent. We then monitored epidermal interphase cells in3-d-old etiolated seedlings at focal planes ranging fromthe cortex to approximately 0.6 to 1 mm below the PM. Atthe latter subcortical focal planes (at 0.8 mmbelow the PMfocal plane), we observed evidence for migration alongAFs by some SmaCCs for periods lasting from 2 s tomore than 30 s at a density of 2.46 0.55 particles per 100mm2 under control conditions (Fig. 3A; Supplemental Fig.S4, F–H; Supplemental Movies S3 and S4). Unlike theMT-associated SmaCCs/MASCs that display alternatingsaltatory and smooth and steady movement with anaverage velocity of 4.06 mm min–1 (n = 122 SmaCCs;Supplemental Fig. S4, B–D), the subcortical SmaCCs/MASCs displayed highly erratic movement with an av-erage velocity of 59.27 mmmin–1 (n = 150 smaCCs; Fig. 3,B and D). The measurement of the subcortical SmaCCs/MASCs was done in conditions where seedlings weretreated with isoxaben (100 nM) and oryzalin (20 mM),which results in removal of cortical SmaCCs/MASCs,thereby ensuring that the analyzed compartments aresubcortical. These velocities are in close agreement withpreviously published velocities of particles that trackalong AFs (Boevink et al., 1998). Furthermore, treatmentwith LatB abolished the movement of the subcorticalCesA vesicles (Fig. 3C). Removal of cortical SmaCCsresulted in a significant increase in the number of sub-cortical CesA compartments (Fig. 3E), suggesting eitherthat the MTs and actin cytoskeleton may compete forSmaCC interaction or that SmaCCs might be handed offfrom the actin to the MT cytoskeleton. These resultssupport the existence of CesA-containing vesicle com-partments that interact with both MTs and the actincytoskeleton.

Stabilization of Cortical AFs Influences the LinearMovement of CesA Particles in the PM

Because cortical MTs guide PM-localized CesA par-ticles (Paredez et al., 2006) and AFs coalign with corticalMTs for short periods of time (Sampathkumar et al.,2011), it is plausible that the PM-tracking CesAs andcortical AFs can come into contact and that the AFscould exert an influence on CesA tracking behavior atthe PM.To first investigate whether cortical AFs can cooccur

with tracks of PM-located CesA particles, we imaged cellsin etiolated dual-labeled GFP:FABD and tdTomatoRFP:CesA6 seedlings. Interestingly, in several instances percell, we observed coalignment between cortical AFs and

linear tracks of CesA particles at the PM in time averageimages (Supplemental Fig. S3E; Supplemental Movie S5).The cooccurrence of the AFs and the CesA trajectoriestypically lasted from several seconds up to more than 40 s(Supplemental Fig. S3E; Supplemental Movie S5). Timeseries overlays and kymograph analyses confirmed thatthe PM-localized CesAs that were analyzed followedlinear trajectories and moved at constant velocity, beingunaffected during instances of actin colocalization(Supplemental Fig. S3, F and G). However, the align-ment of cortical AFs and CesA tracks only took place atfew regions at any given point of time in the cell.

To investigate the potential functional relationshipof the observed alignment of cortical AFs and CesAtracks, we exposed GFP:CesA3-expressing seedlings tojasplakinolide (5 mM for 3 h), an actin-stabilizing drug,and then observed the behavior of the CesAs. TheCesA particles were distributed throughout the PM,similar to mock-treated cells, but when we assembledtime average images to trace CesA migration, we ob-served disorganized trajectories and impaired Golgimovement (Supplemental Fig. S3C; Supplemental MovieS6). Considering that actin stabilization also influencesthe MT organization (Sampathkumar et al., 2011), wehypothesize that the observed changes in CesA trajec-tories are a consequence of the changes in MT organi-zation facilitated by the stabilized actin cytoskeleton.

The Bulk of CesA Delivery to the PM in Hypocotyl CellsAre Not Associated with Paused Golgi Bodies

Given that the actin cytoskeleton facilitates the dis-tribution of CesA-containing Golgi bodies and that it isregarded as an important structure for exocytosis andendocytosis in many organisms, including tip-growingcells in higher plants (Moscatelli et al., 2012), we aimedto explore how defects in the actin cytoskeleton affectCesA delivery rates to the PM. The delivery of CesAparticles to the PM has been described to derive frompausing Golgi bodies at the cortex (Crowell et al.,2009), a conclusion derived from seven observations incells at the base of the hypocotyl. This technique is,however, tedious due to the identification of new CesAinsertion events in an already existing pool of CesAparticles. In observations of CesA delivery in hypocotylcells (Gutierrez et al., 2009; Bringmann et al., 2012),Golgi bodies were occasionally observed at the locationand time of CesA delivery, but frequently no Golgi bodywas apparent. To put these observations on a quantita-tive basis, we analyzed CesA delivery events in hypo-cotyl cells using a photobleaching assay and asked howfrequently organelles consistent with Golgi bodies werepresent when newly arrived tdTomato:CesA6 was firstdetected at a stable position in the plane of the PM (Fig.4A; Supplemental Movie S7). We counted 60 deliveryevents following photobleaching (n = three cells fromthree seedlings) and observed associated Golgi in 38(63%) cases. Of these events, 18 (30% of all observedevents) occurred following sustained Golgi pausing




















(.8 s of immobility). No Golgi bodies were observed inthe vicinity at the time of CesA delivery in 22 (36%)events. Thus, it is clear that while pausing Golgi bodiesprecedes some insertion events, the bulk of deliveryevents in hypocotyl cells either involve rapidly movingGolgi or vesicles no longer associated with Golgi(Supplemental Fig. S5A; Supplemental Movie S7).

MT-Localized Insertion of CesA Particles into the PM IsIndependent of the Actin Cytoskeleton

In addition to its role in guiding CesA particles atthe PM, the cortical MTs also define preferred insertionsites of CesAs into the PM (Gutierrez et al., 2009). Thebrief coordinated actions between MTs and the actincytoskeleton (Sampathkumar et al., 2011), and the

Figure 3. Small subcortical CesA compartment movement is dependent on actin cytoskeleton A, Focal planes below the cellcortex displayed erratic and rapid movement of SmaCCs along the actin cytoskeleton in 3-d-old etiolated dual-labeled GFP:FABDand tdTomatoRFP:CesA6 hypocotyl cells. Green enclosures show rapid movement of the subcorticular CesA compartment alongan AF (red arrowhead). B, Kymographs of the subcorticular CesA compartment moving along the actin cytoskeleton in A. C, LatBtreatment abolishes movement of the subcortical CesA compartments. Bars = 5 mm. D, Histogram showing the velocity of sub-corticular CesA compartments (n = five seedlings, 134 particles). E, Differences in density of CesA compartments at focal planebelow the cortex after different drug treatments (n = five seedlings for each treatment). Error bars represent SE. Asterisks showsignificant difference (P , 0.05) between the wild type and the treatments.


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observation that AFs can coalign with CesA trajecto-ries, prompted us to ask whether the positional infor-mation for CesA insertion is affected when the actincytoskeleton is impaired. To test this possibility, wetreated seedlings expressing GFP:CesA3 and mCherryfused to a tubulin marker (mCherry:TUA5; Gutierrezet al., 2009) with 1 mM LatB for a period of 4 h andphotobleached the GFP channel at the PM. We sub-sequently analyzed the positions of CesA delivery tothe PM with respect to cortical MTs as previously

described (Gutierrez et al., 2009; Bringmann et al., 2012).Fifty delivery events were observed in the LatB-treatedseedlings, of which 38 (76%) occurred in locations de-fined by MT label (44% of the image area, P , 0.001,binomial test; Fig. 4, C and D; Supplemental Movie S8).Surprisingly, these results are very similar to thosereported for untreated seedlings (Gutierrez et al., 2009;Bringmann et al., 2012). Furthermore, observation athigher resolution using TEM showed electron-densevesicles at the cortex adjacent to MTs in the act2act7

Figure 4. Localized insertion of CesA particles in the PM is independent of the actin cytoskeleton. A and B, Different types ofCesA insertion mechanisms. A, CesA insertion can occur without appearance of Golgi body in the frames prior to insertion.Right section shows kymograph along the cyan line. B, CesA insertion can occur from Golgi body that moves rapidly. Rightsection shows kymograph along the cyan line. Red arrowheads indicate Golgi body; yellow arrowheads indicate CesA particleat the PM. C, Single image frame showing CesA insertions (white arrowheads) on MTs in a GFP:CesA3 TUA5:mCherryRFP dual-labeled hypocotyl cell from LatB-treated 3-d-old etiolated seedlings. D, Map of CesA delivery events in LatB-treated seedlings(C). Sites were mapped onto a 6-min average projection of MT signal. Red plus symbols indicate CesA insertion sites. E, TEMsections of 5-d-old act2 act7 double mutant in which vesicular compartments (yellow arrowheads) can be seen in closeproximity to cortical MT sites (red arrows). CW, Cell wall. Bars = 500 nm. F, Histogram showing quantification of averagedistance between vesicles and the nearest MT in the wild type and act2 act7 double mutant. Error bars represent SE (P . 0.05,Student’s t test; n = 161 vesicles for the wild type and n = 273 for act2 act7).






mutant seedlings (Fig. 4, E and F). We did not observeany significant differences in the average distance be-tween cortical vesicles to the closest MT between theColumbia ecotype and act2act7 (Fig. 4F; cortical vesicles600 nm or less below the PMwere included in analysis).In this analysis, we distinguished between clathrin-coated vesicle, dense vesicle, and other secretory vesi-cle (Supplemental Fig. S4A); however, in no cases didwe observe any differences in MT associations. Weconclude that the organization of the actin cytoskeletondoes not influence the positional insertion of CesAs nextto MTs.

The Organization of the Actin Cytoskeleton InfluencesExocytic Rates of CesAs

Next, we explored if and how the actin cytoskeletoninfluences the rate of CesA delivery to the PM. Thisrate can be determined in cells containing GFP-labeledCesA by photobleaching a region of the PM containingactive CesA complexes and measuring the number ofnewly delivered CesA particles per unit area per unittime (Gutierrez et al., 2009). We initially performedthis procedure in cells with and without disrupted AFsto determine if normal actin organization is necessaryfor efficient delivery of complexes to the PM. However,this technique has a notable complication. CytoplasmicGFP:CesA protein, mostly residing in Golgi bodies nearthe PM, are susceptible to bleaching; therefore, someCesA cargo destined for the PM would be invisible andcould potentially cause the rate of CesA delivery to beunderestimated. This source of error is likely greater incells with a compromised actin cytoskeleton because cy-toplasmic streaming is severely inhibited. With robuststreaming in normal cells, the local cytoplasmic pool inthe bleach region is rapidly replaced with unbleachedCesA protein. To address this issue in actin-compromisedcells (act2act7 double mutants and LatB-treated seed-lings), we performed two types of measurements. First,we photobleached relatively small regions of 36 mm2 tominimize bleaching of the cytoplasmic GFP:CesA pool(Supplemental Fig. S5B) and measured recovery of opti-cally resolved GFP:CesA punctae that met the criteria ofactive CesA complexes (linear motility at slow and steadyvelocity). Only punctae within a central 28-mm2 subre-gion were measured to avoid false positives due to activeCesA complexes migrating into the region of observation(Supplemental Fig. S5B). We observed an initial lag phasein CesA recovery, but after 2 min, we observed a gradualand linear increase in CesA recovery in all genotypes andtreatments (Supplemental Fig. S5, C and D). If bleachedCesAs contribute significantly to the inserted pool ofCesAs in the actin deficient cells and the insertion rateis constant, a nonlinear pattern of recovery would beexpected. This is because bleached CesAs would initiallycontribute more to the insertions, whereas newlysynthesized CesAs would subsequently become moreprevalent. We therefore concentrated on the linear por-tion of the recovery curves for further analysis. It should

be noted that there was not a complete recovery of theCesA fluorescence during the observed time frame, in-cluding for the wild type (Supplemental Fig. S5C), in-dicating bleaching of the sample by imaging duringrecovery or the existence of a pool of signal that recoverson a longer time frame. The rate of recovery was re-duced by nearly 50% in the act2act7mutants (4.246 1.59punctae mm–2 h–1; n = six cells) and LatB-treated seed-lings (5.05 6 1.29 punctae mm–2 h–1; n = six cells)compared with control seedlings (11.2 6 2.57 punc-tae mm–2 h–1; n = six cells; Fig. 5A; Supplemental S5C).These results might be obtained either by a decrease inthe delivery rate of new label to the PM or by an in-crease in the rate of their removal. To isolate the de-livery rate, individual CesA deliveries were measuredat three time intervals during the liner phase of re-covery using the criteria described by Gutierrez et al.(2009; Fig. 5B). During recovery, label density at the PMwas low enough to permit this more precise analysis,whereas density at the end of recovery was too high toassay these events with confidence. We observed thatnew deliveries were, in fact, significantly higher in thewild type than in either act2act7 mutants or in LatB-treated cells (Fig. 5C). Furthermore, the consistent deliv-ery rates measured at each of the three time intervalsprovided further evidence that the measurements werenot significantly influenced by delivery of bleachedcomplexes at this stage of recovery.

Because the reduced delivery rate could be a conse-quence of fewer underlyingGolgi in the actin-compromisedcells, we measured the density of the labeled CesAs atthe cortex in frames that just preceded photobleachingin the observed regions. If, in fact, the observed regionsin the actin-compromised cells were devoid of Golgiwithin the range that vesicles can be efficiently deliv-ered to the PM, then we might expect to see that thestarting density of CesAs was lower in the actin-compromised regions. No significant difference (P =0.1895, Student’s t test; P = 0.25, Mann-Whitney U test)in mean CesA particle density was observed betweenthe mutants (1.09 6 0.09 particles mm–2; n = five cells),LatB-treated seedlings (1.005 6 0.19 particles mm–2; n =five cells), and control seedlings (1.34 6 0.08 particlesmm–2; n = five cells; Supplemental Fig. S5C) in regionsthat were used for fluorescence recovery after photo-bleaching (FRAP) analyses. Thus, there was no evidencethat the regions chosen for analysis were depleted of CesAsin the actin-compromised cells prior to photobleaching.

The observation that the densities of CesAs at the PMwere similar, together with our observation that CesAdelivery was significantly lower in actin-compromisedcells, has interesting implications for both the inter-nalization of CesA complexes and the lifetime of CesAcomplexes at the PM. If we assume that the observed cellswere at a steady state, i.e. delivery rates are equal to in-ternalization rates, than maintenance of the same densityat the PM implies that the internalization rate declinesalong with the delivery rate in actin-compromised cells.Likewise, the lifetimes of the CesAs at the membranemust also be longer. We calculated the lifetime of a


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typical CesA particle in act2act7 double mutants to beapproximately 15.42 min (1.09 particles mm–2/4.24 61.59 events mm–2 h–1) and in the LatB-treated seedlings tobe approximately 10.5 min (1.005 particles mm–2/5.05 61.29 events mm–2 h–1), whereas the lifetime of a typ-ical CesA in the control cells was approximately 7.15min (1.34 particles mm–2/11.2 6 2.57 events mm–2 h–1;Fig. 5D).If CesA internalization is reduced in actin-compromised

cells, then other measures of membrane and proteininternalization might also be affected. To test if thismight be true, we used the endocytic marker FM4-64and stained light-grown hypocotyl cells of wild-type,act2act7 double mutant, and LatB-treated seedlings. Aftera period of 10 min, we quantified the amount of FM4-64-localized vesicles in the cells and found that there was asignificant reduction in the number of FM4-64-markedendosomes in the act2act7 double mutant (4.48 6 1.217vesicles per 100 mm2; P = 0.04907, Student’s t test) andLatB-treated (2.99 6 0.83 vesicles per 100 mm2; P =0.00021, Student’s t test) cells compared with the wild-type cells (5.946 1.47 vesicles per 100 mm2; Supplemental

Fig. S5, E–H). Thus, the actin organization is importantfor efficient exocytic and endocytic exchange at the PMand therefore has an influence on the lifetime of a typicalCesA particle in the PM.

DISCUSSION

Actin is a major component of the cytoskeleton andis involved in vesicle trafficking and cargo distributionin eukaryotic cells (Mooren et al., 2012). We show thatwhile the actin cytoskeleton is not essential for exo-cytosis and endocytosis in interphase plant cells, anintact actin organization influences the rate of CesAdelivery and the time a typical CesA spends in the PM.In addition, the actin cytoskeleton does not seem toimpact specific positional insertion of the CesAs intothe PM.

In plant cells, the bulk of research regarding the roleof the actin cytoskeleton in membrane exchange at thePM comes from studies on tip-growing cells. For ex-ample, treatment of pollen tubes and root hairs with

Figure 5. The actin cytoskeleton is important, but not essential, for CesA vesicle trafficking to the PM. A, Box plot showing ratesof recovery of CesA punctae in wild-type, act2 act7 double mutant, and LatB-treated seedlings. Asterisks show significantdifference (P , 0.0003, Student’s t test) between wild-type and actin-compromised conditions (n = six cells for each condition;total area of 170 mm for each condition). B, Frames representing delivery of CesA particle. Arrowheads mark appearance ofCesA particle and steady movement and lower section shows kymograph of typical CesA insertion event following FRAP. Bars =1 mm. C, Histogram showing the number of CesA delivery events after photobleaching in wild-type, act2 act7, and LatB-treatedseedlings during the indicated time frames. CesA insertions were defined as in Gutierrez et al. (2009). Asterisks show significantdifference (P , 0.047, Student’s t test). D, Average lifetime of a CesA complex in wild-type, act2 act7, and LatB-treatedseedlings (n = six cells for each condition). Error bars represent SD, and asterisks show significant difference (P , 0.005, Stu-dent’s t test) between the wild type and the mutant/treatment.







actin-depolymerizing drugs inhibit growth and cyto-plasmic streaming (Miller et al., 1999; Vidali et al.,2001; Ketelaar et al., 2003). The importance of the actinorganization for tip-growing cells is therefore wellestablished. However, with the notable exceptions ofearly endocytic sterol trafficking (Grebe et al., 2003) andcellular CesA distribution (Crowell et al., 2009; Gutierrezet al., 2009), the role of the actin cytoskeleton in inter-phase cells is less well explored. In addition, only veryfew studies have substantiated the observations withrobust quantitative analyses for specific PM components.

The AFs can roughly be divided in two populations.One is characterized by highly dynamic fine corticalAFs, and the other displays less dynamic and thickerAF bundles at subcortical focal planes (Staiger et al.,2009; Sampathkumar et al., 2011). While the precisefunctions of these populations are not clarified, a re-cent study demonstrated differential Golgi motilityalong the two populations (Akkerman et al., 2011).Golgi bodies associated with fine AFs at the cell cortexdisplayed slow nondirectional movement, perhaps en-gaged in exocytic activities, and subcortical Golgi bodiesassociated with thicker actin bundles displayed unidi-rectional movement that may signify long-distance trans-port. These data explain the subcortical CesA-containingGolgi aggregates that accumulated in response to actinimpairment. Moreover, closer observation of Golgi inactin-compromised cells using TEM revealed hyper-accumulation of vesicles adjacent to Golgi, a patternconsistent with continued vesicular biogenesis fromthe Golgi but a lack of transport away from Golgi dueto defects in actin-mediated mobility.

Association of post-Golgi CesA-containing compart-ments, i.e. SmaCCs/MASCs, was previously observedat cortical MTs in stressed cells (Crowell et al., 2009;Gutierrez et al., 2009), where they undergo tip-tracking(Gutierrez et al., 2009) motility driven by MT depoly-merization (Crowell et al., 2009; Gutierrez et al., 2009).MT association and tip-tracking events were also ob-served in unstressed cells for brief durations (Gutierrezet al., 2009), and delivery of CesAs was found to bestrongly associated with the positions of cortical MTs.In this study, subcortical imaging revealed that a pop-ulation of these vesicles also migrates along AFs andcables. While we do not conclude that this migrationoccurs through a direct association between the smaCCsand the AFs, it is important to note that the movement ofthe subcortical smaCCs was substantially reduced whenthe actin cytoskeleton was impaired. In addition, whenactin was disassembled with inhibitors, SmaCCs showedincreased association with MTs, an outcome that mightindicate interruption of an exchange of SmaCCs betweenthe actin and MT cytoskeletons or perhaps stimulation ofSmaCC interaction with MTs, similarly as observed byimposition of osmotic stress (Crowell et al., 2009; Gutierrezet al., 2009).

At least a subset of SmaCCs/MASCs appears to beinvolved in exocytosis of the CesA complexes. Gutierrezet al. (2009) showed that the compartments could deliverCesA complexes to the PM both in growing cells and

after washout of mannitol in osmotically challengedcells. Furthermore, only a small population of the com-partments colocalized with the lipophilic tracer FM4-64after short-term treatment (Gutierrez et al., 2009), a resultconfirmed by Sánchez-Rodríguez et al. (2012), who alsoobserved only a small overlap in FM4-64 distributionand a vesicle population containing the chitinase-likeprotein, which largely colocalized with the SmaCCs/MASCs. Taken together, these observations sug-gested that MTs position CesA insertion at a localscale, perhaps by interacting directly with secretorycompartments.

If SmaCCs/MASCs are involved in exocytosis, andconsidering that disruption of the actin cytoskeletonleads to a larger population of MT-associated com-partments, it is not difficult to envision that the AFsmay not influence the localized insertion of the CesAsinto the PM. Hence, the actin cytoskeleton would pro-mote cellular distribution of the compartments, whereasthe cortical MT-associated compartments would signifypreparation for CesA delivery to the PM. Impairment ofthe actin cytoskeleton would therefore not disturb thepreferential insertion of CesAs adjacent to the MTs. Re-cent reports also show that actin depolymerization didnot affect targeted accumulation of several polar markersin the endodermis (Alassimone et al., 2010). By scoringthe sources of CesA delivery, we found that most CesAswere delivered in regions where no Golgi bodies wereobserved prior to insertion. We hypothesize that thesource of these deliveries are through Golgi-derivedvesicles distributed by streaming in both the corticaland subcortical planes.

By quantifying the CesA density and delivery rate atthe PM, we estimated the average lifetime of a CesAparticle in the PM. Assuming that the density of theCesA particles is at steady state in both control andLatB-treated seedlings, we found that the lifetime of atypical CesA is around 7 to 8 min and that the lifetimeis longer in actin-defective cells. These data are in agree-ment with earlier estimates using amino acid pulse-chaseexperiments, coupled with protein synthesis inhibitors, incotton (Gossypium hirsutum; Jacob-Wilk et al., 2006). If ourassumption about steady state is correct, then the endo-cytic rate in the actin-deficient cells would also be re-duced. In mammals and yeast (Saccharomyces cerevisiae), itis well established that the actin cytoskeleton is importantfor endocytic activities (Engqvist-Goldstein and Drubin,2003). However, this relationship is less clear in plant cells.Studies on endocytosis of the auxin efflux carrier PIN1showed that the removal of PIN1 from the membranewas unaffected in actin-compromised root cells (Bouttéet al., 2006). By contrast, recycling of sterols and certaincell wall components is perturbed upon actin depoly-merization in roots (Baluska et al., 2002; Grebe et al.,2003). Furthermore, live-cell imaging studies of GFP-tagged endocytic markers in Arabidopsis roots showedthat endocytic vesicles could form in actin-impaired cells(Konopka et al., 2008). However, the movement of thesevesicles away from the site of internalization was mostclearly affected, and the majority of the GFP-tagged


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endocytic markers remained immobile for long pe-riods of time upon treatment with either LatB or with2,3-butanedione monoxime, which inhibits myosinactivity (Konopka et al., 2008). Our observation usingthe lipophilic tracer FM4-64 in interphase cells of hy-pocotyls suggests that endocytosis is affected in actin-perturbed cells. While the nature of this perturbanceremains to be investigated, this could contribute to theincreased lifetime of the CesA particles at the PM thatwe estimated in the actin-compromised cells.The actin-deficient seedlings contained reduced levels

of cellulose and displayed uneven wall thickness intransverse sections. While these two measures arenot directly connected, i.e. many other cell wall com-ponents contribute to cell wall thickness, it is likelythat the impaired actin cytoskeleton delivers materialunevenly to the apoplast. We also report that CesAs inthese seedlings stay at the PM for a longer time com-pared with the wild type. Arguably, if the CesAs stayat the PM for a longer time and the velocities aresimilar (and thus the presumed rates of synthesis),then each complex should produce more cellulose,which in the end would result in similar levels of cel-lulose as in the wild type. An obvious question is then,why do we see lower levels of cellulose? One likelyreason could be that the actin-deficient cells on averagecontain lower levels of PM-based CesA molecules. Whilewe ensured that the regions, and even cells, we used forour FRAP analyses contained similar CesA densities, it isimportant to note that this may not be the case for all thecells in the seedling. Hence, a lower overall number ofactive CesAs in many of the actin-deficient cells wouldalso lead to reduced cellulose levels. Another reasoncould be that the CesA complexes perhaps are active fora certain amount of time. In such a scenario, the CesAswould move at a constant speed for a certain distanceand then perhaps become deactivated and wait for in-ternalization. The delayed endocytosis observed in theactin-deficient cells could cause CesAs to be retained inthe PM after deactivation. Perhaps such a deactivationcould involve posttranslational modifications, such asphosphorylation. The CesA complexes can be phos-phorylated, and changes in phosphorylation can causeaberrant behavior of the CesAs (Chen et al., 2010; Bischoffet al., 2011). Although the distribution of CesA lifetimes atthe PM remain to be determined, it seems reasonable topropose that CesA internalization is regulated, both toavoid internalization before cellulose synthesis has beenaccomplished and as ameans to control microfibril length.However, we did not observe any significant increase inthe number of stalled CesAs in the actin-deficient cellswhen compared with wild-type cells. While this does notcompletely rule out that a certain fraction of CesAs aremoving slower, we favor the first scenario in which theactin-impaired cells contain lower levels of active CesAsover the whole of the cell.The actin cytoskeleton plays a crucial role in regu-

lating cell shape, from bacteria to animals to higherplants (Huang et al., 2012; White and Gober, 2012). Inbacteria, the actin-like protein MreB appears to a play

role that is somewhat related to that of the MT cyto-skeleton in high plants; both form cortical arrays thatplay a role in positioning cell wall biosynthetic enzymesand patterning the spatial organization of the cell wall(Huang et al., 2012; White and Gober, 2012). Here, wefound that the actin cytoskeleton in Arabidopsis alsoregulates aspects of cell wall synthesis by regulating thespatial distribution of the CesAs at a cell-wide level andby governing CesA lifetime in the PM. Hence, whereasthe actin cytoskeleton directly controls cell shape in ani-mal cells, the bacterial and plant actin-related cytoskele-ton appears to indirectly manage cell shape by controllingcell wall deposition.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) Columbia ecotype seedlings were grown asdescribed in Sampathkumar et al. (2011). Homozygous act2-1 act 7-1 (Gillilandet al., 2002) double mutant seeds were kindly provided by Richard Meagher(University of Georgia), and prc1-1 (Fagard et al., 2000) was a kind gift fromHerman Höfte. The mCherry:TUA5 (Gutierrez et al., 2009), FABD:GFP (Ketelaaret al., 2004), CesA6:YFP (Paredez et al., 2006), and CesA3:GFP (Desprez et al.,2007) lines have been described as indicated. All plants used for the imaging areF2 segregating lines and seedlings were selected based on phenotype and pres-ence of either one or both fluorescent markers.

Constructs

To generate the tdTomato::CesA6 construct, the Gateway cassette wasamplified from pMDC32 (Curtis and Grossniklaus, 2003) with primers 59-ACCGGTTATCAAACAAGTTTGTACAAAAAAGC-39 and 59-ACCGGTAA-CCACTTTGTACAAGAAAGCTGA-39, containing flanking AgeI sites, andcloned into pGEM-T Easy (Promega). The resulting plasmid was cut with AgeIand ligated into the compatible SgrAI site of CesA6-pBluescript SK+ (Paredezet al., 2006), which is located at the N terminus of CesA6. The tdTomato genewas amplified from pRSET-B tdTomato (Shaner et al., 2004) with primers59-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGG-GCGAGGA-39 and 59-GGGGACCACTTTGTACAAGAAAGCTGGGTCCT-TGTACAGCTCGTCCATCCGT-39 and cloned into CesA6-pBluescript SK+ viaBP and LR reactions. The resulting plasmid was cut with SacI and XmaI andligated into pCAMBIA2300. The tdTomato::CesA6 construct was then intro-duced into prc1-1 plants by Agrobacterium tumefaciens-mediated transformationas described previously (Paredez et al., 2006).

Drug Treatments

For biochemical analysis, TEM, and lignin staining, seedlings were ger-minated and grown on Murashige and Skoog media plates supplementedwith inhibitors, i.e. LatB (100 nM), oryzalin (500 nM), and isoxaben (2 nM). Forshort-term treatment, seedlings were incubated in 2 mL of water containinginhibitors (1 mM LatB, 100 nM isoxaben, or 20 mM oryzalin) in 12-well cell cultureplates in darkness. The seedlings were subsequently mounted for confocal imagingas described in Sampathkumar et al. (2011). Stock solutions of LatB, isoxaben, andoryzalin were dissolved in methanol or in dimethyl sulfoxide, and working stockswere made freshly by further dilution in water. FM4-64 was dissolved in waterand used at a concentration of 20 mM.

TEM

Five-day-old seedlings were high-pressure frozen, freeze substituted, em-bedded, sectioned, and viewed according to McFarlane et al. (2008). Briefly,samples were cryofixed using a Leica HPM 100 in B-type sample holders (TedPella) using 1-hexadecene (Sigma) as a cryoprotectant. Samples were freezesubstituted in 2% (w/v) osmium tetroxide (Electron Microscopy Sciences) and8% (v/v) 2,2-dimethoxypropane (Sigma) in acetone for 5 d at –78°C, then





slowly warmed to room temperature over 2 d and infiltrated with Spurr’sresin (Reynolds, 1963) over 4 d. Samples were sectioned to approximately 70nm using a Leica UCT microtome, suspended on copper grids (Gilder) coatedwith 0.3% (w/v) formvar (Electron Microscopy Sciences), stained with 2% (w/v)uranyl acetate in 70% (v/v) methanol and Reynolds’ lead citrate (Spurr, 1969),and then viewed with a Hitachi H7600 equipped with an Advanced MicroscopyTechniques Advantage CCD camera (Hamamatsu ORCA) at an acceleratingvoltage of 80 kV.

Cellulose Measurement

Cellulose was measured from 5-d-old etiolated seedlings. Fresh plant materialwas harvested in 2-mL Eppendorf tubes and homogenized with a Retsch mill. Thepowdered plant material was washed two times with 70% (v/v) aqueous ethanoland centrifuged. The supernatantwas discarded, and after air drying the pellet, 1mLof methanol:chloroform (1:1, v/v) was added and sonicated for 10 min and spundown. The supernatant was discarded, and the remaining pellet was washed twicewith acetone and air dried for 1 h to obtain cell wallmaterial. The crystalline cellulosecontent was determined using a modified protocol of Updegraff (1969) described inNeumetzler et al. (2012). Data were collected from four technical replicates for eachtreatment and mutant lines. Statistical significance between the wild type and othertreatments was determined using Student’s t test.

Lignin Staining

To analyze lignification pattern, Arabidopsiswild-type, drug-treated, andmutant5-d-old seedlingswere harvestedunder sterile conditions. Seedlingswereplacedontoamicroscope slide and incubatedwith twodrops of freshly prepared phloroglucinol-HCl solution (25 mg phloroglucinol, 25 mL 100% [v/v] methanol, and 25 mL 37%[v/v] HCL) for 5 min before observation under a light microscope.

Calcofluor Staining

Sectioning and staining of roots with calcofluor was performed according toFagard et al. (2000).

Microscopy

Light microscopy was performed using a Leica Stereomicroscope (LeicaMZ12.5, Leica DFC420 digital camera). Seedlings expressing GFP:CesA3, YFP:CesA6, GFP:FABD, and all the dual-labeled lines of FABD:GFP, tdTomato:CesA6 and GFP:CesA, mCherry:TUA5 were imaged on confocal microscopeequipped with a Yokogawa spinning disc head fitted to a Nikon Ti-E invertedmicroscope as described in Sampathkumar et al. (2011). Photobleaching wasachieved using a FRAP/photoactivation system (Roper Scientific) as describedin Gutierrez et al. (2009). Images of seedlings stained with FM4-64 was ac-quired on a Zeiss LSM780, fluorescence was excited using a 514-nm laser, andemission recoded through a 603- to 651-nm filter.

Image Analysis and Processing

All images were processed and analyzed using the ImageJ software.Background correction was performed using the “Subtract Background” tool(rolling ball radius, 30–40 pixels), and StackReg was used to correct focal drift.Kymograph analysis for measurement of CesA and SmaCC velocity was alsodone using ImageJ as described in Gutierrez et al. (2009).

Measurement of Cell Wall Thickness

Length of three random points on the wall was measured using ImageJ, andaverage SD was calculated within these three values as a measure of variation incell wall thickness. Seventy-five to 80 cell walls each for the different conditionsand genotypes were measured. Arbitrary units were used as a measurement forthe relative cell wall thickness within one image to account for differences insectioning planes. Significance was estimated using Student’s t test.

Density Measurement of CesAs at the PM

First frame of a time series data of the PM focal plane was used for quanti-fying number of CesAs. Regions of cell that contained an even distribution of

CesAs at the PM were chosen to avoid complexities arising due to differences inunderlying Golgi numbers in the mutant and LatB-treated cells when comparedwith the wild type. A total area of 432 mm2 was analyzed for the wild-type,act2act7 mutant, and LatB-treated seedlings. The total density was calculated asthe number of particles per 100 mm2 based on 190 CesA particles in control, 186in act2act7mutant, and 173 in LatB-treated seedlings (n = six cells in six seedlingsfor each condition). Significance was estimated using Student’s t test.

Density Measurement of smaCCs

SmaCCs were identified as described in Gutierrez et al. (2009) at differentfocal planes. Cortical smaCCs were scored based on motility of the particlesdescribed previously. Subcortical smaCCs were identified based on focalplane (0.6 to 1 mm below the PM) and on the erratic movement of the particlesassessed in kymographs. Particle density was calculated as number of parti-cles per 100 mm2 (total area of 500 mm2; n = five cells in five seedlings for allconditions). Significance was estimated using Student’s t test.

FM4-64 Staining and Density Measurements

Five-day-old light-grown wild type and act2act7 mutants were incubatedfor 10 min in 20 mM FM4-64. Epidermal hypocotyl cells were imaged under aZeiss LSM780 confocal microscope as indicated above. The density of inter-nalized FM4-64 was determined by counting the number of FM4-64-labeledvesicles in control (total area of 14,011 mm2; n = 25 cells), LatB-treated (totalarea of 19,770 mm2; n = 34 cells), and act2act7 (total area of 16,072 mm2; n = 23cells) seedlings. In total, 2,089 FM4-64-labeled particles were counted. Totaldensity was calculated as number of particles per 100 mm2. Significance wasestimated using Student’s t test.

Delivery Rate Analysis of CesAs

The PM focal plane of wild-type, act2act7 mutant, and LatB-treated seed-lings expressing CesA6:GFP were photobleached using the 491-nm laser. Afterbleach, the new CesA insertions events in the PM plane were identified incontrol (total area, 171 mm2; 151 insertion events), LatB (total area, 171 mm2; 97insertion events), and act2act7 (total area, 171 mm2; 101 insertion events). Wescored the insertion events based on the following criteria. First, prior to CesAdelivery, the immediate region surrounding the delivery should be devoid ofany fluorescent particles, and second, subsequent frames were examined usingkymographs for characteristic CesA tracking behavior. The images were an-alyzed for a new insertion for periods of 10 to 13 min after bleaching. Five cellsin five different seedlings for control and six cells from six different seedlingsfor act2act7 and LatB-treated seedlings were analyzed. Significance was esti-mated using Student’s t test.

Lifetime of CesA Complex

The lifetime of CesA complexes was estimated by dividing CesA particledensity by the rate of CesA insertion.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Actin defects affect Golgi motility and cellulosedeposition.

Supplemental Figure S2. Expression patterns of ACTIN genes in Arabi-dopsis.

Supplemental Figure S3. AFs and bundles can align with CesA trajectoriesat the PM and affect the distribution of CesA molecules.

Supplemental Figure S4. Movement of MT- and AF-associated SmaCCs.

Supplemental Figure S5. Global view of CesA insertion patterns.

Supplemental Movie S1. AF dynamics in the wild type and act2act7 mu-tant.

Supplemental Movie S2. CesA distribution is affected in act2act7 mutant.

Supplemental Movie S3. Tracking of SmaCCs/MASCs along AFs.


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Supplemental Movie S4. Tracking of SmaCCs/MASCs along AFs.

Supplemental Movie S5. AFs dynamically coalign with CesA trajectories.

Supplemental Movie S6. Stabilization of AF with jasplakinolide affectsCesA trajectories.

Supplemental Movie S7. Photobleaching reveals bulk of the CesA deliverydoes not precede sustained pausing of Golgi bodies.

Supplemental Movie S8. MT-localized delivery of CesA occurs indepen-dent of the actin cytoskeleton.

Received January 26, 2013; accepted April 18, 2013; published April 19, 2013.

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patterning and lifetime of plasma membrane-localized ...control seedlings (c), seedlings grown on...

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