Low Water Potential and At14a-Like1 (AFL1) Effects onEndocytosis and Actin Filament Organization1[OPEN]

M. Nagaraj Kumar, Yu-Chiuan Bau, Toshisangba Longkumer, and Paul E. Verslues2,3

Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan

ORCID IDs: 0000-0002-6096-4034 (M.N.K.); 0000-0002-7290-5429 (Y.-C.B.); 0000-0002-7057-1197 (T.L.); 0000-0001-5340-6010 (P.E.V.).

At14a-Like1 (AFL1) is a stress-induced protein of unknown function that promotes growth during low water potential stress anddrought. Previous analysis indicated that AFL1 may have functions related to endocytosis and regulation of actin filamentorganization, processes for which the effects of low water potential are little known. We found that low water potential led to adecrease in endocytosis, as measured by uptake of the membrane-impermeable dye FM4-64. Ectopic expression of AFL1reversed the decrease in FM4-64 uptake seen in wild type, while reduced AFL1 expression led to further inhibition of FM4-64 uptake. Increased AFL1 also made FM4-64 uptake less sensitive to the actin filament disruptor Latrunculin B (LatB). LatBdecreased AFL1-Clathrin Light Chain colocalization, further indicating that effects of AFL1 on endocytosis may be related toactin filament organization or stability. Consistent with this hypothesis, ectopic AFL1 expression made actin filaments lesssensitive to disruption by LatB or Cytochalasin D and led to increased actin filament skewness and decreased occupancy,indicative of more bundled actin filaments. This latter effect could be partially mimicked by the actin filament stabilizerJasplakinolide (JASP). However, AFL1 did not substantially inhibit actin filament dynamics, indicating that AFL1 acts via adifferent mechanism than JASP-induced stabilization. AFL1 partially colocalized with actin filaments but not with microtubules,further indicating actin-filament–related function of AFL1. These data provide insight into endocytosis and actin filamentresponses to low water potential stress and demonstrate an involvement of AFL1 in these key cellular processes.

Even a moderate severity of water limitation duringdrought (moderate decline in water potential [cw]) al-ters plant development and causes a wide range ofcellular changes. These responses to moderate severitylow cw are distinct from mechanisms involved in sur-vival of severe low cw and dehydration (Skirycz andInzé, 2010; Clauw et al., 2016). The plasma membraneand cell wall are sites of many processes related togrowth and stress resistance. Thus, trafficking mecha-nisms that control the composition of the plasmamembrane can impact abiotic stress response. Exam-ples of such mechanisms include regulated endocytosisof aquaporins to control membrane water permeability(Luu et al., 2012; Hachez et al., 2014; Chevalier andChaumont, 2015) and endocytosis of abscisic acid

(ABA) transporters and ABA receptors to control theirabundance on the plasma membrane (Belda-Palazonet al., 2016; Park et al., 2016; Yu et al., 2016). Outsideof these examples there is relatively little data on howdrought stress, especially longer-term moderate sever-ity stress, affects endocytosis, and little is known ofstress-responsive proteins that regulate endocytosis.

Similarly, actin filaments are important for growthand morphogenesis (Szymanski and Staiger, 2018) andhave been proposed to act as sensors or transducers ofexternal signals along the plasma membrane (Staigeret al., 2009). Thus, actin filaments also are likely tohave roles in responses to drought and other environ-mental stresses. Consistent with this idea, actin filamentstabilization could increase survival of severe salt stress(Wang et al., 2010), and actin filament binding proteinshave been shown to affect stomatal regulation (Liu andLuan, 1998; Zhao et al., 2011), cell swelling in hypo-osmotic media (Liu et al., 2013), and pathogen re-sponses (Henty-Ridilla et al., 2013; Li et al., 2015). Also,pharmacological disruption of actin filaments alteredthe abundance of ABA-associated proteins (Takác et al.,2017). Conversely, actin filament organization may beaffected by leaf dehydration (�Sniegowska-�Swierk et al.,2016). As with endocytosis, there is relatively little dataon how actin filament organization and dynamicsare affected by longer-term moderate low cw stresswhere plants have time to adjust cellular processes andacclimate to low cw. Questions about stress effectson endocytosis and actin filaments are likely tointersect each other as actin filament disruption byLatrunculin B (LatB) or other pharmacological agents

1This work was supported by Academia Sinica (postdoctoral fel-lowship) and the Institute of Plant and Microbial Biology, AcademiaSinica.

2Author for contact: paulv@gate.sinica.edu.tw.3Senior author.The 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:Paul E. Verslues (paulv@gate.sinica.edu.tw).

M.N.K. performed experiments and analyzed data; Y.-C.B. andT.L. performed experiments; P.E.V. conceived research, analyzeddata, and prepared the manuscript with assistance from M.N.K.and Y.-C.B; P.E.V. agrees to serve as the author responsible for con-tact and ensures communication.

[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.18.01314

1594 Plant Physiology�, April 2019, Vol. 179, pp. 1594–1607, www.plantphysiol.org � 2019 American Society of Plant Biologists. All Rights Reserved.

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impairs endocytosis in plants (Šamaj et al., 2004). Inother organisms, the connection of actin filaments toendocytosis is relatively well known; however, in plants,this is unclear as a number of endocytosis or actinfilament-related proteins are not present or have dif-fering function (Šamaj et al., 2004).Previous work in our laboratory found that ectopic

expression of the stress-induced protein At14a-Like1(AFL1) led to enhanced growth maintenance and in-creased accumulation of the compatible solute Produring low cw (Kumar et al., 2015). Another studypublished at the same time indicated that over-expression of At14a, which is nearly identical to AFL1,could increase osmotic stress tolerance of suspension-cultured cells (Wang et al., 2015). At14a was alsoassociated with susceptibility to Agrobacterium tumefaciens-mediated transformation (Sardesai et al., 2013).At14a was first identified by immunoscreening an

Arabidopsis (Arabidopsis thaliana) expression libraryusing b-integrin-specific antisera (Nagpal and Quatrano,1999). However, the integrin similarity of At14a andAFL1 is limited to a small domain (now annotated as“Domain of Unknown Function 677”). Although somestudies have suggested At14a (and AFL1) to bemembrane-spanning proteins similar to mammalianintegrins (Lü et al., 2012; Sardesai et al., 2013; Langhanset al., 2017), we found that AFL1 is a peripheral mem-brane protein associated with the plasma membraneand endoplasmic reticulum (ER; Kumar et al., 2015).AFL1 interacted with AP2-2a, an adaptor protein in-volved in cargo selection and clathrin-coated vesicleformation (Kumar et al., 2015). Interestingly, structuralpredictions found similarity of AFL1 to amphiphysin, oneof several types of BAR domain protein that can inducemembrane curvature (Shen et al., 2012), as well as mi-crofilament binding proteins actinin and spectrin. Mam-malian amphiphysin, along with other BAR domainproteins, may connect clathrin-dependent endocytosis toactin microfilaments (Mooren et al., 2012). Amphiphysin,actinin, and spectrin have no clear orthologs in plants;conversely, AFL1 has no clear metazoan ortholog. Simi-larly, others have predicted that the C-terminal region ofAt14a has some similarity to human Myosin XVIIIB,while the At14a N-terminal domain is weakly related tohuman dynein H chain 7 and laminin (Langhans et al.,2017). In addition to our previous work, one other studysuggested that At14a affected both microtubule and actinfilament organization in protoplasts (Lü et al., 2012). De-spite these efforts, whether AFL1 affects endocytosis andcytoskeleton organization is unclear.We investigated the effect of low cw and AFL1 on

endocytosis and cytoskeleton organization usingmethods well established in our laboratory to repro-ducibly impose moderate severity low cw stress overextended periods of time. This was combined withquantification of bulk endocytosis (FM4-64 uptake) andcytoskeleton organization. We also quantified AFL1colocalization with actin filaments, microtubules, andclathrin light chain (CLC). Moderate severity low cwdecreased FM4-64 uptake in wild type. This effect was

counteracted by ectopic expression of AFL1 and exac-erbated by decreased AFL1 expression. AFL1 madeFM4-64 uptake more resistant to disruption of actinfilaments by LatB, altered actin filament organiza-tion under low cw, and partially colocalized with actinfilaments. These data clarify the effects of low cw onthese key cellular processes and give new evidence thatAFL1 is involved in endocytosis and actin filamentorganization.

RESULTS

FM4-64 Uptake Decreases during Low cw Acclimation

Normalized FM4-64 dye uptake (Bashline et al., 2013)was used to access the overall effect of low cw on en-docytosis. FM4-64 uptake was assayed under un-stressed conditions or after short- (6 h) and longer-term(96 h) exposure to a moderate-severity low cw (20.7MPa). Low cw treatments were imposed using highMrpolyethylene glycol (PEG), which cannot penetrate thecell wall and thus causes cytorrhysis, as typically ob-served for plants in drying soil, and not plasmolysis, asseen in solutions of low Mr osmoticum (Verslues et al.,2006). Over the 96-h period used in this study, Arabi-dopsis seedlings can acclimate to20.7 MPa to maintainwater content and have growth at ;30% of the levelseen in unstressed plants (Verslues, 2010; Kumar et al.,2015; Bhaskara et al., 2017).In wild type, we found that FM4-64 uptake decreased

by .40% after 96 h at low cw (Fig. 1A). It was inter-esting to note that the greatest reduction in FM4-64uptake occurred not at 6 h after transfer to low cw,where the plants may still be experiencing reduced tur-gor, but rather at 96 h, where water content and turgorare similar to that in the unstressed control (Verslues,2010). These data contrast with other studies of short-term dehydration or salt stress where increased endo-cytosiswas suggested to be away to decreasemembranearea during cell shrinkage (Zwiewka et al., 2015). Ourdata instead indicated that endocytosismay be regulatedin response to lowcw rather than being solely affected byphysical factors such as changes in turgor or cell volume.

AFL1 Promotes FM4-64 Uptake at Low cw and Makes itMore Resistant to Inhibition by LatB

To investigate the effect of AFL1 on FM4-64 uptake,we first assayed two transgenic lines that express35S:AFL1-FLAG (hereafter referred to as “35S:AFL1”)and have severalfold increase in AFL1 protein level(Supplemental Fig. S1A; Kumar et al., 2015). We foundthat 35S:AFL1 expression could counteract the decreasein FM4-64 uptake caused by low cw. At 6 and 96 h oflow cw treatment, 35S:AFL1 increased FM4-64 uptakeby 25% to 35% (Fig. 1, B and C). Because the 35S:AFL1data were normalized to wild type within each timepoint, the relative increase in FM4-64 uptake in 35S:

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Figure 1. AFL1 expression level influences the rate of endocytosis as measured by FM4-64 uptake and counteracts the inhibitoryeffect of low cw on endocytosis. A, Effect of low cw on FM4-64 uptake in root cells of wild type (W.T.). FM4-64 uptake wasmeasured in four independent experiments and the low cw (20.7 MPa for 6 or 96 h) data were normalized to the unstressedcontrol in each experiment. In each experiment, normalized FM4-64 uptake was measured for 10–20 cells. Data shown are themeans 6 SE (n = 4) of the normalization results from each experiment. Asterisks (*) indicate significant difference from the un-stressed control by one-sample Student’s t-test (P# 0.05). B, Effect of 35S:AFL1 and 100 nM LatB treatment on FM4-64 uptake inthe unstressed control and after 6 or 96 h of exposure to20.7 MPa. Data were analyzed as described in (A) except that the 35S:AFL1 and LatB datawere normalized versus wild type for each time point. Data shown aremeans6 SE (n = 3–4) of the results fromthree to four independent experiments with FM4-64 uptake measured for 10–20 cells in each experiment. Asterisks (*) indicatesignificant difference versus thewild type byone-sample Student’s t-test (P# 0.05). C, Representative images of FM4-64 uptake inroot cells of unstressed or stress-treated (20.7 MPa, 96 h) seedlings with or without 1-h treatment with 100 nM LatB. Scalebars = 20mm. D, FM4-64 uptake in root cells of AFL1 DEX-inducible RNAi lines (AFL1 K.D.) and empty vector (E.V.) control. Twoindependent RNAi lines were used, and combined data from both lines are shown. Data normalization and replication are asdescribed in (B), where two independent experimentswere performed; except that in this case, each individual measurement wasnormalized versus the mean FM4-64 uptake of the E.V. at that time point (n = 40–75). Asterisks (*) indicate significant differenceversus the wild type by one-sample Student’s t-test (P # 0.05). The E.V. + DEX versus AFL1 K.D. + DEX and AFL1 K.D. with orwithout DEX (significant difference indicated by yellow asterisk) were also compared by Student’s t-test. E, Effect of low cw onFM4-64 uptake in root cells of the E.V. control line. The E.V. line was tested both with andwithout DEX treatment. The 6- and 96-hlow cw treatments were normalized versus the unstressed control (time 0) data. Data shown are the means6 SE of individual cells(n = 45–60) combined from two independent experiments. Asterisks (*) indicate significant difference from the unstressed controlby one-sample Student’s t-test (P# 0.05). FM4-64 uptake was not significantly affected byDEX at either 6 or 96 h (Student’s t-test,P # 0.05).

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AFL1 in the 96-h low cw treatment was essentially areversal of the reduction in FM4-64 uptake seen in wildtype (compare Fig. 1, A and B). Conversely, dexa-methasone (DEX)-inducible RNA interference (RNAi)knockdown of AFL1 (hereafter referred to as “AFL1 K.D.”) decreased FM4-64 uptake in the unstressed controlas well as at 6 and 96 h after transfer to low cw (Fig. 1D).The knockdown lines had only a partial decrease in en-docytosis, consistent with the fact that AFL1 proteinlevel was only moderately decreased (Supplemental Fig.S1B). DEX treatment of the empty vector line had a smallbut significant effect on endocytosis (Fig. 1D). However,the DEX-treatedAFL1 K.D. line had a significantly largereffect under all conditions tested (Fig. 1D). When themock- or DEX-treated empty vector lines’ FM4-64 up-take at 6 or 96 h of stress was normalized to the un-stressed control, both had an essentially identical effectof stress that was similar to that seen in wild type(Fig. 1E, compare to Fig. 1A). Both the 35S:AFL1 andAFL1 K.D. data were consistent with AFL1 acting topromote endocytosis, as quantified by FM4-64 uptake.LatB was used to see if the low cw- and AFL1-induced

changes in FM4-64 uptakewere affected by actinfilaments.LatB binds to globular actin and inhibits its incorporationinto actin filaments (Holzinger and Blaas, 2016). In wildtype, LatB treatment reduced FM4-64 uptake by 30% to40% in both the control and low cw treatments (Fig. 1, Band C). Interestingly, the same LatB treatment caused nodecrease in FM4-64 uptake of 35S:AFL1 under either con-trol or low cw stress (Fig. 1, B and C).The effect of LatB on AFL1-CLC colocalization was

also tested as an indication of whether AFL1 effects onendocytosis could be related to actin filaments. LatBtreatment decreased AFL1-CLC colocalization in theunstressed control and blocked the stress-induced in-crease in AFL1-CLC colocalization seen at 24 or 96 hafter transfer to low cw (Fig. 2A). At 6 h after transfer,AFL1-CLC colocalization was variable between cellsand the effect of LatB was marginally nonsignificant. Inthe unstressed control, LatB clearly decreased theamount of CLC present along the along the plasmamembrane (Fig. 2B). Foci of AFL1-CLC colocalizationalong the plasma membrane could be observed (someprominent examples of such foci are indicated by ar-rows in Fig. 2B), consistent with our previous results(Kumar et al., 2015). Whether stress or LatB treatmentsaffected the frequency of such foci could not be reliablydetermined, as there was, in many cases, a nearly con-tinuous band of AFL1-CLC colocalization along the cellperiphery. This made it difficult to reproducibly defineindividual foci of colocalization. Despite this uncer-tainty, the LatB data together indicated that AFL1 ef-fects on endocytosis may be related to actin filaments.

Ectopic Expression of AFL1 Leads to More AggregatedActin Filaments at Low cw

To more directly determine the effects of low cwand AFL1 on actin filaments, 35S:AFL1 (line 8-1;

Supplemental Fig. S1) was crossed with a lineexpressing green fluorescent protein (GFP)-taggedFimbrin Actin Binding Domain 2 (fABD2; Sheahanet al., 2004). Note that in experiments including actinfilament visualization, the GFP-fABD2 line is referredto as “wild type” and GFP-fABD2 with AFL1 ectopicexpression is referred to as “35S:AFL1.” Actin filamentorganization was quantified based on the degree ofskewness, which indicates actin filament bundling oraggregation, and occupancy, which measures the dis-persion of the GFP-fABD2 signal (Higaki et al., 2010;Henty et al., 2011).Both low cw and 35S:AFL1 affected actin filament

organization. In unstressed plants (Fig. 3A, time 0), leafcells had low skewness and high occupancy, indicativeof a relatively dispersed actin filament array, while rootcells had a substantially higher skewness and hypo-cotyl cells were intermediate. Low cw increased skew-ness and decreased occupancy in all cell types (Fig. 3A),although the timing and extent of the effect differedbetween cell types. Interestingly, 35S:AFL1 increasedskewness and decreased occupancy at low cw but hadno significant effect in the unstressed control (Fig. 3A).A similar trend was also observed in basal hypocotylcells (Supplemental Fig. S2). Thick actin cables weremore prevalent in 35S:AFL1 compared to wild type(Fig. 3B). These data indicated that increased expressionof AFL1 led to more bundled actin filament arrays andthis effect was most prominent during low cw stress.The skewness and occupancy values we observed inunstressed wild type were overall similar to someprevious reports (Higaki et al., 2010; Cai et al., 2014),but lower than others (for example Henty et al., 2011; Liet al., 2012), possibly because of our use of light-grownseedlings rather than dark-grown seedlings, use ofmedia without added Suc, or imaging of young grow-ing leaves rather than mature leaves.These differences in actin filament organization

caused by 35S:AFL1 could indicate a specific effect ofAFL1 or a generally increased actin filament stability,which then indirectly leads tomore extensive bundling.We tested this possibility by treating wild type and 35S:AFL1 with a low concentration (100 nM) of Jasplaki-nolide (JASP). JASP promotes actin polymerization andfilament stability (Holzinger and Blaas, 2016). In un-stressed wild type, JASP treatment led to increasedskewness and decreased occupancy similar to the effectof 35S:AFL1 at low cw (Fig. 3C). In 35S:AFL1, JASPtreatment increased skewness and decreased occu-pancy in the unstressed control but had no additionaleffect at low cw (Fig. 3C). Inspection of individual im-ages (representative examples shown in Fig. 3D) con-firmed that both JASP and 35S:AFL1 increased theprevalence of large actin structures. However, the pat-terns were not identical in that 35S:AFL1 cells often hadlong, mostly longitudinal, actin bundles, whereas JASPtreatment produced many types of actin aggregates,including large actin foci with many branches (Fig. 3D).This was consistent with a previous report that treat-ment of hypocotyl cells with higher concentration of

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JASP (5 mM) produced short, thick actin filament ag-gregates that were less dynamic than untreated actinfilaments (Sampathkumar et al., 2011).

PearsonCorrelation Coefficient (PCC) analysis over atime series of images was used to determine how lowcw and 35S:AFL1 affected the dynamic rearrangementof actin filaments. In wild-type apical hypocotyl cells,there was a decrease in actin dynamics at 6 h of low cwtreatment (indicated by slower decline of PCC; Fig. 4A);however, by 96 h, the PCC profile returned to that ofunstressed plants. The profiles of PCC decline we

observed in wild type were of similar magnitude andpattern to those observed in previous studies (Vidaliet al., 2010; Cai et al., 2014; Li et al., 2015). In the un-stressed control, 35S:AFL1 had significantly faster PCCdecline than wild type (Fig. 4B), indicating more dy-namic rearrangement of actin filaments. After 96 h atlow cw, 35S:AFL1 had the opposite (but less dramatic)effect to decrease actin filament dynamics (Fig. 4B).Together, the data indicated that 35S:AFL1 could havelarge effects of actin filament organization while notsubstantially suppressing actin filament dynamics (or

Figure 2. Effect of LatB on AFL1-CLC colocali-zation. A, PCC analysis of root cells exposed tothe indicated duration of low cw (20.7 MPa)stress and treated with mock solution or 100 nMLatB for 1 h. In each plot, the box contains the25th–75th percentiles of data points, whiskersindicate the 10th–90th percentiles, and outlyingdata points are shown as gray circles. Gray linein each box indicates the mean, whereas theblack line indicates the median (in some casesthe black median line is obscured by the meanline). The P value for the comparison of mock toLatB treatment is shown in each graph. Data aremeans6 SE (n = 32–35 cells for unstressed mockand LatB treatments, 20–40 cells for stress mockand LatB treatments) combined from two inde-pendent experiments. B, Representative imagesof AFL1-CLC colocalization. For each treatment,the top image shows the YFP-AFL1 localization,middle image shows CLC-mOrange, and thelower image (labeled “Co” for colocalization) isthe merged YFP-AFL1 and CLC-mOrange im-ages to show colocalization. Areas of AFL1-CLCcolocalization are indicated by blue and whitecolor. Yellow arrows indicate examples of AFL1-CLC colocalized foci along the plasma mem-brane. Scale bars = 20 mm.

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Figure 3. Ectopic expression of AFL1 (35S:AFL1) leads to more aggregated actin filaments at low cw. A, Quantitative analysis ofactin filament skewness and occupancy in the indicated cell types for unstressed seedlings (time 0) or seedlings exposed tomoderate severity low cw (20.7 MPa) for 6 and 96 h. Data are means 6 SE (n = 10–15) combined from two independent ex-periments. Black asterisks (*) inside the wild-type bars indicate a significant difference compared to unstressed wild type(ANOVA, P# 0.05). Red asterisks above the 35S:AFL1 bars indicate a significant difference between wild type and 35S:AFL1 atthat time point. B, Representative images of cells used in the skewness and occupancy measurements. Images shown are fromunstressed plants and plants exposed to 20.7 MPa for 96 h. Images show the increased prevalence of thick actin cables in 35S:AFL1 at low cw, particularly in leaf tissue, consistent with the increased skewness and decreased occupancy shown in (A). W.T. =wild type. Scale bars = 20 mm. C, Quantitative analysis of JASP effect on actin filament skewness and occupancy in apical hy-pocotyl cells of unstressed seedlings (time 0) or seedlings exposed to moderate severity low cw (20.7 MPa) for 6 and 96 h.Seedlings were treated with 100 nM JASP for 1 h or mock treatment. Data are means 6 SE (n = 12–16) combined from threeindependent experiments. Letters on top of each bar indicate significantly different groups (ANOVA, P# 0.05) within each timepoint. For the wild-type (W.T.) mock and 35S:AFL1mock data, asterisks within the bars indicated significant difference comparedto time 0 (ANOVA, P # 0.05). D, Representative images of cells from unstressed control (time 0) or 96-h stress treatment for thedata shown in (C). W.T. = wild type. Scale bars = 20 mm.

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even increasing dynamics in the unstressed control). Incontrast, JASP treatment has been shown to largelyblock the decline in PCC over a similar time course(Vidali et al., 2010). This, along with the different ap-pearance of actin filaments in JASP-treated versus 35S:AFL1 plants, indicated that the mechanism by which35S:AFL1 promotes a more bundled and less dispersedactin filament organization is likely to be different, ormore specific, than that of JASP.

35S:AFL1 Plants Are More Resistant to the Effects of LatBand Cytochalasin D on Actin Filaments, But Are LessEffected in Microtubule Stability

We also observed the effects of two actin filamentdisruptors, LatB and Cytochalasin D (CytD), on actinfilaments in wild type and 35S:AFL1. While LatB in-hibits polymerization by binding actin monomers,CytD inhibits polymerization by binding to the barbedend of actin filaments. Both compounds caused exten-sive disruption of actin filaments in wild type (Fig. 5, Aand B). In contrast, 35S:AFL1 maintained more exten-sive actin filament arrays in the presence of either LatBor CytD (Fig. 5, A and B). This was consistent with theresults in Figure 1B showing that FM4-64 uptake in 35S:AFL1 was not affected by LatB. As our attempt to crossAFL1 K.D. lines with the fABD marker line were un-successful (AFL1 knockdown could no longer be ob-served after crossing), Phalloidin staining was used toinvestigate actin filament organization 35S:AFL1 andAFL1 K.D. Phalloidin staining of 35S:AFL1 indicated

more intact actin filaments after LatB treatment(Supplemental Fig. S3A), consistent with the resultsusing the fABD visualization of actin filaments (Fig. 5).AFL1 K.D. did not differ from wild type without DEXinduction (Supplemental Fig. S3B), but had less exten-sive actin filaments than the empty vector control aftertreatment with DEX and 50-nM LatB (Supplemental Fig.S3C). Also consistent with these results, 35S:AFL1plants had increased resistance to LatB inhibition ofroot elongation (Fig. 5C).

In contrast, there was no difference in root elongationsensitivity to oryzalin, which blocks tubulin polym-erization and causes loss of microtubule organiza-tion (Fig. 5C). We also found that plants expressing35S:yellow fluorescent protien (YFP)-AFL1 andmCherry-tagged MAP4 Microtubule Binding Do-main (mCherry-MTUB) had a similar loss of micro-tubule organization upon oryzalin treatment asplants expressing mCherry-MTUB in the wild-typebackground (Fig. 5D). The combined data of theseexperiments showed that AFL1 clearly affected actinfilament stability but did not have a similar effect onmicrotubules.

AFL1 Partially Colocalizes with Actin Filaments But Notwith Microtubules

The above results raise the question of whether AFL1directly regulates actin organization by binding to actinfilaments. Cosedimentation assays conducted withpurified recombinant AFL1 and in vitro-polymerized

Figure 4. Actin filament dynamics in wild type and 35S:AFL1 under control or low cw treatments. A, Quantification of actinfilament dynamics by PCC analysis in apical hypocotyl cells of wild-type seedlings in the unstressed control or after 6- and 96-hexposure to moderate severity low cw (20.7 MPa). Data are means 6 SE (n = 17–22) combined from three independent exper-iments with two seedlings and 3–4 cells per seedling analyzed in each experiment. Asterisk indicates significant differencecompared to the control (ANOVA, P# 0.05). B, Comparison of PCC profiles for wild type and 35S:AFL1 in the unstressed controland20.7 MPa low cw stress for 6 or 96 h. Data are from cells in the apical region of the hypocotyl. Wild-type data are the sameas in (A) and are replotted here for clarity of presentation. Data are means 6 SE (n = 17222) combined from three indepen-dent experiments as described for (A). Red asterisks indicate significant differences between wild type and 35S:AFL1 (ANOVA,P # 0.05).

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actin filaments found no evidence that AFL1 coulddirectly interact with actin filaments (SupplementalFig. S4). However, the results were complicated bythe fact that AFL1 was only partially soluble underconditions that allow actin filament polymerization.While these results suggested that AFL1 does notdirectly bind actin filaments, we cannot rule outmore specific AFL1 binding, such as binding to fila-ment ends, which would be difficult to detect in thistype of assay. We also emphasize that these data donot exclude other possibilities, such as a requirementfor additional proteins or posttranslational modifi-cation of AFL1 to mediate AFL1 association withactin filaments.As an alternative approach to investigate possible

AFL1-cytoskeleton association, we constructed linesexpressing YFP-AFL1 and mCherry-MAP4 ormCherry-tagged fABD2 (fABD2-mCherry) to assayAFL1-actin filament colocalization or YFP-AFL1 and

mCherry-MTUB for AFL1-microtubule colocalization.Projected images of Z stacks across the plasma mem-brane, cell cortex, and cytoplasm in leaf cells showedfoci of AFL1 colocalization with actin filaments alongwith extensive colocalization of AFL1 with thick actincables (Fig. 6A; examples of AFL1-fABD2 colocalizationfoci are indicated by arrows). AFL1 did not colocalizewith fine actin filaments except for the aforementionedfoci. Cells where thick actin cables were visible hadmore extensive AFL1-fABD2 colocalization than cellswhere only dispersed actin filaments were visible(Fig. 6A). In the Z-stack images, the colocalization ob-served may have included both plasma membrane andendomembrane (most likely ER)-associated AFL1. Wealso examined single optical slices through the cell in-terior to more specifically examine AFL1-actin filamentcolocalization along the cell periphery, possibly asso-ciated with the plasma membrane (Supplemental Fig.S5). In these images also, we found extensive AFL-actin

Figure 5. Ectopic AFL1 expression (35S:AFL1) alters actin filament response to LatB and CytD. A, Representative images of actinfilaments in leaf cells of wild type and 35S:AFL1 seedlings (actin filaments visualized by expression of GFP-fABD2) after mocktreatment or treatment with 100 nM LatB for 1 h. Scale bars = 20 mm. B, Representative images of actin filaments in leaf cells ofwild type and 35S:AFL1 seedlings after mock treatment or treatmentwith 100 nM CytD for 1 h. Scale bars = 20mm. C, Response ofwild-type and 35S:AFL1 root elongation to LatB or oryzalin. Four-d-old seedlings were transferred to plates containing the in-dicated concentrations of LatB or oryzalin and root elongation over the subsequent eight days measured. Data are means 6 SE

(n = 16) combined from two independent experiments. Asterisks (*) indicate significant differences (P # 0.05) of 35S:AFL1compared to wild type (no significant differences were observed for the oryzalin treatments). D, Representative images of mi-crotubule organization in leaf cells fromwild type and 35S:YFP-AFL1 plants expressing the mCherry-MTUBmicrotubule marker.Seven-d-old seedlings were sprayed with 20 mM oryzalin or a mock control 1 h before imaging. Scale bars = 20 mm.

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colocalization and many foci of colocalization along thecell periphery.

To further quantify AFL1-actin filament colocaliza-tion along the cell periphery, and to check colocaliza-tion in the same cell type used for FM4-64 uptake andCLC colocalization assays, we imaged single opticalslices through root cells and used PCC analysis toquantify the extent of colocalization. AFL1-fABD2colocalization PCC values were relatively high in theunstressed control, decreased at 6 h after transfer to lowcw, and then partially recovered at 96 h (Fig. 7A). Somefoci of AFL1-actin filament colocalization could be ob-served along the periphery of cells (see control images

in Fig. 7B), although the diffuse band of actin filamentsaround the cell periphery often obscured such indi-vidual foci. The AFL1-fABD2 PCC values observed inroot cells were overall similar to those observed in leafcells (compare Fig. 7A to Fig. 6A and Supplemental Fig.S5A). Interestingly, the decreased AFL1-actin filamentcolocalization at low cw differed from AFL1-CLCcolocalization, which increased under low cw (compareFig. 7A to Fig. 2).

In contrast, there was less colocalization betweenAFL1 andmicrotubules. Average PCC values for AFL1-MTUB colocalization were near 0 (indicating only ran-dom overlap of the YFP-AFL1 and mCherry-MTUBsignals) in the unstressed control for both leaf androot cells. (Figs. 6B and 7; Supplemental Fig. S5B). Inroot cells, the AFL1-MTUB colocalization increasedslightly 6 h after transfer to low cw (Fig. 7A). However,the colocalization observed at this time was not alongmicrotubule strands, and at this time after transfer tolow cw, it is expected that microtubules are at leastpartially disorganized. We also did not observe foci ofAFL1-MTUB colocalization comparable to the foci ofAFL1-fABD2 colocalization. In cases of relatively highAFL1-MTUB PCC values, the overlapping signal oc-curred in aggregates, which were more prevalent at 6 hafter transfer to low cw, and not along intact microtu-bules (examples of such aggregates can be seen inFig. 6B and Supplemental Fig. S5B). These microtubuleobservations were consistent with the oryzalin data,indicating that AFL1 had little effect on microtubules.The AFL1-MTUB data also provide an importantcomparison to the AFL1-fABD2 data, showing that thepattern of AFL1 actin filament colocalization is specificand likely to indicate a functionally important associ-ation. At the same time, the AFL1-fABD2 colocalizationpatterns, more extensive colocalization of AFL1 withCLC than with actin filaments, and lack of detectableAFL1 actin filament binding in vitro all suggest thatAFL1 does not indiscriminately associate with actinfilaments but rather has a specific pattern of colocali-zation that may depend on the presence of other actin-associated proteins.

DISCUSSION

There is little information on how drought acclima-tion influences endocytosis and actin filament organi-zation or of the proteins involved in the effects of lowcw stress on these key cellular processes. Plant-specificproteins are likely to be involved in regulating theseprocesses, but are little known. Our demonstration thatplants exposed to moderate-severity low cw for an ex-tended period of time have decreased endocytosis (asmeasured by FM4-64 uptake) and altered actin filamentorganization show new cellular aspects of low cw anddrought acclimation. AFL1 promoted endocytosis andinfluenced actin filament organization under low cwand also had a specific pattern of colocalization withactin filaments. Together with previous reports of

Figure 6. Colocalization of AFL1with actin filaments andmicrotubulesin leaf cells. A, Representative images of YFP-AFL1 and actin microfil-aments (visualized by fABD2-mCherry) in leaf cells of unstressedseedlings or 96 h after transfer to 20.7 MPa stress treatment. In themerged images (“Co-localization”), areas of AFL1-fABD2 colocaliza-tion are indicated by blue and white color. Yellow arrows indicate ex-amples of AFL1-fABD2 colocalized foci. Quantification of the extent ofcolocalization by PCC is indicated in each merged image. Green boxesindicate the area of interest used to calculate PCC. Scale bars = 20 mm.B, Representative images of YFP-AFL1 and microtubules (visualized bymCherry-MTUB) in leaf cells of unstressed seedlings or 96 h aftertransfer to 20.7 MPa stress treatment. Data presentation are as de-scribed in (A).

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increased AFL1 protein abundance under low cw,enhanced growth maintenance of 35S:AFL1 linesunder low cw, and interaction of AFL1 with end-ocytosis-related proteins (Kumar et al., 2015), thedata presented here indicate that AFL1 has a role incoordinating endocytic trafficking and actin filamentorganization with environmental signals such as lowcw.Endocytosis is a major mechanism to control plasma

membrane organization and protein content (Baisaet al., 2013; Baral et al., 2015; Fan et al., 2015). Becauseof this, it also influences many sensing and signalingpathways at the plasma membrane. Our observation ofdecreased bulk endocytosis rates under low cw may atfirst seem to contradict previous reports of increasedendocytosis in response to salt or osmotic stress(Zwiewka et al., 2015; Xia et al., 2016). However, thesestudies only examined the first few hours of osmoticstress where turgor is reduced and used lowMr solutesthat cause plasmolysis (shrinkage of the plasma mem-brane from the cell wall). In contrast, we found signif-icant reduction of FM4-64 uptake at 96 h after transfer

to low cw. At this time point, and given the moderatestress severity used (20.7 MPa), turgor pressure ispositive and has largely recovered for initial reductionof turgor that occurs in the first hours after transfer tolow cw (Verslues, 2010). Thus, the reduced endocytosiswe observed is unlikely to be a direct result of change inturgor or cell shrinkage but more likely to result fromspecific regulatory mechanisms controlling endocyto-sis. From our observations and relevant literature, wecan hypothesize that in plants there may be both a di-rect effect of cell shrinkage to stimulate endocytosis aswell as additional regulatory mechanisms that can alterendocytosis independently of turgor or cell volumechange. This hypothesis applies to bulk endocytosisand does not exclude that individual proteins havedifferent responses depending on their function.These data also demonstrate that AFL1 effects en-

docytosis. Increased AFL1 expression promoted FM4-64 uptake during low cw stress, while decreased AFL1expression inhibited FM4-64 uptake in all conditionstested. Whether this involves AFL1 interaction withAP-2a or interactions with other plasma membrane

Figure 7. Quantitative analysis of AFL1 colocalization with actin filaments andmicrotubules in root cells. A, PCC analysis of rootcells in the unstressed control or after exposure to the indicated duration of low cw (20.7 MPa) stress. In each plot, the boxcontains the 25th–75th percentiles of data points, whiskers indicate the 10th–90th percentiles, and outlying data points are shownas circles. Red line in each box indicates the mean, whereas the black line indicates the median (in some cases the black medianline is obscured by the mean line). Red asterisks indicated significant difference (P # 0.05) compared to the time-0 unstressedcontrol. Data are means 6 SE (n = 40–67 for AFL1-fABD2 colocalization and 34-45 for AFL1-MTUB colocalization) combinedfrom three independent experiments. B, Representative images of YFP-AFL1 and actin microfilaments (visualized by fABD2-mCherry) andmicrotubules (visualized bymCherry-MTUB) in root cells of unstressed seedlings or 96 h after transfer to20.7MPastress treatment. In the merged images (“Co-localization”), areas of colocalization are indicated by blue and white color. Yellowarrows indicate examples of AFL1-fABD2 colocalized foci. Quantification of the extent of colocalization by PCC is indicated ineach merged image. Green boxes indicate the area of interest used to calculate PCC. Scale bars = 20 mm.

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proteins or lipids is under investigation. Also, given thesubstantial effect of AFL1 on FM4-64 uptake, it will beof interest to use lines with increased or reduced AFL1expression to identify proteins whose plasma mem-brane abundance is controlled by AFL1 and lowcw-dependent mechanisms. This could reveal furtherstress-responsive plasma membrane proteins involvedin low cw response.

For AFL1, it is tempting to speculate that its effects onendocytosis and actin filaments are linked. The abilityof AFL1 to stabilize FM4-64 uptake against LatB inhi-bition and the foci of AFL1-actin filament colocalizationseen along the cell periphery all seem consistent withthis idea. Although compelling, such a hypothesis re-quires further investigation as our data also indicatethat AFL1 may not interact directly with actin filamentsbut rather needs additional proteins or posttransla-tional modifications to mediate its effect on actin fila-ment organization. The colocalization of AFL1 andactin exhibited two distinct patterns. While the smallpuncta of colocalization along the plasma membranesuggest an endocytosis or trafficking-related function,the colocalization along thick actin filaments is remi-niscent of proteins that link ER to actin filaments (seeCao et al., 2016). We previously observed ER-like pat-terns of AFL1 localization and found AFL1 in bothplasma membrane and endomembrane fractions(Kumar et al., 2015). However, it was unclear whetherAFL1 was inside the ER or associated with the cyto-plasmic side of the ER membrane. Given the pattern ofAFL1-actin filament colocalization, it seems likely thatAFL1 is also present on the cytoplasmic side of the ERmembrane. Interestingly, there is recent evidence thatER-associated proteins can directly participate in en-docytosis (Stefano et al., 2018). Whether AFL1 is in-volved in such a mechanism, as well as the mechanismconnecting AFL1 to actin filaments, is under investi-gation in our laboratory.

As our interest in AFL1 stems, in part, from its abilityto promote growth at low cw (Kumar et al., 2015), thequestion of whether AFL1 promotes growth via its ef-fect on endocytosis (which could alter the plasmamembrane protein profile), or via its effect on actin fil-ament organization (which could alter a range of traf-ficking and organelle positioning functions), is alsopertinent for further study. These possibilities are notmutually exclusive, and the possibility that more-indirect mechanisms are involved should be kept inmind. For example, AFL1 may affect cytokinin signal-ing or response (Sardesai et al., 2013; Kumar et al.,2015), and it has been recently reported that cytoki-nins promote actin bundling associated with rapid cellelongation in roots (Takatsuka et al., 2018). Conversely,AFL1 could be involved in mediating the effect of cy-tokinin on actin filament bundling.

More broadly, our observations on AFL1 illustratehow endocytosis and actin filament organization areless understood in plants than in yeast or metazoans. Inyeasts, which have turgor pressure, endocytosis isdependent on microfilaments (Aghamohammadzadeh

and Ayscough, 2009) and patches of actin polymeriza-tion generate force to drive membrane invaginationagainst the outward force of turgor pressure (Carlssonand Bayly, 2014; Lewellyn et al., 2015). In metazoancells, actin filaments are not always required for endo-cytosis but are required when the membrane is undertension (Aghamohammadzadeh and Ayscough, 2009;Boulant et al., 2011;Mooren et al., 2012). Plant cells havehigh turgor pressure and thus, similar to yeast, could behypothesized to require actin patches and actin fila-ment polymerization to drive membrane invagination(Mooren et al., 2012); however, such actin patches havenot been reported in plants, and the mechanisms bywhich actin filaments are connected to endocytosis inplants are little known. Mammalian actin-related pro-tein (ARP)2 and ARP3 coordinate actin polymerizationat endocytosis sites; however, in plants, these proteinshave different localization patterns and are not clearlyrelated to endocytosis (Konopka et al., 2008; Zhanget al., 2013). Plant-specific proteins, such as AFL1,which have roles in these basic cellular processes re-main largely uncharacterized. Further characterizationof AFL1 and the mechanisms by which it associateswith actin filaments and influences endocytosis mayhelp reveal plant-specific aspects of these basic cellularprocesses while also revealing important aspects ofdrought resistance.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

In a previous study, we demonstrated that 35S:YFP-AFL1 and 35S:AFL1-FLAG plants have essentially identical phenotypes (Kumar et al., 2015). FM4-64uptake experiments were conducted using lines with high expression of 35S:AFL1-FLAG (lines 4-2 and 8-1, Supplemental Fig. S1A; Kumar et al., 2015), andline 8-1 was crossed with 35S:GFP-fABD2 (kindly provided by ChristopherStaiger, Purdue University) to visualize actin filaments. Colocalization of AFL1and CLC was assayed using a 35S:YFP-AFL1/CLC-mOrange line previouslygenerated by Kumar et al. (2015). Lines with DEX-inducible RNAi knockdownof AFL1 were described in Kumar et al. (2015). Extraction and immunoblotdetection of AFL1 in transgenic plants was conducted as described in Kumaret al. (2015). Transgenic lines used for AFL1 colocalization with actin filamentsor microtubules are described below in the section "AFL1 Colocalization withActin Filaments and Microtubules".

Plants were routinely propagated for seed production in a growth room at23°C and 16-h light period. For plate experiments, seeds were sterilized andplated on agar plates followed by stratification for 3–4 d at 4°C and then placedvertically in the growth chamber at 23°C and continuous light (70–100 mmolphotons m22 s21) as previously described for experiments in our laboratory(Kumar et al., 2015; Bhaskara et al., 2017). The standard (unstressed) growthmedia consisted of half-strength Murashige and Skoog (MS) salts, 2 mM 2-(N-morpholino)ethanesulfonic acid buffer (pH 5.7), and 1.5% (w/v) agar withno sugar added.

Low cw Stress, Pharmacological Treatments, andGrowth Assays

Low cw stress was imposed by transferring 7-d-old seedlings (for actin fil-ament organization, FM4-64, and colocalization experiments) to 20.7 MPaPEG-8000 infused agar plates prepared using an established protocol (Verslueset al., 2006). To assay root elongation response to LatB or oryzalin, 4-d-oldseedlings were transferred to plates containing the indicated concentrations ofLatB or oryzalin, and root elongation was measured over the subsequent 7 d.Stocks of LatB (Sigma-Aldrich) and oryzalin (Sigma-Aldrich) were made in

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ethanol and stored at220°C. For actin filament observations and colocalizationexperiments, 100 nM LatB was sprayed onto seedlings 1 h before experimentalobservations were conducted (Staiger et al., 2009). Mock control plates weresprayed with 0.02% ethanol solution. For CytD (Sigma-Aldrich) and JASP(Sigma-Aldrich), stocks were made in dimethyl sulfoxide (DMSO) and stored at220°C. For observing their effect on actin organization, 100 nMCytD or JASP (or0.5% DMSO as a mock control) was sprayed onto seedlings 1 h before obser-vation. For observing the effect of oryzalin on microtubules, 20 mM oryzalin (orsame concentration of ethanol without oryzalin as a mock control) was sprayedonto seedlings 1 h before microtubule observation.

For dexamethasone (DEX) treatment, 4-d-old seedlings were transferred toplates containing 10mMDEX and grown for another 3 d. On d 7, seedlings weretransferred to20.7 MPa PEG-infused plates containing 10 mMDEX or the freshhigh cw control plates containing DEX. At the time of transfer and every daythereafter, 30 mM DEX (or mock solution for the minus DEX control) wassprayed on the seedlings to maintain induction of the RNAi construct.

Analysis of FM4-64 Uptake, AFL1-CLC Colocalization, andActin Filament Organization and Dynamics

An LSM-510 Meta confocal microscope (Zeiss) was used to observe actinfilament organization in the hypocotyl, root elongation zone, and leaves. GFP-fABD2 was excited at 488 nm and emission detected at 500–550 nm. Forcolocalization experiments, EYFP-AFL1 was imaged using excitation at 488 nmand emission at 560–575 nm, while CLC-mOrange was imaged using excitationat 514 nm and emission detected at 530–590 nm. Previous analysis showed thatthere was no bleed-through in signal between the YFP-AFL1 and CLC-mOrange images (Kumar et al., 2015). To quantify the extent of colocaliza-tion, areas of interest encompassing 1–2 cells (in root) or one cell or portion ofcell (leaf) were selected and PCC calculated using the Zeiss LSM-510 analysissoftware in Expert Mode. For quantification of actin filament skewness andoccupancy, cells were imaged using a fixed exposure time and gain settings setfor all images from each treatment and genotype and all time points of theexperiment. A maximum intensity projection of 15–20 image stacks was sub-jected for image processing and analysis in the software ImageJ (National In-stitutes of Health) performed as described by Higaki et al. (2010). For eachgenotype and treatment, 10–15 seedlings were analyzed over two or more in-dependent experiments.

For analysis of actin filament dynamics, imaging was conducted using anLSM-880 (Zeiss), which allowed imaging at lower laser power to avoidbleaching. Individual cells from the apical hypocotyl region were imaged every2 s over a 60-s time course. To quantify actin filament dynamics, 18–20 cells(combined from two to three independent experiments) from the apical hy-pocotyl region were used to calculate actin filament dynamics following theprotocols in Li et al. (2015) and Vidali et al. (2010). FIJI ImageJ with theColoc2 Plug-in was used to calculate a PCC between the time-0 image versuseach subsequent image over the 60-s time course. The acquisition parameterssuch as laser power and gain settings were standardized and the same settingsused for all actin dynamics experiments.

For assays of FM4-64 uptake, a stock solution of FM4-64 (Merck) was pre-pared in DMSO and aliquots stored at220°C. At the time of analysis, the stocksolution was diluted to 2 mM with half-strength MS medium for analysis ofunstressed seedlings or with a20.7 MPa solution of PEG-8000 in half-strengthMS for analysis of seedlings growing on low cw agar plates. Intact seedlingswere removed from agar plates and incubated in 2-mM FM4-64 solution in amicrofuge tube for 3 min on ice. After incubation, seedlings were washed withmedia lacking FM4-64, mounted on a glass slide and further incubated for12 min before imaging. Fully expanded root epidermal cells (20–40 mm fromthe root tip) were imaged. Imaging of FM4-64 uptake used excitation at 488 nmand emission at 575–610 nm. Normalized FM4-64 internalization was quanti-fied using ImageJ according to Bashline et al. (2013).

Actin filaments in leaves were stained with Alexa Fluor phalloidin (ThermoFisher Scientific) as described in Panteris et al. (2006) and Yang et al. (2011), butwith slight modification. Seven-d-old seedlings of AFL1 K.D. and empty vectorcontrol line were treatedwith DEX ormock control as described above and thentreated with 50 nM LatB or mock control for 1 h. 35S:AFL1 lines were treatedwith 100 nM LatB ormock control in the samemanner as for fABD imaging. Thiswas followed by incubation with 300 mM M-maleimidobenzoyl-N-hydrox-ysuccinimide ester in 50 mM piperazine-N,N9-bis(2-ethanesulfonic acid), 5 mM

magnesium sulfate, and 5 mM EGTA, pH 6.8 (PME buffer) plus 0.1% (v/v)Triton X-100 and 2% (v/v) DMSO for 30 min at room temperature in darkness.The samples were rinsed once with PME buffer and fixed with 2%

paraformaldehyde in PME buffer for 1 h. Samples were then rinsed with PMEbuffer and incubated with 200-nM Alexa-488 phalloidin in PME buffer at 4°Covernight in darkness. Images were collected using an LSM-510 Meta confocalmicroscope (Zeiss) with filters and other settings as described in Panteris et al.(2006) and Yang et al. (2011).

Expression and Purification of Recombinant AFL1

BL21(DE3) competent cells were freshly transformed with pET28-AFL1plasmid (to express His-tag AFL1). The LB-recovered primary transformantwas used to directly inoculate an overnight Luria-Bertani broth (Miller) pre-culture. We noted that overgrowth of the overnight preculture would severelyimpair the recombinant protein expression. A 3.5-mL overnight preculture wasused to inoculate 350 mL of fresh Luria-Bertani broth supplemented with50 mg/L of kanamycin sulfate. The culture was grown in a 37°C shaker incu-bator until OD600 nm reached 0.4–0.6 and was induced by addition of 1.0-mM

Isopropyl-b-D-thiogalactoside. The cells were grown for an additional 4 h at thesame culture condition before harvest. The cell pellet was resuspended in 6 mLof prechilled lysis buffer (50mMpotassiumP at pH 7.4, 200-mMNaCl, 10% [v/v]glycerol, 5-mM b-mercaptoethanol, 13 cOmplete, EDTA-free Protease InhibitorCocktail [Roche]) supplemented with 1% (v/v) IGEPAL CA-630 and 10 mM

Imidazole-chloride at pH 8.0, followed by sonication on ice using a 1/4-inchmicrotip controlled by the S-4000 ultrasonic processor (Misonix Sonicators). Thecell lysates were clarified by high-speed centrifugation at 13,000g for 30 min at4°C (Avanti J-26 XP centrifuge with JA-25.50 rotor; Beckman Coulter), followedby a 0.45-mm filtration (Millex-HP, 33 mm, polyethersulfone; Merck Millipore)before applying to a 1.53 10 cm gravity Econo-column (Bio-Rad Laboratories)packed with 2 mL of His-60 Ni Superflow Resin (Takara Bio USA). The lysatewas incubatedwith resin at 4°C for 2 h using end-over-end gentle agitation. Theresin was washed by 15 column volumes of lysis buffer, followed by 15 columnvolumes of wash buffer (lysis buffer supplemented with 49 mM Imidazole-chloride at pH 8.0) before elution. AFL1 was eluted with 20 mL of HisB elu-tion buffer (50 mM potassium P at pH 7.4, 200 mM NaCl, 10% [v/v] glycerol,5 mM b-mercaptoethanol, 0.1% [v/v] IGEPAL CA-630, 400 mM Imidazole-chloride at pH 8.0) and was concentrated using a 10-kD molecular-mass cut-off Amicon Ultra Filter (Merck Millipore) before liquid N snap-freezing andstoring at 280°C.

All purification fractions were examined using denaturing SDS-PAGE fol-lowed by Coomassie blue R-250 staining. The protein concentration was de-termined using the Bradford method (Bio-Rad Protein Assay Dye ReagentConcentrate; Bio-Rad Laboratories). This expression and purification procedurewould typically generate ;3 mg AFL1 recombinant protein from 350 mL ofculture.

AFL1:Filamentous Actin Cosedimentation Assay

All the components in the Actin Binding Protein Biochem Kit, Muscle Actin(BK001; Cytoskeleton) were reconstituted and handled following the manu-facturer’s instructions. The frozen AFL1 stock was slow-thawed on ice beforeconducting buffer exchange by Zeba Spin Desalting Column (7-kD molecular-mass cut-off; Thermo Fisher Scientific), which was pre-equilibrated with themodified actin buffer 20 mM Tris-chloride at pH 8.0, 200-mM potassium chlo-ride, 1.8 mM calcium chloride, 2.0 mM magnesium chloride, and 5 mM

b-mercaptoethanol (K200 buffer) or with other samples kept in the HisB elutionbuffer. The lyophilized actin was freshly reconstituted on ice and polymerizedinto filamentous actin at room temperature before use. The bovine serum al-bumin negative control and AFL1 were clarified using ultracentrifugation at150,000g for 60 min at 4°C (Optima MAX-XP ultracentrifuge with TLA-120.2rotor, Beckman Coulter). The AFL1 stock concentration in elution buffer andK200 buffer were 8.00–10.24 and 3.83–5.39 mg/mL, respectively. Protein andbuffer components were assembled following the manufacturer’s instructions,incubated at 24°C for 30 min and sedimented by ultracentrifugation at 150,000gfor 90min at 24°C. The pellets were resuspendedwith 1% SDS. The supernatantand pellet fractions were examined by denaturing SDS-PAGE followed by ei-ther Coomassie blue R-250 staining (3.2% of the reaction loaded) or antipoly-His immunoblot (1.6% of the reaction loaded). For immunoblot detection ofAFL1, protein was blotted onto polyvinylidene difluoride membrane by wet-tank procedure at 30 V for 16 h at 4°C. Membranes were blocked using 5%nonfat milk and probed with antipoly-His primary antibody (cat. no. H1029;Sigma-Aldrich) at 1:3,000 dilution followed by secondary antibody (rabbit anti-mouse IgG H&L [HRP], AbCam ab6728) at 1:10,000 dilution. Immunoreactivebands were visualized by the enhanced chemiluminescence method (Pierce

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ECLWestern Blotting Substrate, Thermo Fisher Scientific) on a charge-coupleddevice imaging system (UVP ChemiDoc-It 510, Analytik Jena).

AFL1 Colocalization with Actin Filamentsand Microtubules

Visualization of actin used previously described vectors (Ivanov andHarrison, 2014) in which expression of mCherry-tagged versions of eitherfABD2 (fABD2-mCherry) actin filament marker (Sheahan et al., 2004) or theMAP4 microtubule binding domain (mCherry-MTUB) microtubule marker(Marc et al., 1998) is driven by the Arabidopsis (Arabidopsis thaliana) Ubiquitin10promoter. Vectors were obtained from Addgene (pCMU-ACTFr, Addgene cat.no. 61191; pCMU-MTUBr, Addgene cat. no. 61196) and used to transform Col-0wild type. 35S:EYFP-AFL1 (line number 442/1-1 described in Kumar et al.,2015) was crossed with the fABD2-mCherry or mCherry-MTUB transgenicsand double homozygous plants isolated. An LSM-880 confocal microscope(Zeiss) was used to observe the mCherry signal from both fABD2 and MTUBusing excitation/emission wavelengths of 561 nm/630–650 nm, whereas AFL1was observed by the excitation/emission wavelengths of 488 nm/500–550 nm.To quantify colocalization in root cells, a single image plane from both channelswas captured by 403 apochromatic objective of the LSM-880 microscope. Re-gions of interest (where the focal plane went through the interior of one or morecells) were selected and PCC calculated using the Zeiss LSM-510 analysissoftware in Expert Mode. For leaf cells, PCC was calculated for both single-plane images as well as maximum projections of 15–20 Z-stack images.

Statistical Analysis

All experimental data shown were collected from two to four independentbiological experiments. Significant differences were determined by analysis ofvariance (ANOVA), implemented in the software SigmaPlot 12 (Systat Soft-ware), or by Student’s t-test.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Immunoblot assay of AFL1 protein levels inAFL1 RNAi (AFL1 K.D.) and overexpression lines.

Supplemental Figure S2. Actin filament skewness and occupancy of basalhypocotyl cells.

Supplemental Figure S3. Phalloidin staining of filamentous actin inAFL1 K.D. and 35S:AFL1 lines.

Supplemental Figure S4. AFL1 has little or no direct binding to actin fil-aments in vitro.

Supplemental Figure S5. AFL1-fABD2 or AFL1-MTUB colocalization inleaf cells.

ACKNOWLEDGMENTS

We thank Christopher Staiger (Purdue University) for the GFP-fABD2 line,Sebastian Bednarek (University of Wisconsin-Madison) for the CLC-mOrangeline, J.-Y. Huang andM.-J. Fang for microscopy assistance, the live cell imagingcore laboratory of the Institute of Plant and Microbial Biology for use ofequipment, Dr. Wei Siao for useful discussion, and Trent Z. Chang andShih-Shan Huang for laboratory assistance.

Received October 18, 2018; accepted January 31, 2019; published February 6,2019.

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