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Originally published In Press as doi:10.1074/jbc.M106628200 on November 2, 2001

J. Biol. Chem., Vol. 277, Issue 2, 1301-1309, January 11, 2002
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The Human EMAP-like Protein-70 (ELP70) Is a Microtubule Destabilizer That Localizes to the Mitotic Apparatus*

Bernd Eichenmüller, Patrick Everley, Jean Palange, Denise Lepley, and Kathy A. SuprenantDagger

From the Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045

Received for publication, July 16, 2001, and in revised form, October 17, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report, we show that the echinoderm microtubule (MT)-associated protein (EMAP) and related EMAP-like proteins (ELPs) share a similar domain organization with a highly conserved hydrophobic ELP (HELP) domain and a large tryptophan-aspartic acid (WD) repeat domain. To determine the function of mammalian ELPs, we generated antibodies against a 70-kDa human ELP and showed that ELP70 coassembled with MTs in HeLa cell extracts and colocalized with MTs in the mitotic apparatus. To determine whether ELP70 bound to MTs directly, human ELP70 was expressed and purified to homogeneity from baculovirus-infected Sf9 cells. Purified ELP70 bound to purified MTs with a stoichiometry of 0.40 ± 0.04 mol of ELP70/mol of tubulin dimer and with an intrinsic dissociation constant of 0.44 ± 0.13 µM. Using a nucleated assembly assay and video-enhanced differential interference contrast microscopy, we demonstrated that ELP70 reduced seeded nucleation, reduced the growth rate, and promoted MT catastrophes in a concentration-dependent manner. As a result, ELP70-containing MTs were significantly shorter than MTs assembled from tubulin alone. These data indicate that ELP70 is a novel MT destabilizer. A lateral destabilization model is presented to describe ELP70's effects on microtubules.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell motility, cell morphogenesis, spindle formation, and chromosome movements require the dynamic turnover of microtubules (MTs).1 This non-equilibrium behavior is an intrinsic property of MTs driven by the irreversible hydrolysis of tubulin-liganded GTP and the structural polarity of the tubulin heterodimer (1, 2). Individual MTs can switch between extended periods of slow growth and rapid shrinkage. The transitions between these two states, known as catastrophe and rescue, are quite sudden and are believed to be stochastic events (3, 4). This dynamic behavior of MTs is best described by the dynamic instability model and is generally associated with the behavior of MT plus-ends nucleated at the centrosome (5, 6).

MT dynamics in living cells are highly regulated. For example, MTs in vivo polymerize more rapidly and undergo more frequent catastrophe and rescue events than do MTs assembled from pure tubulin in vitro (7). These results indicate that there are cellular factors that regulate individual parameters of MT dynamics (1). In addition to having cell type or tissue specific factors (8), the cell must regulate the activity of each factor during cell division (9, 10), during cell differentiation (11), and within different regions of the cell (12).

Recently, several proteins that destabilize MTs have been characterized. In yeast, Kar3 is a minus-end-directed kinesin that localizes to spindle poles in vivo and destabilizes MTs at their minus ends in vitro (13, 14). Op18/stathmin is a low molecular weight tubulin-binding protein that destabilizes MTs in tissue culture cells (15) and in Xenopus egg extracts (16). XKCM1, a member of the Xenopus Kin I kinesin family, promotes catastrophes by favoring a conformational change at the MT ends that leads to protofilament peeling and MT shortening (17, 18). Katanin uses the energy of ATP hydrolysis to sever MTs at the centrosomes (19, 20). It is clear that several proteins with different modis operandi destabilize MTs in the cell.

One of the most abundant microtubule-associated proteins (MAPs) in dividing sea urchin embryos is the echinoderm MT-associated protein (EMAP) (21-23). EMAP is a 75-kDa, WD repeat protein with little sequence similarity to other well-characterized MAP families (24, 25). In early embryos, EMAP localizes to the MTs of the first cleavage mitotic apparatus where it is hyperphosphorylated (26-28). Although the function of EMAP in cells is unknown, its evolutionary conservation suggests that it plays an important role in MT assembly and function in a variety of cells (25). Here, we characterize a 70-kDa human EMAP-like protein (ELP70), show that it is a novel MT destabilizer, and suggest that it is an important regulator of MT dynamics during the cell cycle.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Reagents-- HeLa cells (American Type Culture Collection) were cultured at 37 °C in 5% CO2 with Dulbecco's modified Eagle's medium/F-12 Hams' plus 2 mM L-glutamine, 10% fetal bovine serum (Invitrogen), 10,000 units/ml (1670 units/mg) penicillin, and 10 mg/ml streptomycin.

Paclitaxel (Taxol) was a gift of the Drug Synthesis and/Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, NCI, National Institutes of Health. GTP (tetralithium salt) was purchased from Roche Molecular Biochemicals and was stored as a stock solution in distilled water at -80 °C.

Monoclonal anti-beta -tubulin (TUB 2.1) was from the Sigma Chemical Corp. Cy2- and Cy3-conjugated donkey anti-mouse and rabbit secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc.

Expression and Purification of Recombinant ELP70-- ELP70 was expressed as a 6His fusion protein in baculovirus-infected Sf9 cells. The full-length ELP70 coding sequence was inserted into the KpnI and NdeI restriction sites of the baculovirus transfer vector pAcHLT-C (BD PharMingen Inc.). Sf9 insect cells were transfected with this construct and the linearized BaculoGold baculovirus DNA. Active viruses generated by homologous recombination (Ac6HisELP70) were isolated as individual plaques that were subsequently amplified and used for infection of Sf9 cells.

Sf9 cell lysates were prepared at 72 h post-infection by sonication in 50 mM Na2HPO4/NaH2PO4, 0.5 M NaCl, pH 7.5, 200 µM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 10 µg/ml pepstatin A, and 20 µg/ml aprotinin. The lysate was centrifuged (10 min at 39,000 × g), and 6HisELP70 was purified from the supernatant fluids by cobalt-affinity chromatography (TALON metal affinity resin, CLONTECH Laboratory Inc.) and size exclusion chromatography (BIO-GEL P60, Bio-Rad Inc.). 6His ELP70 was concentrated by vacuum dialysis into 80 mM PIPES/K+, 1 mM MgSO4, 1 mM EGTA, pH 6.8 (BRB80). The final concentration of ELP70 was ~0.35 g/liter (4.7 µM).

ELP70 MT Binding Assays-- Tubulin was purified to homogeneity from bovine brain by three cycles of MT assembly followed by phosphocellulose chromatography (29, 30). MTs were polymerized at 28 °C by the stepwise addition of paclitaxel to a final concentration of 20 µM. A constant amount of paclitaxel-stabilized MTs (10 µg) was mixed with ELP70 in BRB80 containing 1 mM GTP and 20 µM paclitaxel. After 30 min, MTs were pelleted at 100,000 × g (10 min). Bound and free ELP70 was quantitated from Coomassie Blue-stained gels as described below. Purified ELP70, for which the protein mass had been determined by a BCA assay (see below), was used as an internal standard on the same gel. Control experiments were carried out with identical concentrations of ELP70 in the absence of MTs. Densitometric analysis of the gels was carried out with a Hewlett-Packard ScanJet and quantitated using IMAGE version 1.6.1 (National Institutes of Health, available at rsb.info.nih.gov/nih-image). To obtain the number of ELP70 molecules, the area under each protein peak was converted to total mass units and divided by 70,000, the relative molecular mass of ELP70.

MT Dynamics Assay-- The behavior of individual MTs nucleated from Chlamydomonas flagellar axonemes was visualized using video-enhanced differential interference contrast (VE-DIC) microscopy as described previously (31). The microscope stage was maintained at 37 °C with an air curtain incubator, and the samples were preincubated for 30 min to reach a steady state prior to data collection. Data were collected during the following 30-45 min.

A Nikon Optiphot microscope equipped with rectified DIC optics and high extinction HN-32 polarizers was used to visualize individual MTs. The light beam from a 100-watt mercury lamp passed through a fiber optic light scrambler (Technical Video, Ltd.), a 546-DF 24-nm interference filter and KG-5 heat-absorbing filter, and a 1.4-numerical aperture (NA) condenser, and images were formed with a 60× PlanApo 1.4-NA objective. Background-subtracted and contrast-enhanced images were captured by a Hamamatsu C2400 Newvicon camera and Argus-10 image processor. Super VHS format recordings were digitized with a Video Van Gogh board (Tekmatic Systems Inc.) and analyzed with the Real Time Measurement software (kindly provided by Ted Salmon, University of North Carolina, Chapel Hill, NC). MT growth and shortening rates were analyzed by a Student's t test.

Affinity-purified ELP70 Antibodies-- For protein expression, an ELP70 cDNA encoding amino acids 31-648 was cloned into the Xho/BamHI site of the pET14b vector (Novagen, Inc.) and was transfected into the lysogenic Escherichia coli strain BL21(DE3) plysS (32). Following isopropyl-1-thio-beta -D-galactopyranoside induction, inclusion bodies containing the expressed protein were isolated and fractionated on a preparative SDS-polyacrylamide gel. Rabbit polyclonal antibodies were generated against the electro-eluted ELP70 (Cocalico Biologicals, Inc.).

Affinity-purified antibodies were isolated by column chromatography (33). Briefly, purified inclusion bodies were solubilized in 0.1 M sodium borate, pH 9.0, 0.5 M NaCl, and 0.5% SDS and coupled to CNBr-activated Sepharose 4B (Amersham Biosciences, Inc.) (34). Anti-ELP70 antiserum (UK176) was diluted 1:1 in TBS (15 mM NaCl, 20 mM Tris-HCl, pH 7.4), sterile-filtered (0.2 µm), and passed over the column five times. After washing as described previously (33), antibodies were eluted with 0.2 M glycine-HCl, pH 2.0, or 6 M guanidine HCl in TBS. Antibody-containing eluates were dialyzed into TBS, concentrated with a Centricon 30 (Amicon Corp.), made 50% in glycerol, and stored at -20 °C.

HeLa MT Preparation-- MTs were assembled and purified from 5 g of frozen HeLa cells as described previously (35). Briefly, HeLa cells were homogenized in 2 volumes of BRB80 containing a protease inhibitor mixture that contained 158 mg/ml benzamidine, 10 mg/ml leupeptin, 2 mg/ml pepstatin, 1 mg/ml aprotinin, 1 mg/ml antipain, 1 mg/ml chymostatin, and 0.5 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 18,000 rpm (Beckman JA 20 rotor) for 10 min at 2 °C. The pellet was discarded, and the supernatant was centrifuged at 45,000 rpm (Beckman Ti65 rotor) for 90 min at 2 °C. Paclitaxel and GTP were added to the supernatant at a final concentration of 20 µM and 0.5 mM, respectively. The cytosolic extract was briefly warmed to 37 °C (3-5 min) and then chilled for 15 min on ice. The cytosolic extract was transferred to a chilled centrifuge tube and underlayed with ice-cold BRB80 containing 10% w/v sucrose, 20 µM paclitaxel, and 0.5 mM GTP. MTs were pelleted through the sucrose cushion by centrifugation at 18,000 rpm (Beckman JA20 rotor) for 30 min at 2 °C. MT proteins were analyzed by SDS-PAGE and immunoblotting.

Immunofluorescence Microscopy-- HeLa cells were fixed with 3% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) in PBS (15 min at room temperature). After fixation, cells were rinsed three times (~5 min each) in PBS and permeabilized with 0.15% (w/v) saponin in PBS (5 min). Fixed and permeabilized cells were incubated in primary antibodies diluted into PBS with 0.15%(w/v) saponin (aldehyde fixation) for 1 h at 37 °C. After washing five times with PBS, the primary antibodies were detected Cy3- or Cy2-conjugated donkey anti-mouse or anti-rabbit secondary antibodies diluted in PBS containing saponin. After 1 h at 37 °C coverslips were rinsed five times in PBS and mounted on microscope slides in a solution comprised of 90% (v/v) glycerol, 10% (v/v) PBS, pH 9.0, and 5% (w/v) n-propyl gallate.

For immunofluorescence microscopy, cells were viewed with a Nikon Optiphot microscope, and images were recorded on T-MAX 400 film. Negatives were digitized, and images were contrast-enhanced with Adobe Photoshop (Adobe Photosystems, Inc.).

RNA Blot Analysis-- Random-priming of an ELP70 retina cDNA (H92185) generated a [32P]CTP-labeled ELP70 probe that was hybridized to a human cancer cell line Multiple Tissue Northern blots (CLONTECH Laboratories Inc.).

SDS-PAGE and Western Blotting-- Proteins were analyzed on SDS-denaturing polyacrylamide gels stained with Coomassie Blue (36). Gels were scanned and quantitated by densitometry using the IMAGE software, version 1.61 (rsb.info.nih.gov/nih-image). For standardization, known amounts ELP70 were run on the same gel.

Proteins were electrophoretically transferred from SDS-PAGE gels to polyvinylidene difluoride membranes (Millipore, Inc.) (37), probed with affinity-purified anti-ELP70 antibodies, and visualized by chemiluminescence with alkaline phosphatase-conjugated secondary antibodies (Tropix Inc.).

Determination of Protein Concentration-- Protein concentration was determined by the bicinchoninic acid (BCA) assay (Pierce Inc.) using bovine serum albumin as a standard.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Conservation of a Hydrophobic EMAP-like Protein (HELP) Domain-- The echinoderm MT-associated protein (EMAP) is a member of a family of EMAP-like proteins (ELPs) found in many metazoans and at least one protozoan (Fig. 1). In humans, there are three EMAP-like proteins (ELPs): ELP70, ELP79/EMAPL, and ELP120/ropp120 (GenBankTM accession numbers AF103939, U97018, and AAG09279). The gene for ELP79, the first EMAP-like protein (EMAPL) identified in humans, was positionally cloned from the Usher syndrome 1A locus at 14q32 (38). The role played by ELP79 in Ushers is unknown at this time, but cytoskeletal defects are believed to be responsible for the retinitis pigmentosa, vestibular dysfunction, and deafness, characteristic of this disorder (39, 40). ELP70 was identified and cloned by homology to sea urchin EMAP and is the most similar to sea urchin EMAP (41), sharing 57% sequence identity and 77% sequence similarity over the length of the polypeptide. At 981 amino acids in length, ELP120/ropp120 is the largest member of the ELP family in humans. ELP120/ropp120 is less conserved over the full-length of the polypeptide, but only because there are long NH2- and COOH-terminal sequences not found in either ELP79 or ELP70. Interestingly, ELP120/ropp120 was originally identified as the restrictedly overexpressed proliferation-associated protein (ropp), which is dramatically overexpressed during mitosis (42).


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  		Fig. 1.   a, ClustalW analysis of the HELP domain. Conserved amino acids are boxed with identical amino acids shaded dark gray and similar amino acids shaded light gray. The consensus sequence is shown below the alignment. Abbreviations used: H. sapiens (Hs), S. purpuratus (Sp), L. variegatus (Lv), D. melanogaster (Dm), C. elegans (Ce), and E. octocarinatus (Eo). b, Sweet/Eisenberg hydrophobicity plot of the HELP domain (amino acids 51-93) of Hs ELP70. c, domain organization of ELP70. The HELP domain resides within the amino terminus (box). Over 75% of the polypeptide is comprised of a series of eleven possible WD repeats (ovals). The positions of these repeats are at amino acids 157, 203, 250, 292, 335, 375, 416, 455, 503, 570, and 615. This WD repeat domain is predicted to be slightly acidic, whereas the amino terminus of ELP70 is predicted to be basic.

In addition to the human ELPs, complete ELP-coding sequences are predicted for the ciliate Euplotes octocarinatus (AJ251505), the sea urchins, Strongylocentrotus purpuratus (U15551) and Lytechinus variegatus (AF136234), the fruit fly, Drosophila melanogaster (CT32830), and the nematode, Caenorhabditis elegans (Z92833).

A comparison of eight ELP sequences revealed a highly conserved 43-amino acid domain that begins with an invariant proline residue (Fig. 1, a and b). This hydrophobic ELP (HELP) domain is 97% conserved between human and sea urchin ELPs and has not been detected in other known proteins. This high degree of conservation among ELP family members indicates that the HELP domain is likely to be critical to ELP function. For example, a preliminary study has shown that the NH2 terminus of sea urchin EMAP, which contains the HELP domain, is sufficient for MT binding in vitro (43).

The NH2-terminal region of ELP70 is also very rich in the amino acids proline, serine, and arginine. Although there is no striking sequence homology between this region and the known MT binding domains of other MAPs, serine- and proline-rich domains have been identified in MAP-4 (44). In addition, all members of the ELP family share a conserved SGGG motif (amino acids 310-314, ELP70) perhaps analogous to the PGGG repeat domain of MAP-2, MAP-4, and tau (45, 46). Unlike the latter MAPs, the SGGG motif is only found once in the ELP sequence.

Over 75% of the ELP70 polypeptide is comprised of a series of 11 domains that satisfy the regular expression pattern of a WD repeat domain (Fig. 1c). WD repeats are found in a variety of proteins involved with RNA processing, transcriptional regulation, cytoskeleton assembly, and vesicular trafficking (bmerc-www.bu.edu/wdrepeat). The crystal structure of the Gbeta subunit of the heterotrimeric G protein, transducin, revealed that a WD repeat adopts a beta -propeller fold comprised of a four-stranded anti-parallel beta -sheet (47). By analogy to beta -transducin, ELP70 may fold into an 11-bladed propeller structure.

ELP70 Is Expressed in Cancer Cell Lines-- ELP70 is expressed in a variety of normal tissues from a single gene located on chromosome 19 (41). To determine if ELP70 was expressed in human cancer cell lines, we probed a Northern blot with a 550-bp probe corresponding to the COOH terminus of ELP70 (Fig. 2). The ELP70 probe recognized a major 2.5-kb band in HeLa (S3), chronic myelogenous leukemia (K-562), lymphoblastic leukemia (MOLT-4), colorectal adenocarcinoma (SW480), lung carcinoma (A549), and melanoma (G361) cells. The ELP70 probe also hybridized to a 3.2-kb major band in promyelocytic leukemia (HL-60) and Burkitt's lymphoma (Raji), indicating that there may be tissue-specific alternative splicing of ELP70 transcripts. The 3.2-kb band was also observed in HeLa, colorectal adenocarcinoma, lung carcinoma, and melanoma cells but at much lower levels (data not shown). These results are interesting in light of the fact that, in normal human tissues, the 3.2-kb band is only detected in brain and spinal cord (41). This raises the possibility that ELP70 RNA processing is altered in tumor cells.


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  		Fig. 2.   Northern blot containing 2 µg of poly(A)+RNA per lane from eight different human cancer cell lines. Lanes 1-8 contain, in order, RNA from human promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361 cells. A 3.2- and 2.5-kb band hybridized with the human ELP70 probe. The human beta -actin probe hybridized with a 2.0-kb band.

Endogenous ELP70 Cosediments and Colocalizes with HeLa Cell MTs-- Because ELP70 transcripts were abundant in HeLa cells, we used antibodies generated against a bacterially expressed ELP70 fusion protein to determine if ELP70 proteins were associated with HeLa MTs. Affinity-purified ELP70 antibodies recognized a polypeptide with a molecular mass consistent with the conceptually translated ELP70 cDNA (70 kDa) and not the related ELP79 (79 kDa) or ELP120 (120 kDa) cDNAs (Fig. 3).


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  		Fig. 3.   SDS-polyacrylamide gel (GEL) and immunoblot (BLOT) showing that ELP70 was enriched in a paclitaxel-stabilized HeLa MT preparations. Mr, molecular weight markers × 10-3; lane 1, HeLa cell extract (150,000 × g supernatant); lane 2, MT-depleted supernatant; lane 3, HeLa MT pellet. The arrow points to the 70-kDa ELP70 polypeptide. Equal amounts of protein (60 µg) were loaded in each lane.

To determine whether HeLa ELP70 was an MT-binding protein, endogenous HeLa MTs were analyzed by SDS-PAGE. The immunoblot shown in Fig. 3 shows that the 70-kDa ELP70 polypeptide was greatly enriched in the MT pellet and depleted in the MT-depleted supernatant. These results are consistent with the ELP70 polypeptide being an MT-binding protein in HeLa cells.

Because ELP70 is associated with MTs in vitro, the affinity-purified antibodies were used to examine the intracellular distribution of ELP70 in dividing HeLa cells. The images presented in Fig. 4 show that ELP70 was concentrated in the region of the mitotic spindle apparatus.


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  		Fig. 4.   Localization of ELP70 to the region of the HeLa mitotic apparatus (a, b, and c) by indirect immunofluorescence. HeLa cells were double-stained with affinity-purified antibodies against ELP70 (a) or tubulin (b). The ELP70 staining (red) and tubulin staining (green) were overlaid in panel c (yellow). Bar = 10 µm.

ELP70 Binds to MTs in a Concentration-dependent Manner-- To determine whether ELP70 bound directly to MTs, a 6His-ELP70 fusion protein was purified from baculovirus-infected Sf9 insect cells and incubated with paclitaxel-stabilized MTs (Fig. 5). Purified ELP70 bound directly to purified bovine brain MTs. In similar assays no ELP70 sedimented in the presence of actin filaments, intermediate filaments, or in the absence of MTs.


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  		Fig. 5.   ELP70 binds to MTs in vitro. a, stages in the expression and purification of 6HisELP70 shown on a Coomassie Blue-stained SDS-polyacrylamide gel. Lanes 2-5, Sf9 proteins at 0, 24, 48, and 72 h post-infection with a recombinant baculovirus expressing ELP70. 6HisELP70 was purified from baculovirus-infected Sf9 cell lysates (lane 6) by immobilized metal affinity chromatography (lane 7) and gel filtration chromatography (lane 8). Purified ELP70 is shown in lane 8, indicated by the arrow. Molecular weight standards (Mr) × 10-3 are shown in lane 1. b, immunoblot of Sf9 cell extracts showing time course of ELP70 expression, visualized with an affinity-purified anti-ELP70 antibody. c, an MT pull-down assay was used to assess the binding of ELP70 to MTs. ELP70 (3 µg) was incubated in the presence (+) and absence (-) of paclitaxel-stabilized bovine brain MTs (10 µg). Proteins remaining in the MT-depleted supernatant (S) and MT pellet (P) are shown.

The results of several binding assays with a constant amount of paclitaxel-stabilized MTs indicated that ELP70 bound to MTs in a concentration-dependent manner. From these data, an equilibrium-binding isotherm was obtained (Fig. 6). ELP70 binding to bovine brain MTs was saturated at a stoichiometry of 0.40 ± 0.04 mol of ELP70/mol of tubulin dimer and the intrinsic dissociation constant (Kd) for ELP70 binding to MTs was determined to be 0.44 ± 0.13 µM. This level of saturation indicated that ELP70 binds along the length of the MT polymer. In support of this conclusion, we found no evidence that ELP70 bound preferentially to MT ends. The same quantity of ELP70 copelleted with longer MTs as with an equal mass of shorter MTs produced by shearing with a syringe (data not shown).


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  		Fig. 6.   Binding isotherm for 6HisELP70 to MTs. A two-parameter Langmuir-type equilibrium equation given below was fitted to the set of experimental data, assuming that there was only one saturable binding site for ELP70 on the tubulin dimer, q = (qmax × c)/(Kd + c). In this equation, q denotes the amount of EMAP that is bound to tubulin heterodimers and c is the concentration of free EMAP in solution. Kd and qmax are the model parameters obtained from the fitting, representing the dissociation constant and the amount of ELP70 bound to tubulin at the saturation level.

6HisELP70 Alters MT Dynamics in Vitro by Reducing Seeded Nucleation, Promoting Catastrophes, and Reducing the Growth Rate-- To examine the effect of ELP70 on MT dynamics in vitro, individual MTs nucleated from axonemes were visualized by VE-DIC microscopy. As shown in Fig. 7, seeded nucleation was dependent upon the ELP70 concentration. The number of MTs emanating from the plus-ends of individual axonemes was reduced from an average value of 4.67 ± 1.44 MTs per axoneme (no ELP70) (n = 15, where n = number of axonemes) to 3.43 ± 1.17 MTs per axoneme (0. 5 µM ELP70) (n = 21) and 2.23 ± 0.93 MTs per axoneme (0.8 µM ELP70) (n = 13). The reduction in seeded nucleation was statistically significant (p < 0.05) at both concentrations of ELP70.


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  		Fig. 7.   ELP70 reduced seeded nucleation in a concentration-dependent manner. MTs emanating from individual axonemes were counted, and the average numbers are presented with standard deviations. The number of MTs growing from the plus-ends of individual axonemes was reduced from an average value of 4.67 ± 1.44 MTs per axoneme (no ELP70) (n = 15, where n = number of axonemes) to 3.43 ± 1.17 MTs per axoneme (0. 5 µM ELP70) (n = 21) and 2.23 ± 0.93 MTs per axoneme (0.8 µM ELP70) (n = 13).

In addition to reducing seeded nucleation, plus-end MT lengths were dramatically shorter (Fig. 8, Table I). In the presence of 0.8 µM ELP70, more than 55% of the MTs were shorter than 3 µm (compared with none without ELP70) and MTs rarely exceeded a length of 6 µm. At these concentrations of ELP70 and tubulin, there were very few minus-end MTs that could be measured. At higher ratios of ELP70 to tubulin, the MTs were too few and too short to gather meaningful data regarding further increases in the frequency of catastrophe or reduction of growth rate.


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  		Fig. 8.   Histograms of MT length in a nucleated assembly assay in the presence of ELP70. The increase in the catastrophe events paired with the reduction in elongation rate resulted in a decrease in MT length. The percentage of MTs at a given length is shown for 16 µM tubulin, 0 µM ELP70 (n = 58 MTs measured); 16 µM tubulin, 0.5 µM ELP70 (n = 72); and 16 µM tubulin, 0.8 µM ELP70 (n = 41).

                              
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Table I
ELP70 reduces microtubule elongation velocity and increases catastrophes at the plus-ends of microtubules

Individual parameters of dynamic instability such as the elongation and shortening rates as well as catastrophe and rescue frequencies were analyzed at video frame rates. As shown in Tables I and II, ELP70 had major effects on the dynamic behavior of MTs. The elongation rate was reduced significantly in the presence of sub-stoichiometric levels of ELP70. For example, the addition of 0.5 µM ELP70 to 16 µM tubulin significantly reduced the elongation rate from 0.94 ± 0.24 µm/min to 0.71 ± 0.17 µm/min (p < 0.001). Increasing the concentration of ELP70 from 0.5 to 0.8 µM resulted in an additional significant decrease in the elongation rate from 0.71 ± 0.17 to 0.64 ± 0.21 µm/min (p < 0.05). The ELP70-dependent reduction in the elongation rate was concentration-dependent and was more apparent at a tubulin concentration of 13 µM, where the addition of 0.5 µM ELP70 decreased the elongation rate from 0.78 ± 0.23 to 0.47 ± 0.15 µm/min (p < 0.001). In contrast to the effects on elongation, ELP70 appeared to have little effect on the velocity of shortening.

These ELP70-dependent effects on the elongation rate were specific to the plus-ends of the MTs. Minus-end MTs grew at the same rate in the presence and absence of ELP70 (Table II).

                              
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Table II
Parameters for microtubule dynamics at the minus-ends of microtubules

ELP70 also increased the transition from a growing MT to shortening MT (catastrophe frequency), in a concentration-dependent manner. For example, in the presence of 0.8 µM ELP70 and 16 µM tubulin, plus-end catastrophes were observed every 140 s (40 catastrophes in 5581 s of elongation) compared with one catastrophe every 300 s for 16 µM tubulin alone (50 catastrophes in 15,000 s of elongation). This is a 2.2-fold increase in the catastrophe frequency. An increase in the catastrophe frequency was observed for the minus-end MTs as well.

Although the frequency of catastrophe increased at both the plus- and minus-ends of MTs in the presence of ELP70, there was no apparent effect on the shortening rates at either the plus- or minus-ends. In addition, there was no apparent effect on the transition from a shortening MT to a growing MT (rescue frequency) at the plus-end. Generally, plus-end MTs, polymerized at these tubulin concentrations, shortened all the way back to the nucleation site, and rescue events were observed very infrequently. Hence, statistically significant numbers for the frequency of rescue cannot be reported. Although only a few rescue events were observed at the minus-ends, it is noteworthy that rescues appeared to be inhibited in the presence of 0.5 µM ELP70.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To identify MT proteins important in mitosis, biochemists have fractionated dividing cells, such as mammalian cells grown in culture, or fertilized sea urchin eggs. More than two decades ago, several abundant MT-associated proteins (MAPs) were identified in HeLa cells and classified as polypeptides that associate stoichiometrically with MTs through repetitive cycles of MT assembly and disassembly (48, 49). The most abundant HeLa MAPs identified in this manner were the 210-kDa MAP-4 analog (50) and the Epithelial-MAP of 115 kDa (ensconsin) (51, 52). Following the identification of these MAPs, the Rebhun laboratory succeeded in purifying an abundant MAP from sea urchin mitotic apparatuses (21). This 75- to 80-kDa polypeptide was subsequently named the echinoderm MT-associated protein (EMAP), because it was the most abundant MAP detected in sea urchin, starfish, and sand dollar eggs (27). In this report, we show that an EMAP-like protein, ELP70, is present in HeLa cells and that this protein is an MT destabilizer that localizes to the mitotic apparatus.

Novel EMAP-like Protein (ELP) Is Conserved in Metazoans and a Protozoan-- The high degree of sequence similarity among ELP family members indicates that all members of the ELP family may have similar functions. All of the current experimental data indicate that ELP family members have MT-related functions. Here we show that Homo sapiens ELP70 is an MT-associated protein. In addition, sea urchin, sand dollar, and starfish EMAPs all bind MTs in vitro and in vivo (27) and sea urchin EMAP has been shown to modify the assembly dynamics of MTs in vitro (53). C. elegans ELP-1 also cosediments with endogenous paclitaxel-stabilized worm MTs and is expressed in MT-rich touch neurons (54). In addition, human ELP120/ropp120 is overexpressed during mitosis (48), and Euplotes ELP is annotated as an abundant ciliary protein in GenBankTM (AJ251505).

ELP70 Destabilizes MTs in Vitro-- ELP70 is an MT-binding protein as demonstrated by in vitro MT binding assays, cosedimentation of endogenous ELP70 with HeLa MTs, and localization of ELP70 to the mitotic apparatus. In vitro analysis of MT dynamics in the presence and absence of ELP70 reveals that ELP70 is an MT destabilizer. Under nucleated assembly conditions, ELP70 generated fewer and shorter MTs compared with controls with tubulin alone, predominantly by promoting catastrophes and by reducing the elongation rate of MTs at their plus ends. For example, at 16 µM tubulin, 0.8 µM ELP70 reduced the elongation rate from 0.94 to 0.64 µm/min at the plus-end, but had negligible effects at the minus-end. At 16 µM tubulin, 0.8 µM ELP70 increased the frequency of catastrophe 2-fold at the plus-end. The net result was that MTs assembled in the presence of ELP70 were 3-fold shorter than those assembled from tubulin alone. In addition, there were fewer numbers of MT nucleated from axonemes in the presence of ELP70.

Sea urchin EMAP and mammalian ELP70 differ in their effects on MT dynamics. The most obvious difference is that MTs assembled in the presence of EMAP are slightly longer than MTs assembled from tubulin alone (53), and MTs assembled in the presence of ELP70 are significantly shorter (this report). The increase in MT length in the presence of EMAP is brought about by a slight, 1- to 2-fold increase in the elongation rate at the plus-end. In addition, sea urchin EMAP does not affect the frequency of catastrophe, but rather inhibits the frequency of rescue 8-fold at the plus-end. For example, at a tubulin concentration of 21 µM, rescues were recorded once every 50 s; in contrast, at the same tubulin concentration in the presence of 2.2 µM EMAP, rescues occurred every 385 s (53). In contrast, ELP70 produces a small, 2-fold inhibition of rescue at the minus-end and little detectable effect on the frequency of rescue at the plus-end (this report). However, at the tubulin concentrations used in the assay of ELP70 (13 and 16 µM tubulin), rescues were infrequent, and therefore, statistically significant numbers of rescues were never obtained. Therefore, it is still formally possible that ELP70 inhibits rescue events.

Both sea urchin EMAP and mammalian ELP70 bind MTs in a concentration-dependent manner. Sea urchin EMAP binds to MTs with a molar stoichiometry of 0.7 mol of EMAP per mol of tubulin dimer with an intrinsic dissociation constant of 0.18 µM (43). In this report we show that human ELP70 saturates the MT binding sites at a molar ratio of 0.4 mol of ELP70 per mol of tubulin dimer with an intrinsic dissociation constant of 0.44 µM. The binding data of EMAP and ELP70 are on the same order of magnitude, and it is premature to suggest that they have different binding sites on the MT.

There were some procedural differences in the assays used to characterize sea urchin EMAP and human ELP70; however, it is unclear how these differences contributed to the differences in their effects on MT dynamics. For example, sea urchin EMAP is approximately twice as soluble in an MT-assembly buffer as human 6hisELP70. Therefore, MT assembly assays in the presence of sea urchin EMAP (53) were carried out at slightly higher molar ratios of EMAP to tubulin dimers than were the ELP70 studies (this report). In addition, the effects of human ELP70 on bovine brain tubulin assembly was assayed at 37 °C following a 30-min preincubation to reach a steady state (this report). Because sea urchin EMAP was purified from a marine poikilotherm that normally exists at a temperature of 9-12 °C, EMAP's effects on MT dynamics were assayed at 23 °C, a temperature that supported the assembly of porcine brain tubulin but did not precipitate EMAP (53). Although these assay conditions may have affected the results obtained, it is more likely that EMAP and members of the ELP family have evolved with different effects on MT formation and function. A careful molecular dissection of the functional domains of ELP70 and related proteins should reveal domains critical for each activity.

How Does ELP70 Generate Shorter MTs?-- ELP70 MTs were shorter due to the combined effects of reducing the elongation rate and increasing the catastrophe frequency. Catastrophes are driven by GTP hydrolysis (55, 56) and are correlated with the formation of protofilament "peels" as the MT rapidly shortens (57, 58). Therefore, models for catastrophic shortening include both structural and chemical transitions that occur specifically at the MT ends (1, 59). For example, Op18/stathmin is proposed to induce MT catastrophes, specifically at the plus-ends, by stimulating GTP hydrolysis (60). It is clear from the high resolution model of an MT that GTP hydrolysis by the plus-end exposed beta -tubulin will have little effect on the lateral interactions of the alpha -subunits exposed at the minus ends (61). Therefore, Op18's effects on the plus-ends only are entirely consistent with this model. In contrast, XKCM1 induces catastrophes at both plus- and minus-MT ends in the absence of GTP hydrolysis (18). XKCM1 does not bind along the length of the MT but rather associates specifically with both MT ends. When incubated with frog MTs, XKCM1 induces protofilaments to change from a linear to a curled configuration. These results indicate that XKCM1 can induce a conformational change in the tubulin subunits along a protofilament.

ELP70 may destabilize MTs by a third mechanism. The stoichiometry of binding to paclitaxel-stabilized MTs indicates that ELP70 binds along the length of the MT, rather than the MT ends. This alone indicates that it is unlikely that ELP70 binding results in a chemical transition at either the plus- or minus-ends of the MT. It is also unlikely that ELP70 affects the catastrophe frequency by sequestering tubulin dimers. For this to be true, 0.5 mol of ELP70 would need to bind more than 3 mol of tubulin dimer to reach a free tubulin concentration that resulted in a similar elongation rate (0.71 µm/min at 16 µM tubulin/0.5 µM ELP70 versus 0.78 µm/min at 13 µM tubulin). Yet, the strong increase in the frequency of catastrophe would not be explained. In addition, if ELP70 sequestered tubulin, the growth rates at both the plus- and the minus-end should have been affected. Because that was not the case, sequestration seems unlikely.

Lateral Destabilization Model-- These data favor a structural model, and we propose that ELP70 destabilizes MTs by an interdimer association of ELP70 with the tubulin subunits in adjoining protofilaments. Specifically, we propose that ELP70 binds to the MT through its HELP domain and that this HELP domain binds to the lateral contacts between protofilaments and acts as a wedge that weakens the interactions between the M loop and the neighboring S3 helix/H1-S2 loop (59, 61). These lateral contacts between tubulin subunits are critical interactions during MT polymerization. For example, mutations or phylogenetic variations that alter the cold stability of the MT polymer map to the lateral contacts between protofilaments (62, 63). Once the lateral contacts are weakened, GDP-tubulin subunits near the ends of the polymer could relax into their curved state (64); a phenomenon that could produce the peeling protofilaments characteristic of a depolymerizing MT (57, 65, 66). In addition, ELP70 may induce a conformational change in the tubulin dimer that doesn't favor the curvature of the extended protofilament sheet associated with MT growth (67-69). We predict that MTs assembled in the presence of ELP70 will have smaller extensions at the plus-ends and slower elongation rates.

Finally, why did ELP70 affect the elongation rate specifically at the MT plus-ends? The simplest explanation for this effect is that, in general, minus-ends appear to be more stable, because there is no GTP cap at the minus-end of the MT (70). Moreover, there are structural considerations that indicate that the alpha -alpha lateral interactions, which are exposed at the minus-ends, are intrinsically much stronger than the beta -beta interactions (61). In addition to these considerations, ELP70 may affect the plus-end elongation rate simply by binding preferably to interdimer beta  subunits throughout the lattice. Because the alpha -subunits are exposed at the minus-end, ELP70 may not effect the elongation rate at this end. These are testable speculations.

ELP70 Function in Vivo?-- During the cell cycle, ELP70 is concentrated in the region of the mitotic apparatus, a location that indicates that ELP70 may have an MT-related function during mitosis. It is tempting to speculate that ELP70 is in part responsible for the increase in MT dynamics and changes in MT polymer levels that occur during the transition from G2 to mitosis (71, 72). Specifically, the frequency of catastrophe increases and the frequency of rescue decreases as cultured mammalian cells enter mitosis (73). In this regard, ELP70 may be the factor responsible for the change in the catastrophe frequency. In addition, ELP70 may not act alone. We have proposed that ELP70 destabilizes the lateral protofilament interactions in an MT, and in doing so, ELP70 may "prime" an MT for rapid depolymerization in association with other destabilizers (15-18, 19, 60). Whether ELP70 regulates MT dynamics in vivo is an important area for future research.

    FOOTNOTES

* This work was supported by National Science Foundation (NSF) Grant MCB-9982377 (to K. A. S.), a Deutsche Forschungsgemeinschaft Ei407/2-1 post-doctoral fellowship (to B. E.), and an NSF Research Experience for Undergraduates award (to P. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF103939, U97018, and AAG09279.

Dagger To whom correspondence should be addressed: Dept. of Molecular Biosciences, 1200 Sunnyside Ave., University of Kansas, Lawrence, KS 66045. Tel.: 785-864-4580; Fax: 785-864-5321; E-mail: ksupre@ukans.edu.

Published, JBC Papers in Press, November 2, 2001, DOI 10.1074/jbc.M106628200

    ABBREVIATIONS

The abbreviations used are: MT, microtubule(s); MAP, microtubule-associated protein; EMAP, echinoderm MT-associated protein; ELP, EMAP-like protein; HELP, hydrophobic ELP; ELP70, 70-kDa human EMAP-like protein; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; ropp, restrictedly overexpressed proliferation-associated protein; VE-DIC, video-enhanced differential interference contrast microscopy; WD, tryptophan-aspartic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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