Calcium/calmodulin affects microtubule stability in lysed protoplasts

RICHARD J. CYR

Department of Biology, Pennsylvania State University, University Park, PA 16802, USA

Summary

Microtubules (Mts) are found in four distinct arrays appearing sequentially in a cell-cycle-dependent fashion within the cells of higher plants. Addition- ally, the cortical Mts of non-cycling cells are spatially altered in a variety of differentiated states. Infor- mation regarding the molecular details underlying these Mt-reorientation events in plant cells is scarce. Moreover, it is unclear how cytoskeletal behavior integrates with the myriad of other cellular activities that are altered concomitantly in both differentiating and cycling cells. Data are presented herein to

Introduction

The plant cytoskeleton, particularly the microtubule (Mt) component, plays several important roles during growth and development. These roles include, but are not limited to, participation in nuclear positioning (Mineyuki and Furuya, 1986; Mineyuki and Palevitz, 19901, karyokinesis (Baskin and Cande, 19901, cytokinesis (Gunning, 1982), and cellulose orientation (Seagull, 1989). Interestingly, the heterogeneity in Mt usage correlates with the sequential appearance and dissolution of at least four Mt arrays within the cell. The different arrays are known as the preprophase band, the spindle apparatus, the phrag- moplast, and the cortical array (Seagull, 1989). The events in which these arrays participate are fundamentally understood; however, information on the molecular details of their activities is limited. Additionally, although rearrangement of Mts seems to be a central aspect of cytoskeletal function in plant cells, little is known about the factors governing their spatial and temporal place- ment within the various arrays. In order to appreciate the factors affecting Mt appearance it is appropriate to first consider what is known about the biochemistry of Mts.

Mts are composed primarily of tubulin (Fosket, 19891, which is capable of self-assemblage into Mts. It is believed that cellular Mts are not static structures, but rather are constantly growing and shrinking. The state of 'dynamic instability' is thought to affect the spatial organization of Mts by selective stabilization of Mts within the cell (Kirschner and Mitchison, 1986). Presently, it is unclear what role, if any, dynamic instability plays in the formation of plant Mt arrays. However, because the different arrays appear to arise (at least in part) de novo it seems likely that factors that stabilize as well as destabilize Mts will be important in the sequential appearance and dissolution of the various arrays. There- Journal of Cell Science 100, 311-317 (1991) Printed in Great Britain 0 The Company of Biologists Limited 1991

indicate that calcium, in the form of a Ca2+/ calmodulin complex, can alter the behavior of Mts in lysed carrot protoplasts. Mechanistically, we show that Ca2+ /calmodulin most likely interacts with Mts via associations with microtubule associated pro- teins (MAPS). These results are discussed with reference to how Ca2+ may alter the dynamic behavior of Mts during growth and development.

Key words: microtubules, cytoskeleton, calcium, calmodulin.

fore, it is important that factors influencing the stability of Mts within the cells of higher plants be identified.

Tubulin is known to exist in different isoforms within plant cells (Hussey et al. 1988) and it is possible that these isoforms may differentially affect the assembly state of Mts (Sullivan, 1988); however, this is yet to be documented in plant cells. Besides tubulin, the role that other molecules may play in the formation of Mts must be considered. For example, Mts contain other proteins termed microtubule associated proteins (MAPs) which bind to and affect the behavior of Mts in vitro (Olmsted, 1986; Tucker, 1990; Cyr, 1991). This class of proteins has been identified in higher plants where they are known to affect both the morphology and the stability state of in vitro assembled Mts (Cyr and Palevitz, 1989). It is likely that MAPs play a role in the organization of Mts within the cell and, therefore, it is necessary to consider how they, and tubulin, are integrated into cellular function. For example, what cues are Mt proteins responsive to in order to ensure their proper function at the appropriate time and place within the cell?

It has been suggested that plant cells use calcium as a second messenger for a number of processes of develop- mental importance (Hepler and Wayne, 1985; Allan and Hepler, 1989). Some of these processes include events in which Mts are known to participate, most notably karyokinesis (Hepler, 1989). Additionally, it was pre- viously demonstrated that cortical Mts are sensitive to calcium (Cyr et al. 1987). However, relatively high concentrations of calcium were used to disrupt Mts and therefore the physiological relevance of the observation was uncertain. The present study was initiated to identify conditions that would alter the sensitivity of Mts towards calcium. The experiments reported here were prompted by the observation that calcium often functions via inter- mediates such as calmodulin, a ubiquitous calcium

binding protein (Allan and Hepler, 1989). Moreover,calmodulin has been shown to affect the sensitivity state ofMts in animals cells (Keith et al. 1983), but until now itwas unclear if a similar effect could be demonstrated inplants. The present data show that calmodulin alters thesensitivity of Mts to calcium by at least two orders ofmagnitude. This finding is discussed in terms of howcalcium might function within the growing and differen-tiating cell to affect cytoskeletal behavior.

Materials and methods

Plant materialCarrot (Daucus carota) cell suspensions were cultured aspreviously described (Cyr and Palevitz, 1989). Cells wereconverted into protoplasts using standard enzymatic methods(Hahne et al. 1983). Incubations in enzymes did not exceed 3h.After conversion, protoplasts were filtered through cotton,collected by centrifugation at 300 g for 5 min, and washed twice inPM buffer (50 mM Pipes (pH 6.9), 1 HIM MgSO4,1 HIM EGTA) withmannitol added to 0.4 M as an osmoticum. PM buffer sup-plemented with mannitol is referred to as PMM.

Lysing of protoplastsProtoplasts, suspended in PMM were typically settled onto poly-L-lysine coated slides (applied as a lmgml"1 solution;Mr=300xl03; Sigma Chemicals, St Louis, MO) for 5min. Excesssolution was removed by capillary action and the detergent lysisbuffer (PMM, 10 jug ml each of the following protease inhibitors;antipain, aprotinin, chymostatin, leupeptin, and pepstatin-CHAPs detergent added to 10 mM) was applied for 5 min. Forvarious subsequent treatments, the slides were then washed 3 xin 50 mM Pipes (pH6.9) and lmM MgSO4, then exposed to thetreatment. All calcium and/or calcium/calmodulin treatmentswere carried out in Pipes (pH6.9) and lmM MgSCi. AnEGTA/calcium buffer system was employed when the requiredcalcium level was less than 100 J.IM.

For some experiments the protoplasts were lysed in solution,the last change of PMM was removed and a small volume of lysisbuffer added for 5 min. Lysed cytoskeletons were collected bycentrifugation {500 g, 5 min), washed once in a large volume ofPipes (pH 6.9) and 1 mM MgSO,j, pelleted again and then exposedto various treatments. The reaction was again centrifuged and thesupernatants removed. The pellet was washed in a large volumeof P M + 10JUM taxol and centrifuged in a microfuge at 15 000 g for10 min. The resulting pellet was solubilized with 100 j.i\ of 8 M ureaand the urea-insoluble material removed by centrifugation anddiscarded.

Immunolocalization of MtsMts were visualized using a modification of established pro-cedures (Lloyd et al. 1980). Following treatments, the lysedprotoplasts (on poly-L-lysine-coated slides) were fixed for 20 minwith 4 % formaldehyde (made fresh from paraformaldehyde) and0.1% glutaraldehyde in PM buffer supplemented with 1%glycerol. The fix was removed, the lysed cells washed withphosphate-buffered saline (PBS), next blocked with 3% BSAdissolved in PBS and then the primary anti-tubulin antibody(anti-/? tubulin; Amersham Corp., Arlington Heights, IL, or ananti-soy tubulin: Cyr et al. 1987) was added and incubated for45 min. After rinsing for 15 min in PBS, an appropriate secondaryantibody, conjugated with FITC, was added and incubated for45 min followed by a 15 min wash in PBS. The slides weremounted in 4 M glycerol, lOOmM Tris, pH9.0, lmgrnl"1 phenyl-enediamine, lmgml"1 Hoescht 33258 (Calbiochem, La Jolla,CA). The slides were viewed with a Zeiss Axioskop (Carl Zeiss,Thornwood, NY) equipped with a 150 W xenon epifluorescentilluminator and a 100 x objective. Photomicrographs wereobtained using Tri-X film, which was exposed and developednominally.

Image analysisMicrotubule frequency was quantified by capturing the fluor-escent images using a Silicon Intensified Tube camera (Hama-matsu Photronics, Hamamatsu City, Japan). The images weredigitized using a PC-Vision Plus video digitizer (ImagingTechnology Inc. Woburn, MA. USA) mounted in a CompuAdd286-12 microcomputer (Compuadd Corp., Austin, TX. USA). Theimages were displayed on a Sony PVM 1344Q (Sony Corp. ofAmerica, Denver, CO. USA) and analyzed with the aid of Canopysoftware (Rich, 1990). Briefly, an area of interest was defined andoverlayed onto the digitized image of the protoplast. Using athreshold feature of the image analysis software, all grey valuescorresponding to Mt images were converted to white anddisplayed. A routine was executed that computed the number ofblack, white, and total pixels in the area of interest. Thepercentage of white pixels then corresponded to the relative Mtfrequency. A minimum of two treatment slides were made foreach data set and the results averaged. The selection of cells forquantification was based on a fixed sampling pattern that wasconsistent for all slides and treatments.

Isolation of calmodulin and construction of a calmodulinaffinity columnBovine brain calmodulin was isolated from fresh calf brains usingpublished procedures (Gopalakrishna and Anderson, 1982).Carrot calmodulin was isolated from carrot cell suspensions usingmodifications of published procedures (Gopalakrishna and Ander-son, 1982; Charbonneau et al. 1983). Briefly, carrot cell suspen-sions were homogenized and the total proteins subjected toammonium sulphate precipitation at pH 7.5. After centrifugation,the supernatant was titrated to pH4.1 and the precipitatedcalmodulin collected by centrifugation. The isoelectric-precipi-tated calmodulin was resolubilized at pH7.5 and subsequentlypurified to apparent homogeneity (data not shown) using phenyl-agarose affinity chromatography.

To prepare the calmodulin for immobilization onto agarose theproteins were dialyzed against 0.1 M NaHCO3, pH8.5. The carrotand bovine calmodulins (1 mg each) were then mixed and added tolml of Affi-gel 15 (prepared according to the manufacturer'sinstruction; Bio-Rad Laboratories, Richmond, CA) and allowed tobind overnight at 4°C. Two batches were prepared, one in thepresence (2niM) and one in the absence of Ca2+. Analysis of thesolution after binding showed that >90 % of the applied proteinwas immobilized onto the agarose. The final column, containing2 ml of gel was then used for affinity purification of calmodulinbinding proteins.

Results

Rationale for using a lysed cell systemCarrot protoplasts were selected as an experimental modelfor two reasons. First, the cell wall is capable ofsequestrating large amounts of calcium (DeMarty et al.1984) while protoplasts afford a model system that permitscalcium concentrations to be known with a reasonabledegree of certainty. Second, plant protoplasts are easier tolyse than are walled cells and a lysed cell model spans thegap between whole-cell behavior and traditional biochem-istry.

An EGTA extractable component is required for Ca2+ todepolymerize MtsCalcium depolymerizes plant Mts (Cyr et al. 1987).However, the biochemical basis of this effect in plant cellsis unclear. Calmodulin has been implicated as a molecularparticipant in cell types where the effect of calcium uponMts has been extensively examined. Evidence to dateindicates that calcium first binds to calmodulin, thecalcium/calmodulin complex in turn, interacts with Mts toaffect their stability. Often, the binding of calmodulin to a

312 R. J. Cyr

Fig. 1. EGTA alters Mt sensitivity to subsequent calciumtreatment. Protoplasts extracted with lysis buffer containingEGTA possess readily demonstrable Mt arrays (A). If, calcium(1 min) is included in the lysis buffer few Mts are present (B).However, when protoplasts are extracted in lysis buffercontaining EGTA, rinsed in calcium free-buffers, then exposedto high levels of calcium (1 HIM), Mts are easily demonstrated(C). Scale bar, 5/tm.

variety of intracellular target sites requires the presenceof calcium (Daview and Klee, 1981). Therefore, EGTA (acalcium chelator) has been used to experimentally disasso-ciate calmodulin from its site of action. The present studyalso exploited this phenomenon by investigating ifpreextraction of lysed protoplasts with EGTA altered thesensitivity of Mts to subsequent exposure to calcium.

In agreement with previous reports (Cyr et al. 1987)Fig. IB illustrates that the inclusion of 1 mM free calciumin protoplast lysis buffer results in the destabilization ofMts. In order to ascertain if this effect might becalmodulin-dependent, protoplasts were first extracted inlysis buffer containing EGTA. The lysis buffer was

Fig. 2. Calmodulin resensitizes the Mts to calcium in EGTA-treated lysed protoplasts. Protoplasts lysed in the presence ofEGTA were rinsed with low calcium buffer (as in Fig. 1C), thenexposed to IOJIM calmodulin+500;<M calcium; Mts aremarkedly affected (A). Protoplasts, similarly lysed and rinsed,were exposed to 10 j.tM calmodulin alone without an obviousaffect upon Mt images (B). Scale bar, 5/jm.

subsequently removed; the extracted protoplasts washedin EGTA-free buffer, and calcium then added to 1 mM.Under these conditions, as shown in Fig. 1C, relativelylittle effect upon Mts could be observed (compare with acontrol cell shown in Fig. 1A), demonstrating that Mtsensitivity to calcium requires an EGTA-sensitive inter-mediate.

Addition of exogenous calmodulin restores the ability ofCa2+ to destabilize MtsThe finding that Mt sensitivity to calcium requires anEGTA-sensitive intermediate implicates calmodulin inthe process of calcium-induced destabilization. If so, thenapplication of exogenous calmodulin should restore cal-cium sensitivity. When carrot protoplasts are lysed in thepresence of EGTA and washed in EGTA-free buffer andsubsequently exposed to 500 f-iM free calcium there is noobvious effect upon the Mts (data not shown). However,Fig. 2A shows that if similarly treated protoplasts areexposed to 500 f/M calcium and calmodulin, the Mts aremarkedly affected. Moreover, Fig. 2B shows that calmodu-lin alone does not discernibly effect the distribution of Mts,indicating that Ca2+/calmodulin as a complex affects Mtsin this lysed cell model.

Although phragmoplasts, cortical arrays, and prepro-phase bands appeared affected by the action of Ca2+/calmodulin it was observed, as shown in Fig. 3A, that Mtsassociated with condensed chromosomes (and presumablyspindle components) are relatively resistant. Only meta-

Calcium/calmodulin and microtubules 313

Table 1.

Fig. 3. Mts associated with metaphase chromosomes arerelatively insensitive to Ca2+/calmodulin. Protoplasts werecollected by centrifugation and resuspended in lysis buffercontaining EGTA, the cytoskeletons were collected bycentrifugation, rinsed in low calcium buffer, and treated with20 ;IM carrot calmodulin and 500 ;JM calcium. A small samplewas taken for in situ localization of Mts, which revealed onlyspindle Mts to be intact; arrow (A). The preparations were alsostained with the chromatin binding dye Hoescht 33258 tovisualize chromosomes (B). Scale bar=5/<m.

phase figures were noted and therefore it is uncertain ifthe kinetochore and interzonal Mts are equally stableunder these conditions.

Microtubule frequency in the lysed cell model can bequantifiedIn order to study the effect of Ca2+/calmodulin upon Mtstability with greater precision, it was necessary toquantify Mt frequency in the lysed cell model. This wasaccomplished using computer-assisted image analysis.Computer-assisted fluorimetry enables the assignment ofnumerical values that approximate the frequency of Mtsas assessed using immunocytochemical localization. Ad-ditionally, it is possible to statistically analyze treatedcells by adopting a consistent search pattern for eachtreatment slide. Table 1 lists these quantitative results,which were found to be consistent with our qualitativeassessment (compare with Figs 1, 2). Furthermore, theability to apply statistical analysis permits the degree ofsignificance to be addressed.

In order to corroborate and extend the microscopicassessment of the effect of calmodulin upon Mt stability,an independent biochemical assay was used. This assayrelies upon the observation that assembled Mts andunassembled soluble protein can be separated by differen-tial centrifugation techniques because the Mt polymer is

Lysis regime

EGTACa2+

EGTA followedby Ca2+

EGTA followedby calmodulin

EGTA followedby Ca2+/calmodulin

EGTA followedby Ca2+/calmodulin

EGTA followedby Ca2+/calmodulin

[Calcium]

<0.01flMlniMlmM

<0.01 /«M

500 /jut

10 JIM

[Calmodulin]

NDNDND

20 IIM

20fiM

5/IM

5/1M

RelativeMt frequency

41.5±2.55.5±1.638±2.9

34±2.6

3.2±1.0

17±2.6

16±2.0

Protoplasts were treated as outlined in the legend for Fig. 1. Therelative frequency refer;images within the area

s to the percentage of pixels occupied by Mtof analysis. All data are ±s.E. ND, not done.

1 2 3 4 5 6 7 8 9

Fig. 4. Sedimentation analysis indicates Ca2+/calmodulindepolymerizes Mts. Protoplasts were lysed and treated as inFig. 3. The cytoskeletons treated with Ca2+/calmodulin werethen collected by centrifugation at 50 000 g for 30 min and thesupernatants removed. The pellets were solubilized using 8 Murea. Equal volumes of supernatants and solubilized pelletswere separated by SDS-PAGE followed by immunoblotanalysis using anti-tubulin antibodies. Both the pellets (equalloadings; lanes 1-4) and the supernatants (equal loadings;lanes 5-8) were separated by SDS-PAGE followed byimmunoblot analysis using anti-soybean tubulin antisera.Protein A/alkaline phosphatase was used as the reportermolecule. Lanes 1 (pellet) and 5 (supernatant), PM buffer only.Lanes 2 (pellet) and 6 (supernatant), 20;IM calmodulin only.Lanes 3 (pellet) and 7 (supernatant), 500 JJM calcium only.Lanes 4 (pellet) and 8 (supernatant), 500 //M calcium and 20 /.IMcalmodulin. Lane 9, purified carrot tubulin (2 jig).

denser than the nonassembled tubulin subunits. Proto-plasts were prepared and lysed as before in the presence ofEGTA. Following a brief wash, the lysed cells were treatedwith calcium (in the presence and absence of calmodulin).Following centrifugation, the pellets were subjected toimmunoblot analysis using anti-tubulin antibodies. Fig. 4(lane 4) shows that a small amount of tubulin sedimentedfollowing Ca2+/calmodulin treatment, indicating thatlittle tubulin was polymerized following exposure to theCa2+/calmodulin complex; moreover, more tubulin wasnoted in the supernatant following this treatment (Fig. 4,lane 8). Little effects were noted with uncomplexed Ca2+

or calmodulin (Fig. 4, lanes 2 and 3).

Ca2+/calmodulin interacts with MAPs to affect MtstabilityThe above data indicates that Ca2+/calmodulin caninteract with the cortical microtubules of carrot cells toalter the stability of Mts. What is the nature of this

314 R. J. Cyr

Lysis regime

EGTA, NaCl elution, then Ca2H

EGTA, NaCl elution, then Ca2H

EGTA, NaCl elution, then Ca2"

Protoplasts were treated as outlined in

Vcalmodulinh/calmodulinH/calmodulin

Fig. 5. Values

Table[Calcium]

500/IM500 jiM500 ;IM

as in Table 1.

2[Calmodulin]

20 JIM

20 jiM20 /(M

[NaCl]

0.1 M0.2M

0.3 M

Relative Mt frequency

2.0±0.614.5±3.118.5+3.1

Fig. 5. MAPs are involved in Ca2+/calmodulin destabilizationof Mts in lysed protoplasts. Protoplasts were lysed in theabsence of calcium, then extracted using 0.3 M NaCl, andwashed 3x in EGTA-free buffer. They were then exposed to500 jiM calcium+20/(M calmodulin; Mts are still demonstrable(A). Protoplasts were next extracted and treated as in A, but inthe presence of Mt-binding proteins (B).

interaction? One possibility would be for Ca2+/calmodulinto interact directly with the polymerized tubulin. A secondpossibility is for Ca2+/calmodulin to interact with a non-tubulin component of the Mts. To help distinguish betweenthese possibilities, lysed protoplasts were treated withincreasing concentrations of NaCl and upon salt removalthe cells were exposed to Ca2+/calmodulin. Table 2, aswell as Fig. 5A, illustrates that with increasing concen-trations of salt pretreatment there is a significant decreasein the ability of Ca2+/calmodulin to destabilize Mts. Theseresults suggested that Mts might possess a salt-labilecomponent which interacts with Ca2+/calmodulin to alterMt behavior.

MAPs are a prominent non-tubulin component of Mts.These proteins are known to associate with tubulin viaionic interactions. The finding that Ca2+ action in thelysed cell model also requires a salt-sensitive componentsuggests that Ca2+/calmodulin affects Mt stability (atleast in part) via an interaction with MAPs. Thispossibility was examined by extracting lysed protoplasts

with EGTA as well as with salt. Next, carrot microtubulebinding proteins, prepared as previously described (Cyrand Palevitz, 1989), were added with Ca2+/calmodulin.Microtubule images were not observed, as depicted inFig. 5B, under these conditions.

Polypeptides that have similar Mr values to knownMAPs bind calmodulin in vitroThe finding that the Ca2+/calmodulin effect upon Mts inlysed protoplasts could be modulated by salt treatmentand that Mt-binding proteins can restore sensitivitysuggests that MAPs may be involved in mediating theinteraction of Ca2+/calmodulin to Mts.

To obtain further evidence for this association, calmodu-lin was immobilized onto agarose and used as an affinitymatrix. After equilibration with lmM Ca2+, a proteinfraction harboring at least two carrot MAPs possessing Mrvalues of 76 000 and 129 000 was applied in the presence oflmM Ca2+. The column was washed extensively withlmM Ca2+, then lmM Ca2+ plus 100mM KC1, and lastlyeluted with EGTA. The eluted peak was collected andanalyzed by SDS-PAGE as shown in Fig. 6; numerouspolypeptides are observed. Interestingly, two of these havesimilar Mr values to those previously reported to bind Mts(Mr=76000 and 129000; Cyr and Palevitz, 1989).

Discussion

The data show that Ca2+, in the form of a Ca2 +/calmodulin complex, affects the stability of cortical Mts inlysed carrot protoplasts. The role of cortical Mts indirecting the orientation of cellulose microfibrils (amorphogenically important event; Seagull, 1989), and thefact that calcium is thought to act as a regulator of cellularactivity (including actions of morphogenic relevance;Hepler and Wayne, 1985), make it compelling to consider arole for Ca2+/calmodulin in affecting Mt dynamics withinthe intact cell. Although additional physiological studiesto delineate such an in vivo role for Ca /calmodulin areclearly needed, it is appropriate to consider the impli-cations of the present findings.

It is known that the various Mt arrays participate in anumber of important cellular processes at various pointsin the cell cycle; however, it is unknown how the cellcontrols the appearance and positioning of Mts atappropriate times. In order to manage these differentarrays the cell must possess mechanisms by which Mts areintegrated with global cellular activities during the cellcycle. Cellular signals must exist by which Mts respond atthe appropriate time, and in the correct manner, to carryout their allotted tasks. What might these signals be?

Calcium is believed to be an important regulatory ion inplant cells (Hepler and Wayne, 1985), although otherregulatory moieties undoubtedly exist. The present dataindicate that Mts are sensitive to this ion at physiologicalconcentrations and because calmodulin is required toachieve this sensitivity the cell may have an additional

Calcium/calmodulin and microtubules 315

1 2 3 4Fig. 6. Polypeptides having similar Mr values to known carrotMAPs bind to calmodulin in vitro. A protein fraction obtainedusing a CM-Trisacryl column was applied (lane 2), in thepresence of lmM Ca2+, to a lml column to which calmodulinhad been covalently bound to agarose. The unbound proteinswere collected (lane 3). The column was extensively washedwith Ca2+, and Ca2+/KCl-containing buffers until the OD280 ofthe washing buffers reached the baseline. The column wasthen eluted with EGTA-containing buffer and the elution peakwas collected (lane 4). The solid circles denote the twopolypeptides having similar migration characteristics as twoMt-binding proteins (Cyr and Palevitz, 1989). The open squaredenotes the polypeptide showing a Mr value of approximately52 000. Lane 1 contains relative molecular mass standards;205000: 116000: 97 400: 66000: 45 000: 29000. The gel was a10 % SDS-PAGE gel stained with Coomassie blue.

level of regulatory modulation at its disposal to affect Mtbehavior, e.g. control over calmodulin levels. Indeed,calmodulin levels are reported to be variable within aplant. The higher levels reported in apical meristematicregions (Allan and Trewavas, 1985) may reflect a role forcalmodulin in cell division processes, including theinfluence upon Mt dynamics.

In considering the mechanism(s) by which Ca2+/calmodulin may alter Mt behavior in the intact cell thepresent data obtained from lysed cells must be carefullyinterpreted. There are at least three possible alternativesto explain the current data. First, Ca2+/calmodulin mayfunction to increase the rate of Mt shrinkage, perhaps by'decapping' a stabilized Mt. If the event is considered interms of dynamic instability then the number of cata-strophic decay events would be increased in the presence ofthis complex. A second possibility is that Ca2+/calmodulinaffects the bridging of Mts to the plasma membrane or todetergent, insoluble factors still present after lysis.Indeed, there are suggestions in the literature that thecortical Mts are stabilized by a physical bridge to theplasma membrane (Kakimota and Shiboaka, 1986).Although this second scenario may be operative, it doesnot explain our current data as we have found that lysedprotoplasts, containing Mt arrays stabilized by the drugtaxol, are unaffected by Ca2+/calmodulin (data not

shown). A third possibility is that Ca2+/calmodulin affectsMt arrays by increasing the overall dynamic state ofindividual Mts. In this situation the rate of dimer additionmay increase as well as the frequency of decay events. Theuse of a lysed cell model does not permit discriminationbetween this third possibility and the first because afterlysis the soluble dimer pool will be diluted below the levelnecessary for rapid addition, i.e. below the criticalconcentration. The first or third possibility are the mostlikely direct mechanistic routes followed by the Ca2+/calmodulin complex to alter the presentation of Mts inlysed cells and these possibilities are currently underinvestigation.

The finding that maximal effect of Ca2+/calmodulinrequires the presence of a salt-sensitive component and,furthermore, that Mt-binding proteins can restore sensi-tivity, implicates MAPs in the process as well. CertainMAPs, termed STOPs (Stable Tubule Only Proteins), havean affinity for calmodulin (Margolis et al. 1986). Othershave shown that Mts assembled in the presence of STOPsare relatively stable; however, upon addition of Ca2+/calmodulin the stabilized Mts become destabilized (Job etal. 1981). STOP proteins have been purified usingcalmodulin affinity chromatography (Margolis etal. 1986),and we have undertaken preliminary work to ascertain ifsimilar proteins exist in carrot cells. To do this we testedthe ability of the proteins that were isolated from ourcalmodulin affinity column to affect Mt stability in aCa2+/calmodulin-dependent fashion. Although these pro-teins were able to support the assembly of tubulin (5 J/M),the Mts so formed were cold labile (data not shown).Although STOP proteins do not appear to reside in thesecarrot cell preparations we believe that calmodulinaffinity columns will aid in determining the composition ofother carrot Mt proteins involved in the association ofcalmodulin to Mts. Our current working hypothesis is thatboth calmodulin, and one or more MAPs, are probableparticipants in an integrative pathway linking Mtfunctions (via changes in dynamic behavior) to requisitecellular activities.

The finding that spindle Mts are apparently insensitiveto the destabilizing action of Ca2+/calmodulin is notsurprising as previous immunolocalization data showcalmodulin to be bound to these Mts both in plants(Vantard et al. 1985; Wick et al. 1985) and animals (Welshet al. 1978). The significance of this interaction is unclear.Some think calmodulin may assist in the depolymeriz-ation of Mts in kinetochore fibers as the chromosomesmigrate during anaphase (Vantard et al. 1985). However,another possibility is that calmodulin may actuallystabilize Mts in kinetochore fibers (Sweet et al. 1988). Inview of the differential stability of mitotic Mts it iscompelling to consider that one mechanism by whichcortical Mts (including the preprophase band) disappearprior to, or during, mitosis involves the differentialregulation by a Ca2+/calmodulin complex.

Previous immunolocalization data in plant cells haveshown calmodulin to be localized to the spindle apparatus(Vantard et al. 1985; Wick et al. 1985). Additionally, Wicket al. (1985), have reported its presence in the phragmo-plast, and occasionally in the preprophase band. Calmodu-lin has not been immunolocalized in the interphasecortical arrays, possibly because interphase Mts withbound calmodulin are unstable, as the data hereindemonstrate. Therefore, it may be problematic to demon-strate the association of calmodulin with Mts that aredestabilized by the Ca2+/calmodulin complex.

316 R. J. Cyr

The author would like to thank Randy Wayne for providingadvice as well as the computer program used to computeCa2+/EGTA buffers and to Deborah Fisher for helpful commentsduring the preparation of this manuscript. This work wassupported by the U.S. Department of Agriculture-CompetitiveResearch Grants Office Grant 89-37261-4591.

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(Received 16 May 1991 - Accepted, in revised form. 1 July 1991)

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