tests were performed with a higher E cut value (900 eV) and led to the same results.

The Al substrate was described by an asymmetric monoclinic slab of 4 atomic layers. Atoms of the two bottom layers were kept at their position in the bulk during all computations. The atoms of the two top layers of the slab and of the adsorbed molecules were optimized. Atomic positions were relaxed with the conjugate gradient algorithm until forces on each moving atom were less than 0.02 eV Å À1 . The vacuum region was set at 18 Å (equivalent to 8 atomic layers thick) to minimize the interactions in the z direction between periodic images of the molecule/substrate system. The molecules were adsorbed on the top layer of the slab with different surface coverages. The lowest coverage (y = 0.2) was selected to minimize the interaction between the adsorbed molecule and its periodic images on a (5 Â 6) array (an Al surface area of 212 Å 2 ). The lateral dimension of the supercell was then successively decreased with one 8HQ molecule per unit cell to mimic the increase of the surface coverage. The surface coverage y = 1 was reached when the Al(111) surface was fully covered by the 8HQ molecules or derivatives (Fig. 2, y = 1). Five supercells containing 30, 20, 12, 9 and 6 aluminum surface atoms were used to simulate y = 0.20, 0.33, 0.5, 0.66 and 1, respectively. The k-point grid for the reciprocal Brillouin zone sampling was increased accordingly (see Table 1).

For the study of the dissociation of the O 2 molecule on the Al(111) surface covered by organic layers (y = 0.66 and y = 1), a (6 Â 6) array was chosen for the metallic slab (144 atoms) with a sampling of (3 Â 3 Â 1) k-points in the Brillouin zone.

van der Waals interactions were included according to the DFT-D2 method 37 implemented in VASP. As in our previous work on the adsorption at low coverage, 20 only interactions at the molecule/aluminum interface and between molecules in Fig. 2 Adsorption topologies and electronic density variation (Dr) at two coverages (y = 0.66 and y = 1). The 8HQ molecule (w); and the tautomer (t). Top: Top view; and bottom: side view.

the organic layer were considered. The total energy was therefore calculated for the (slab + molecules) system by:

E DFT-D tot = E DFT
tot + E vdW slab/mol + E vdW mol/mol (1) with E vdW slab/mol the vdW interactions at the aluminum/molecule interface and E vdW mol/mol the vdW interactions between the molecules. From the total energies, two energetic features were calculated to quantify the molecule/surface interactions. The first one, E ads , characterized the simultaneous adsorption of all the molecules from their free state. The adsorption energy on the Al(111) surface was thus calculated as:

E ads = E slab+mol À E slab/opt À E mol/opt (2)
where E slab+mol is the total energy of the system with the molecule adsorbed on the Al(111) surface, E slab/opt is the energy of the bare relaxed Al(111) slab calculated without dispersive corrections and E mol/opt is the energy of the free molecule optimized in vacuum. The binding energy, E bind , characterized the strength of the bonding at the molecule/aluminum interface without taking into account energetic trends for the deformation of the substrate and the adsorbate induced by the formation of the layer on Al(111). The binding energy was then defined as:

E bind = E slab+mol À E slab/sp À E mol-layer/sp ( 3
)
where E slab+mol is the total energy of the system with the molecule adsorbed on the Al(111) surface and E slab/sp and E mol-layer/sp are the total energies of the isolated slab and isolated deformed molecular layer at their geometry after adsorption, respectively. Therefore, the deformation energies of the substrate E deform slab and of the molecule E deform mol during the adsorption process were evaluated. The substrate deformation energy E deform slab was calculated as:

E deform slab = E slab/sp À E slab/opt ( 4
)
where E slab/sp and E slab/opt are the total energy of the isolated slab at the geometry after adsorption and the total energy of the relaxed slab, respectively. The molecule deformation energy E deform mol was determined as:

E deform mol = E mol/sp À E mol/opt ( 5
)
where E mol/sp and E mol/opt are the total energy of the isolated molecule at the geometry after adsorption (one molecule per unit cell) and of the optimized free molecule, respectively. Finally, the interactions between molecules in the layer were evaluated from the calculation of the cohesive energy per molecule in the organic layer E coh layer by:

E coh layer = E mol-layer/sp À E mol/sp (6)
where E mol-layer/sp is the total energy of the isolated layer and E mol/sp is the energy of the isolated molecule at the geometry after adsorption without interaction with the periodic images (large unit cell).

To quantify the electronic changes in the systems, the net charge variation (Dq x ) was determined on each atom by: Dq x = q ads x À q free x (7) where q free x and q ads x are the charges on each atom x (Bader's population analysis on VASP charge densities 38 ) of the molecule and the slab before (optimized geometries of the free systems) and after adsorption. The total molecular charge variation DQ mol was calculated as:

DQ mol = Q ads mol À Q free mol ( 8
)
To clearly observe the charge transfer, the electron density variation Dr was plotted:

Dr = r slab+mol À (r slab/sp + r mol-layer/sp ) (9)
where r slab+mol is the electron density distribution on the optimized system, and r slab/sp and r mol-layer/sp are the electron density distributions on the substrate and molecular layer at the geometry after adsorption, respectively.

3 Results and discussion

Adsorption of 8HQ species on the Al(111) surface

We took into account experimental results presented in the literature with bulk 8HQ or in polar solvent. 8HQ molecules are associated as dimer molecules (with hydrogen bonds between two monomers) in the bulk 8HQ crystal. 39 When the crystal in powder is introduced in a polar solvent, which is the case when studying corrosion inhibitors in aqueous solution, the dimer molecules are dissociated and 8HQ molecules (monomers) are the main species in the solution. [40][41][42] We thus studied the interaction of 8HQ monomers with the Al(111) surface. Preliminary calculations were also performed on the adsorption of the 8HQ dimer on the Al(111) surface and results are presented in the ESI. † To mimic the different chemical forms of the molecule present in aqueous solution at different pH, we looked at the pK a values of the 8HQ molecule. Many organic molecules can gain and/or lose a proton in aqueous solutions by reacting with water molecules. These phenomena are dependent on the pH of the solution. The pK a of a molecule gives information on its ability to be protonated or deprotonated at a certain pH and, for a given molecule, two pK a values indicate the presence of two different functional groups that can be protonated or deprotonated. The pK a values of the 8HQ molecule are 5.13 and 9.89. 40 In neutral solution,

3 Â 3 Â 1 3 Â 3 Â 1 5 Â 5 Â 1 5 Â 5 Â 1
O atom from a bridge site on the Al(111) surface for y = 0.2 to 0.66 to a top site for y = 1 (Fig. 2). For y = 1, the O, N, C2 and C4 atoms of the four molecules are located on top of Al atoms. of the bonding, the binding energies (per molecule) are significantly larger (by about 0.5 eV for Z up to more than 2 eV for t) than the adsorption ones that are drastically reduced (in absolute value) by the positive (destabilizing) deformation energies. From y = 0.2 to 0.66, the molecule deformation energies are strong with values of 0.55 AE 0.01 eV (d), 0.71 AE 0.03 eV (Z), 1.57 AE 0.01 eV (w) and 1.96 AE 0.01 eV (t). At these coverages, the slab deformation energies when adsorbing w, t and Z are rather weak (about 0.13 eV for w, 0.10 eV for Z and 0.25 eV for t), whereas when d is adsorbed, the slab deformation energy reaches 0.45 eV.

As previously explained, in addition to the deformation energies, the adsorption energy takes also into account the interactions between the molecules in the organic layer. The cohesive energy E coh layer of the layers formed on the Al(111) surface at different coverages is shown in Fig. 4. The energies were calculated by only taking into account the interactions between the molecules in the layer and not the deformation of the molecules during the adsorption process (eqn ( 6)). The results show a change for y = 0.5 to 1 with negative values of E coh layer that indicate stabilizing interactions between molecules. At low coverages, the molecules are not close enough to be interacting (E coh layer E 0). At full coverage, E coh layer , that quantifies the interactions in the layer, represents 6% of the adsorption energy E ads for the dehydrogenated species, 18% for the tautomer and up to 30% and 32% for the native 8HQ and the hydrogenated species.

To sum up all these energetic features, it can be written:

E ads = E bind + E deform mol + E deform slab + E coh layer
In this equation, it must be underlined that the molecule and slab deformation energies are unfavorable for a stable layer on the Al(111) surface, and in contrast, the molecule/surface binding energy and the intermolecular cohesion energy are the driving force of the adsorption process.

3.1.2 van der Waals dispersive forces. Only van der Waals interactions between the organic layer and the Al(111) surface and between the molecules in the layer were taken into account. Fig. 5(a) presents the total van der Waals energy in the systems. Fig. 5(b) and(c) show the variation of the vdW energies as a function of the coverage y at the molecule/ substrate interface and between molecules in the layer, respectively. The native 8HQ molecule, the tautomer and the hydrogenated species show similar trends and this can be linked to comparable topology on the surface (topology parallel to the surface). For these three species, the total vdW energy decreases slightly with increasing coverage showing that the van der Waals interactions increase. The average value is À1.63 AE 0.06 eV. For the dehydrogenated molecules, the value is À1.06 AE 0.04 eV from y = 0.2 to 0.66 and À1.43 eV at full coverage (y = 1). From y = 0.2 to 0.66, the vdW energy at the molecule/substrate interface is À1.24 AE 0.05 eV for native 8HQ, the tautomer and the hydrogenated species and À0.96 AE 0.03 eV for y = 1 (Fig. 5(b)). The decrease of the interactions (increase of the van der Waals energy) can be attributed to the topological change occurring at full coverage, where the molecules become tilted on the Al(111) surface. Thus, the contact between the molecules and the substrate is reduced and vdW interactions at the interface are limited. For the dehydrogenated molecule, a small variation of the vdW energy at the interface is observed (À0.78 AE 0.06 eV), explained by the fact that this species presents the same adsorption topology independently of the coverage. The vdW energy between the molecules in the organic layer shows a limited variation from y = 0.2 to 0.5 (Fig. 5(c)). The energies are À0.34 AE 0.04 eV for 8HQ, the tautomer and the hydrogenated species and À0.28 AE 0.01 eV for the dehydrogenated molecule. For low coverages, the vdW interactions between the molecules are minimized due to the large distance between the molecules. At high coverages (y Z 0.5), the great number of molecules on the Al(111) surface reduces the distance between the molecules and hence increases the vdW interactions between them. For all the molecules, the vdW energies in the organic layer are À0.43 AE 0.07 eV and À0.67 AE 0.08 eV for y = 0.66 and y = 1, respectively. The decrease of the contribution of the vdW energy at the interface, when y increases, is compensated for by the increase of the vdW contribution between the molecules in the layer, in particular at full coverage. Besides the formation of covalent bonds between the molecules and the Al(111) surface, these results underline a strong contribution of vdW forces in the adsorption process.

Charge transfers, dipole moments and work function changes

Charge transfers. As defined previously, the global charge variation DQ mol is calculated by the difference between the total number of electrons on the adsorbed molecules and that of the free species calculated in vacuum. Strong electronic transfers from the substrate to the adsorbates were determined. Once adsorbed on Al(111), the w, t, Z and d molecules were negatively charged. Fig. 6 shows the global charge variation on the molecules as a function of the coverage y. Independently of the coverage, the global charge variations in the molecules are À0.84 AE 0.11 e, À1.18 AE 0.09 e, À1.40 AE 0.07 e and À1.50 AE 0.09 e for the hydrogenated, the dehydrogenated, the native 8HQ and the tautomer species, respectively. Table 3 where m z (mol) and m z (slab) are the components in the direction normal to the surface of the dipole moments for the molecular layer and the substrate at their geometry after adsorption, respectively. Thus Dm z is the dipole moment induced at the interface by the electronic redistribution due to chemical interactions between the molecules and the substrate. This decomposition of m z (mol/slab) is artificial because all the components are closely linked. However, analyzing the variation of each component gives insight in the origin of the work function changes that we describe below. Fig. 7(b) and (c) show the variation of m z (mol) and Dm z as a function of the coverage y for all the adsorbed species on the Al(111) surface. The change of these two quantities can be attributed to the deformation and/or the orientation of the molecule which depend on the density of the molecular layer. The molecular dipolar component m z (mol) is oriented from the molecule down to the substrate and it has the same sign and the same decreasing trend with the coverage as m z (mol/slab). However, the component induced by the charge transfer Dm z is oriented oppositely (from the surface to the molecule, Dm z ) to m z (mol), except for the hydrogenated molecule Z (see Fig. 7(c)). For all the adsorbed molecules (native 8HQ and its derivatives) the value of m z (slab) varies from 0.01 D to 0.05 D. This is negligible when compared to m z (mol) and Dm z . The global interface dipole moment m z (mol/slab) is thus an equilibrium between the contribution of the molecular layer and the contribution at the interface related to the charge transfer.

presents the charge

To conclude, the magnitude of the molecular permanent dipole (m z (mol)) and the variation of the dipole moment at the interface (Dm z ) induced by electronic transfers are significant in the adsorption of 8HQ and derivatives on Al(111). Similar conclusions were reported for the adsorption of the Al(8HQ) 3 complex on cobalt, 16 whereas the dipole moment was dominated only by the molecular dipole moment in the adsorption of the Al(8HQ) 3 complex on magnesium 17 and aluminum 18,19 surfaces.

Work function changes.

The work function is one of the properties affected by electronic changes due to the adsorption of organic molecules on metallic surfaces and the interface dipole plays a crucial role in the work function shift. In the present work, the work function was calculated from the difference between the Fermi energy (E F ) and the asymptotic electrostatic potential energy (V N ) in the middle of the vacuum of the simulation cell:

f = V N À E F .
The work function change during the adsorption process was given by:

Df = f(mol/slab) À f(slab)
where f(mol/slab) and f(slab) are the work function of the molecule/metal systems and the bare Al(111) surface, respectively.

Fig. 8(a) shows the work function variation as a function of the surface coverage y. Unlike the adsorption energy and the electronic transfers, which show a low dependence on the coverage y, the work function change is strongly sensitive to the adsorption topology of the molecules, which depends on the coverage. In all cases, the adsorption of the 8HQ molecule and its derivatives on the Al(111) surface reduces significantly the work function of the substrate (Df r 0) with a change of the work function between 0.1 and 1.4 eV. These values are in agreement with experimental work function changes (0.8 to 1.5 eV) and DFT computed values (0.9 to 1.7 eV) determined for the adsorption of the Al(8HQ) 3 complex on an aluminum surface. 18,19 The shift of the work function Df as a function of y is attributed to the electron redistribution due to the molecules' chemisorption on Al(111) and to the charge transfers between the substrate and the adsorbates. Moreover, the polarization effects due to van der Waals forces change with the topology of the adsorbate and also modify the work function of the system. It is known as ''the push back effect'' 23,44,45 where the electron cloud of the metal is compressed by the electron cloud of the adsorbed molecule.

For y = 0.2 to 0.66, the dehydrogenated species d shows the largest change of the work function followed by the hydrogenated species Z, the tautomer t and finally the native 8HQ molecule w(Df(d) r Df(Z) r Df(t) r Df(w)). It is not possible to find any correlation between the total electronic transfer from the substrate to the adsorbed molecules and the work function changes because of different adsorption geometries of w,t, Z and d species.

For y = 1, a different order in the ranking of the work function change induced by the adsorbed molecules is observed and the largest work function change is for the tautomer/Al(111) system (Df(Z) r Df(d) r Df(w) r Df(t)). In that case where all the molecules have similar adsorption geometries, the work function variation varies as the reverse of the total electronic transfer (DQ mol = À0.95, À1.10, À1.47, À1.54 e for Z, d, w and t respectively). To conclude, the work function change depends on the nature of the adsorbate and on the coverage.

The relation between the work function variation and the interface dipole moment m z (mol/slab) was also investigated. The correlation between the work function variation and the interface dipole moment m z (mol/slab) follows a linear variation as proposed by the Helmholtz model: Df = À4pym z (in atomic units), with the slope of the lines increasing with y.

Adsorption of molecular oxygen on the protected Al(111) surface

In order to verify the efficiency of these protective layers against oxygen reduction which is the cathodic reaction involved in the corrosion process, the interaction of molecular oxygen with the covered Al(111) surface was also investigated for two surface coverages: (i) y = 0.66 where the Al surface is totally covered by molecules parallel to the surface for the 8HQ and tautomer species and by molecules tilted on the surface in the case of the dehydrogenated form, and (ii) y = 1 where all the organic species are tilted on the metallic surface. The interaction of O 2 on the Al(111) surface covered with the hydrogenated species was not studied because it will be shown in the following that the presence of a hydroxyl -OH or an amino -NH chemical group on the organic molecule favors the dissociation of the O 2 molecule.

For y = 0.66, the main results concerning the geometrical changes in the layer (w, t or d species) due to interactions with O 2 are presented in Table 4. For the native 8HQ molecule, a spontaneous dissociation of the O 2 molecule is obtained when it is positioned on top of the N atom of the adsorbed molecule. The resulting geometry is shown in Fig. 9(a À1.73 e. Finally, the total energy variation in the system during the O 2 reduction process is À8.16 eV. This value was calculated as the difference between the total energy of the system after O 2 adsorption and the sum of the total energy of the 8HQ/Al(111) system and the free O 2 molecule.

The interaction of the O 2 molecule on the Al(111) surface covered by the tautomer t for y = 0.66 is similar to the case of w with a lower total energy variation (À7. process, the molecules are strongly deformed, which reduces the adsorption energies. Among the 8HQ molecule and its derivatives, the dehydrogenated species has the strongest coupling with the Al(111) surface and adopts similar adsorption topologies independently of the coverage. In contrast, the native 8HQ, tautomer and hydrogenated species exhibit a structural change in their adsorption topologies between low and high coverage. The strongest bonding on Al(111) of the dehydrogenated species, present in alkaline aqueous solutions, is in agreement with recent experimental results (electrochemical measurements and surface analysis) showing that the 8HQ molecule is especially efficient in alkaline solutions (these results are unpublished because studies are still in progress). For all the coverages and all the adsorbed species, localized electronic transfers from the substrate to the adsorbates are observed. The electronic density redistribution due to the adsorption of the molecules induces the reduction of the work function of the metal. The increase of the coverage leads to the strongest change of the work function. This variation is linked to the formation of a dipole at the molecule/metal interface and of a dipole in the organic layer.

To demonstrate that the formation of a complete layer of 8HQ molecules or derivatives on Al(111) can inhibit aluminum corrosion, molecular oxygen reduction on the covered aluminum surface is also investigated. This reaction is one of the elementary reactions occuring during the corrosion process of aluminum. At low coverage, dissociation of the O 2 molecule occurs on uncovered areas of the Al(111) surface. At full coverage (y = 1), O 2 dissociation does not happen in the presence of the organic layers thus preventing aluminum atoms from further oxidation (no electronic transfer). 8HQ layers on Al(111) form a protective barrier against corrosion of the Al substrate. 1 and also presented in the manuscript) but with H bonding between the two species. The total adsorption energy is -2.82 eV (taking as the reference the free dimer species) or -1.41 eV/molecule if we divide the latter value by the number of monomers in the dimer.

In our manuscript, for the adsorption of the monomer species, i.e. adsorption of only one type of species for each calculation, the adsorption energy of the dehydrogenated δ molecules is -3.44 eV/molecule independently of the coverage and the adsorption energy of the hydrogenated species is -1.71 eV/molecule independently of the coverage. So the binding of only one type of species is stronger than the adsorption energy of the same species in the modified dimer (averaged at -1.41 eV per molecule). For the conformation in Figure 1 (C3), the total adsorption energy is -2.12 eV for the dimer (taking as the reference the free dimer species) and then -1.06 eV/molecule, and the binding energy per 8HQ

Fig. 3 (

 3 a) presents the variation of the adsorption energy E ads with the coverage y of the 8HQ molecule and its tautomer, hydrogenated and dehydrogenated forms on the Al(111) surface. Negative adsorption energies are obtained for all species and coverages, indicating the formation of a stable organic layer on Al(111). From y = 0.2 to 0.66, constant adsorption energies of À1.12 AE 0.04 eV for native 8HQ, À1.66 AE 0.07 eV for the hydrogenated molecule, À2.41 AE 0.02 eV for the tautomer, and À3.44 AE 0.02 eV for the dehydrogenated species are calculated. For y = 1, the native 8HQ, the hydrogenated, the tautomer and the dehydrogenated molecules have an adsorption energy of À1.00 eV, À1.40 eV, À2.27 eV and À3.60 eV, respectively. However the adsorption energy values includes not only the bonding of the molecule to the surface but also the deformation of the molecule and of the slab due to the adsorption process. The binding energy, E bind , shown in Fig.3(b), only quantifies the bonding at the molecule/metal interface without taking into account the energetic cost of the deformation of the slab and the deformation of molecules in the organic layer, and the interactions between molecules. Due to the covalent character

Fig. 4

 4 Fig. 4 Cohesive energies in the layers for different coverages y: The 8HQ molecule (w); the tautomer (t); dehydrogenated 8HQ (d); and hydroge nated 8HQ (Z).

Fig. 5

 5 Fig. 5 Contribution of the dispersion forces at different coverages y for the adsorption of the molecules on the Al(111) surface. The 8HQ molecule (w); the tautomer (t); dehydrogenated 8HQ (d); and hydrogenated 8HQ (Z). (a) Total vdW energies, (b) vdW energies at the molecule/substrate inter face, and (c) vdW energies in the organic layer.

Fig. 6

 6 Fig. 6 Global charge variation as function of the coverage y of the 8HQ molecule and its derivatives after adsorption on the Al(111) surface. The 8HQ molecule (w); the tautomer (t); dehydrogenated 8HQ (d); and hydro genated 8HQ (Z).

Fig. 8

 8 Fig. 8 Work function change (Df) as a function of the surface coverage and relationships between the work function change (Df) and the dipole moment (m z (slab/mol)) at each coverage y. The 8HQ molecule (w); the tautomer (t); dehydrogenated 8HQ (d); and hydrogenated 8HQ (Z).

Fig. 8 (

 8 b) presents the work function change Df as a function of m z (mol/slab) at different coverages (from y = 0.2 to 1).

  ). In the free O 2 molecule, the calculated O-O distance is 1.23 Å. After the adsorption on the partially 8HQ-covered surface (y = 0.66), the O-O distance is increased to 2.83 Å. In fact, one of the O atoms (O a ), initially placed at more than 2 Å from the hydroxyl group of the 8HQ molecule, captures the H atom of this group with a final O a -H distance of 0.98 Å. This atom (O a ) is adsorbed on-top of an Al atom with a distance O a -Al of 1.76 Å. The second O atom (O b ) is adsorbed on a threefold symmetry site (hcp) of the surface not covered by the molecules with an averaged O b -Al distance of 1.84 Å. Due to this reaction, the native 8HQ molecule becomes dehydrogenated by losing the H atom of the hydroxyl group (Fig. 9) and adopts a geometry with three covalent bonds (between O, N, C4 atoms and Al atoms) similar to the adsorption geometry of d at low coverage 20 (presented in Table 4 as d 2 ). The net charges of the O a and O b atoms after the dissociation of the O a -O b molecule are respectively À1.52 e and

  19 eV). The final geometry is shown in Fig. 9. After the dissociation, the O a -O b distance is 2.81 Å. One of the O atoms (O a ) has taken the H atom of the NH group of the tautomer and is adsorbed on top of an Al atom in the Al(111) surface with an O a -Al distance of 1.75 Å. The length of the O a -H bond is 0.98 Å. The second O atom (O b ) of the oxygen molecule is adsorbed on a hcp site of the Al(111) surface with an average O b -Al distance of 1.84 Å. One of the four tautomer molecules in the simulation cell is thus dehydrogenated during the O 2 reduction. Again, the bond

1 (

 1 dimer). Others conformations are chemisorbed conformations with the formation of covalent bonds between the molecules and the Al surface atoms. It indicates the strong affinity of the 8HQ molecule with aluminum. Two conformations are shown in Figure C2) and (C3). The most stable adsorption topology of the dimer on the Al(111) surface led to a chemical modification of the organic species (Figure 1 (C2)): one H atom of the hydroxyl group of one 8HQ molecule is transferred to the other 8HQ molecule. The 8HQ dimer becomes thus one hydrogenated and one dehydrogenated species on Al(111) with conformations of the two molecules on the Al(111) surface similar to the conformation of the dehydrogenated δ and hydrogenated η monomers at low coverage (see Table

Table 1

 1 Grid of k points for molecule/slab calculations according to the coverage y

	Coverage y	0.20	0.33	0.5	0.66	1
	Number of atoms in the surface	30	20	12	9	6
	Number of atoms in the unit cell	120	80	48	36	24
	Surface area (Å 2 )	212	141	85	64	42
	k Points

Table 2

 2 Distances in Å between the Al surface atoms and the atoms of the 8HQ molecule and its derivatives after adsorption for different coverages y

	Coverage	O Al	N Al	C2 Al	C4 Al
	8HQ
	y 0.2	2.659	1.968	2.948	2.189
	y 0.3	2.701	1.967	2.957	2.191
	y 0.5	2.762	1.969	3.000	2.188
	y 0.66	2.745	1.966	2.905	2.184
	y 1	2.073	1.916	2.178	3.684
	Tautomer
	y 0.2	1.838	2.223	3.154	2.182
	y 0.3	1.841	2.224	3.153	2.187
	y 0.5	1.844	2.223	3.160	2.184
	y 0.66	1.852	2.217	3.146	2.189
	y 1	1.814	2.095	2.168	3.707
	Hydrogenated 8HQ
	y 0.2	3.021	2.314	3.281	2.176
	y 0.3	3.019	2.311	3.271	2.179
	y 0.5	3.026	2.311	3.279	2.176
	y 0.66	3.091	2.324	3.284	2.187
	y 1	2.263	2.198	2.219	3.808
	Dehydrogenated 8HQ
	y 0.2	1.914	2.048	3.373	4.710
	y 0.3	1.914	2.044	3.402	4.402
	y 0.5	1.907	2.050	3.421	4.929
	y 0.66	1.917	2.032	3.453	4.902
	y 1	1.822	2.119	3.510	4.492

Table 4

 4 Geometrical parameters of the molecules in the native 8HQ, tautomer and dehydrogenated layers adsorbed on Al(111) for a coverage y = 0.66 before and after reduction of an O 2 molecule

			d C2C3	d C3C4	d C4C10	d C10C5	d C5C6	d C6C7	d C7C8	d C8C9	d C9C10	d C9N	d C2N	d C8O	d OH	d NH
	8HQ
	Free mol.	1.417	1.381	1.418	1.419	1.383	1.415	1.385	1.431	1.430	1.361	1.325	1.355	0.990	2.051
	y 0.66	1.368	1.472	1.476	1.405	1.401	1.400	1.399	1.408	1.413	1.419	1.415	1.375	0.990	2.140
	y 0.66 + O 2	1.366	1.470	1.477	1.402	1.401	1.396	1.412	1.425	1.421	1.415	1.401	1.348	2.222	2.300
	Tauto
	Free mol.	1.404	1.388	1.421	1.406	1.402	1.401	1.426	1.466	1.434	1.350	1.338	1.270	2.064	1.041
	y 0.66	1.352	1.472	1.480	1.406	1.399	1.399	1.408	1.411	1.406	1.442	1.439	1.352	2.354	1.026
	y 0.66 + O 2	1.366	1.471	1.479	1.402	1.401	1.397	1.410	1.423	1.422	1.410	1.408	1.355	1.936	2.722
	Deh. 8HQ
	Free mol.	1.410	1.384	1.414	1.426	1.399	1.387	1.456	1.496	1.431	1.344	1.334	1.249
	d 2	20	1.368	1.465	1.475	1.403	1.401	1.397	1.413	1.425	1.421	1.408	1.406	1.346
	y 0.66	1.401	1.390	1.420	1.419	1.389	1.412	1.390	1.417	1.425	1.375	1.349	1.366
	y 0.66 + O 2	1.401	1.390	1.420	1.418	1.388	1.412	1.388	1.414	1.425	1.375	1.348	1.365

Table 1 :

 1 H bond length in Å between the atoms of the 8HQ dimer assemblies after adsorption on Al(111), adsorption and binding energies in eV. Conformations H bond length E ads/dimer E ads/8HQmolecule E bind/8HQmolecule surface than in the case of the dimer (Binding energy: -2.90 eV/molecule for the 8HQ monomer and -1.21 eV/8HQmolecule when the 8HQ is in the form of the

	C1 on Al(111)	OH N: 1.99	-2.42	-1.21	-1.21
	C2 on Al(111)	OH O: 1.78	-2.82	-1.41	/
		NH O: 1.95
	C3 on Al(111)	CH N: 2.33	-2.12	-1.06	-2.88
	χ 8HQ monomer on Al(111)	/	/	-1.11	-2.90
	δ 8HQ monomer on Al(111)	/	/	-3.44	-4.20
	η 8HQ monomer on Al(111)	/	/	-1.71	-2.31
	stronger to the

Acknowledgements

This work was performed using HPC resources from CALMIP (Grant p12174) and from CINES (Grant c2013097076). It was supported by the National Research Agency (ANR number ANR-2011 JS08 015 01). The authors thank Nathalie Tarrat from CEMES, Toulouse, for very fruitful discussions.

Conflicts of interest

There are no conflicts to declare.

Electronic Supplementary Information (ESI) Corrosion protection of Al(111) by

8-hydroxyquinoline: a comprehensive DFT study

Dimer adsorption

We tested additional initial geometries including the adsorption of dimer assemblies of 8HQ molecules. The optimized geometries are presented in Figure 1.

We calculated the total adsorption energy of the dimer, the adsorption energy per 8HQ molecule and the binding energy.

(1)

monomer on the Al(111) is -2.88 eV/molecule. The geometries of the 8HQ molecules in the adsorbed dimer species are the same as the geometry of one 8HQ monomer adsorbed on Al(111). In the work presented in the revised manuscript, the adsorption energy for one 8HQ monomer is about -1.11 eV/molecule independently of the surface coverage.

The 8HQ monomer binds slightly stronger to the surface than in the case of the dimer (Binding energy: -2.90 eV/molecule for the 8HQ monomer and -2.88 eV/8HQmolecule when the 8HQ is in the form of the dimer). These results show that the association of the molecules as dimers on the Al(111) surface is thus not favored over the adsorption of monomer species, presented in the article.


Christine Blaineau

Magali Tessier

Pascal Dubessay

Lena Tasse

Lucien Crobu

Michel Pagès

Patrick Bastien

Michel Page

A Novel Microtubule-Depolymerizing Kinesin Involved in Length Control of a Eukaryotic Flagellum

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The recent completion of their genome-sequencing programs [START_REF] Ivens | The genome of the kinetoplastid parasite, Leishmania major[END_REF][START_REF] Berriman | The genome of the African trypanosome Trypanosoma brucei[END_REF] also revealed that they exhibit an unusually high complement of kinesins, with 54 putative kinesins, of which five are undoubtedly related to the microtubule (MT)-depolymerizing kinesin-13 family ( [START_REF] Wickstead | A ''holistic'' kinesin phylogeny reveals new kinesin families and predicts protein functions[END_REF] and unpublished data). The first protein of this kinesin-13 family on which we focused was a mitotic-centromere-associated kinesin (MCAK)-like protein that, as expected, localized to the nucleus and was involved in mitosis [START_REF] Dubessay | Cell cycle-dependent expression regulation by the proteasome pathway and characterization of the nuclear targeting signal of a Leishmania major Kin-13 kinesin[END_REF]. The second one, also annotated as MCAK-like in the genome database GeneDB (http://www.genedb.org) and here termed LmjKIN13-2, is encoded by gene LmjF13.0130 (EMBL accession number CT005252.1). It is a 730 amino acid protein that contains the highly conserved kinesin motor domain in internal position (residues 40-356 according to Pfam, score 8.2e-112). The alignment of the motor-domain sequence with that of nine other kinesin-13 members revealed the conservation of residues and motifs previously identified as strictly specific of the kinesin-13 family and involved in their depolymerizing activity (Figure S1 in the Supplemental Data available online) [START_REF] Ogawa | A common mechanism for microtubule destabilizers-M type kinesins stabilize curling of the protofilament using the class-specific neck and loops[END_REF][START_REF] Shipley | Structure of a kinesin microtubule depolymerization machine[END_REF], in particular the KVD site (here KLD) necessary for MT depolymerization and the KEC site apparently essential for binding to MTs. This clearly classifies this protein among kinesin-13 members and makes it different from previously identified flagellar kinesins-kinesin-II, the ubiquitous molecular-motor-driving anterograde intraflagellar transport (IFT) [START_REF] Rosenbaum | Intraflagellar transport[END_REF], and KLP1, essential for flagellar motility [START_REF] Yokoyama | Regulation of flagellar dynein activity by a central pair kinesin[END_REF]that both belong to other kinesin families and are also present in trypanosomatids. Surprisingly, however, the sequence of the ''neck'' domain of the protein, adjacent to the motor domain and considered to be conserved in kinesin-13s [START_REF] Ovechkina | K-loop insertion restores microtubule depolymerizing activity of a ''neckless'' MCAK mutant[END_REF], is not conserved here. Sequence alignments show that this supposedly family-specific neck sequence is not present in the two other kinesin-13s from protozoa that have been published [START_REF] Dubessay | Cell cycle-dependent expression regulation by the proteasome pathway and characterization of the nuclear targeting signal of a Leishmania major Kin-13 kinesin[END_REF][START_REF] Shipley | Structure of a kinesin microtubule depolymerization machine[END_REF], in the kinesin-13 from Chlamydomonas, or in the other kinesin-13s of Leishmania (Figure S1, unpublished data). A recent phylogenetic analysis of the kinesin superfamily could distinguish two groups in kinesin-13s: the ''animal-specific'' MCAK/KIF2 subfamily and the ubiquitous and more ''ancestral'' KIF24 subfamily [START_REF] Miki | Analysis of the kinesin superfamily: Insights into structure and function[END_REF]. All protistan members of the kinesin-13 family included in this phylogenetic study belong to the latter. Our own alignment of KIF24 with MCAK subfamily members again failed to identify a conserved neck sequence in KIF24 (not shown), suggesting that this feature is actually not part of the KIF24 subfamily.

The next most closely related family to kinesin-13s is the kinesin-8 family, which exhibits both a plus-enddirected MT-depolymerase activity and a translocation activity but does not possess the kinesin-13-specific motifs. Interestingly, whereas the less ''ancestral'' yeast Saccharomyces cerevisiae lacks kinesin-13 family members and hence appears to only rely upon kinesin-8s for MT depolymerization [START_REF] Walczak | Kinesin-8s: Motoring and depolymerizing[END_REF], L. major lacks kinesin-8 members [START_REF] Wickstead | A ''holistic'' kinesin phylogeny reveals new kinesin families and predicts protein functions[END_REF] but has more kinesin-13s-suggesting that different organisms have evolved varying strategies for performing similar cell-biological functions relying upon kinesins.

LmjKIN13-2 Localizes to the Flagellum

The LmjKin13-2 gene was introduced into the expression vectors pTH6nGFPc and pTH6cGFPn [START_REF] Dubessay | Cell cycle-dependent expression regulation by the proteasome pathway and characterization of the nuclear targeting signal of a Leishmania major Kin-13 kinesin[END_REF]. After transfection into L. major cells, both vectors are maintained episomally and allow the constitutive expression of a recombinant protein bearing the GFP either at the N-or the C-terminal end. Surprisingly, expression of both GFP-fused proteins allowed their visualization essentially at the distal tip and the basis of the flagellum and, when more pronounced, along the length of the flagellum, the cytoplasm being only slightly decorated (Figure 1, Movie S1). When we substituted the GFP with the less bulky c-Myc tag, the localization, revealed by immunofluorescence, proved similar, whether the tag was in the N-or C-terminal position (Figure S2). The recombinant protein was never observed at the nucleus level, particularly at the mitotic spindle (Figure S3), despite its primary annotation as an MCAK. Moreover, no phenotypic changes concerning mitosis or in vitro cellular growth were observed, in L. major cells overexpressing the protein or in T. brucei cells subjected to RNAi (see below), strongly suggesting that LmjKIN13-2 is not involved in mitotic division.

LmjKIN13-2 Overexpression Induces Short-13 Flagellum Phenotypes

The second remarkable observation with this recombinant expression was that more than 90% of the cells exhibited a phenotype consisting of a significant reduction of the flagellar length (FL) (Figures 1, 2A, and2B). Thus, the FL in the mid-log growth phase was reduced to 52% and 70% of its value in the wild-type strain in the cell lines expressing LmjKIN13-2-GFPc and GFPn-LmjKIN13-2, respectively (p < 0.0001 in both cases) (Table S1). This was completed by a modification of the cell morphology, with a short, and often stumpy, cell body (Figure 1). Identical phenotypes were obtained with the c-Myc-tagged protein and, more importantly, with the nontagged full-length recombinant protein (Table S1). This shows that the phenotype was not caused by protein-function impairment due to end tagging. Also of note is the fact that flagellum motility was retained in the mutant cells (Movie S1). All these data strongly suggest that LmjKIN13-2 is a flagellar protein involved in FL control.

Flagella are dynamic organelles that undergo continuous turnover [START_REF] Marshall | Intraflagellar transport balances continuous turnover of outer doublet microtubules: Implications for flagellar length control[END_REF]. FL is thought to be primarily controlled through changes in the ratio of IFT-dependent tubulin assembly versus disassembly and hence through the regulation of IFT [START_REF] Marshall | Cellular length control systems[END_REF][START_REF] Marshall | Intraflagellar transport balances continuous turnover of outer doublet microtubules: Implications for flagellar length control[END_REF][START_REF] Rosenbaum | Organelle size regulation: Length matters[END_REF]. This model was supported by the short-flagellum phenotypes obtained a variety of systems including trypanosomes, after mutations affecting IFT proteins (reviewed in [START_REF] Scholey | Intraflagellar transport[END_REF][START_REF] Kohl | Novel roles for the flagellum in cell morphogenesis and cytokinesis of trypanosomes[END_REF]). Nevertheless, this does not preclude a complementary regulation model, also partly supported by the analysis of shortflagellum mutants [START_REF] Erdman | Interacting protein kinases involved in the regulation of flagellar length[END_REF][START_REF] Asleson | Genetic analysis of flagellar length control in Chlamydomonas reinhardtii: A new longflagella locus and extragenic suppressor mutations[END_REF][START_REF] Cuvillier | LdARL-3A, a Leishmania promastigote-specific ADPribosylation factor-like protein, is essential for flagellum integrity[END_REF][START_REF] Marshall | Size control in dynamic organelles[END_REF][START_REF] Berman | A novel MAP kinase regulates flagellar length in Chlamydomonas[END_REF][START_REF] Wiese | Protein kinase involved in flagellar-length control[END_REF][START_REF] Wilson | Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas reinhardtii[END_REF][START_REF] Bengs | LmxMPK9, a mitogen-activated protein kinase homologue affects flagellar length in Leishmania mexicana[END_REF] and based on a signaling pathway yet to be identified (see below). Here, we report the effect of a protein that, being a kinesin-13, can directly promote flagellar disassembly by catalyzing MT depolymerization [START_REF] Wordeman | Microtubule-depolymerizing kinesins[END_REF].

Flagellum Shortening Is Due to the Depolymerizing Activity of LmjKIN13-2 All members of the kinesin-13 family that could be tested to date, and particularly their representative members, XKCM1 in Xenopus [START_REF] Desai | Kin I kinesins are microtubule-destabilizing enzymes[END_REF][START_REF] Kline-Smith | The microtubuledestabilizing kinesin XKCM1 regulates microtubule dynamic instability in cells[END_REF] and MCAK in mammals [START_REF] Wordeman | Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis[END_REF][START_REF] Hunter | The kinesin-related protein MCAK is a microtubule depolymerase that forms an ATPhydrolyzing complex at microtubule ends[END_REF], have been shown to effectively depolymerize MTs in vitro. The same has been found for the protozoan PfKinI of Plasmodium falciparum [START_REF] Shipley | Structure of a kinesin microtubule depolymerization machine[END_REF] as well as for the mammalian KIF2A, which apparently plays a nonmitotic role in the development of the nervous system by suppressing extension of superfluous branches at the cell edge of postmitotic neurons [START_REF] Homma | Kinesin superfamily protein 2A (KIF2A) functions in suppression of collateral branch extension[END_REF]. In the kinesin-13 family, at least two class-specific motifs have been described within the motor domain as essential for MT-depolymerizing activity. The most significant one is the KVD finger. Mutational studies on the PfKinI catalytical core, exchanging KVD for three alanine residues, completely abolished depolymerizing activity, whereas the ATPase and MT-binding activities were intact. A second class-specific set of residues, the KEC motif, was also shown to be essential for depolymerization through MT binding [START_REF] Shipley | Structure of a kinesin microtubule depolymerization machine[END_REF].

We therefore mutagenized these two sites in LmjKIN13-2 and expressed the mutant proteins as GFP-fusion proteins in L. major. The replacement of the first motif (here KLD, position 75 of the motor domain [MD]) by three alanines was sufficient to restore wildtype morphology (Figures 2C and3A, Table S1). This mutation also caused the loss of of the mutated protein in the flagellum, this protein being located only at the flagellar base. Alanine replacement in the KEC motif (position 292 of the MD) partially restored the wild-type phenotype and gave the same localization as the KLD mutation (Figure 3B, Table S1). With each of these mutations, the flagellum was significantly longer than the short-flagellum phenotype caused by the expression of the full-length recombinant kinesin (p < 0.0001). These results show that MT depolymerization is the basis for the observed effect of LmjKIN13-2 on FL.

Because the neck of kinesin-13s is known to be essential for efficient MT depolymerization in mammalian MCAKs [START_REF] Ogawa | A common mechanism for microtubule destabilizers-M type kinesins stabilize curling of the protofilament using the class-specific neck and loops[END_REF][START_REF] Ovechkina | K-loop insertion restores microtubule depolymerizing activity of a ''neckless'' MCAK mutant[END_REF], we also constructed a mutant where most of the N-terminal part of LmjKIN13-2 (residues 1-30) was deleted. A GFP-fused version of this mutant localized to the flagellum like the full-length recombinant LmjKIN13-2 (not shown), and its overexpression also yielded a short-flagellum phenotype (Table S1). This suggests that, as suspected for P. falciparum PfKinI [START_REF] Moores | A mechanism for microtubule depolymerization by KinI kinesins[END_REF], this domain may not be essential for the depolymerizing activity of this kinesin-a hypothesis that might apply to other ''ancestral'' kinesin-13s of the KIF24 subfamily.

RNAi Knockdown of the Ortholog of LmjKIN13-2 Induces an Increase in Flagellar Length RNA interference (RNAi) is not functional in Leishmania but is efficient in T. brucei. Therefore, we constructed an RNAi vector to inhibit the expression of Tb11.02. 2260, the exact ortholog of LmjKIN13-2 in T. brucei (see Supplemental Data). From 4 days of induction, a significant increase of the mean FL was observed as compared with noninduced transformants (Figure 4). No in vitro cell-growth impairment was noted (not shown). In order to control for possible off-target effects of the RNAi, we performed a second RNAi experiment directed to another portion of the gene that does not overlap with the portion used in the first experiment. A similar FL increase was then obtained (Figure S4). These data strongly suggest that LmjKIN13-2 activity is present at steady state and therefore coexists with constitutive disassembly as well as IFT in a complex FL-control process. This also supposes some degree of regulation of this activity in order to maintain a balance between shortening and lengthening of the flagellum.

Long-flagellum phenotypes have been reported previously, essentially following mutations of protein-kinase genes [START_REF] Erdman | Interacting protein kinases involved in the regulation of flagellar length[END_REF][START_REF] Asleson | Genetic analysis of flagellar length control in Chlamydomonas reinhardtii: A new longflagella locus and extragenic suppressor mutations[END_REF][START_REF] Berman | A novel MAP kinase regulates flagellar length in Chlamydomonas[END_REF][START_REF] Wilson | Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas reinhardtii[END_REF][START_REF] Bengs | LmxMPK9, a mitogen-activated protein kinase homologue affects flagellar length in Leishmania mexicana[END_REF]. Thus, the alternative model of FL control exposed above has gained support from the identification of a MAP-kinase cascade where loss of function yielded flagella of altered length, particularly in Leishmania [START_REF] Erdman | Interacting protein kinases involved in the regulation of flagellar length[END_REF][START_REF] Berman | A novel MAP kinase regulates flagellar length in Chlamydomonas[END_REF][START_REF] Wiese | Protein kinase involved in flagellar-length control[END_REF][START_REF] Bengs | LmxMPK9, a mitogen-activated protein kinase homologue affects flagellar length in Leishmania mexicana[END_REF]. Similar data have been obtained in Chlamydomonas with a NIMA-related kinase that was found to regulate FL by promoting flagellar disassembly [START_REF] Bradley | A NIMA-related kinase, Cnk2p, regulates both flagellar length and cell size in Chlamydomonas[END_REF]. Nevertheless, the substrates of these kinases remain unknown. Kinesins are well known to be subject to regulation via specific phosphorylation events [START_REF] Wordeman | Microtubule-depolymerizing kinesins[END_REF]. Hence, although this remains speculative, a future working hypothesis would be that LmjKIN13-2 be a substrate of such a regulating cascade.

The data presented here are novel in two ways. They are the first report of an MCAK-like protein localized to a eukaryotic flagellum. More importantly, they also constitute the first report of an effector protein that would be directly involved in the process of FL control, through the depolymerization of axoneme MTs. Obviously, more data need to be gathered before a complete picture of the part played by this protein in FL regulation emerges-e.g., its interactions with other known flagellar proteins or whether it is truly involved in a feedback system. Still, kinesin-13 family members are ubiquitous proteins with a conserved MT-depolymerizing function in almost all eukaryotes studied to date. Kinesin LmjKIN13-2 might thus be one of the missing pieces in the FL-regulation puzzle. Indeed, our data do not contradict but complement the existing FL-control models [START_REF] Marshall | Cellular length control systems[END_REF][START_REF] Rosenbaum | Organelle size regulation: Length matters[END_REF] in an aspect based upon a direct intervention in MT dynamics.

Supplemental Data

Experimental Procedures, four figures, one table, and one movie are available at http://www.current-biology.com/cgi/content/full/17/9/ 778/DC1/.

Figure 1 .

 1 Figure 1. LmjKIN13-2 Shows an Unusual Localization at the Flagellum and Induces Short-Flagellum Phenotypes (A) Typical morphology of a wild-type L. major ''Fiedlin'' promastigote cell in log-phase growth viewed in phase-contrast microscopy. The scale bar represents 10 mm. (B) Images of L. major cells expressing LmjKIN13-2-GFPc (the protein fused to GFP at its C-terminal end) viewed in fluorescence (left) and phase-contrast (right) microscopy. LmjKIN13-2-GFPc (green) localizes essentially at the flagellum. The nucleus (N) and the kinetoplast (K), which is the single mitochodrial DNA located near the basal body of the flagellum, are stained with DAPI (blue). The mutant cells exhibit demonstrative morphological phenotypes, with a shortened flagellum and a short and often ovoid cell body, as well as, in some cells, an elongation of the posterior end (thick arrowhead). The scale bar represents 10 mm. (C) Images of L. major cells expressing GFPn-LmjKIN13-2. Phenotypes in this line were similar to those observed in (B). The cells exhibiting the most intense flagellar fluorescence were mainly predividing and dividing cells (visible here from the presence of the nascent daughter flagellum, nf). N indicates the nucleus and K indicates the kinetoplast. The scale bar represents 10 mm.

Figure 2 .

 2 Figure 2. Histograms of Flagellar Lengths in Wild-Type and Mutant L. major Promastigote Cells (A) Wild-type Friedlin cell line. The abscissa shows groups of flagellar lengths in microns and the ordinate shows the number of cells as percentages. Typically, 200 flagella were measured for each cell line. All flagella were measured at the same stage of growth (see Supplemental Data). (B) Transformant cloned cell line expressing LmjKIN13-2-GFPc, showing a very high proportion of short flagella. (C) Transformant cloned cell line expressing LmjKIN13-2-GFPc mutated at the KLD site (DKLD/AAA), showing restoration of the wildtype phenotype.

Figure 3 .

 3 Figure 3. Mutations of LmjKIN13-2 at the KLD and KEC Sites Restore the Wild-Type Phenotype (A) Images of L. major cells expressing LmjKIN13-2-GFPc mutated at the KLD site (DKLD/AAA) viewed in phase-contrast (left) and fluorescence (right) microscopy. Promastigotes exhibit typical wild-type long flagella and slender cell bodies. LmjKIN13-2-GFPc (green) did not decorate the flagellum but instead showed a cytoplasmic localization with preferential accumulation at the anterior pole of the cell. K indicates the DAPI-stained (blue) kinetoplast DNA and N indicates DAPI-stained nucleus. The scale bar represents 10 mm. (B) Images of L. major cells expressing LmjKIN13-2-GFPc mutated at the KEC site (DKEC/AAA). The wild-type morphology appeared to be restored, and the phenotype is as in (A). A loss of localization at the flagellum was also noted. The scale bar represents 10 mm.

Figure 4 .

 4 Figure 4. RNAi Knockdown of the Ortholog of LmjKIN13-2 in Trypanosoma brucei Induces Flagellar Lengthening (A) Histogram of flagellar lengths in tetracyclin-induced (black bars) and noninduced (white bars) T. brucei transfected procyclic cells. Abscissa and ordinate are as in Figure 2. The flagellar measurements shown here were made after 6 days of induction. Two hundred cells were examined for each experimental condition. The mean flagellar lengths for tetracyclin-induced versus noninduced transformants were 22.5 6 2.9 mm and 19.3 6 2.7 mm (p < 0.0001). (B) Northern-blot analysis of RNAi of Tb11.02.2260, the ortholog of LmjKIN13-2, in T. brucei procyclic cells. Total RNA (10 mg/lane) isolated from transfected procyclic cells grown in the absence (2) or presence (+) of tetracyclin (1 mg/ml) for 5 days was electrophoresed, blotted, and probed at high stringency with a 454 bp and a 653 bp DNA fragment of the Tb11.02.2260 gene (kinesin-13, top) and the T. brucei GPI8 anchor-subunit gene Tb10.61.3060 (control, bottom), respectively.

Acknowledgments M.T. is a recipient of a fellowship from the French Ministry of National Education and Research (MENESR). We thank Fre ´de ´ric Bringaud (Universite Victor Segalen Bordeaux 2) for providing the T. brucei 29-13 cell line as well as advice for the RNAi. We acknowledge TrypanoFAN (director Mark Field) through whom we were able to get the RNAi vector. We also wish to thank Keith Gull (University of Oxford) for his generous gift of monoclonal antibodies KMX and ROD-1. We are finally grateful to Gilles Labesse (Centre de Biologie Structurale, Institut National de la Sante ´et de la Recherche Me ´dicale U554, Montpellier) for the bioinformatic analysis of LmjKIN13-2 and to Juliette Van Dijk (Centre de Recherche en Biochimie Macromole

´culaire, Centre National de Recherche Scientifique, Montpellier) for fruitful discussions.