extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

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Interaction Kinetics of Reversible Inhibitors and Substrates with Acetylcholinesterase and Its Fasciculin 2 Complex*

Fasciculin 2 (Fas2), a three-fingered peptide of 61 amino acids, binds tightly to the peripheral site of acetylcholinesterases (AChE; EC 3.1.1.7), occluding the entry portal into the active center gorge of the enzyme and inhibiting its catalytic activity. We investigated the mechanism of Fas2 inhibition by studying hydrolysis of cationic and neutral substrates and by determining the kinetics of interaction for fast equilibrating cationic and neutral reversible inhibitors with the AChE·Fas2 complex and free AChE. Catalytic parameters, derived by eliminating residual Fas2-resistant activity, reveal that Fas2 reducesk cat/K m up to 106-fold for cationic substrates and less than 103-fold for neutral substrates. Rate constants for association of reversible inhibitors with the active center of the AChE·Fas2 complex were reduced about 104-fold for both cationic and neutral inhibitors, while dissociation rate constants were reduced 102-to 103-fold, compared with AChE alone. Rates of ligand association with both AChE and AChE·Fas2 complex were dependent on the protonation state of ionizable ligands but were also markedly reduced by protonation of enzyme residue(s) with pK a of 6.1–6.2. Linear free energy relationships between the equilibrium constant and the kinetic constants show that Fas2, presumably through an allosteric influence, markedly alters the position of the transition state in the reaction pathway. Since Fas2 complexation introduces an energetic barrier for hydrolysis of substrates that exceeds that found for association of reversible ligands, Fas2 influences catalytic parameters by a more complex mechanism than simple restriction of diffusional entry and exit from the active center. Conformational flexibility appears critical for facilitating ligand passage in the narrow active center gorge for both AChE and the AChE·Fas2 complex.

The interaction between acetylcholinesterase (EC 3.1.1.7) (AChE) 1 and fasciculin 2 (Fas2) is a well studied example of a tight binding peptide-protein complex. The bound "three-fin-gered" peptide toxin, isolated from venom of Elapidae snakes, resides at the opening of the AChE active center gorge, thus sealing off the entry to the gorge and resulting in virtually complete inhibition of hydrolysis of the neurotransmitter acetylcholine. Static snapshots of the complex structure revealed in crystals of mouse AChE⅐Fas2 (1), Torpedo californica AChE⅐Fas2 (2) and human AChE⅐Fas2 (3) complexes indicate that the "solvent-accessible" surfaces of two molecules overlap in a nearly continuous area of more than 1000 Å 2 around the entry to the AChE active center gorge. The complex, however, retains residual catalytic activity whose magnitude was reported to vary between species, being about 3% in human AChE (4) and about 0.09% in mouse AChE (5), when measured with acetylthiocholine (ATCh). Inhibition of residual catalytic activity of both complexes by conjugation of the active center serine by either organophosphate or trifluoroacetophenone (TFK) inhibitors, showed only a 1-order of magnitude reduction in reaction rates of cationic inhibitors for the AChE⅐Fas2 complex when compared with uncomplexed AChE (5). Rates of association and dissociation of reversible ligand N-methylacridinium were found, however, to be reduced by 3-4 orders of magnitude in the AChE⅐Fas2 complex (6).
Recently, Mallender et al. (7) found that human AChE contained Fas2-resistant esterase activity in purified preparations. Monitoring the phosphorylation rates of fluorescent organophosphates, which exhibit negligible dephosphorylation of the enzyme, enabled these investigators to show that reactions of organophosphates with AChE⅐Fas2 were 2-3 orders of magnitude slower than those for uncomplexed AChE, a reduction greater than observed in earlier studies (4). The predominant residual catalytic activity of the human AChE⅐Fas2 complex appeared to be due to contaminating Fas2-resistant activity (7). Although mouse AChE⅐Fas2 complex appeared to contain only 0.09% activity resistant to Fas2 inhibition (5,8), comparatively high turnover rates of a fasciculin-resistant enzyme may also complicate our analysis of ligand entry into the active center gorge.
By employing an irreversible inhibitor to inhibit selectively the residual Fas2 resistant activity, we demonstrate here that mouse AChE⅐Fas2 complex retains only about 0.004% catalytic activity at 1 mM ATCh instead of the previously reported 0.09% (5). Stopped-flow techniques with fluorescent detection enabled us to determine rates of association and dissociation of several reversible inhibitors that equilibrate with AChE in a subsecond time frame. Furthermore, we have developed a competitive binding assay utilizing a fluorescent carbamate, M7C, to measure binding rates of nonfluorescent inhibitors to both mouse AChE and the AChE⅐Fas2 complex. Utilizing these procedures, we present a quantitative characterization of interaction of * This work was supported by United States Public Health Service Grants GM18360 and DAMD 17-1-8014 (to P. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

MATERIALS AND METHODS
Enzyme-Wild-type mouse AChE was expressed in HEK-293 cells stably transfected with cDNA encoding a monomeric form of the enzyme truncated at its carboxyl terminus at position 548. The enzyme secreted into the medium was purified to homogeneity on affinity columns as described previously (9).
Enzyme Activity-Hydrolysis of ATCh, PTCh, BTCh, S-PhAc, and pNPAc was measured spectrophotometrically at 412 nm for released thiocholine (10) or at 405 nm for p-nitrophenol. Activities of AChE⅐Fas2 complexes were measured following a 10-min incubation of 640 nM AChE with 1.3 M Fas2 to form the complex and 30-min incubation of the complex with 530 nM S P -3,3-dimethylbutyl methylphosphonothiocholine to inhibit hydrolytic activity not originating from the complex. The very slow reaction of the charged organophosphate with the active center of the Fas2⅐AChE complex gives rise to selective inhibition. Kinetic constants for hydrolysis of the above substrates by AChE and AChE⅐Fas2 complex were determined as described previously (5).
Enzyme Inhibition-The time course of AChE inhibition by trifluoroacetophenones was monitored by measuring ATCh hydrolysis in aliquots from the reaction mixture. Typically, 0.96-ml aliquots were drawn from 10 ml of the reaction mixture and mixed with 30 l of DTNB (10 mM) and 10 l of ATCh (100 mM) at specified time intervals to stop inhibition and determine residual enzyme activity. Both the inhibitory reaction and the assessment of residual activity were conducted in 0.1 M phosphate buffer, pH 7.0. First order rate constants for inhibition (k) were obtained by fitting fractional enzyme inhibition to the following equation, where f, f 0 , and f ϱ are fractions of enzyme inhibited at times t, t ϭ 0, and t ϭ ϱ, respectively. Second order inhibition rate constants were derived from plotting the first order rate constants for inhibition against inhibitor concentration and extrapolating to infinite dilution. Stopped-flow Kinetic Assays-Association and dissociation rate constants for fast equilibrating fluorescent inhibitors were determined using a SX.18 MV stopped-flow instrument equipped with fluorescence detection (Applied Photophysics). Excitation wavelengths were selected to achieve maximum signal amplitude. Acridine, 9-aminoacridine, and N-methyl acridinium were excited at wavelengths between 250 and 260 nm, coumarin at 220 nm, and propidium at 288 nm. For experiments with nonfluorescent inhibitors BW286C51, edrophonium, and decamethonium, but also for selected fluorescent ligands, AChE or AChE⅐Fas2 complex was excited at 276 nm (11). Quenching of ligand or AChE fluorescence upon complex formation was monitored using a 305-nm cut-off filter, and for 9-aminoacridine a 335-nm cut-off filter was used. Tacrine association was detected by excitation at 323 nm, with a 335-nm cut-off filter for emission. Linear plots of observed first order rate constants versus ligand concentrations gave second order associa-tion rate constants from the slope of the line and a first order dissociation rate constant from the ordinate intercept (11,12).
M7C Reaction Assay-The time courses of TFK, (Ϫ)-huperzine, and BW286C51 association with AChE and the AChE⅐Fas2 complex were followed using N,N-dimethyl carbamoyl N-methyl-7-hydroxy-quinolinium (M7C). This nonfluorescent dimethyl carbamoyl ester reacts covalently with the AChE active serine releasing a fluorescent N-methyl-7-quinolinium leaving group (M7H) ( excitation ϭ 410 nm; emission ϭ 510 nm; Ref. 13), thus allowing one to monitor both the rate of M7C reaction with the serine and the number of binding sites. The rate of binding of nonfluorescent covalent inhibitors to AChEs was followed by monitoring a decrease in number of AChE binding sites available for M7C reaction resulting from prior covalent reaction with the nonfluorescent inhibitor. Forty l of 1.0 mM M7C was added to 0.4-ml aliquots drawn at different time intervals from 4 ml of reaction mixture containing buffered solution of an AChE (10 -70 nM) with or without Fas2 (650 nM) and the nonfluorescent inhibitor. Aliquots were quickly transferred into a spectrofluorometric cuvette, and the fluorescence increase was followed at emission ϭ 510 nm ( excitation ϭ 410 nm) for 20 s in the absence of Fas2 or for 100 s in the presence of Fas2. The fluorescence signal exponentially approached a maximum value, which was diminished with increasing times of prior exposure to the nonfluorescent inhibitor, indicating progress of its reaction. The first order rate constant of binding of nonfluorescent inhibitors was determined by fitting the time dependence of relative change in maximal fluorescence to Equation 1.
Equilibrium Binding Measurements-Equilibrium dissociation constants for several fluorescent reversible ligands were determined by titration with increasing concentrations of AChE or AChE⅐Fas2 complex, resulting in quenching of ligand fluorescence. Concentrations of free fluorophores during titrations were determined from corresponding calibration curves constructed for each fluorophore, limiting fluorescence quenching in the bound state. Corrections were made for small changes in reaction volume due to the addition of titrant. Equilibrium dissociation constants (K d ) were determined from the reciprocal of the slopes of Scatchard plots.
Determination of Ligand pK a Values-UV-visible spectra of fast equilibrating reversible AChE inhibitors with ionizable functional groups were recorded in buffers of varying pH. Changes of peak absorbances as a function of buffer pH were analyzed using the following equations (rearranged from Ref. 14), where A min and A max are minimal and maximal peak absorbances and K a is the equilibrium proton dissociation constant. The following buffers were used: 0.10 M citric acid plus 0.20 M sodium phosphate (pH 2.6 -7.8) and 0.050 M sodium phosphate plus sodium pyrophosphate (pH 5.0 -12).

M7C Reaction
Assay-Time courses of the M7C reaction with the AChE and the AChE⅐Fas2 complex are shown in Fig. 1. M7C added at a concentration of 100 M binds to AChE at a rate too fast for measurement by conventional mixing and detection by spectrofluorometry. Hence, the carbamoylation step is reflected in a rapid burst of fluorescence. The reaction of M7C with the AChE⅐Fas2 complex was slower but was virtually complete within 100 s. The final fluorescence intensities upon carbamoylation and release of M7H were linearly dependent on AChE and AChE⅐Fas2 concentrations (Fig. 1, B and C).
The rapid and stoichiometric reaction of M7C with AChE or the AChE⅐Fas2 complex enabled us to develop a fluorescent assay for monitoring of binding rates for nonfluorescent ligands. The assay is based on consecutive, rapid M7C titrations of residual catalytically active AChE during the course of its reaction with nonfluorescent ligands. The first order rate constants for reaction were determined by fitting to Equation 1 the fluorescence intensities obtained in subsequent titrations. Good agreement between rate constants for ligand reaction measured from the decrement in the M7C reaction and ligand inhibition of ATCh hydrolysis is evident in Table I.

Hydrolysis of Substrates by AChE and AChE⅐Fas2
Complex-Catalytic hydrolysis of three cationic (ATCh, PTCh, and BTCh) and two neutral (S-PhAc and pNPAc) esters by AChE and AChE⅐Fas2 was compared. Substrate concentration dependences of AChE hydrolysis deviate from Michaelis kinetics at millimolar substrate concentrations, with ATCh, PTCh, and BTCh inhibiting their own hydrolysis. The kinetics of hydrolysis is characterized by substrate inhibition constants (K ss ) and relative activities of enzyme-substrate ternary complexes (b), in addition to Michaelis constants (K m ) and maximum turnover rates (k cat ) (Table II; Refs. 16 and 17). The three cationic substrates have similar Michaelis constants (15)(16)(17)(18), yet the commitment to catalysis (defined as a ratio of the enzyme acylation rate constant and dissociation rate constant of the Michaelis complex (cf. Ref. 19)) and the formation of the tetrahedral transition state, reflected in k cat , appear severely restricted for BTCh and other substrates containing a large acyl group (17,19).
Hydrolysis of neutral substrates by AChE obeyed Michaelis kinetics (Table II), but with K m values up to 2 orders of magnitude larger than for cationic substrates, thus emphasizing the importance of electrostatic interactions in formation of Michaelis complexes. k cat of S-PhAc was similar to k cat values for charged substrates and significantly greater than k cat for pNPAc. A similar relationship between catalytic constants for PhAc and pNPAc has been well described for Electrophorus electricus AChE (20).
Although the AChE⅐Fas2 complex hydrolyzed all substrates significantly more slowly than AChE, cationic substrates with high turnover rates were most affected. Hydrolysis of both cationic and neutral substrates by AChE⅐Fas2 obeyed Michaelis kinetics. Our earlier published curve of ATCh hydrolysis by AChE⅐Fas2 was biphasic, indicating substrate activation (5). The first phase, due to the presence of residual, Fas2-resistant, esterase activity, was inhibited selectively using the organophosphate, S P -3,3-dimethylbutyl methylphosphonothiocholine. Hence with this correction, the ATCh hydrolysis profile for the Fas2⅐AChE complex shows typical Michaelis kinetics. k cat /K m values for ATCh and PTCh were reduced by 5-6 orders of magnitude, whereas k cat /K m for BTCh was reduced by only 3 orders of magnitude. Neutral substrates S-PhAc and pNPAc exhibited much smaller reductions in k cat /K m (680 and 96 times, respectively). The reduction in k cat /K m for the five substrates by AChE⅐Fas2 resulted in distinct clusters of values for charged and neutral substrates. Values of 1.2-4.7 ϫ 10 3 M Ϫ1 s Ϫ1 for neutral substrates approach the k cat /K m value for benzoylthiocholine hydrolysis by AChE (1.1 ϫ 10 4 M Ϫ1 s Ϫ1 ; Ref. 17).
k cat /K m values for neutral substrates with the AChE⅐Fas2 complex were 1-2 orders of magnitude greater than for the charged substrates, suggesting that Fas2 association may impart a more restrictive barrier in the AChE⅐Fas2 complex to diffusional entry and acylation by cationic substrates. The reduction in values of k cat /K m was reflected in both an increase in K m and reduction in k cat . Whereas for neutral substrates and free AChE, the large K m values were limiting, the situation is reversed for the AChE⅐Fas2 complex, where large K m values primarily limit hydrolysis of cationic substrates. k cat values for the AChE⅐Fas2 complex were significantly reduced for all substrates. In addition, k cat values for the AChE⅐Fas2 complex correlate with the size of the acyl moiety in the substrate, suggesting that the steric occlusion by acyl pocket residues also limits substrate hydrolysis for the AChE⅐Fas2 complex. Coulombic repulsion from arginines and lysines of bound Fas2 could also interfere with the orientation of thiocholine in the transition state and its exit from the gorge. Both the reversible binding steps and turnover of two reactive cationic substrates, ATCh and PTCh, appeared to be reduced to the greatest extent in the AChE⅐Fas2 complex, suggesting a similar influence of the acyl pocket on the stability of the Michaelis complexes and tetrahedral transition states. This was not the case for neutral substrates, where k cat alone is affected.
Kinetics of Binding of Reversible Ligands to AChE-The second order association and the first order dissociation rate constants for two substituted trifluoroacetophenones and 10 noncovalent reversible ligands to AChE and AChE⅐Fas2 complex are listed in Table III. The constants for slow equilibrating   inhibitors such as the trifluoroacetophenones and (Ϫ)-huperzine A were determined using the M7C reaction assay, whereas for the faster equilibrating inhibitors, stopped-flow techniques with fluorescence monitoring were used (Figs. 2 and 3). Rates of interaction for BW286C51 were analyzed by both techniques. The association rate constants of the 10 ligands with AChE covered a narrow range of less than 2 orders of magnitude (4.5 ϫ 10 7 to 2.8 ϫ 10 9 M Ϫ1 s Ϫ1 ), and only (Ϫ)-huperzine A had a significantly slower association rate constant. Since several fast equilibrating ligands from Table III bear ionizable groups, their protonation states at pH 7.0 were analyzed by determining their pK a values (Table IV). These pK a values indicate that, , where s is substrate concentration, K ss is the substrate inhibition or activation constant, and b is a measure of relative activity of ternary enzyme-substrate complexes. ATCh, K ss ϭ 15 mM, b ϭ 0.23 (data of Radić et al. (16)); PTCh, K ss ϭ 1.3 mM, b ϭ 0.21; BTCh, K ss ϭ 7.1 mM, b ϭ 0.48 (data of Hosea et al. (17). Shown are means from 2-4 experiments. The typical S.E. of determination was 30 -40% of the mean value.  Fig. 3. The typical S.E. was 30 -40% of the mean value of constants. The equilibrium dissociation constant (K d ) was derived as the ratio of k Ϫ1 /k 1 . n.d., not determined due to slow rates of dissociation (TFK ϩ and TFK 0 ) or dissociation rates too rapid for stopped-flow measurements (propidium).
in addition to quaternary ligands, tacrine and 9-aminoacridine will be positively charged at pH 7.0; acridine will be neutral and coumarin exists as approximately a 3:1 ratio of neutral to anionic species. The fastest association rate constants were observed for quaternary ammonium ligands bearing a permanent cation (k 1 ϭ 1.3 ϫ 10 8 to 2.8 ϫ 10 9 M Ϫ1 s Ϫ1 ). Protonated cationic ligands, tacrine and 9-aminoacridine (k 1 ϭ 1.3 ϫ 10 8 to 1.5 ϫ 10 8 M Ϫ1 s Ϫ1 ), approached the lower end of the range of rates for quaternary compounds, while neutral ligands (acridine and coumarin) had the slowest association rate constants (k 1 ϭ 4.5 ϫ 10 7 to 1.2 ϫ 10 8 M Ϫ1 s Ϫ1 ). Association rate constants for peripheral site ligands, propidium and coumarin, exhibited the same rates as those ligands associating with the active center, despite the greater surface accessibility of the peripheral site.
The rate constants for dissociation of the 12 ligands extending over 7 orders of magnitude (1.8 ϫ 10 Ϫ5 to 4.2 ϫ 10 2 s Ϫ1 ), spanned a greater range than the association rate constants. Trifluoroacetophenones were the slowest dissociating ligands. These slowly reversible ligands interact covalently at the esteratic site and noncovalently in the choline binding site (12,21). Comparison of quaternary, protonated, and neutral ligands binding at the active center reveals that apart from trifluoroacetophenones and (Ϫ)-huperzine A, cationic bisquaternary BW286C51 and the cationic tertiary amines, tacrine and 9-aminoacridine, had the slowest dissociation rate constants.
Kinetics of Binding of Reversible Ligands to the AChE⅐Fas2 Complex-Ligand interactions with the AChE⅐Fas2 complex reveal marked reductions in both association and dissociation rate constants, compared with free AChE (Table III). The most affected should be the peripheral site ligands, propidium and coumarin, whose binding is competitive with Fas2 and the bisquaternary ligands, decamethonium and BW286C51, which share a portion of the peripheral binding site with Fas2. The 8-order of magnitude slower association rate constants of BW286C51 and decamethonium accompanied with only 1-3order of magnitude slower dissociation constants indicate that these bisquaternary ligands will form ternary complex with mAChE⅐Fas2.
By contrast, the interaction of coumarin with the AChE⅐Fas2 complex was altered in a different manner than for propidium, BW286C51, and decamethonium. The small reduction in the coumarin association rate constant is characteristic of active center ligands. Coumarin's dissociation rate constant was, however, reduced significantly more than those for the above ligands. Taken together, the comparative kinetics indicate the existence of a secondary binding site for coumarin in the active center gorge of the AChE⅐Fas2 complex. Unlike other peripheral site ligands coumarin appears small enough to enter the active center gorge in AChE. Previous mutagenesis and molecular modeling studies also suggest coumarin binding to the active center when the peripheral site is occupied (22)(23)(24).
For the eight remaining active center ligands, the association rate constants with AChE⅐Fas2 were about 4 orders of magnitude smaller than the corresponding constants for AChE, irrespective of ligand charge or size. Their dissociation rate constants were reduced by only 1-3 orders of magnitude. The greater reduction of association than dissociation rate constants leads to an increase in K d for active center ligands in the AChE⅐Fas2 complex typically by 1 order of magnitude but in one case by 2 orders. The K d for coumarin, with its presumed occupation of the active center in the AChE⅐Fas2 complex is actually slightly decreased.
Equilibrium Binding Measurements-Equilibrium dissociation constants for several reversible ligands, determined also by fluorescence titrations (Fig. 4), are listed in Table V and show good agreement with corresponding constants derived from the ratios of dissociation to association reaction rate constants (cf . Table III).
pH Dependence for Interaction of Reversible Ligands with AChE and the AChE⅐Fas2 Complex-AChE and several reversible ligands from Table III bear ionizable groups, and their mutual interactions may depend on pH of the reaction medium. Interaction of a reversible ligand and enzyme, both bearing ionizable groups, can be described as follows.
It can be shown that for association between ligand and enzyme in the above scheme, the following equation holds, where k 1 and k 1 max are second order association rate constants for ligand and enzyme at a given pH and a maximal rate constant of the reactive species, respectively. Factor b is a measure of the difference in the second order rate constants for reaction between enzyme and protonated versus nonprotonated forms of ligand. The species EH ϩ is assumed not to bind ligand. K a E and K a L are equilibrium dissociation constants for a proton from ionizable groups in the enzyme and ligand, respectively.
The pH dependence of reaction rate constants of four ligands with AChE was studied in the range of pH 4.5 to 11.0 (Table  VI). Association rate constants (k 1 ) for all ligands decreased at  pH values below 6.0 by 1-3 orders of magnitude; the greatest reduction was observed for quaternary cation edrophonium. Analysis of dependences of association rate constants for edrophonium and 9-aminoacridine on pH, using the model from Scheme 1 and the corresponding Equation 4, is shown in Fig.  5A. Association of both ligands in the low pH range depends on ionization of the same group, presumably in AChE, with a pK a E value of 6.1-6.2. The AChE group with pK a of 6.2 was implicated as critical for carbamoylation and phosphorylation of AChE by inhibitors containing quaternary ammonium in the leaving group (25). The reduction of association constants for edrophonium and coumarin in the pH range above pH 7.0 is consistent with their deprotonation (cf. Table IV). The asymmetric shape of the pH dependence curve for edrophonium, reflected in a value of b larger than 0 (b ϭ 0.14), may suggest that zwitterionic edrophonium species formed by deprotonation of the hydroxyl group associates with enzyme with k 1 max of 1.7 ϫ 10 7 M Ϫ1 s Ϫ1 , which is about 7-fold slower than k 1 max for cationic edrophonium.
On the other hand reduction of association constants of 9-aminoacridine is reflected in a pK a L value of 10.3. This value is close to both the pK a value of 9-aminoacridine (10.1) and the pK a value found for carbamoylation and phosphorylation of human AChE (10.25;Ref. 25) and therefore could be either ligand-or enzyme-related. Minimal variations of dissociation rate constants of cationic ligands (Table VI) in the pH interval studied suggests an absence of a pH related conformational change in the active center gorge. Protonation of acridine slowed the dissociation rate 10-fold, emphasizing the role of electrostatic interactions in stabilization of cationic ligands in the AChE active center gorge.
The pH dependence of 9-aminoacridine association with AChE⅐Fas2 complex at low pH is very similar to the dependence observed for association with AChE alone, and reflected in pK a E value of 6.1 (Fig. 5B). At the high pH, the association rate constants were only slightly reduced, much less than observed for association with AChE alone. Thus, both protonated and neutral 9-aminoacridine appear to associate with AChE⅐Fas2 at similar association rates. In addition, protonation of a basic AChE residue(s), with a presumed pK a of ϳ10.3 (Fig. 5A), may be affected by bound Fas2 or is no longer a rate-limiting factor in ligand association. The deviations of experimental points from the calculated curve observed in Fig. 5B at intermediate and high pH appeared reproducible and are possibly an indication of the interacting species being more complex than described in Scheme 1. Rates of 9-aminoacridine dissociation from AChE⅐Fas2 complex were largely independent on pH. DISCUSSION Our comparison of the interaction kinetics of five substrates and 12 reversible inhibitors between the AChE⅐Fas2 complex and free AChE (Fig. 6) showed second order reaction rate constants for substrates and association rate constants for reversible ligands to be markedly reduced for the AChE⅐Fas2 complex. When the contribution of the fasciculin-resistant enzyme species is accounted for, the magnitude of the rate reductions approaches 4 -6 orders of magnitude.
Fas2-resistant Esterase-A Fas2-resistant esterase in quantities consisting of 0.09% of the total activity is retained and eluted from the affinity columns. Characterization of this species is problematic due to its comparatively small quantities. Its specificity for cationic organophosphates differs from mouse butyrylcholinesterase and suggests that it may be a proteolysis product or a spontaneous modification of AChE. Allowing for the contaminant, we now estimate the catalytic efficiency for ATCh with the AChE⅐Fas2 complex to be 0.004% of uncomplexed AChE activity instead of the previously reported 0.09% (5). Mallender et al. (7) found that a large fraction of catalytic activity of the human AChE⅐Fas2 complex, that represented 2-31% of uncomplexed human AChE activity, was in fact contaminating, Fas2-resistant esterase activity. Their findings prompted us to develop multiple approaches to examine whether mouse AChE, despite its low residual activity in the presence of Fas2, also contained a Fas2-resistant component.
Development of a novel competitive binding assay based on

FIG. 4. Scatchard plots for fluorescence titrations of 9-aminoacridine (circles) and tacrine (triangles) by AChE (closed symbols) and the AChE⅐Fas2 complex (open symbols).
Concentration of both ligands was 10 nM in titration with 5-500 nM AChE and was 50 nM (tacrine) or 100 nM (9-aminoacridine) in titration with 25-3000 nM AChE⅐Fas2 complex. Inset, decreases in fluorescent emission spectrum of 9-aminoacridine during titration with increasing concentrations of AChE and AChE⅐Fas2 complex. Shown are 10 nM 9-aminoacridine titrated with concentrations between 0 and 500 nM AChE and 100 nM 9-aminoacridine titrated with concentrations between 0 and 3000 nM AChE⅐Fas2 complex.  Aminoacridine  38  760  Tacrine  44  800  Coumarin 14,000 ND fluorescent product of the carbamoyl ester, M7C, enabled us to determine rate constants for binding of nonfluorescent ligands to mouse AChE⅐Fas2 complex and to unliganded mouse AChE. The carbamoylation reaction weights each active center equivalently and avoids amplification from the high turnover by residual enzyme not inhibited by Fas2. In addition, incubation of a mixture of the AChE⅐Fas2 complex and Fas2-resistant esterase with a bulky organophosphate selectively inhibits the latter, enabling one to account for the disparity of findings with previous studies. Finally, employing stopped-flow kinetics with direct fluorescent detection of the complexes allowed us to separate association and dissociation reaction rate constants for binding of several rapidly equilibrating reversible inhibitors to both free AChE and the AChE⅐Fas2 complex.
Influence of Fasciculin on Hydrolysis of Substrates-Analysis of the kinetics indicates that inhibition of AChE activity by Fas2 is not due solely to a diminished rate of substrate entry into the active center in the AChE⅐Fas2 complex. The commonly considered reaction steps in substrate hydrolysis by AChE (14,28,29) are shown in Scheme 2, where enzyme (E) and substrate (S) associate to form a Michaelis complex (ES). In turn, the complex is converted to an un-stable acyl enzyme (EA) with loss of the substrate leaving group (P 1 ). EA is hydrolyzed to free enzyme and acyl group of substrate (P 2 ). The corresponding catalytic parameters, K m , k cat , and k cat /K m are described as follows.
Second order rate constants for reaction of cationic substrates with AChE⅐Fas2, reflected in k cat /K m values between 60 and 100 M Ϫ1 s Ϫ1 , are slower than rate constants for entry of reversible ligands into the AChE⅐Fas2 complex. The exceptionally slow association rates of BW286C51 and decamethonium are most easily explained by partial overlap of their binding site with the site of Fas2 residence due to their extended conformation (cf. Fig. 6). Thus, additional conformational constraints apply to their association rates. Furthermore, the reduction of nearly 6 orders of magnitude in k cat /K m for ATCh and PTCh exceeds the reduction in association rate constants for the reversible cationic inhibitors with the AChE⅐Fas2 complex. Catalyzed hydrolysis by AChE⅐Fas2 for various substrates yielded sets of limiting values for k cat and for k cat /K m . The similarity of k cat values for charged (ATCh) and two neutral acetate esters (S-PhAc and pNPAc) suggests that the rate of deacylation of EA (k 3 ) may be involved in rate limitation for the chemical part of the catalytic reaction under the influence of bound Fas2. The second order rate constants of association were fitted to Equation 4 with the following parameters. A, , pK a E ϭ 6.2, pK a L ϭ 7.6, b ϭ 0.19 Ϯ 0.03 (for edrophonium) and k 1 max ϭ (1.4 Ϯ 0.1) ϫ 10 8 M Ϫ1 s Ϫ1 , pK a E ϭ 6.1, pK a L ϭ 10.3, b ϭ 0 (for 9-aminoacridine). B, k 1 max ϭ (5.2 Ϯ 0.2) ϫ 10 4 M Ϫ1 s Ϫ1 , pK a E ϭ 6.1, pK a L ϭ 11.9, b ϭ 0 (for 9-aminoacridine).

TABLE VI
Effect of pH on association and dissociation rate constants for selected reversible ligands and mouse AChE Reactions were monitored in 0.1 M citrate-0.2 M phosphate buffers (pH 4.5-7.0), 0.1 M phosphate buffers (pH 5.5-8.0), and 0.050 M phosphatepyrophosphate buffers (pH 6.0 -9.0). Values of constants measured in different buffers of the same pH were similar and were therefore averaged. Experiments were performed in duplicate. pH 9-Aminoacridine Acridine Edrophonium Coumarin Parallel reductions in k 2 by Fas2 are also likely, since the inhibition rates of several organophosphates were shown to be reduced in the AChE⅐Fas2 complex by several orders of magnitude more than the rates of association of reversible inhibitors (6). 2 Furthermore, a second clustering of k cat /K m values for hydrolysis of charged acyl thiocholines by AChE⅐Fas2 (ϳ83 M Ϫ1 s Ϫ1 ) as opposed to neutral acetates (ϳ3000 M Ϫ1 s Ϫ1 ) indicates that the corresponding k Ϫ1 and k 2 constants are of the same magnitude and, consequently, together with k 1 both influence values of k cat /K m . This follows because the reduction of k cat /K m for ATCh, imposed by Fas2, is larger than reductions observed in rates of association of reversible inhibitors. Charge on the leaving group influences both reactions, described by k 1 and k 2 , which both participate in limiting the rates of hydrolysis by the AChE⅐Fas2 complex. The absence of the selective steric constraints of the acyl pocket in the AChE⅐Fas2 complex (Table II) further suggests that the leaving group of the substrate is limiting for catalytic throughput (k cat /K m ) in the AChE⅐Fas2 complex. Thus, in addition to the pronounced effects of bound Fas2 on rates of ligand entry into AChE, reflected in k 1 , an allosteric interaction between the Fas2 binding site and the active center appears to affect the efficiency of AChE catalysis primarily through reduction of both k 3 and k 2 .
Influence of Fasciculin on the Binding of Reversible Ligands-Our compilation of reaction rate constants for diverse reversible inhibitors of AChE permits one to dissect the factors controlling ligand entry to the gorge and steric constraints within the gorge. The affinities of these inhibitors are governed by two factors. First, the association rate constants for all but one ligand studied approach the diffusion-limited range, ensuring rapid formation of inhibitory complexes. Similar rates of association for ligands binding at the peripheral site and active center reflect minimal steric constraints for entry into the narrow, 18 -20 Å in depth active center gorge (cf. Fig. 6) for those ligands not exceeding a critical volume (propidium) or shape ((Ϫ)-huperzine A).
Second, the residence times of ligands in the stabilized complexes with AChE varied over 7 orders of magnitude. Previ-ously reported rate constants for ligand association with AChE, limited to slow dissociating fasciculins, huprines, trifluoroacetophenones, huperzine A, ambenonium, and bisquaternary o-CIB-BQ (Table VII), and only three fast equilibrating ligands, N-methylacridinium, 7-hydroxyquinolinium, and propidium, were in good agreement with the constants reported here (cf .  Tables III and VII The binding of several ligands, such as edrophonium and decamethonium, that lack a spectral overlap with tryptophan fluorescence emission nonetheless results in diminished AChE fluorescence in the ligand-enzyme complex. Since fluorescence resonance energy transfer is not possible in this situation, the quantum yields of several tryptophans have been affected by ligand occupation of the enzyme active center. This observation not only increases the range of ligands whose association with the enzyme can be measured directly but also indicates that the microenvironments of several aromatic residues are perturbed in the complex. The implications of this finding will be addressed in subsequent studies. Linear Free Energy Relationships-Reduced association and dissociation rate constants for ligands interacting with the AChE⅐Fas2 complex reveal significant steric hindrance for entry of ligands into the gorge. The different magnitude of the steric intervention on association and dissociation steps, however, suggests that Fas2 alters the position of the transition state relative to the free and bound species. This is evident from the strikingly different linear free energy relationships between association or dissociation rate constants of the ligands and their equilibrium dissociation constants for AChE and AChE⅐Fas2 complex (Fig. 7).
Relating free energy changes in the transition state to those in the ground states for the various ligands enables one to better describe the energetic pathway for ligand entry and exit from the gorge for AChE and the AChE⅐Fas2 complex (37-39). Thus, for a series of ligands, L, the following linear free energy 2 Radić and Taylor, manuscript in preparation. relationships can be developed.
⌬G ‡ A,L and ⌬G ‡ D,L are the activation free energies for association and dissociation, ⌬G 0 L is the respective equilibrium free energy for each ligand, and ⌬G ‡ 0 is a constant, intrinsic activation barrier. The slope of the relationships is related to ⌽, an TABLE VII Literature values of kinetic constants for interaction of reversible inhibitors with acetylcholinesterase Second order association rate constants (k 1 ) and first order dissociation rate constants (k Ϫ1 ) were determined under the following experimental conditions: 50 mM sodium phosphate buffer, pH 7.2, with ϳ100 mM NaCl and 25 M Triton X-100 (12), 100 mM phosphate buffer, pH 7.0, with 0.01% bovine serum albumin (5, 31), 10 mM Tris/HCl buffer, pH 7.5, with 100 mM NaCl and 0.1 mg/ml bovine serum albumin (30), 20 mM phosphate buffer, pH 7.0, with 0.02% Triton X-100 (4,32), 100 mM phosphate buffer, pH 7.0, with 1.0% Triton X-100 (33) estimate of the position of the transition state for ligand association, which can vary between 0 and 1. Analysis of the data in Fig. 7 shows a ⌽ value of 0.093 for ligand association with AChE and 0.93 for ligand association with AChE⅐Fas2. For dissociation, 1 Ϫ ⌽ is 0.91 for AChE and 0.07 for the AChE⅐Fas2 complex. In each case, huperzine was not included in the analysis, since it was an obvious exception to the correlations, which may relate to its slow association rate. The slow rate of trifluoroacetophenone dissociation and the mutually exclusive binding between propidium and Fas2 preclude the analysis of their interactions with the AChE⅐Fas2 complex.
The capacity of Fas2 to alter the relationship between the transition and ground states points to the role of the Fas2 in influencing the energetics of the pathway for small ligand entry into the gorge. In the absence of Fas2, the activation barrier for formation of ligand-AChE complexes correlates with the stabilization energy of the complex as ligand structure is varied. In the presence of Fas2, the transition barrier energetics correlate with the energies of the free species. Hence, diffusional entry of the ligand to the gorge requires bypassing the cationic bound Fas2, and this becomes the new energetic barrier limiting for the interaction. In the ligand-bound AChE⅐Fas2 complex, escape of the ligand from its residence site deep in the gorge no longer is the rate limitation for dissociation. This altered energetic requirement arises from not simply the charge and steric barriers imparted by the Fas2 molecule per se (6) but also the capacity of Fas2 to restrict conformational flexibility of the ligand binding site, deep in the AChE gorge.
Factors Controlling Kinetics of Ligand Interaction-By using stopped-flow instrumentation, we examined interaction kinetics for rapid and slow equilibrating ligands with selectivity for the active and the peripheral sites. It has been suggested (7, 40 -42) that binding of cationic substrates to peripheral site of mammalian, fish, or insect AChE effectively increases the local substrate concentration available for binding to the active center and thus enhances catalytic efficiency. Such a mechanism seems plausible where substrates would have to traverse an energy barrier imposed by a narrow gorge to reach to the site of catalysis in the active center. As would be expected, the kinetic barriers imposed by the pre-Michaelis complex are minimal, since active center ligands and peripheral site ligands associate with AChE at comparable rates. The modular analysis of molecular traffic through the AChE active center (41) suggested distinct limits for diffusion of neutral and cationic ligands, differing by about 1 order of magnitude. Our determined average second order association rate constants of 8.9 ϫ 10 8 M Ϫ1 s Ϫ1 (for cationic ligands) and 8.2 ϫ 10 7 M Ϫ1 s Ϫ1 (for neutral ligands) are in accord with the prediction.
Protonation of neutral ligands (9-aminoacridine, tacrine) only modestly affects association rates with AChE and the AChE⅐Fas2 complex, perhaps reflecting a balance between rate acceleration by the addition of a charge and deceleration from the increased hydrated molecular volume. The hydrated cation structure, however, decreases the dissociation rates, as evidenced by a comparison of N-methylacridinium and acridine with 9-aminoacridine and tacrine as well as by pH-dependent decrease of the dissociation rate of acridine. Net neutralization of charged edrophonium by turning it into zwitterion, as well as the addition of negative charge to neutral coumarin markedly reduced their association rate constants, as reported for 1-methyl 7-hydroxyquinolinium (36). We find that protonation of enzyme group(s) of pK a 6.1-6.2 critically decreases association rates of positively charged ligands with both AChE and the AChE⅐Fas2 complex in addition to reduction of phosphorylation and carbamoylation rates of the AChE active serine (25).
Rapid conformational isomerizations may control the opening times of the active center gorge to admit ligands (43). In addition to pathway restrictions imposed by charge and steric constraints of Fas2, these isomerizations could be far less frequent in AChE⅐Fas2 due to restrictions in the breathing motions of domains of AChE immobilized by Fas2. Fas2 at the gorge entry can also be expected to influence allosterically the conformation of residues controlling catalysis and ligand dissociation at the base of the gorge.
Comparative analysis of the kinetics of ligand entry and exit from the active center gorge for free AChE and AChE⅐Fas2 complex reveals that the steric influences of Fas2 at the gorge entry are insufficient to explain the differences in linear free energy relationships between the kinetic and equilibrium constants for free AChE and the Fas2⅐AChE complex (Fig. 7).  Table III. Quaternary ammonium, cationic inhibitors are noted by circles, protonated cationic inhibitors by diamonds, and uncharged inhibitors by squares. Lines were generated by linear regression of Equations 8 and 9. (Ϫ)-Huperzine A (inhibitor number 4) was not included in the calculation. A, linear free energy relationship for dissociation from AChE and AChE⅐Fas2. Calculated slopes were (1.0 Ϯ 0.1) for AChE, and (0.074 Ϯ 0.13) for AChE⅐Fas2. B, linear free energy relationship for association with AChE and AChE⅐Fas2. Calculated slopes were Ϫ(0.093 Ϯ 0.056) for AChE, and Ϫ(0.93 Ϯ 0.13) for AChE⅐Fas2.


extfieldsofstudy: Biology Medicine

text:
Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein–tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.

Introduction
Most intracellular short-lived proteins are selectively degraded by the ubiquitin-proteosome pathway (Hershko and Ciechanover, 1998), while most long-lived proteins are degraded in the lysosome. Macroautophagy, usually referred to simply as autophagy, is an intracellular bulk degradation system in which cytoplasmic components, including organelles, are directed to the lysosome/vacuole by a membrane-mediated process (Seglen and Bohley, 1992;Dunn, 1994). Cytoplasmic constituents are first enclosed by double-or multiple-membrane structures called autophagosomes. Eventually, autolysosomes are generated by the fusion of the outer membranes of the autophagosomes and lysosomes. Lysosomal hydrolases degrade the cytoplasm-derived contents of the autophagosome, together with its inner membrane. Autophagy is regulated by nutrients and hormones, and has been suggested to be essential for cellular homeostasis (Mortimore and Pösö, 1987). In addition, autophagy is thought to be crucial for various physiological processes, such as cellular remodeling (Bolender and Weibel, 1973;Masaki et al., 1987), differentiation (Tsukada and Ohsumi, 1993), production of pulmonary surfactant (Hariri et al., 2000), and nonapoptotic cell death during embryogenesis (Clarke, 1990). Defective autophagy may contribute to the pathogenesis of mammary tumors (Liang et al., 1999) and a specific type of myopathy (Nishino et al., 2000;Tanaka et al., 2000).
Autophagosomes are thought to be derived from capshaped cisternae known as isolation membranes. Electron microscopic analysis suggested that the isolation membranes appear to elongate with curvature and finally close to form autophagosomes with nearly constant diameter, 0.5-1.5 m (Pfeifer, 1987;Seglen and Bohley, 1992). Development of the cisternae is a quite unique process compared with other known intracellular membrane dynamics. However, little is known about how the cup-shaped isolation membranes are formed.
In the yeast Saccharomyces cerevisiae , autophagy-defective ( apg and aut ) mutants were isolated (Tsukada and Ohsumi, 1993;Thumm et al., 1994), and most of them were suggested to have defects in autophagosome formation (Klionsky and Ohsumi, 1999). We recently found that a novel protein conjugation system is essential for yeast autophagy (Mizushima et al., 1998a). Two proteins, Apg12 and Apg5, are covalently attached in a manner similar to the ubiquitin conjugation system. The COOH-terminal glycine of Apg12 forms an isopeptide bond with the ⑀ -amino group of a lysine residue in Apg5. This conjugation reaction requires ATP and two enzymes: Apg7 and Apg10 (Kim et al., 1999;Shintani et al., 1999;Tanida et al., 1999;Yuan et al., 1999). These discoveries expanded the field of ubiquitin-like protein conjugation systems (Hochstrasser, 2000). The Apg12-Apg5 conjugate further interacts noncovalently with Apg16 . Analyses of an apg5 null mutant and a temperature-sensitive mutant suggested that Apg5 is required for formation of autophagosomes (George et al., 2000). However, its subcellular localization and molecular function are unknown.
We also demonstrated that the Apg12 conjugation system is conserved in human (Mizushima et al., 1998b). In the present study, to examine the role of Apg5, we created an Apg5 null mutant cell by the gene targeting method using mouse embryonic stem (ES) 1 cells. The resulting clone clearly demonstrated that Apg5 is essential also for autophagy in mammals. By generating various stable transformants, we investigated the subcellular localization and function of Apg5, and the role of its modification by Apg12. This study also enabled us to identify the isolation membrane at early stages and visualize its development into autophagosome.

Plasmids
Mouse Apg5 cDNA was obtained by reverse transcription-PCR based on the sequences of expressed sequence tag clones (mv76e10.r1, me31a04.r1, mj23e09.r1). The cDNA sequence was deposited in the DDBJ/EMBL/ GenBank databases (AB048349), which encodes for a 275 amino acids protein that is 97% identical to human Apg5 (Hammond et al., 1998;Mizushima et al., 1998b). Mouse APG5 genomic clones were isolated from a 129/Sv genomic library using the mouse Apg5 cDNA as a probe. A targeting vector was constructed by replacing a 5.6-kb BamHI-SpeI fragment including the putative second (containing the first ATG) and third exons with the neo-resistant cassette from pMCI-Neo. The herpes simplex thymidine kinase gene was inserted downstream of the short arm for negative selection against random integration of the vector (see Fig. 1). The mouse Apg5 cDNA was also subcloned into the SmaI site of a mammalian expression vector pCI-neo (Promega). The green fluorescent protein (GFP)-tagged Apg5 expression vector has been described previously (Mizushima et al., 1998b). Replacement of Lys130 to Arg (K130R) was performed using a Quick Change Site-directed Mutagenesis Kit (Stratagene).

ES Cell Culture
R1 ES cells (a generous gift from Dr. Andras Nagy, Samuel Lunenfeld Research Institute, Toronto, Canada) were cultured on mitomycin C-treated embryonic fibroblasts, STO feeder cells (Lexicon Genetics Inc.), or gelatinized dish in a complete ES medium: high glucose Dulbecco's modified Eagle's medium supplemented with 20% FCS, 2 mM L -glutamine, 1 ϫ nonessential amino acids (GIBCO BRL), 1 M 2-mercaptoethanol, antibiotics, and 1,000 U/ml leukemia inhibitory factor (Life Technologies, Inc.). For amino acid starvation, cells were cultured in Hanks' solution containing 10 mM Hepes, pH 7.5 (without amino acid and FCS).

Production of APG5 Ϫ / Ϫ ES Cells and Stable Transformants
The linearized targeting vector (30 g) was transfected into 10 7 R1 ES cells by electroporation using a Gene Pulser (Bio-Rad Laboratories) set at 270 V and 500 F. The 0.25 mg/ml G418-and gancyclovir-selected clones (96 clones) were examined, and homologous recombination was detected in 13 clones. These clones were also tested for single integration by Southern blot analysis with a neo probe. To obtain APG5 Ϫ / Ϫ cells, an APG5 ϩ / Ϫ clone (#33) was grown in the ES medium containing 8 mg/ml G418. 3 of 123 clones selected were APG5 Ϫ / Ϫ (A11, B19, B22). To obtain stable transformants, 8 ϫ 10 6 ES cells were electroporated with 20 g of circular or linearized expression vector, and with 2 g of pPGKpurobpA when required. Cells were selected in the presence of 0.5-1 mg/ml G418 or 5-10 g/ml puromycin.

Antibodies
Anti-Apg5 antibody (SO4) against glutathione-S-transferase-fused human Apg5 was prepared by immunization of rabbits and affinity purified. Anti-mouse Apg12 antibodies (NM2) against a synthetic peptide corresponding to the NH 2 -terminal 14 amino acids of mouse Apg12 and an additional Cys (MSEDSEVVLQLPSAC) was prepared by immunization of rabbits as previously described  and affinity purified. Anti-LC3 antibody against recombinant LC3 , anti-rat Lgp85 antibody (Okazaki et al., 1992), and anti-rat aldolase antibody (Kominami et al., 1983) were previously described. For immunofluorescence microscopy, fluorolink Cy5-labeled goat anti-rabbit IgG antibody (Amersham Pharmacia Biotech) was used. Rabbit polyclonal anti-GFP antibody (CLONTECH Laboratories, Inc.) was used for immunoelectron microscopy.

Preparation of Whole-Cell Lysates and Western Blotting
Whole-cell lysates were prepared with a lysis buffer (2% NP-40, 0.2% SDS in PBS supplemented with protease inhibitors). Western blotting was performed as previously described (Mizushima et al., 1998b).

Electron Microscopy
Conventional electron microscopy was performed as described previously , except that ES cells grown on gelatinized plastic coverslips were prefixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 2 h. For immunoelectron microscopy, the pre-embedding silver enhancement immunogold method was performed as previously described , with a slight modification. ES cells cultured on gelatincoated plastic coverslips were fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, for 2 h. The cells were washed in the buffer three times, dipped in phosphate buffer (PB) containing 15% glycerol and 35% sucrose, frozen and thawed using liquid nitrogen, and then incubated in PB containing 0.005% saponin, 10% BSA, 10% normal goat serum, and 0.1% cold water fish skin gelatin for blocking for 30 min. Cells were then treated with rabbit IgG against GFP (diluted 500 ϫ ) in the blocking solution, overnight. Then, the cells were washed in PB containing 0.005% saponin for 10 min six times, and incubated with goat anti-rabbit IgG that was conjugated to colloidal gold (1.4-nm diameter) in the blocking solution for 2 h. Cells were washed with PB for 10 min six times, and fixed with 1% glutaraldehyde in PB for 10 min. After washing, the gold labeling was intensified by using a silver enhancement kit for 6 min at 20 Њ C in the dark. After washing in distilled water, cells were post-fixed in 0.5% OsO 4 for 90 min at 4 Њ C, washed in distilled water, incubated with 50% ethanol for 10 min, and stained with 2% uranyl acetate in 70% ethanol for 2 h. The cells were further dehydrated with a graded series of ethanol and embedded in epoxy resin. Ultra-thin sections were doubly stained with uranyl acetate and lead citrate.
Morphometric analysis was performed with NIH image software. 30 cell sections were analyzed. Autophagosomes were defined as double-or multiple-membrane structures surrounding undigested cytoplasmic constituents. Autolysosomes were defined as single membrane structures containing cytoplasmic components at various stages of degradation.

Bulk Protein Degradation Assay
Degradation of long-lived proteins was measured based on a standard method (Ogier-Denis et al., 1996). ES cells maintained on gelatinized plates without feeder cells were plated at 2 ϫ 10 5 cells/ well in gelatinized 24-well plates and cultured in complete ES medium for 24 h. Cells were then labeled for 24 h with the same medium containing 1.5 Ci/ml L -[ 14 C] valine (Moravek Biochemicals Inc.). After three rinses with PBS, cells were incubated in either complete ES medium or Hanks' solution containing 0.1% BSA and 10 mM cold valine. When required, 1 mM chloroquine, 0.1 M bafilomycin A 1 , or 10 mM 3-methyladenine (3-MA) was added. After the 1-h incubation, the medium was replaced with identical fresh medium and incubated for additional 2 h. The medium was precipitated in 10% TCA, and TCA-soluble radioactivity was measured. Total-cell radioactivity was measured after lysis with 0.1 M NaOH. [ 14 C] Valine release was calculated as a percentage of the radioactivity in the TCA-soluble supernatant to the total cell radioactivity.

Fluorescence Microscopy
ES cells grown on gelatinized coverslips were fixed and, if necessary, stained with affinity-purified anti-mouse Apg12 antibody (200 ϫ dilution) or anti-LC3 antibody (200 ϫ dilution), and then examined under a fluorescence laser scanning confocal microscope, LSM510 (Carl Zeiss, Inc.), as previously described . Time-lapse video microscopy was performed at 37 Њ C with a DeltaVision microscope system (Applied Precision Inc.) equipped with a ⌬ TC3 culture dish system (Bioptechs) for temperature control.

Online Supplemental Material
A supplemental figure is supplied in which subcellular fractionation analysis demonstrates that most Apg12-Apg5 conjugates are recovered in the cytosolic fraction (Fig. S1). The QuickTime movies from which the images of Fig. 7 A were taken are also available. APG5 Ϫ / Ϫ cells expressing GFP-Apg5 (videos 1-3) or GFP-Apg5K130R (video 4) were cultured in Hanks' solution for 1 h, and GFP images were taken every 3.8 s. They are animated at 15 images/s. Online supplemental materials can be found at http: //www.jcb.org/cgi/content/full/152/4/657/DC1.

Production of Apg5-deficient Cells
To produce a null mutation in the mouse APG5 gene, we targeted the mouse APG5 gene of R1 ES cells, using a targeting vector shown in Fig. 1 A. By subjecting an APG5 ϩ / Ϫ ES clone (#33) to growth selection in an elevated concentration of G418, three Apg5-deficient ( APG5 Ϫ / Ϫ ) clones (A11, B19, and B22) were obtained (Fig. 1 B). An anti-Apg5 antibody recognized a 56-kD protein in the wild-type ES cells (Fig. 1 C). In addition, a very faint band at 30 kD, corresponding to Apg5 monomer, was observed. The 56-kD band was also reactive with anti-mouse Apg12 antibodies (data not shown). These data suggest that Apg5 exists almost completely in the Apg12-conjugated form in the wildtype ES cells. The amount of the Apg12-Apg5 conjugate decreased in the heterozygous ( APG5 ϩ / Ϫ ) mutant, and was not detected at all in the three independent APG5 Ϫ / Ϫ clones ( Fig. 1 C). Taken together, these data confirm that the targeted ES cells contain a null mutation in the APG5 gene.
We then generated several stable transformants by transfecting A11 cells with Apg5 cDNAs (Fig. 1 D). In two transformants with wild-type Apg5 cDNA (WT1 and WT13), exogenous Apg5, of which expression was a little weaker than in wild-type cells, was conjugated with Apg12 almost completely. The Apg5 K130R mutant was unable to be conjugated (KR27), confirming our previous observation that Lys130 is an acceptor residue for Apg12 conjugation (Mizushima et al., 1998b). GFP-fused Apg5 was expressed (GFP24) at a similar level as in wild-type cells, most of which is conjugated with Apg12.

APG5 Ϫ / Ϫ Cells Exhibit a Block in the Autophagic Pathway
APG5 Ϫ / Ϫ cells were found to grow at the same rate as wild-type cells and form colonies with normal morphology (data not shown). Since three APG5 Ϫ / Ϫ clones (A11, B19, and B22) showed similar phenotypes, we used A11 for further studies unless otherwise indicated.
Autophagy can be well induced by amino acid starvation in wild-type ES cells (Fig. 2 A) as other cultured cells. 2 h after amino acid withdrawal, autophagic vacuoles occupied ف 1.1% of the total cytoplasmic volume (Fig. 2  E). Autolysosomes were observed more frequently than autophagosomes (autophagosome 0.33%, autolysosome 0.77%). In contrast, no autolysosomes were observed at all in APG5 Ϫ / Ϫ cells, even under amino acid starvation conditions (Fig. 2, B and E). Some autophagosome-like structures were induced by starvation (Fig. 2, C and D), but these structures were significantly fewer in number than autophagosomes in wild-type cells (Fig. 2 E). Other organelles, such as mitochondria, the endoplasmic reticulum, the Golgi apparatus, lysosomes, and multivesicular bodies, appeared normal. These results suggest that, in APG5 Ϫ / Ϫ cells, autophagic degradation does not occur and autophagosome formation is impaired.

Bulk Protein Degradation Is Reduced in Apg5-deficient Cells
Autophagy is thought to be a major pathway for degradation of cytoplasmic proteins. We assessed bulk degradation of long-lived proteins by measuring the release of TCA-soluble [ 14 C] valine from cells. Lysosomal protein degradation was estimated by the difference between the [ 14 C] valine release from cells treated with and without lysosomotropic reagents such as chloroquine and bafilomycin A 1 , a vacuolar H ϩ -ATPase inhibitor. Lysosomal protein degradation induced by amino acid starvation in APG5 Ϫ / Ϫ cells was significantly reduced to less than half of that in APG5 ϩ / ϩ and APG5 ϩ / Ϫ cells (Fig. 3, A and B). Apg5 cDNA transformants (WT1, WT13, and GFP24) restored protein degradation, confirming the specificity of Apg5 deficiency (Fig. 3 B). Cells grown in nutrient-rich medium showed a very low level of basal lysosomal degradation, with no significant difference between APG5 ϩ / ϩ and APG5 Ϫ / Ϫ cells. These data indicate that autophagy is indeed a major route for lysosomal protein degradation. In addition, in the KR27 clone, protein degradation was not restored, suggesting that Apg12 conjugation of Apg5 is essential for autophagic degradation.
3-MA, a commonly used inhibitor of autophagic sequestration (Seglen and Gordon, 1982), reduced bulk degradation during amino acid deprivation even more markedly than did the disruption of the APG5 gene (Fig. 3 A). Furthermore, the effect of 3-MA was still observed in autophagy-negative APG5 Ϫ / Ϫ cells, suggesting that 3-MA may have some additional effects on protein degradation other than autophagy.

Apg5 Colocalizes with Punctate LC3 Structures
Since the electron microscopic analysis of APG5 Ϫ / Ϫ cells suggested that Apg5 acts at an early stage of autophagosome formation, we examined the subcellular distribution of Apg5 in wild-type ES cells. The Apg12-Apg5 conjugates were recovered primarily in the cytosolic fraction, with a very small portion found in the membrane fraction (see Fig. S1 in the Online Supplement). The total amount and subcellular distribution of the Apg12-Apg5 conjugates did not change significantly after amino acid starvation.
We further examined the localization of Apg5 by confocal microscopy. Bulk protein degradation (Fig. 3 B) and autolysosome formation (see Fig. 6) were restored in the GFP24 clone, indicating that GFP-fused Apg5 (GFP-Apg5) was functional and that the physiological localization of Apg5 can be indicated by observing the GFP signal. Under nutrient-rich conditions, most GFP-Apg5 was found to distribute evenly throughout the cytoplasm, with few punctate spots (Fig. 4, 0 min). 30 min after the medium was replaced with amino acid-free medium, the number of the punctate structures increased, which continued at 60 and 120 min. These GFP-Apg5 punctate spots were overlapped with anti-Apg12 antibody staining, indicating that GFP-Apg5 on these structures were conjugated with Apg12 (Fig. 5 A). 3-MA and wortmannin, an inhibitor of phosphatidylinositol 3-kinases, which were known to inhibit autophagy, suppressed GFP-Apg5 spot induction during amino acid deprivation (Fig. 4). APG5 ϩ / ϩ cells stably expressing GFP alone displayed only cytoplasmic staining (data not shown).
We next counterstained endogenous LC3 to use as a specific marker for autophagosome. LC3 is a mammalian homologue of Aut7/Apg8 (Lang et al., 1998;Kirisako et al., 1999) and is the only mammalian protein that has been identified to localize to the autophagosome membrane as well as to the cytosol . In cells grown in complete medium, LC3 was stained as a cytoplasmic pattern with very fine punctate structures throughout the cytoplasm (Fig. 5 B), as reported previously . Amino acid starvation induced large dots of LC3 staining, which could represent autophagic vacuoles (Fig. 5 C). In the starved cells, GFP-Apg5 well colocalized with LC3 structures (Fig. 5, C-G), although some GFP-Apg5 spots were very weak for LC3 staining (Fig. 5, F and G, arrows). On the other hand, there were many spots that were positive for LC3 staining but negative for GFP-Apg5. The colocalization of Apg5 with part of LC3 suggests that Apg12-Apg5 is targeted to certain restricted structures in the autophagic pathway.

Apg5 Localizes to Isolation Membranes
To examine the localization of Apg5 in more detail, we carried out immunoelectron microscopy using an anti-GFP antibody. Under nonstarvation conditions, silver-enhanced gold particles showed mostly cytoplasmic distribution (data not shown). In cells subjected to a 2-h starvation period, gold particles were found extensively associated with isolation membranes (Fig. 6, A, B, and G). Their distribution was asymmetric; most of the gold particles were found on the outer side of isolation membranes, with a few on the inner membrane. In contrast to the isolation membrane, almost no GFP-Apg5 signal was detected on the autophagosome and autolysosome membranes (Fig. 6, A-C, I, and J). Apg5 associated with an isolation membrane that was close to completing circularization (Fig. 6 H), suggesting that Apg12-Apg5 detaches from the membrane immediately before or after autophagosome formation is completed.
In addition to typical isolation membranes, there were some smaller structures that were labeled throughout with gold particles (Fig. 6, B and C, double arrows, and E and F). These single membrane-bound compartments adopted a crescent shape and were induced by amino acid starvation. Occasionally, small vesicles were observed inside these compartments. The curved morphology suggests that these structures represent the early stage of the development of isolation membrane.
To determine whether the isolation membranes are derived from the newly generated, Apg5-associated small compartment, we carried out time-lapse video microscopy. Small dots of GFP-Apg5 appeared and elongated slowly, and then bent and became semi-spherical structures (Fig. 7 A and videos 1-3). Finally, the GFP signal disappeared when the structure was close to forming a complete spherical body. Such a pattern of development is consistent with the electron microscopic images (Fig. 6, E-J). The time-lapse microscopy showed that the GFP-Apg5 spots were transient, appearing at random times during amino acid starvation and disappearing immediately before or after circularization, never forming any stable structures (Fig. 7 B). The average lifetime of the GFP-Apg5 structures was 9.7 Ϯ 1.8 min.

Apg12 Conjugation Is Not Required for Membrane Targeting of Apg5, but Is Essential for Membrane Elongation
To investigate the role of Apg12 conjugation of Apg5, we generated an APG5 Ϫ/Ϫ clone expressing GFP-Apg5 K130R (GKR-1). Western blot analysis confirmed that GFP-  Apg5 K130R was not conjugated with Apg12 (data not shown). In amino acid-starved GKR-1 cells, numerous punctate GFP signals were observed (Fig. 5 H). The GFP-Apg5 K130R dots were slightly increased in number, about twice as many as those in starved wild-type (GFP24) cells.
Apg12 was not recruited to the GFP-Apg5 K130R spots (data not shown). Immunoelectron microscopy demonstrated that the crescent-shaped compartments were generated, to which GFP-Apg5 K130R localized (Fig. 6 D). These results clearly indicate that Apg12 conjugation is not required for membrane association of Apg5. However, autolysosomes were not detected in starved GKR-1 cells. Time-lapse video microscopy demonstrated that the GFP-Apg5 K130R -associated spots did not mature into cup-shaped structures and basically remained as punctate spots (Fig. 7 A and video 4). These results suggest that Apg12 conjugation is required for involvement of Apg5 in elongation of the membrane to form cup-shaped isolation membrane and autophagosome. This is in agreement with the data that bulk protein degradation was not restored in KR27 cells (Fig. 3 B).

Targeting of LC3 to Isolation Membrane Depends on Apg12-Apg5
We observed some autophagosome-like structures in amino acid-starved APG5 Ϫ/Ϫ cells (Fig. 2). We thus determined whether LC3 normally localized to these membranes. In amino acid-starved wild-type ES cells, LC3 staining was observed on both inner and outer membranes of autophagosomes (Fig. 8, A and B) and isolation membranes (C), as well as in the cytosol. However, no LC3 Figure 6. GFP-Apg5 is present on isolation membranes. GFP24 (A-C, E-J) or GKR-1 (D) cells were cultured in Hanks' solution for 2 h and fixed. The localization of GFP-Apg5 was examined by silver-enhanced immunogold electron microscopy using an anti-GFP antibody. In B, an isolation membrane is enclosing a mitochondrion. The isolation membranes (arrows), autophagosomes (open arrowheads) and autolysosomes (closed arrowheads) are indicated. Double arrows indicate small membrane compartments to which GFP-Apg5 extensively localizes. The typical images of these structures are shown at higher magnification (E-J). Bars, 1 m.
staining was detected on the autophagosome-like structures in APG5 Ϫ/Ϫ cells (Fig. 8, D and E). In immunofluorescence microscopy, large dots of LC3 staining were not observed in starved APG5 Ϫ/Ϫ cells (data not shown), in contrast to wild-type cells (Fig. 5 C). We also examined the localization of LC3 in the GKR-1 clone. While GFP-Apg5 K130R localized to punctate spots as mentioned above, LC3 did not colocalize with the GFP-Apg5 K130R spots and retained its cytoplasmic staining during starvation (Fig. 5  H). Taken together, these results suggest that Apg5 and its modification with Apg12 play important roles in recruitment of LC3 to isolation membranes.
We previously demonstrated that LC3 is post-translationally modified to generate LC3-I, and then LC3-II. LC3 on autophagic vacuoles is in the LC3-II form, while LC3-I is cytosolic . In amino acid-starved ES cells, significant amounts of LC3-II were generated. However, LC3-II was not detected in APG5 Ϫ/Ϫ cells (Fig. 8 F). LC3-II generation was restored in the stable transformants with wild-type Apg5 cDNA, but not with the Apg5 K130R mutant. These biochemical data confirmed that without Apg12-Apg5, LC3 could not target to the membranes.

Discussion
We have genetically dissected autophagic process in yeast and now are a getting better understanding of the process. However, many questions remain to be addressed. How the isolation membranes are formed is one of the important yet poorly understood subjects. In this study, we demonstrated that the cup-shaped isolation membranes are developed from the crescent-shaped small membrane compartments using Apg5 as a marker molecule in mouse cells. Serial sections revealed that the crescent-shaped structures were not parts of mature isolation membranes (data not shown). These structures have never been recognized unless labeled with GFP-Apg5, but they are likely to be direct precursors of the autophagosome, as they were shown to mature into cup-shaped structures (Fig. 7). We were able to visualize the autophagosome formation in real time for the first time. The results directly evidenced the elongation model of isolation membrane, which has been hypothesized hitherto based on electron microscopic observations. The video microscopic analysis clearly demonstrated that the cisternae membranes elongate, bend, and form spherical autophagosomes, which is a quite unique membrane movement within cells. The molecular mechanism for such membrane dynamics is interesting, and studies on Apg proteins on the membrane would provide us further insights.
The existence of autophagosome precursors was proposed earlier by Seglen (1987) and Fengsrud et al. (1995). They observed thick, osmiophilic membrane multilayers in isolated hepatocytes by conventional electron microscopy, and postulated that they were autophagosome precursors. These structures, termed phagophores, were morphologically different in some respects from the Apg5-associated compartment. However, these two structures might be essentially the same, with such differences due to different fixation methods and different cell types. Our direct evidence indicated that isolation membranes are maturated from the small compartments, not simply derived from large preexisting membranes, although we do not know at this stage whether the small compartments are formed de novo or derived from some membrane sources. Many questions regarding autophagosome formation would be clarified by using Apg5 as a marker for autophagosome precursors. For example, our results indicate that phosphatidylinositol 3-kinase activity is required for generation of the Apg5-associated small compartment rather than its maturation into autophagosome (Fig. 4). Isolation and characterization of these compartments would provide evidences about the origin of autophagosomal membrane, which has been the subject of controversy (Hirsimäki and Reunanen, 1980;Dunn, 1990;Yamamoto et al., 1990;Ueno et al., 1991).
During development of the isolation membranes, the distribution pattern of Apg5 becomes very characteristic. Most Apg5 associates with the outer side of the isolation membranes (Fig. 6), in contrast to the symmetrical distribution of LC3 (Fig. 8). Previous freeze-replica ultrastructural analysis pointed out differences between the outer and inner membranes of autophagosomes (Réz and Meldolesi, 1980;Hirsimäki et al., 1982;Baba et al., 1995). The inner membrane is almost free of intramembrane particles, while the outer membrane has a small, but significant, number of particles. The unique distribution of Apg5 is the first evidence that the molecular composition of the two membranes is different. It further suggests that such molecular asymmetry is already generated before completion of autophagosome formation. Apg5 must dissociate from the membrane immediately before or after completion of autophagosome formation, since almost no Apg5 was detected on the autophagosomes and autolysosomes ( Fig. 9 A).
Based on the localization of Apg5, we postulate that Apg5 functions during development of isolation membranes. This hypothesis was supported by the phenotype of the Apg5 null mutant cells: the number of the isolation membranes and autophagosomes is reduced, and the rarely observed isolation membrane-and autophagosome-like structures are not labeled with LC3. We also examined the role of Apg12 conjugation by generating the stable clones expressing only conjugation-deficient Apg5 K130R . Some ubiquitin-like modification systems affect intracellular localization of substrate proteins. For example, protein ubiquitination is accompanied by proteasome targeting or endocytosis (Hershko and Ciechanover, 1998). SUMO-1 modification of Ran-GAP1 leads it to the nuclear pore complex (Hochstrasser, 2000). However, our data suggest that Apg12 is not required for membrane association of Apg5. Rather, Apg12 conjugation of Apg5 is required for dynamic change of membrane morphology. We concluded that Apg5, by forming a conjugate with Apg12, directs elongation of the isolation membranes from the small crescent compartments (Fig. 9 B). How Apg5 is involved in membrane elongation is not known, but at least there are two possibilities. First, Apg12-Apg5 transports membranes or lipids from somewhere to the isolation membranes. Second, Apg12-Apg5 association with the membrane regulates acquisition of additional membranes. We showed in the present study that cytosolic LC3 targets to the isolation membranes in an Apg12-Apg5-dependent manner (Fig. 9 B). Therefore, membrane elongation would be related to recruitment of LC3 to the membrane. Whether there is a direct interaction between Apg12-Apg5 and LC3 is not clear; coimmunoprecipitation of these molecules has thus far been unsuccessful (data not shown).
This study provided us the first mammalian clone with genetically impaired autophagic activity. Although some autophagosome-like structures are occasionally seen in APG5 Ϫ/Ϫ cells, LC3 staining revealed that they are not normal autophagosomes, suggesting that impaired autophagosome formation results in a complete block in the autophagic pathway. Based on the data in Fig. 3, autophagy would account for %07-06ف of starvation-induced lysosomal protein degradation in ES cells. Lysosomal degradation is still clearly enhanced by starvation in APG5 Ϫ/Ϫ cells. The process responsible for this is unknown. So far, at least two pathways other than macroautophagy have been proposed for delivery of cytoplasmic constituents to lysosomes. One is microautophagy, in which small portions of cytoplasm are sequestered into lysosomes by invagination of the lysosomal membrane (Marzella and Glaumann, 1987). The other is chaperon-mediated autophagy, proposed by Dice (1990). Although it remains to be determined, these pathways may account for the remaining degradation activity of APG5 Ϫ/Ϫ cells. In any case, this clone would be useful to study physiological meanings of autophagy in mammals, which would be more complex than in unicellular eukaryotes.
In conclusion, our present study suggests that Apg12-Apg5 localizes to autophagosome precursors, and plays an essential role in their development into autophagosomes, in cooperation with LC3. Establishment of the Apg5 null mutant cells not only demonstrated the indispensable role of Apg5, but also provided us new insights into the early stages of autophagosome formation that have been poorly understood both in yeast and mammals even since the discovery of autophagy in the 1960s. Figure 9. Model of subcellular localization of Apg12-Apg5 and LC3, and role of Apg12 conjugation in autophagosome formation. (A) Apg12-Apg5 conjugate localizes to the crescent-shaped autophagosome precursors. While these structures elongate and maturate into cup-shaped isolation membranes, LC3 is recruited to the membrane in the Apg5-dependent manner and Apg5 changes its localization to the outer side of the membrane. Apg5 plays an essential role in this membrane development. Immediately before or after the completion of autophagosome formation, Apg5 detaches from the membrane. Some LC3 also dissociate from the autophagosomal membrane thereafter. (B) Apg12 conjugation is not required for membrane targeting of Apg5, but it is essential for maturation of the isolation membrane into autophagosome and recruitment of LC3 to the membrane.


extfieldsofstudy: Medicine Biology

text:
Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans

Centrosomes mature as cells enter mitosis, accumulating γ-tubulin and other pericentriolar material (PCM) components. This occurs concomitant with an increase in the number of centrosomally organized microtubules (MTs). Here, we use RNA-mediated interference (RNAi) to examine the role of the aurora-A kinase, AIR-1, during centrosome maturation in Caenorhabditis elegans. In air-1(RNAi) embryos, centrosomes separate normally, an event that occurs before maturation in C. elegans. After nuclear envelope breakdown, the separated centrosomes collapse together, and spindle assembly fails. In mitotic air-1(RNAi) embryos, centrosomal α-tubulin fluorescence intensity accumulates to only 40% of wild-type levels, suggesting a defect in the maturation process. Consistent with this hypothesis, we find that AIR-1 is required for the increase in centrosomal γ-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis. Furthermore, the AIR-1–dependent increase in centrosomal γ-tubulin does not require MTs. These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation.

Introduction
In metazoans, centrosomes consist of a pair of centrioles surrounded by electron-dense pericentriolar material (PCM)* that directs the assembly of microtubules (MTs). During cell division, centrosomes undergo a series of structural changes (for review see Fry et al., 2000). During the G2/M transition, coincident with the activation of Cdk1, centrosomes mature, accumulating ␥ -tubulin and other PCM components and increasing in size and nucleating capacity (for review see Palazzo et al., 2000). Centrosomes separate before spindle assembly with timing that varies among cell types (Callaini et al., 1997;Keating and White, 1998;Fry et al., 2000) and organize the poles of the mitotic spindle after the breakdown of the nuclear envelope. The regulation of these cell cycle-dependent events remains largely unknown, although mitotic kinases likely play a key role (Fry et al., 2000;Nigg, 2001).
In addition to responding to cell cycle transitions, centrosomes also contribute to cell cycle progression. Recent experiments have shown that centrosomes are required for cells to enter S phase (Hinchcliffe et al., 2001;Khodjakov and Rieder, 2001). In addition, Cdk1 and polo-like kinase, two mitotic kinases that participate in a positive feedback loop regulating mitotic entry, accumulate at centrosomes (Glover et al., 1998;Ohi and Gould, 1999;Nigg, 2001). This has led to the speculation that centrosomes might accelerate the G2/M transition by facilitating the rapid coactivation of cell cycle regulators in proximity to target proteins involved in spindle assembly (Ohi and Gould, 1999).
In vertebrate cells, centrosome maturation occurs late in the G2/M transition. The amount of ␥ -tubulin at centrosomes increases 3-5-fold in late prophase (Khodjakov and Rieder, 1999), coincident with an increase in the size of centrosomal asters (Palazzo et al., 2000). Polo-like kinases have been directly implicated in PCM recruitment during maturation (Glover et al., 1998). Mutants in Drosophila polo fail to recruit ␥ -tubulin and the centrosomal protein CP190 (Sunkel and Glover, 1988;Donaldson et al., 2001), and injection of antibodies to Plk-1 into immortalized human tissue culture cells results in small centrosomes that fail to recruit ␥ -tubulin and MPM-2 phosphoepitopes (Lane and Nigg, 1996).
Aurora-A serine/threonine kinases are a recently emerging family of mitotic kinases that localize to centrosomes. Aurora-A kinases have been implicated in centrosome sepa-ration and spindle assembly (for review see Bischoff and Plowman, 1999;Giet and Prigent, 1999;Nigg, 2001). Interestingly, aurora-A is amplified frequently in human malignancies, and its overexpression can transform cells (Bischoff and Plowman, 1999). The activity of aurora-A kinase peaks during the G2/M transition (for review see Bischoff and Plowman, 1999), making it an attractive candidate for regulating centrosome maturation.
To analyze the role of aurora-A in centrosome maturation, we focus on the first mitotic division of the Caenorhabditis elegans embryo. A major experimental advantage of C. elegans is the ability to specifically destroy the mRNA transcript derived from any gene by dsRNA-mediated interference (RNAi) (Montgomery and Fire, 1998). Injection of dsRNA into adult hermaphrodites results in the formation of oocytes containing cytoplasm essentially cleared of the targeted protein within 20-30 h. Here, we combine RNAi of aurora-A with live and fixed assays for centrosome assembly and function to reveal a fundamental role for aurora-A in centrosome maturation.

AIR-1 localizes to centrosomes and is required for spindle assembly
The C. elegans homologue of aurora-A, AIR-1, localizes to centrosomes and is required for normal spindle assembly (Schumacher et al., 1998). To investigate the role of aurora-A in the centrosome cycle, we first analyzed its localization during the first embryonic division. We raised an antibody to AIR-1 that detects a single band of ف 40 kD on Western blots. This band is reduced Ͼ 90% in air-1(RNAi) worms ( Fig. 1 A), confirming the specificity of our antibody. During fertilization, a sperm-derived centrosome enters the egg (which lacks centrosomes) and duplicates, resulting in two small centrosomes positioned between the sperm pronucleus and the cortex. AIR-1 localizes to centrosomes in early embryos ( Fig. 1 B , top) and also weakly to astral and cytoplasmic MTs. In wild-type, the maternal pronucleus migrates toward the sperm pronucleus as the chromosomes condense. The pronuclei fuse, their nuclear envelopes break down, and the first mitotic spindle assembles. In mitotic embryos, AIR-1 concentrates in the centers of the asters (Fig. 1 B, bottom) in a donut-shaped region peripheral to ␥ -tubulin staining (Fig. 1 C) and also localizes along the base of astral MTs. The pattern of localization of AIR-1 is specific, since it is not observed in air-1(RNAi) embryos.
In air-1(RNAi) embryos judged to be mitotic by the presence of condensed chromosomes, normal spindles were never observed. Instead, a pair of closely apposed centrosomal asters was associated with the condensed chromosomes ( Fig. 1 D). These results confirm an essential role for AIR-1 in spindle assembly and are reminiscent of observations in Xenopus and Drosophila where inhibition of aurora-A function results in monopolar spindles with unseparated centrosomes (Glover et al., 1995;Roghi et al., 1998).

AIR-1 is required to maintain centrosome separation during spindle assembly
To examine the kinetics of centrosome separation, we filmed air-1(RNAi) embryos expressing GFP-␣ -tubulin ( Fig. 2, A and B). In wild-type, the two centrosomes are located on opposite sides of the nucleus and maintain a constant separation just before and after nuclear envelope breakdown (NEBD). The distance between the centrosomes begins to increase late in metaphase and contin-  . AIR-1 is required to maintain centrosome separation during spindle assembly. (A) Panels summarize time-lapse recordings of wild-type (left) and air-1(RNAi) embryos (right) expressing GFP-␣-tubulin. Times are seconds after NEBD. In wild-type (left), unseparated asters (Ϫ446 s, arrow) associate with the sperm pronucleus. (Ϫ174 s) The asters separate and position themselves on opposite sides of the sperm pronucleus. The maternal pronucleus is also indicated (arrowhead). (ϩ0 s) The pronuclei meet, move with the asters (arrows) to the center of the embryo, and the nuclear envelope breaks down. (ϩ145) After NEBD, the asters increase in size and become the poles of the mitotic spindle. In air-1(RNAi) embryos (right), the asters (arrows) separate as in wild-type (Ϫ563 s and Ϫ282 s). The maternal pronucleus (arrowhead) is out of focus. After NEBD, the centrosomal asters (arrows) collapse toward each other till they sit side by side (ϩ97 s and ϩ177 s). See also videos 1 and 2 available at http://www.jcb.org/cgi/ content/full/jcb. ues to increase as the spindle elongates during anaphase (Oegema et al., 2001). In air-1(RNAi) embryos, centrosomal asters were detected associated with sperm pronuclei as early as 600 s before NEBD (Fig. 2 A, Ϫ 563 s). The asters remained associated with the sperm pronucleus and always separated ( Fig. 2 A, Ϫ 282 s). Cytoplasmic MTs depolymerized in both wild-type ( Fig. 2 A, Ϫ 446 compared with ϩ 0 s) and air-1(RNAi) embryos (Fig. 2 A, Ϫ 563 s compared with ϩ 97 s) as they entered mitosis. However, subsequent to NEBD the asters in air-1(RNAi) embryos collapsed together (collapse occurs between ϩ 97 and ϩ 177 s; Fig. 2 A), resulting in two small relatively bright asters lying side by side. In some cases, the asters moved apart slightly during late anaphase/telophase. In other examples, the two asters remained unseparated and moved around the embryo or rocked back and forth coincident with the onset of cortical contractions. These results suggest that AIR-1 is not necessary for initial separation of centrosomes in C. elegans but is required to maintain centrosome separation during spindle assembly.
We noticed that mitotic centrosomal asters in air-1(RNAi) embryos appeared less robust than in wild-type. To quantify this, we measured centrosomal ␣ -tubulin fluorescence (Fig. 2 C). In wild-type, centrosomal ␣ -tubulin fluorescence increased during the interval between 300 s before NEBD and anaphase onset ( ف 155 s after NEBD). In air-1(RNAi) embryos, centrosomal ␣ -tubulin fluorescence also increased but ultimately reached only ف 40% of wildtype levels. This suggests that mitotic centrosomes in air-1(RNAi) embryos organize fewer MTs than in wild-type.

AIR-1 is required to recruit centrosomal ␥ -tubulin during maturation
The small centrosomal asters in mitotic air-1(RNAi) embryos suggested a role for AIR-1 in centrosome maturation. To determine if AIR-1 has a role in the accumulation of centrosomal ␥ -tubulin during this transition, we filmed air-1(RNAi) embryos expressing both GFP histone, as a marker for cell cycle progression, and GFP-␥ -tubulin (Fig. 3 A). ␥ -Tubulin was visible shortly after fertilization at the still tiny centrosomes in both wild-type and air-1(RNAi) embryos (Fig. 3 A, Ϫ 484 s and Ϫ 608 s). The cell cycle progressed in air-1(RNAi) embryos as evidenced by DNA condensation, the release of cortical contraction at the end of pseudocleavage, NEBD, the onset of cortical contractions coincident with the initiation of cytokinesis in wild-type, and reformation of the nuclear envelope. However, the dramatic accumulation of centrosomal ␥ -tubulin that occurs in wild-type during the interval between 300 s before NEBD and anaphase onset (Fig. 3 A, compare -256 s with ϩ 122 s) was completely absent in air-1(RNAi) embryos (Fig. 3 A, right, all panels). Quantification of integrated fluorescence intensity showed that centrosomal ␥ -tubulin fluorescence does not change significantly as air-1(RNAi) embryos enter mitosis (Fig. 3 B). In summary, our results show that the cell cycle progresses in air-1(RNAi) embryos. However, the dramatic increase in centrosomal ␥ -tubulin, a hallmark of centrosome maturation, never occurs.
Interestingly, we also noticed that the interval between NEBD and the onset of cortical contractions was slightly shorter in air-1(RNAi) embryos (168 Ϯ 64 s; Fig. 3 A, air-1(RNAi) , 0 s and ϩ 121 s) than in wild-type (268 Ϯ 13 s; Fig. 3 A, wild-type, 0 s and ϩ 242 s). This result suggested that NEBD might be delayed. Consistent with this, in air-1(RNAi) embryos we often observed fully condensed rodlike chromosomes in nuclei that had not yet broken down (Fig. 3 A; Ϫ132 s; videos 4, 5, and 6 available at http://www.jcb.org/cgi/content/full/jcb.200108051/DC1), whereas in wild-type completion of condensation to form Figure 3. AIR-1 is required for the accumulation of centrosomal ␥-tubulin during maturation. (A) Panels summarize time-lapse recordings of wild-type (left) and air-1(RNAi) embryos (right) expressing GFP histone and GFP-␥-tubulin. Both sequences start at a similar early embryonic stage, judged by the extent of cortical ruffling, the small size of the sperm pronuclei (arrowheads), and the lack of separation of ␥-tubulin-labeled centrosomes (arrow). The sequences end with onset of cytokinesis (wild-type, ϩ242 s) or cortical contractions (air-1(RNAi), ϩ121 s, arrowheads). The duration of both recordings is 21ف min. Times are seconds after NEBD. The times in the corresponding panels differ by 2ف min because NEBD is slightly delayed in air-1(RNAi) embryos. See videos 3-6 available at http://www.jcb.org/cgi/content/full/jcb.200108051/DC1. (Left) In wild-type, the DNA condenses, beginning before the migration of the pronuclei toward each other and continuing during migration. This occurs concomitant with an increase in the amount of centrosomal ␥-tubulin (compare Ϫ256 s with Ϫ88 s). The accumulation of centrosomal ␥-tubulin continues after NEBD as the mitotic spindle assembles (ϩ122 s). (Right) In air-1(RNAi) embryos, the chromosomes condense with timing similar to wild-type, but ␥-tubulin fails to accumulate at centrosomes (arrows, all panels). The migration of the maternal pronucleus toward the sperm pronucleus is lethargic, probably due to the failure of centrosomes to nucleate robust mitotic asters, but the two pronuclei eventually move toward each other, and the nuclear envelope breaks down (0 s). Chromosomes never align properly, but cortical contractions begin coincident with cytokinesis in wild-type embryos (ϩ121 s). Cytokinesis does not succeed in air-1(RNAi) embryos (unpublished data). (B) Kinetic traces of centrosomal ␥-tubulin fluorescence. Centrosomal fluorescence was quantified in 7 wild-type and 10 air-1(RNAi) embryos. Three traces are shown for each. Bar, 10 m (as in Fig. 2). rod-like chromosomes usually occurs coincident with NEBD ( Fig. 3 A, 0 s; video 3 available at http://www.jcb.org/cgi/ content/full/jcb.200108051/DC1). From these results, we conclude that NEBD is delayed by 2-1ف min with respect to other cell cycle events in air-1(RNAi) embryos.

AIR-1 is required to recruit other PCM components during centrosome maturation
The results above showed that a GFP-␥-tubulin fusion protein fails to accumulate at mitotic centrosomes in air-1(RNAi) embryos. To confirm this for endogenous ␥-tubulin, we fixed air-1(RNAi) embryos and stained them for DNA, MTs, ␥-tubulin, and AIR-1 (Fig. 4 A). In mitotic air-1(RNAi) embryos, AIR-1 was depleted to undetectable levels ( Fig. 1 D), and ␥-tubulin staining foci were much smaller than in wildtype ( Fig. 4 A), consistent with the live analysis. Interestingly, each centrosome in the air-1(RNAi) embryos often appeared as a pair of small dots of ␥-tubulin staining (Fig. 4 A, bottom right, inset). These dots are likely to be ␥-tubulin associated with paired centrioles that have not yet separated.
To further investigate the role of AIR-1 in the recruitment of PCM components, we tested whether two additional centrosomal proteins CeGrip, a C. elegans homologue of Spc98p (unpublished data; B. Haberman, personal communication), and ZYG-9 (Matthews et al., 1998) also require AIR-1 to accumulate at mitotic centrosomes. We fixed and stained wildtype and air-1(RNAi) embryos for CeGrip (Fig. 4 B) and ZYG-9 (Fig. 4 C). Centrosomes labeled with either marker also remained tiny in mitotic air-1(RNAi) embryos, demonstrating that AIR-1 is required for the centrosomal accumulation of three PCM components during maturation.

AIR-1-dependent recruitment of centrosomal ␥-tubulin does not require MTs
Our results show that AIR-1 is required for the increase in centrosomally organized MTs and for the accumulation of centrosomal ␥-tubulin as embryos enter mitosis. To test the possibility that the decrease in centrosomally organized MTs  Wild-type (left) and air-1(RNAi) embryos (right) were treated with nocodazole, fixed, and stained for MTs, DNA, ␥-tubulin, and AIR-1. ␥-Tubulin staining is reduced dramatically, and two small foci of ␥-tubulin staining are observed for each centrosome in air-1(RNAi) embryos (compare wild-type and air-1(RNAi) insets). Insets are magnified 5.5-fold. Bar, 10 m.
is responsible for the failure to accumulate centrosomal ␥-tubulin, we treated wild-type and air-1(RNAi) embryos with nocodazole to depolymerize MTs and stained them for MTs, DNA, ␥-tubulin, and AIR-1 (Fig. 5). MTs were completely depolymerized in nocodazole-treated embryos with only centrosomes detectable using an ␣-tubulin antibody (Fig. 5, top). Embryos were treated with nocodazole for 7 min before fixation (see Materials and methods). Since pronuclear migration requires MTs, we analyzed embryos with condensed chromosomes in which the pronuclei were still separated. This separation indicates that MT depolymerization occurred at a stage similar to the Ϫ256 s panel in Fig. 3 A, which is before the accumulation of additional centrosomal ␥-tubulin. Despite complete depolymerization of MTs, centrosomes in wild-type embryos accumulated ␥-tubulin and AIR-1 to normal levels during the 7 min incubation in nocodazole. In contrast, centrosomes in nocodazole-treated air-1(RNAi) embryos remained small, similar to those in untreated air-1(RNAi) embryos. From these results, we conclude that the AIR-1-dependent accumulation of centrosomal ␥-tubulin during maturation does not require MTs.
Cumulatively, our results demonstrate that the aurora-A kinase, AIR-1, is required to execute a MT-independent pathway for the recruitment of PCM components during centrosome maturation. Spindle assembly also fails in air-1(RNAi) embryos. One possibility, suggested by our results, is that centrosome maturation is a prerequisite for spindle assembly. Our kinetic analysis of centrosome dynamics revealed that AIR-1 is not required for centrosome separation but instead is required to prevent the centrosomes from collapsing together after NEBD. In Drosophila and Xenopus, inhibition of aurora-A function results in the generation of monopolar spindles with unseparated centrosomes (Glover et al., 1995;Roghi et al., 1998). Kinetic studies during a single cell cycle will be needed to determine if these phenotypes result from an initial failure of centrosome separation or, as we have found for C. elegans, from a failure to maintain centrosome separation during the early stages of spindle assembly. Differences in the onset of the mitotic phenotype associated with inhibition of aurora-A could also arise from variations in the timing of centrosome separation among cell types. This event occurs relatively early in the mitotic cycle in C. elegans and Drosophila embryos and just before spindle assembly in vertebrate tissue culture cells (Callaini et al., 1997;Keating and White, 1998;Fry et al., 2000).
NEBD is also delayed with respect to other cell cycle events in air-1(RNAi) embryos. One possibility is that aurora-A independently regulates and ensures the coordination of multiple events during mitotic entry including centrosome maturation, spindle assembly, and NEBD. A second possibility, which we favor, is that centrosome maturation is required for spindle assembly and timely NEBD. The accumulation of cell cycle regulators such as polo-like kinases and Cdk1 at centrosomes prompts the speculation that maturation might promote NEBD and spindle assembly by facilitating the rapid coactivation of cell cycle regulators in proximity to target proteins important for these processes. Alternatively, the failure of centrosome maturation might trigger a checkpoint that delays subsequent cell cycle events (Lane and Nigg, 1996).

RNA-mediated interference
For production of dsRNA to air-1 (gcctctcggaaaaggaaagt, ccttgattctggcgatcaat), the primers in parentheses with tails containing T3 and T7 promoters were used to amplify regions from genomic N 2 DNA or the cDNA yk364b4. PCR products were transcribed and double stranded RNA prepared as described (Oegema et al., 2001).

Live imaging and quantification of centrosomal fluorescence
Live analysis and RNAi in strains expressing GFP fusions were performed as described (Oegema et al., 2001). GFP-␣-tubulin and GFP-␥-tubulin fluorescence were quantified from live recordings made using spinning disc confocal and wide-field microscopy, respectively. The bright centrosomal regions in the ␥-tubulin and ␣-tubulin sequences were circled, the average fluorescence intensity in this region and in a similarly sized background region were determined using Metamorph software (Universal Imaging Corp.), and the integrated centrosomal fluorescence was calculated from these values.

Antibodies and fixed imaging
Antibodies to the COOH-terminal 12 amino acids of AIR-1 and the COOH-terminal 17 amino acid of CeGrip and ␥-tubulin were raised, affinity purified, and directly labeled as described (Oegema et al., 2001). Antibodies to ZYG-9 were a gift from A. Pozniakovsky (Max-Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany). Embryos were fixed and processed for immunofluorescence as described (Oegema et al., 2001) and imaged using a 63ϫ, 1.4 NA PlanApochromat lens on a Deltavision microscope. Three-dimensional wide-field data sets were computationally deconvolved and projected (Applied Precision).

Nocodazole treatment
Worms were dissected on slides coated with 1 mg/ml polylysine in egg buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 0.025 mM Hepes, pH 7.4) containing 10 g/ml nocodazole. A coverslip was placed on top, and gentle pressure was applied to crack embryo egg shells. Slides were incubated in the presence of nocodazole for 7 min in a humid chamber before fixation.