Zen-Hong Huang

Gladys Massiera

Laurent Limozin

Paul Boullanger

Marie-Pierre Valignat

Annie Viallat

Sensitive detection of ultra-weak adhesion states of vesicles by interferometric microscopy

We used an original analysis of reflection interference contrast microscopy (RICM) to detect an ultra-weak specific interaction between a glycolipid vesicle and a lectin-coated substrate. The membrane height fluctuations in the contact zone are observed with high illumination aperture; the membrane profile and the membrane-substrate distance are quantitatively determined by using the new analysis, which accounts for multiple interfaces and multiple incidence rays. We showed that this refined version of RICM theory is necessary, specifically in the case of intermediate membrane-substrate distance (~30 nm) and helped to discriminate between ultra-weak interaction and pure gravitational sedimentation

Introduction

Giant unilamellar lipid vesicles (GUVs) are widely used as biomimetic objects able to isolate and to modulate specific aspects of cell mechanics 1,2,3,4, dynamics 5,6,7,8, or membrane organization 9,10 . As their membrane can contain several kinds of lipids bearing charges or specific functions, the vesicles can easily interact with a neighbour substrate, and this interaction may alter the vesicle behaviour. It is thus crucial to unambiguously detect and characterize possible membrane-wall interactions. Indeed, GUVs adhesion onto model substrates has been extensively studied 11,12,13,14,15 , notably because it is a way to gain an insight into the physical basis of cell adhesion 16 . However, the case of very weak interactions is not very documented although in most physical and biophysical studies involving GUVs, the vesicles are located close to a substrate. Moreover, RICM theory and new experimental set ups such as the dual wave length method 17 , while useful to extract In this paper, we use for the first time a refined model of reflection interference contrast microscopy (RICM) to analyse RICM data obtained for vesicles in ultra-weak adhesion with a substrate 18 . This method allows to accurately reconstruct the vertical profile of the membrane close to the contact zone. We show that this method is necessary to enable the detection of ultra-weak adhesion states and to discriminate these states from that induced by pure gravitational settling. We stress that this method applies to vesicles which are not particularly deflated, for which the membrane thermal fluctuations may be of relatively weak amplitude.

RICM is the technique of reference for the observation of vesicle adhesion 17,19,20 . Based on the principle of Newton's rings formation, this method allows the visualization of the vesicle contact zone, and the detection of adhesion patches. Moreover, a simple theory, which relates intensity to height above the substrate, allows a quantitative analysis of the interference pattern and provides both the measurements of height fluctuations of the membrane and the reconstruction of the vertical profile of the membrane at the immediate vicinity of the substrate 15 . The technique has proved to be suitable to characterize membrane spreading times 13,22 membrane thermal fluctuations 21 , membranesubstrate separation and associated adhesion strength 12,13,22 .

However, in the context of the study of very weak adhesion, the theory used routinely for RICM analysis, which assumes two interfaces and normal incidence, is not valid, even when the membranes are roughly flat and parallel to the substrate. On the one hand, as outlined recently 22 , the reflection on the internal side of

Background on vesicle specific adhesion

Vesicle adhesion has been extensively studied both experimentally 24 and theoretically 25 .

Usually, GUVs exhibit key elements of the cell surface involved in adhesion 26 . They are generally prepared with receptors or receptor fragments reconstituted into the membrane and with lipids bearing polymer headgroups of polyethylene-glycols 11,12,13,14,27 which mimic the glycocalix and model the associated generic long range repulsion force. The substrate is constituted by supported lipid bilayers containing homophilic receptors 12,28,29 or coated/grafted with ligands recognizing the receptors 13 .

The specific adhesion of membranes to substrates is governed by an interplay of short range attractions between receptor-ligand pairs and non-specific repulsive interactions such as Helfrich entropic repulsion. As a result of this attraction/repulsion competition, three main behaviours have been reported 16 :

The non-adherent regime, determined by the Recently, adhesion domains were detected in absence of repellers in the case of weak receptor-ligand interactions when the ligands are anchored to a polymeric spacer. Free ligands then provide a membrane-substrate repulsion comparable to that of the usual lipopymers (PEG2000) used as repellers 27 .

The strongly adherent regime induced either by specific or non-specific forces. The contact area has an homogeneous quasi-circular nonfluctuating shape, characterized by a dark ring surrounding the adhesion disc 22 and a membrane-substrate distance 12 h < 10 nm.

In our system, the specific attraction caused by the recognition between a glycolipid and a lectin result in an ultra-weak adhesion state characterized by a small circular homogenous adhesion disk with an intermediate distance to the substrate.

RICM model

We which had never been accounted for so far, even in the most comprehensive models (Wiegand 19 , Gingell 23 ).

In the analysis presented here, the total reflected intensity is expressed as a function of the amplitude of the reflection coefficients of a multifilm structure, R s and R p . It can be written as the sum of a first term, equal to the intensity obtained with unpolarized light and corresponds to the intensity calculated by

Wiegand 19 for plane interfaces and of a second corrective term due to the polarization :

€ I = ( R p 2 + 0 θ max ∫ Rs 2 ) sin θ dθ - (R p * R s + R p R s * 0 θ max ∫ ) sin θ dθ
which can be written as

€ I = I 0 R s -R p 2 sin θ dθ 0 θ max ∫
where the indices -s and -p denote the polarization of the reflected wave perpendicular or parallel, respectively, to the plane of incidence, I 0 is a constant independent of the optical properties of the sample, θ is the illumination angle and θ max the maximal illumination angle. For isotropic multifilm structures, the coefficient of reflection can be evaluated by a 2x2 matrix method, as described by Azzam and Bashara 32 and further, Wiegand 19 .

Here, As shown in Fig. 1a, WGA lectin is a dimer has a highly specific interaction with GlcNAc and (GlcNAc) 2 with two sites of recognition on each subunit. The association constant for the binding of the dimer (GlcNAc) 2 to WGA is rather strong [START_REF] Allen | [END_REF][START_REF] Sela | The Antigens[END_REF] , of the order of 10 4 . The affinity of the monomer GlcNAc for the WGA lectin is about 600 times weaker than that of (GlcNAc) 2 , and is in the range of weak ligand-receptor interaction.

The adhesion strength of glyco-vesicles onto lectin-substrates strongly depends on the accessibility of the lectins grafted on the substrate to the sugar embedded in the vesicle membranes and is believed to be very small in the system presented here, probably due to geometrical considerations, so that vesiclesubstrate interaction is finally ultra-weak.

Lipids and proteins

The lipid Grafting stage. We incubated silanized coverslides in the lectin-PEG hybrid solution for 3h. Then, lectin-grafted coverslides were well rinsed 3 times in PBS buffer (pH = 7.2).

All the preparation stages were performed at room temperature and stored in PBS solution at 4°C. The coverslides were used within the three days following their preparation. A schematic view of glycolipids and the substrate is presented in Fig. 2.

Grafted lectins. Recognition by GlcNAc

We estimated the density of grafted lectins by Massachusetts, USA), using self-written routines. In striking contrast, the contact disc of glycovesicles on silanized substrate (Fig. 3C) as well as that of DOPC vesicles on both silanized and lectin substrates (Fig. 3D and3E) is nonhomogeneous. The 'leopard skin' texture 30 , clearly observed in Fig. 3E However, when the object is not close enough to the substrate no minimum is observed (Fig. 1). In this case, we take for the second We emphasize that this method provides a non-ambiguous measurement of h c , since a whole damped sinusoidal curve is fitted with only one free parameter and we stress the importance of a careful normalization of light intensity curves, which is very sensitive to the damping induced by the numerical aperture.

Results

RICM observation of vesicle adhesion

Non spherical vesicles. When the profile of the vesicle is far from a sphere, as shown in Fig. 6, the fit of the whole I N (r) curve is no longer possible. We then calculate the theoretical curve I th (h) using the experimental INA. We first check that the amplitudes of the damped oscillations observed on experimental curves are similar to that on theoretical curves over at least the first three extrema of light intensity, thus indicating that experimental curves have been correctly normalized. We then assign to the membrane element located at a radial distance r characterized by the light intensity I N (r), the distance h such as I N (r) = I th (h) . The way we reconstructed the profile of the vesicle is illustrated in Fig. 6 on a glyco-vesicle on lectin (vesicle 3). The corresponding vesicle profile is displayed in the insert of Fig. 6. The value h c is determined from the profile extrapolated at r=0.

Membrane fluctuations.

The amplitude of spacing fluctuations (see Table 1) were determined from the probability distribution function of the normalized light intensity, P(I N ), measured in the same way as that shown in Fig. 4B. For the non adherent cases (DOPC on lectin and silane and glyco vesicles on silane), we used the part of the curve at the right of the minimum of I(h) (Fig. 2C) to calculate membrane-substrate distances h and to derive the standard deviation of the hdistribution that we note ω. For the case of glyco-vesicles on lectin, as the fluctuations of light intensity in the contact area are of the order of magnitude of the noise (as typically shown in Fig. 4), the height fluctuations of the membrane with respect to the substrate could not be determined. They are equal or less than that due to the noise (of the order of 7 nm) and are therefore significantly smaller than that observed for the three non adherent cases.

The regime of membrane fluctuations is however dependent on the bending rigidity of the membrane, which can be slightly different for pure DOPC vesicles and glyco-vesicles.

That is why we emphasize that we measured height fluctuations on the same glycomembranes (glyco-vesicles) and that they are significantly different on lectins and on silanes.

Another factor that can affect the regime of fluctuations is the membrane tension.

Membrane -substrate distance

The values of the membrane-substrate distance at the centre of the contact area, h c , obtained from the shape reconstructions are reported in Table 1. It clearly appears that the membranesubstrate spacing is smaller for glyco-vesicles on lectins (value of <h c > equal to 35 nm) than for glyco-vesicles on silanes (average value equal to 84 nm) and for DOPC vesicles on lectins and silanes (value of <h c > equal to 102 nm and 120 nm respectively).

The values we obtained clearly show the existence of a specific interaction between glyco-vesicles and lectin coated subtrates.

Indeed, in this case, altough the average h c value is large compared to tightly adhered membranes and small compared to free membranes, it is compatible with that found on weak-adhesion patches between sialyl-Lewis x ligands and E-selectin receptors (30 nm) 31 .

Moreover, the much higher values obtained for the three control systems can be compared to the spacing estimated for non-adherent vesicles. For a rough estimation, we write that the equilibrium membrane-substrate distance results from the balance between gravitational attraction and Helfrich repulsion 37 . Gravitation is given by F g = Δρ V g, where V is the vesicle The repulsion force writes as:

€ F rep = -A dV H dh = - 2 c A (kT) 2 κ < h > 3
where A is the contact area, V H is the Helfrich repulsive potential, c is a constant ≈ 0.115., κ is the membrane bending energy (we take κ = 4 10 -20 J) 38 , k is the Boltzmann's constant and T is the temperature. By writing that F g + F rep = 0 and by measuring the contact area A, we estimate the equilibrium mean spacing <h>.

Results are presented in Table 1. We clearly see that these values are in reasonable agreement with the experimental ones for non adhering vesicles. In contrast, for glycovesicles on lectins, there is a factor two between predicted (70 nm) and observed (35 nm), thus bringing additional evidence to prove that the membrane-substrate distance is not determined by the gravitation/entropic repulsion balance but involves a supplementary attractive interaction.

Discussion and conclusion

We have worked on DOPC and glyco-vesicles

  the membrane induces an additional phase shift, which depends on the refractive indices of the internal vesicle medium. This shift sensitively changes the relation between membrane height and light intensity in the intermediate range of heights (30-50 nm) where weakly bound membranes are located. On the other hand, height fluctuations are most conveniently measured using a high illumination aperture in order to maximize the illumination. The interest is to reduce exposure time and blurring effects occurring with fast moving membrane parts. The consequence is that, even for relatively small membrane height (h~50nm), one observes a fringe contrast damping with the radial distance due to large angle incident rays 23 . The damping has to be accounted for to describe the variation of the fringes light intensity with distance and to obtain an accurate height reconstruction of the vesicle profile over several fringes. We use in this study a theory of RICM, which accounts for multiple interfaces and multiple incidences rays. One key improvement is to account for interferences between rays of different incident polarization and also to carefully normalize the data. While the interfaces are supposed to be horizontal, the theory applies to the reconstruction of the vesicle profile up to 4-5 fringes, better than the current most elaborated theory 19 . We work on a statistical ensemble of glycolipid and phospholipid vesicles electroformed in standard conditions and used without further treatment (no significant deswelling stage for instance) in order to fit to many experimental situations encountered in vesicle studies. We show the existence of an ultra weak attraction between glycolipid vesicles and lectin surfaces, which we discriminate from the pure settling state presented by glycolipid vesicles on silanized surface and by phospholipid vesicles both on lectin and silanized surfaces.

  balance of gravitational attraction (vesicle weight) and Helfrich repulsion. It is characterized by a strong membrane flickering in the adhesion disc. The contact area is inhomogeneous, and the probability distribution of the membrane-substrate distance in the contact disc, P(h), (estimated from RICM) is Gaussian with a large mean value h > 50nm, and a roughness (root mean square of P(h)) of about 25 nm 30 . The intermediate adherent regime, which occurs in presence of mobile receptors and repulsive lipopolymers. It is characterized by the formation of adhesion plaques, which are related to the lateral segregation of receptorligand pairs 29,31 . . This regime results from the competition between short range ligandreceptor forces and long range glycocalix repulsion, which leads to spontaneous segregation of receptors. The tight adhesion plaques do not show fluctuations, and are associated with a membrane-substrate distance h < 30 nm. In contrast, the non-adherent regions of the contact zone are flickering.

  have analysed RICM images by using a new and recently developed optical model detailed elsewhere 18 . This model quantitatively calculates the intensity reflected by the vesicle interface and by the substrate by taking into account three important specificities of the experimental system. The first one is the superposition of different refractive indices layers formed by the vesicle and the substrate (3 interfaces). The second one is the illumination numerical aperture (INA) of the microscope, responsible for a large angle distribution of incident rays. A summation of the intensity over all angles of illumination is therefore required to compute the light reflected intensity. The third characteristics is the polarization of the light. Indeed, the presence of the antiflex component in the experimental set up induces light polarization,

  a settled vesicle is modelled by a multifilm formed by 3 plane interfaces: the glass-glucose solution, the glucose solution-membrane and the membranesucrose solution. The variation of the intensity of the reflected light (normalized with respect to the minimal and maximal observed intensities) with the distance of the membrane to the substrate, h, is computed with this model, and is displayed in Fig. 1b and 1c for various INAs. A first remark is that the curves clearly depend on the INA even for small h values, and in particular, the value of h at the minimum light intensity increases with the INA as shown in the insert. These effects must therefore be accounted for to accurately determine the vesicle/substrate distance. An important issue shown in Fig. 1b is that at one light intensity correspond two values of h located on two distinct branches. Values on the left branch are typical of adhered membranes whereas large h-values on the right branch are characteristic of non-adhesion states. The assignment of the left value of h is easy for strongly adhered membranes which are very close to the substrate (about 10 nm). Indeed, the bright contact zone is surrounded by a dark ring observed as h increases and reaches 50nm. However, when the membrane/substrate distance is typically of the order of 30 -40 nm, which is the case for weak adhesion, the contact zone is dark and the contrast is not always sufficient to detect whether a darker surrounding ring exists or not. It is then very difficult to know on which branch h must be determined and, therefore, the analysis of the contact area does not allow to conclude about the adhesion state of the membrane. An attempt to overcome this problem is to consider the light intensity I N over a zone much larger than the contact zone, for instance along a radial line passing through the centre of the contact zone and to analyse the variation of I N with the radial distance over several fringes. This is enabled by the model used here, which accurately describes the damped light intensity oscillations due to the INA (Fig.1b). The membrane/substrate distance in the central zone h, which is the only free parameter, can be then determined. This method will be used and detailed in the following.ExperimentalLectin-sugar recognitionGUVs prepared with a mixture of phospholipids and lipids bearing a sugar (Nacetylglucosamine, GlcNAc) are allowed to interact with a substrate coated with lectins from Wheat Germ Agglutinin (WGA). Lectins are sugar-binding proteins which are highly specific for their sugar moieties. Lectins occur ubiquitously in nature from bacteria attachment to host cells or to specific functions in leukocyte adhesion to endothelial cells for instance through sialyl-LewisX recognition.

  measuring the fluorescence of FITC-labelled grafted lectins by confocal microscopy. We found a value in the range 2000 -2500 µm -2 . In order to show that GlcNAc specifically recognizes grafted lectins, we incubated grafted lectin coverslides in a solution (pH = 7.2) of glucose (200 mM) and FITC-labelled GlcNAc (1µM) for 20 min before extensive rinsing. Widefield epifluorescence images disclosed that the substrates exhibited fluorescence contrarily to bare coverslides. Comparison of the fluorescence intensity to that measured from grafted FITC-lectins revealed that about half of grafted lectins are functional for GlcNAc molecules. It is probable however that only a fraction of these functional lectins are accessible to the sugar moiety, when it is born by the lipid membrane. Lectin recognition of glycosites on vesicle membranes The specific recognition of GlcNAc on glycovesicle membranes by lectins in solution was tested by suspending glyco-vesicles and DOPC vesicles in a solution (200 mM glucose) containing FITC-lectins. The membranes of glyco-vesicles were fluorescent, revealing the presence of FITC-lectin fixed onto the glycolipid membrane whereas DOPC vesicles exhibited no fluorescence at their surface. Image acquisition and processing. Confocal fluorescence measurements were realized with a scanning confocal microscope Leitz, with a 1.40 NA 63x objective and blue laser excitation (488 nm). The pinhole was set to impose an optical slice thickness of about 0.4 µm. RICM images were acquired with a Zeiss axiovert 200 inverted microscope (Carl Zeiss, Jena, Germany) equipped with a 63x antiflex objective and a C7780 camera (Hamamatsu, Tokyo, Japan). White light emitted by a HBO lamp was filtered using a green filter (λ=546+/-12 nm). 200 consecutive images (total duration 10 s, individual exposure time 50 ms) of the interference pattern were recorded for different vesicles, located at different positions on the substrate. Raw RICM images were firstly corrected for inhomogeneous illumination by a background subtraction procedure 22 . This yields an image with a uniform background as judged by comparing the intensity distribution in 20x20 pixels regions at the four corners of the image. The interference intensity of such images was considered as measured intensity (I) for further analysis. We normalized the measured interference intensity: I N = (I -I min ) / (I max -I min ). Here I max and I min are the maximum and the minimum light intensity in each fringe pattern and are determined by the whole grey-scale interference pattern. Specific normalization for membrane shape reconstruction will be detailed further. We used phase contrast microscopy (magnification x63) for direct visualization and vesicles radii determination. Image processing and data analysis were done using the image analysis software Image-J (public domain NIH) and/or the generalpurpose mathematical software Igor-pro (Wavemetrix, Portland, OR, USA), IDL (ITT Visual Information Solution, Colorado, USA) and MATLAB (The Mathworks, Inc.

  Typical time sequences of RICM images observed on DOPC and glyco-vesicles settled on lectin and silanized surfaces are displayed in Fig. 3. Comparison of successive images provides an indication of the temporal variation of the contact area. One observes well-defined contact zones, which are determined by the first Newtonian ring. The Newtonian fringes correspond to lines of equal height of the membrane surface above the substrate. We distinguish two behaviours.The first behaviour is observed in the contact disc of glyco-vesicles on a lectin surface (Fig.3A and 3B). It is characterized by an homogenous central dark zone, which is welldefined and stable with time. The central dark zone is always darker than the first fringe. A darker ring around the contact area is however not clearly and systematically observed.

1 µm 2 )

 12 Fig. 4B in the whole contact area are similar: the contact area is flat on average. The small light intensity presented by glycovesicles on lectin substrates compared to the other systems and the associated smaller spatial and dynamic intensity fluctuations are a strong indication for the existence of a weak homogeneous specific adhesion of glycovesicles onto lectin substrates. These results however concern only light intensities. In order to be conclusive, the light intensity has to be converted into a distance to the substrate. It

  reference point the minimum intensity reached in the second dark fringe. This intensity is unambiguously observed and, contrarily to the simplified models, is accurately computed by the model used here, which describes the damped oscillations induced by the INA. Spherical vesicles. The variation of I N along a radial line is reported in Fig. 5 for two glycovesicles of radius equal to 18.5 µm and 12 µm settled on silanes and lectins respectively. The curves are fitted by the model we have described, when a spherical shape of the membrane is assumed. The fit is obtained with a single fitting parameter, the membranesubstrate distance h c at the centre of the contact zone. The two other parameters (INA and radius of the vesicle) are measured independently. As it is clearly seen in Fig. 5, the fit is very good and allows an accurate determination of h c . It is equal to 90 nm and 35 nm respectively for the two displayed vesicles (glyco-vesicles on silanes and on lectins respectively).

  It is directly related to to the reduced volume of the object, which depends on the vesicle preparation and is not strictly controlled. It varies over an assembly of vesicles, even when they are prepared in a single batch. That is why it is important to measure membrane fluctuations and substrate spacings on several vesicles obtained from different batches and to compare the average values obtained on each vesicle-substrate system.

  volume and Δρ is the difference of density between the fluid in the vesicle and the suspending fluid. The Helfrich repulsion is due to the reduction of entropy induced by the presence of the substrate which reduces the amplitude of membrane thermal fluctuations.

Fig. 1 .Fig. 2 .Figure 3 .Fig. 4 .Fig. 5 . 2 Figure 6 .

 1234526 Fig. 1. a) Schematic representation of a vesicle at the vicinity of the substrate, and the reflections occurring at different interfaces. b) and c) Normalized relative intensity versus the substrate/vesicle distance calculated for three different values of INA (corresponding to cones of illuminating light having angles of θ max ; INA = n glucose Sin (θ max ) ). Black circle : normal incidence (INA= 0); Green square : INA = 0.6; Red bold line : = 1 ; The values of the refraction index have been taken equal to : n substrate = 1.525; n glucose = 1.3386; n membrane = 1.486; n sucrose = 1.346; The thickness of the membrane equal to 5 nm and the calcultation were done with a monochromatic wavelength of 546 nm.

Table 1 .

 1 Vesicle radius, area of the contact zone, substrate-membrane spacing in the centre of the contact zone, theoretical membrane-substrate spacing calculated from equilibrium between buoyancy and Helfrich repulsion (averaged ± 5 nm), dynamic roughness for the four studied systems

	Condition		vesicle .	R (µm) A (µm 2 ) hc (nm)	<h> (nm) w (nm)
			1	18.2	46.4	15	110	6.5
			2	18.2	9.4	41	70	8.7
			3	12.1	19.6	30	130	7.4
			4	12.0	7.0	35	90	7.9
			5	14.0	11.8	30	90	7.7
			6	21.9	21.5	40	70	8.5
			7	27.2	24.8	32	60	8.1
			8	11.4	6.1	33	90	7.2
			9	25.9	16.7	47	60	7.3
			10	15.5	6.4	38	70	7.2
			11	33.3	23.6	33	50	6.8
			12	37.9	44.2	45	50	6.9
			13	11.2	4.0	39	80	8.9
			14	23.9	15.6	35	60	6.9
	glyco-vesicle	on	15	29.5	20.8	30	50	7.0
	lectin	coated	16	14.0	7.5	35	80	9.7
	substrate		17	41.0	43.6	40	50	5.8
	Mean value					35	70	7.6
	Standard deviation				7.3	22.6	1.0
			18	19.1	14.9	113	70	8.2
			19	22.1	25.1	93	80	10.8
			20	19.1	11.9	102	70	11.1
			21	14.3	6.1	90	70	10.5
			22	13.5	24.8	98	120	13.4
	DOPC vesicle on	23	13.0	11.6	101	100	14.1
	lectin	coated	24	14.2	37.9	105	130	17.4
	substrate		25	19.1	41.5	115	100	12.3
	mean					102	93	12.2
	Standard deviation				8.8	23.8	2.8

70

Notes and references

Acknowledgments.

We thank V. Rosilio, P. Nassoy and K.

Sengupta for fruitful discussions. We thank A. M. Benoliel for help for confocal experiments. Z. H. Huang gratefully acknowledges funding by the Nanolane Company. Adhesion and Inflammation and LCVN labs belong to the CNRS consortium CellTiss


Agnès Belly

Gilles Bodon

Béatrice Blot

Alexandre Bouron

Rémy Sadoul

Yves Goldberg

B Atrice Blot

CHMP2B mutants linked to frontotemporal dementia impair

Keywords: ESCRT, FTLD, neurodegeneration, synapse, spine head, NeuronStudio Δ

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.

Introduction

The Endosomal Sorting Complexes Required for Transport are a set of four cytoplasmic protein complexes (ESCRT -0 to III), conserved from yeast to human. ESCRTs are central to a growing range of cellular activities, including the delivery of down-regulated surface proteins to the lumen of late endosomes ( ; ), the release of viruses at the plasma Hurley andEmr, 2006 Slagsvold et al., 2006 membrane ( ), cytokinesis of dividing cells ( ), and autophagy of protein von Schwedler et al., 2003 Carlton and[START_REF] Carlton | Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery[END_REF] aggregates ( ). All of these diverse processes involve the budding and fission of cellular membranes, which appear [START_REF] Filimonenko | Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease[END_REF] to be directly catalysed by ESCRTs. Recent results show that ESCRTs are sequentially recruited to the cytosolic side of endosomal or plasma membrane microdomains, until ESCRT-III subunits locally polymerise into filaments. Transient assembly of ESCRT-III filaments on the membrane is a physical agent of membrane deformation and vesiculation ( ; ; [START_REF] Fabrikant | Computational Model of Membrane Fission Catalyzed by ESCRT-III[END_REF]Hanson et al., 2008 Lata et al., ; ; ; ; ). Hence, ESCRT-III polymers are pivotal 2008 [START_REF] Muziol | Structural basis for budding by the ESCRT-III factor CHMP3[END_REF][START_REF] Saksena | Functional reconstitution of ESCRT-III assembly and disassembly[END_REF][START_REF] Wollert | Membrane scission by the ESCRT-III complex[END_REF][START_REF] Wollert | Molecular mechanism of multivesicular body biogenesis by ESCRT complexes[END_REF] effectors of cellular membrane restructuring.

In keeping with its potent effects, the polymerisation of ESCRT-III is normally under tight regulation. The core subunits of ESCRT-III form a family of highly related proteins, the Charged Multivesicular body Proteins (CHMP) 1 7, all of which contain a conserved oligomerisation and lipid binding interface (the ESCRT-III or SNF-7 domain), linked to a C-terminal regulatory region (see ). This Fig. 1 latter mediates both intramolecular inhibition of the ESCRT-III domain and intermolecular interaction with subunit-specific factors that regulate filament dynamics ( ; ; ;

). Deletion [START_REF] Bajorek | Structural basis for ESCRT-III protein autoinhibition[END_REF][START_REF] Muziol | Structural basis for budding by the ESCRT-III factor CHMP3[END_REF][START_REF] Stuchell-Brereton | ESCRT-III recognition by VPS4 ATPases[END_REF][START_REF] Zamborlini | Release of autoinhibition converts ESCRT-III components into potent inhibitors of HIV-1 budding[END_REF] of the regulatory region converts ESCRT-III subunits into dominant negative mutants, able to block ESCRT-dependent processes such as the release of HIV-1 virions from infected cells ( ). [START_REF] Zamborlini | Release of autoinhibition converts ESCRT-III components into potent inhibitors of HIV-1 budding[END_REF] Among ESCRT-III subunits, CHMP2B has received particular attention following the discovery that mutations in the gene CHMP2B underlie a dominantly heritable form of frontotemporal dementia (called FTD-3), a presenile brain disease due to selective degeneration of cortical neurones (

). In a thoroughly studied Danish kindred, the FTD phenotype was tightly linked to a single point [START_REF] Skibinski | Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia[END_REF] mutation at the splice acceptor site of exon 6. The mutation generates two distinct aberrant transcripts, and CHMP2B CHMP2B intron5

, both of which encode proteins with a defective C terminus ( ) (see ). Neurodegeneration has CHMP2B Δ 10 Skibinski et al., 2005 Fig. 1 been hypothesized to result from perturbation of neuronal autophagy by these mutants subunits ( ). Consistent [START_REF] Rusten | ESCRT functions in autophagy and associated disease[END_REF] with the loss of the regulatory domain, in heterologous cells, both CHMP2B and CHMP2B form insoluble polymers, often coating intron5 endosomes, and cause dominant-negative inhibition of ESCRT-dependent processes including autophagosome maturation (Filimonenko et ; ; ). In cultured cortical neurones, overexpression of CHMP2B was shown to induce al., 2007[START_REF] Lee | ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration[END_REF]Skibinski et al., 2005 intron5 retraction of dendritic arbors and subsequent cell death, partly through toxic build-up of autophagosomes ( ; [START_REF] Lee | ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration[END_REF]. Surprisingly, however, CHMP2B failed to increase neuronal death, even though in heterologous cells, its effects on autophagy 2009 10 Δ are similar to those of CHMP2B ( ). Furthermore, knock-down of CHMP2B by RNAi did not modify intron5 [START_REF] Filimonenko | Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease[END_REF] neuronal viability. These observations suggest that disease-linked CHMP2B mutations may affect neuronal physiology by ways other than lethal malfunction of autophagic mechanisms.

Accumulating evidence indicates that in various neurodegenerative diseases neuronal death is a late event, preceded by early disruption of dendritic spines and synapses, which itself is a pivotal pathogenic step ( ; ; [START_REF] Morfini | Axonal Transport Defects in Neurodegenerative Diseases 10.1523/JNEUROSCI.3463-09[END_REF]Wei et al., 2009 Wishart et al., ). In FTD, extensive synaptic loss and reduction of spines have been documented in diseased cortex ( ;2006Ferrer, 1999 Lipton et al., ). Given its role in endosomal traffic and plasma membrane deformation, ESCRT-III may plausibly control aspects of synaptic 2001 biology. Here, we examine the possible impact of CHMP2B mutants and CHMP2B depletion on the development of dendritic spines.

CHMP2B

and CHMP2B could be expressed in hippocampal neurones without causing apparent anomalies in endosomal intron5 10 Δ morphology, autophagosome amount, or cell viability. The mutants potently suppressed the growth of spine heads, strongly lowering the proportion of mushroom spines. Very similar effects were obtained with an siRNA targeting endogenous CHMP2B, consistent with dominant-negative action of the mutants. Physiologically, CHMP2B caused a drop in the frequency and amplitude of spontaneous 10 Δ excitatory synaptic currents, with selective disappearance of large currents. We propose that CHMP2B and potentially ESCRT-III are required for the maturation of dendritic spines, and that pathogenic CHMP2B mutants perturb this process. To the extent that maturation correlates with functional potentiation, these results implicate ESCRT-III in synaptic plasticity. In the protracted course of FTD, CHMP2B mutations may affect synaptic homeostasis and plasticity long before inducing massive neuronal death.

Materials and Methods

Plasmids and antibodies

The mCherry plasmid was a kind gift from P. Dournaud (INSERM, Paris). The LC3-GFP plasmid was a kind gift from T. Yoshimori (National Institute of Genetics, Mishima, Japan). The GFP plasmid was pEGFP-C1 (Clontech). The wild-type human CHMP2B cDNA was cloned from HeLa cells by RT-PCR, in frame with an N-terminal dimeric HA tag, using the following primer pair:

5′ CTCTCGAGGCCACCATGTACCCATACGACGTCCCAGACTACGCTTACCCATACGA C G T C C C A G A C T A C G C T G C G T C C C T C T T C A A G A A G A A 3 ; a n d 5 ;
TACTCGAGCTGCAGTCACTAATCTACTCCTAAAGCCTTGAGT 3;. The CHMP2B mutant cDNA was amplified from the same intron5 cDNA preparation, using an alternate rearward primer (5; TACTCGAGTCACACCTTTCCAGAAATTTCAAT 3;). The two cDNAs were subcloned in pcDNA3.1 between the XhoI sites. To obtain the CHMP2B cDNA, the C-terminal part was first synthesized in vitro using

Neuronal culture and transfection

Primary cultures of hippocampal neurones were prepared according to a modification of the procedure described in (Banker and ). Handling and sacrifice of animals were in conformity with European law (EEC Directive n 86/609) and the internal Goslin, 1998 °regulations of INSERM. Hippocampi were dissected out from E19 rat embryos, digested with trypsin, and hippocampal cells were seeded at a density of 12,000 per cm onto acid-washed glass coverslips (13 mm diameter, Marienfeld, Germany) that had been precoated with 50 2 g/ml poly-D-lysine (Sigma). Cultures were maintained in serum-free Neurobasal medium containing 2 mM glutamine, 1 mM sodium μ pyruvate, 10 g/ml penicillin and streptomycin, and 2 B27 supplement (Invitrogen). For transfection, all plasmids were purified using μ %

the Endotoxin-free purification kit (Quiagen, Hilden, Germany). Preliminary experiments established that CHMP2B toxicity was minimal when transfected at the dose used here. The conditioned culture medium was removed and saved and coverslips were placed in 2 ml Minimal Essential Medium (Invitrogen) containing 2 mM glutamine, 1 mM sodium pyruvate, 15 mM Hepes: NaOH (pH 7.35). 0.5 g μ CHMP2B plasmid (or control vector) was mixed with 2.5 g mCherry, GFP, or LC3-GFP plasmid, diluted into 250 l transfection μ μ medium, and combined with 1.5 l of Plus Reagent and then 4 l Lipofectamine LTX (Invitrogen). The mixture was incubated for 25 min μ " " μ at room temperature, then added to the dishes, which were left to incubate at 37 C for 1h. The transfection solution was removed and the °conditioned medium was returned to the dishes. 20 60 neurones per coverslip were usually transfected.

-

Fixation and immunostaining

The coverslips were rinsed in PBS and incubated for 8 min in PBS containing freshly prepared 4 p-formaldehyde and 4 sucrose at % % room temperature (RT). After 3 4 washes in PBS, the cells were permeabilised by incubating in PBS containing 0.3 Triton X-100 for 10

-% min at RT. After washing, coverslips were pre-incubated in blocking solution (PBS containing 2 goat preimmune serum and 1 bovine % % serum albumin (Sigma-Aldrich)) for 1 h, then in blocking solution containing 0.5 1 g/ml primary antibody (or antibody mixture) at RT.

-μ

After washing, incubation with secondary antibody conjugated to Alexa488 or Alexa594 (Molecular Probes, 0.2 g/ml) and final washes, μ coverslips were gently dried, and mounted on slides in Mowiol medium. The specificity of immunofluorescence was routinely verified by staining control cultures that had been transfected with empty pcDNA vector (for anti-tag antibodies) or by omitting the primary antibody in control incubations. The specificity of CHMP2B antibody was further confirmed by RNAi-mediated extinction of immunofluorescence.

Microscopy

Images of fluorescent neurones were acquired with a Leica SP-E laser scanning confocal microscope equipped with a 100 Leica × Neofluar objective with NA 1.3. Laser power was adjusted to maximize the dynamic range of each sample. For dual-colour samples, the = adjustable spectral window of fluorescence collection was set for each channel with a singly coloured control sample, so that cross-contamination between channels was avoided. Stacks of 0.2 m-spaced optical sections were acquired (averaging over 4 scans per μ pixel, 512 512 pixels) with the required electronic magnification. For spine analysis, any neuron showing signs of neuritic swellings or × retraction was excluded. Dendritic segments located at approximately equivalent distances from the soma were selected, and each of those was scanned at 1.5 electronic zoom.

×

Image analysis

Image files were processed with Metamorph (Molecular Imaging). All figures show maximal intensity projections. For analysis of spines, serial image files corresponding to z-stacks of 20 30 optical sections per dendritic segment were directly processed with -Neuron-Studio, a software package specifically designed for spine detection and analysis ( ; ) [START_REF] Rodriguez | Automated Three-Dimensional Detection and Shape Classification of Dendritic Spines from Fluorescence Microscopy Images[END_REF][START_REF] Rodriguez | Automated Three-Dimensional Detection and Shape Classification of Dendritic Spines from Fluorescence Microscopy Images[END_REF] ). Voxel size was 0.143 0.143 0.200 m. The fluorescence threshold for inclusion in objects was http://www.mssm.edu/cnic/tools.html

× × μ
dynamically set for each local sampling of the dendrite surface by the Isodata segmentation algorithm implemented in NeuronStudio. The seed location for automatic dendrite tracing was typically set near the base of a major proximal dendrite. At bifurcation points, the attach ratio was 1.5 and neurites shorter than 5 m were not retained. After modelling of the dendrite surface, protrusions with a minimum μ volume of 5 voxels (0.020 m ), length comprised between 0.2 m and 3 m, and a maximal width of 3 m were retained as spines.

μ 3 μ μ μ
Following defaults settings of the program and the empirical classification rule defined by ( ), spines with minimum [START_REF] Rodriguez | Automated Three-Dimensional Detection and Shape Classification of Dendritic Spines from Fluorescence Microscopy Images[END_REF] head diameter of 0.35 m and minimum head vs. neck ratio of 1.1 were classified as mushroom spines. Non-mushroom spines with μ minimum volume of 10 voxels (0.040 m ) were classified as stubby spines. All other spines were considered thin. The typical μ3 percentages of the three spine types which we obtained were close to those found by (

). Measurements obtained by [START_REF] Rodriguez | Automated Three-Dimensional Detection and Shape Classification of Dendritic Spines from Fluorescence Microscopy Images[END_REF] NeuronStudio were transferred to a spreadsheet (Excel or Gnumeric) for analysis. Sholl analysis was also performed with NeuronStudio, using single-plane, lower magnification views centred on the cell soma. For each neurone, a series of 2 m-spaced concentric circles μ centred on the soma were drawn as reference marks, starting with a circle just large enough to enclose the cell body. The total length of neurites comprised within two consecutive circles was measured, and the measurement was repeated for increasingly large circle radii.

Stepwise increases in neuritic length were plotted as a function of radial distance from the soma.

Cell degeneration assay

Neurones were transfected with 0.5 g CHMP2B plasmid and 2.5 g mCherry as above. After fixation and mounting onto slides, -°Western immunoblotting Neurones were plated in plating medium at a density of 24, 000 cells per cm onto 35 mm plastic dishes (Nunc) that had been 2 pre-coated with 50 g/ml poly-D-lysine, and grown as described for coverslips. Sister dishes were withdrawn at the indicated time points μ and cells were lysed in lysis buffer containing 1 Triton-X100, 0.2 SDS, 10 mM NaF, 10 mM -glycerophosphate (pH 7.5) and

% % β =
protease inhibitor mixture (Complete, Roche Molecular), and frozen. All lysates from a given culture were loaded on the same 10% SDS-polyacrylamide gel (10 g total protein per lane) and analysed by electrophoresis and immunoblotting with anti-CHMP2B antibody μ and anti-actin antibody. Bands were revealed by chemiluminescence (Pierce Femto reagent). Luminographs were scanned (300 dpi, 8-bit scale) and the resulting image files were analysed with Metamorph. Grey levels measured for CHMP2B bands were normalized to the corresponding actin bands.

Statistical analysis

The statistical significance of overall variations within sets of conditions was determined by one-way ANOVA. The significance of pair-wise differences between conditions was then assessed by post-hoc one-tailed t tests using the Bonferroni method. Unless indicated otherwise, all the reported p values refer to the difference with the control condition. We verified that criteria for robust ANOVA were satisfied; moreover Kruskal-Wallis analysis of mean rank variance yielded the same qualitative results as ANOVA. For analysis of mEPSC frequency, the significance of the transfection effect was confirmed by two-way ANOVA on a balanced subset of mEPSCs, separating the effect of plasmid identity from intrinsic neurone-to-neurone variation due to the variability of innervation in the culture. The significance of differences between multiple frequency distributions was assessed with the test. A post-hoc test of pairwise differences χ 2 used the Marascuilo procedure (NIST Handbook of statistics, ). For Sholl analysis, curves were http://www.itl.nist.gov/div898/handbook// compared by two-way ANOVA, separating the effect of plasmid identity from that of radial distance. Post-hoc comparisons between curves were performed by the Bonferroni method. All calculations were performed with the statistics-oriented spreadsheet Gnumeric ( ) or with OpenStat (

). All error bars show S.E.M. http://projects.gnome.org/gnumeric/ http://statpages.org/miller/openstat/

Results

Expression of CHMP2B variants in hippocampal neurones

To study the neuronal effects of CHMP2B proteins, hippocampal neurones were transfected at 10 DIV with plasmids encoding N-terminally tagged CHMP2B , CHMP2B , or CHMP2B ( ). Using immunofluorescent staining with anti-tag antibody, wt intron5 10 Δ Fig. 1 expression of these plasmids could be reliably detected up to 5 days after transfection. The subcellular distribution of the mutant proteins clearly differed from that of wild-type ( ; , left; , left). Wild-type CHMP2B was homogeneously distributed throughout Fig. 1B independent investigators, we could not detect any autophagy-associated drop in the diffuse pool of LC3-GFP. Thus in the present setting, pathogenic CHMP2B mutants did not significantly enhance autophagosome accumulation. Furthermore, to determine whether the mutant CHMP2B puncta observed in transfected neurones were related to endosomes, transfected neurones were doubly stained with anti-tag antibody, and antibodies raised against the early endosomal marker EEA-1 ( ) or the late endosomal/lysosomal protein Fig. 3, a2 c2 -LAMP-1 ( ), respectively. No colocalisation was observed with these markers. These data suggest that the CHMP2B Fig. 3, d2 f2 aggregates which form under our transfection conditions are not associated with endosomes.

To visualize the morphology of neurones that had been expressing mutant CHMP2B for several days, the cells were co-transfected with CHMP2B constructs and a plasmid encoding fluorescent protein mCherry, allowing staining of the whole cytosol. Five days after transfection, 75 80 of neurones co-transfected with empty vector, CHMP2B , or CHMP2B

, and 55 of neurones co-transfected

-% wt 10 Δ %
with CHMP2B were healthy as judged from the overall integrity of their neurites and the absence of neuritic swellings ( ).

intron5 Fig. S1 These percentages did not change much after 10 days, and were largely sufficient to allow meaningful analysis of dendritic arbors and spines. Only healthy neurones were retained for further analysis. The effect of CHMP2B constructs on dendritic arborisation was determined by Sholl analysis of neuronal morphology. For this, we measured the rate of increase in total neuritic length as a function of radial distance from the cell body ( ). CHMP2B and CHMP2B had no significant effect on arborisation. CHMP2B Fig. S2 wt 10 Δ intron5 induced a moderate (25 ) decrease in average branching rate, but not the large-scale retraction of dendrites that accompanies lethal % perturbations of ESCRT-III ( ). [START_REF] Lee | ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration[END_REF]

CHMP2B mutants affect dendritic spine density and morphology

The dendritic morphology of neurones co-transfected with mCherry and CHMP2B plasmids was examined in detail by 3D confocal imaging of mCherry fluorescence.

shows representative views of neurones transfected with the different CHMP2B Fig. 4A(a d) constructs. Stacks of serial optical sections were produced for each neuron and the NeuronStudio software package [START_REF] Rodriguez | Automated Three-Dimensional Detection and Shape Classification of Dendritic Spines from Fluorescence Microscopy Images[END_REF] ) was then used to generate three-dimensional reconstructions of dendritic arbors and spines, and to count, measure [START_REF] Rodriguez | Automated Three-Dimensional Detection and Shape Classification of Dendritic Spines from Fluorescence Microscopy Images[END_REF] and classify spines (see Materials and Methods).

shows a reconstructed dendritic segment obtained from a typical control neurone, Fig. 4B and the results of automated identification of spine types. The volumes of the reconstructed spines followed a statistical distribution which closely resembled that described in a recent study of hippocampal spine dynamics ( ) ( ). Furthermore, the Yasumatsu et al., 2008 Fig. S3 peak of our distribution (between 0.050 and 0.075 m ) was in the same range as published values (0.058 / 0.034 m ) previously

μ 3 + - μ 3
obtained by electron microscopy for the spines of hippocampal neurones in dissociated culture at 14 DIV ( ). The [START_REF] Boyer | Comparison of Hippocampal Dendritic Spines in Culture and in Brain[END_REF] distribution of spine lengths was also similar to previously published values (

). These data indicate that analysis by NeuronStudio Fig. 4G correctly detected the spine population in our cultured neurones.

The density of spines (pooling all spine types) detected by this technique was determined for each neurone (

). Fig. 5A,B Over-expression of tagged, wild-type CHMP2B did not change the average density compared to control (cotransfection with empty vector). By contrast, expression of CHMP2B raised the average density of spines by 64 compared to control ( , col.3). intron5 % Fig. 5A However, many of these additional spines were very small (see below). Neurones expressing the CHMP2B mutant also had a tendency 10 Δ to increase the density of their protrusions (average density 24 higher), but this trend did not reach significance ( , col. 4). To physiology ( ), we determined how CHMP2B mutants affected the basic morphological categories of spines. [START_REF] Noguchi | Spine-neck geometry determines NMDA receptor-dependent Ca2 signaling in dendrites +[END_REF] Fig. 5I shows that neurones expressing either mutant had an abnormally low fraction of mushroom spines, identified by their head vs. neck width ratio. The fraction of spines with mushroom morphology was decreased by ~50 with both CHMP2B and CHMP2B , as they

A CHMP2B mutant affects miniature excitatory synaptic currents

To determine whether the effects of mutant CHMP2B on spine morphology correlated with functional changes at synapses, we recorded excitatory synaptic currents from cultured hippocampal neurones that had been co-transfected with GFP together with empty vector, or with vectors encoding wild-type CHMP2B or CHMP2B . Transfected cells were identified by GFP fluorescence. We 10 Δ monitored the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) as an indicator of possible effects of plasmids on synaptic strength. In total, 16 out of 18 transfected neurones were synaptically active; the two inactive neurones were discarded from analysis. Sample traces representative of each transfection condition are shown in . A sequence of 128 consecutive Fig. 6A mEPSCs was randomly picked from each recording, and the sequences were pooled according to the transfected plasmid. Mean inter-event intervals were calculated for each pool. Intervals were significantly longer between mEPSCs observed in neurones expressing CHMP2B Δ than in controls (

). Thus, the mean frequency of mEPSCs was lower in CHMP2B expressing cells than in control cells (1.47 10 Fig. 6B 10 Δvs. 1.94 Hz), indicating a decrease in synaptic activity ( ). The mean amplitude was slightly lower in mEPSCs of cells transfected Fig. 6B with CHMP2B

than in those of control-or CHMP2B -transfected neurones (12.6 vs. 13.6 pA) ( ). However, cumulative Δ wt Fig. 6C amplitude histograms indicated that CHMP2B caused a significant reduction in the frequency of large amplitude events, i.e. those belonging to the top quintile ( ). mEPSCs with amplitude larger than 20 pA (approximately twice the median amplitude), which Fig. 6D represent up to 19 of mEPSCs in control neurones, were reduced by a third in neurones expressing CHMP2B

. Conversely, in these % 10 Δ latter cells the proportion of small amplitude events (<5pA) raised from 6.8 to 10 ( ). The drop in the frequency of large % % Fig. 6E currents was not observed in neurones that overexpressed CHMP2B . In view of the known correlation between spine head size and wt amplitude of AMPA receptor-mediated synaptic currents (

), these data are consistent with the morphologically [START_REF] Matsuzaki | Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons[END_REF] observed reduction in mushroom spines. Mutant CHMP2B may cause a reduction in the proportion of the more potent synapses, and an increase in the proportion of weak synapses.

Endogenous CHMP2B is required for maturation of dendritic spines

As dysregulated CHMP2B mutants affect spine morphogenesis, the question arises whether the endogenous normal protein plays some role in this process. Endogenous CHMP2B was clearly detectable in extracts from cultured neurones at 8 DIV, and increased in older cultures, in which synaptogenesis and spine maturation are known to take place (

). Immunofluorescent staining of the neurones with Fig. 7 the anti-CHMP2B antibody showed the protein to be widely distributed throughout soma, axons and dendrites, similar to data obtained with the tagged wild-type protein (

). To disrupt endogenous CHMP2B expression, we transfected cultured hippocampal neurones at Fig. 8 10 DIV with a pSuper-based plasmid vector encoding a previously described CHMP2B-specific shRNA (

). The efficiency [START_REF] Lee | ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration[END_REF] of RNAi was ascertained by Western blotting of transfected BHK cells (

). Transfected neurones could be tracked and outlined by Fig. 8A mCherry, co-expressed from the same plasmid. Immunofluorescent staining with anti-CHMP2B antibody confirmed the efficient down-regulation of endogenous CHMP2B expression in transfected neurones ( , middle panel). In agreement with published results Fig. 8B ( ), the CHMP2B shRNA did not affect neuronal viability. To verify the target specificity of RNAi, control experiments [START_REF] Lee | ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration[END_REF] were performed in which CHMP2B shRNA was co-expressed with an RNAi-resistant CHMP2B mRNA (called CHMP2B ) ( , * Fig. 8B lower panel). Confocal imaging of mCherry and spine analysis were performed as in the case of mutant CHMP2B ( , ). Fig. 8C Fig. 9 Depletion of endogenous CHMP2B increased the average density of spines, and the increase was fully reversed upon rescue by

CHMP2B (

). CHMP2B depletion did not significantly affect the length of spines ( ). In contrast, a highly significant * Fig. 9A,B Fig. 9C, D reduction in mean spine diameter per neurone was observed in shRNA-transfected cells compared to controls; this was rescued to a near-normal value upon co-expression of CHMP2B ( ). Similar to the case of neurones transfected with mutant CHMP2B, the * Fig. 9E,F size distribution of spine heads showed a loss of the large spine subset; this loss was largely reversed by rescue with RNAi-resistant

CHMP2B (

). Spine classification revealed a two-fold drop in the average proportion of mushroom spines, from 17 in control Fig. 9G % neurones to 9 in shRNA-transfected cells (

). This effect is equivalent to that observed upon expression of mutant CHMP2B.

%

Fig. 9H

Upon co-expression of CHMP2B , the average proportion of mushroom spines could be rescued to 13 , at which level the difference with * % control neurones was no longer significant ( ). This demonstrates the specificity of CHMP2B shRNA effects. Taken together, these Fig. 9H results indicate that CHMP2B is required for spine head expansion, a basic structural aspect of synaptic plasticity. Furthermore, the data suggest that human disease-linked CHMP2B mutants act as dominant negatives with respect to CHMP2B function in spine morphogenesis.

To determine whether mutant CHMP2B could perturb the subcellular distribution of the normal endogenous protein, neurones transfected with tagged CHMP2B or CHMP2B were immunostained with both anti-CHMP2B and anti-tag antibodies ( ).

intron5 10 Δ Fig. 10 For this, we took advantage of the fact that the anti-CHMP2B antibody was raised against the wild-type C-terminal region, and therefore does not recognise the mutant proteins. The larger CHMP2B aggregates recruited conspicuous amounts of the endogenous protein, intron5 inducing it to form abnormal clusters (upper panel, arrow). Faint clusters of CHMP2B also occasionally overlapped with CHMP2B 10 Δ puncta (lower panel). However, the major, diffuse pool of endogenous CHMP2B remained unaffected. These results suggest that a fraction of native CHMP2B molecules may be perturbed in their biochemical function by interaction with mutant protein.

Discussion

Here we found that in cultured hippocampal neurones, two FTD-linked CHMP2B mutants strongly perturbed the normal pattern of dendritic spine development. Both mutants caused a striking decrease in the proportion of large spines with mushroom morphology, i.e.

those associated with potentiated synapses. This outcome was not associated with cell death. In keeping with these results, CHMP2B 10 Δ induced a loss of large mEPSCs, namely those reflecting the activity of the strongest synapses. Consistent with the view that CHMP2B mutants act as dominant negatives, depletion of endogenous CHMP2B by RNAi resulted in changes which were very similar to those induced by mutant CHMP2B, and which could be largely suppressed by co-expression of rescuing protein. Together, these data show that normal spine morphogenesis requires CHMP2B, and that abnormal stoichiometry or pathological dysregulation of the protein can deeply affect the structure and function of spines.

Overexpression of CHMP2B in neurones has previously been shown to trigger a specific toxic process, leading to dramatic intron5 retraction of dendritic trees, followed by cell death after 2 3 days; this lethality has been linked to excessive accumulation of autophagosomes ( ; ). However, several lines of evidence indicate that the impairment in spine [START_REF] Lee | ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration[END_REF][START_REF] Lee | Inhibition of autophagy induction delays neuronal cell loss caused by dysfunctional ESCRT-III in frontotemporal dementia[END_REF] maturation described here occurs through a mechanism distinct from this toxic process. First, the changes in spine morphology were observed in healthy neurones. In particular, expression of CHMP2B or knock-down of endogenous CHMP2B potently affected spines, 10 Δ even though these perturbations of CHMP2B activity did not diminish neuronal viability, as expected from published data. Second, alterations of spines did not correlate with changes in dendritic complexity. CHMP2B elicited no detectable loss of dendritic branching, 10 Δ and in our conditions CHMP2B only induced a 25 decrease in branching rate. This reduction remained modest compared with the intron5 % 80 shortening of total dendritic length shown to occur in the early phase of CHMP2B -induced cell death ( ). The % intron5 [START_REF] Lee | ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration[END_REF] CHMP2B shRNA caused a reduction in dendritic branching, but this may have been an off-target effect, as unlike the reduction in mushroom spines it was not suppressed by co-expression of an RNAi-insensitive CHMP2B cDNA (A. Belly et al, data not shown). Third, when expressed in hippocampal neurones, neither of the CHMP2B mutants caused a clear-cut increase in LC3-GFP condensation onto autophagosomes. By contrast, a lethal degree of CHMP2B overexpression has been shown to induce strong clustering of LC3-GFP; intron5 furthermore, preventing autophagosome formation retarded the neuronal death due to CHMP2B ( ; intron5 [START_REF] Lee | ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration[END_REF][START_REF] Lee | Inhibition of autophagy induction delays neuronal cell loss caused by dysfunctional ESCRT-III in frontotemporal dementia[END_REF]. Beyond some threshold level of expression, the ability of CHMP2B mutants to block the ESCRT-dependent fusion of autophagosomes to lysosomes ( ; ) may favour the proliferation of toxic autophagosomes. The difference with [START_REF] Filimonenko | Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease[END_REF][START_REF] Urwin | Disruption of endocytic trafficking in frontotemporal dementia with CHMP2B mutations[END_REF] our results may arise in part from the relatively low level of mutant CHMP2B expression we used in hippocampal neurones. Indeed, with four-fold higher amounts of CHMP2B plasmid, leading to higher expression, we observed toxicity similar to that described by hippocampal neurones, presumably similar non-endosomal aggregates largely predominated. The occurrence of non-endosomal CHMP2B clusters is also consistent with the fact that in vitro, activated ESCRT-III subunits can form large polymers in the absence of lipids (Lata et ). Our attempts to identify further components of the mutant CHMP2B clusters have so far been unsuccessful, except for the al., 2008 finding that endogenous CHMP2B is sometimes concentrated in CHMP2B granules. This suggests that native CHMP2B can be intron5 trapped in polymeric structures together with the mutant. Since much of the endogenous CHMP2B appeared to remain outside of the mutant CHMP2B puncta, sequestering of the native protein in the aggregates is unlikely to explain the apparent dominant negative effect of the mutants, especially in the case of CHMP2B . Mutant molecules may instead perturb or displace their native counterparts in 10 Δ supramolecular structures that cannot be detected by simple light microscopy.

The mechanism(s) through which mutant CHMP2B proteins and CHMP2B depletion affect the morphology of spines will require elucidation. Given the endosomal trafficking function of ESCRT-III, one possibility is that disabling CHMP2B may interfere with the endocytic recycling of some membrane protein critical for spine growth. Examples of such proteins include the EphB and TrkB receptor tyrosine kinases and the GluR2 subunit of AMPA receptors ( ; ). Of note, perturbation of a [START_REF] Henkemeyer | Multiple EphB receptor tyrosine kinases shape dendritic spines in the hippocampus 10.1083/jcb.200306033[END_REF][START_REF] Passafaro | Induction of dendritic spines by an extracellular domain of AMPA receptor subunit GluR2[END_REF] neurotrophin receptor such as TrkB would have implications for both spine morphogenesis and long-term survival of specific neurones.

Alternatively, spine growth may require direct deformation of the dendritic plasma membrane by CHMP2B-containing ESCRT-III filaments ( ; ), or coupling of the actin cytoskeleton to ESCRT-III ( ; [START_REF] Hanson | Plasma membrane deformation by circular arrays of ESCRT-III protein filaments[END_REF][START_REF] Sweeney | The Coiled-Coil Protein Shrub Controls Neuronal Morphogenesis in Drosophila[END_REF][START_REF] Sevrioukov | A mutation in dVps28 reveals a link between a subunit of the endosomal sorting complex required for transport-I complex and the actin cytoskeleton in Drosophila[END_REF]. Identification of CHMP2B-binding dendritic proteins and observation of ESCRT-III dynamics in live neurones may [START_REF] Vaccari | Comparative analysis of ESCRT-I, ESCRT-II and ESCRT-III function in Drosophila by efficient isolation of ESCRT mutants[END_REF] shed light on these possibilities.

Looking for a functional correlate of the CHMP2B effect, we found that this mutant significantly decreased the frequency of 10 Δ mEPSCs. This result indicates that CHMP2B elicits a decrease in synaptic activity. Moreover, the mutant induced a significant drop (32 10 Δ

) in the proportion of large mEPSCs. The two observations are in keeping with the loss of mature post-synaptic spines. In view of the % general correlation between spine size and synaptic potency ( ), the change in synaptic properties is consonant with [START_REF] Matsuzaki | Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons[END_REF] the reduction in the proportion of mushroom spines induced by CHMP2B

. This result raises the intriguing possibility that pathogenic 10 Δ CHMP2B mutants might directly prevent synaptic potentiation and/or weaken previously strong synapses and thereby perturb the functional connectivity of neurones. Further experiments are required to determine the role of CHMP2B and the impact of CHMP2B mutants on activity-dependent changes of spine structure, such as the enlargement associated with long-term potentiation.

While lethal perturbation of autophagy may explain the loss of cortical neurones at the symptomatic stage of FTD-3 (Lee and Gao, ;

), the protracted course of the disease, the lack of toxicity displayed by disease-linked mutants such as CHMP2B 2009 [START_REF] Urwin | Disruption of endocytic trafficking in frontotemporal dementia with CHMP2B mutations[END_REF] , and the low level of mutant CHMP2B mRNA in patients brain lead one to argue that CHMP2B mutants may initially have 10 Δ ' pathogenic effects other than dysfunctional autophagy leading to cell death. Our results suggest that in FTD-3 as in Alzheimer s disease, ' the pathogenic proteins may perturb synaptic plasticity and microstructure, long before overt cell death. A synaptotoxic mechanism would be consistent with observations of synapse loss in FTD ( ; ) and the widespread abnormalities in spine Ferrer, 1999 [START_REF] Lipton | Contribution of Asymmetric Synapse Loss to Lateralizing Clinical Deficits in Frontotemporal Dementias 10.1001/archneur[END_REF] density and morphology seen in diverse neurodegenerative diseases ( ; ). Understanding how defects [START_REF] Fiala | Dendritic spine pathology: cause or consequence of neurological disorders?[END_REF][START_REF] Wishart | Synaptic vulnerability in neurodegenerative disease[END_REF] in the highly conserved CHMP2B protein specifically lead to cortical degeneration may shed light on fundamental mechanisms of normal and pathological neurobiology.

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  and then fused with the CHMP2B cDNA. The cDNAs was subcloned in pcDNA3.1 between the EcoRV and NotI sites. The intron5 pSuper-mCherry plasmid was constructed by removing GFP from pSuper-Neo-GFP (Oligoengine) and replacing it with mCherry. The shRNA targeted the rat CHMP2B sequence at positions 578 596 (5; GACACTACAAACAATGCAG 3;) according to ( ), Lee et al., 2007 and the encoding oligonucleotide was cloned between the Hind III and Bgl II sites of pSuper-mCherry. The RNAi resistant CHMP2B cDNA was generated by introducing four silent mutations in the target sequence (yielding 5; GACGTTACAGACGATGCAA 3;) by means of the Quick-Change mutagenesis kit (Stratagene). All plasmids were verified by sequencing (Cogenics, Meylan, France) and initially tested in transfected BHK cells by immunoblotting and immunofluorescence with anti-tag antibody. The following polyclonal antibodies were purchased from Abcam (Cambridge, UK): anti-CHMP2B, anti-EEA1, anti-LAMP1. Anti-HA tag antibody was from Cell Signaling Technology (Beverly, Massachussets); anti-myc tag antibody from Santa Cruz (California); and anti-actin antibody from Chemicon.

  under an epifluorescence microscope (Zeiss Axiovert 200M) with a 63 objective (1.3 NA) and a rhodamine × filter, and scored as dead or alive according to morphological criteria (see text). The actual presence of co-expressed CHMP2B was verified by immunofluorescent staining with anti-tag antibody, using the GFP filter.ElectrophysiologySpontaneous tetrodotoxin-resistant miniature excitatory postsynaptic currents (mEPSCs) were recorded by means of the whole-cell configuration of the patch clamp method (Hamill et al 1981). The recording medium contained (in mM): 136 NaCl, 5 KCl, 2 CaCl , 1 4 18 puller (Zeitz Instruments, Germany) from thick wall borosilicate glass capillaries (1.5 mm o.d. 0.86 mm i.d) (Clark Electromedical × Instruments, Phymep, France). Filled with a medium consisting of (in mM): 130 Cs-gluconate, 5 EGTA, 1 MgCl , HEPES, 4 Na -ATP, CsOH), they had a resistance of 3 to 4 M . Glass coverslips were put on the stage of an upright Olympus microscope (BX51WI, Ω Olympus, France) equipped with a water immersion objective lens ( 20, N.A. 0.95, Olympus, France). GFP-expressing cells were voltage × clamped at a holding potential of 70 mV and the mEPSCs were recorded and then analyzed off-line by means of the pClamp software -(Molecular Devices, Dipsi, France). Synaptic currents, filtered at 1 kHz, were sampled at 10 kHz on an Axoclamp 200B amplifier (Molecular Devices, Dipsi, France). The experiments were conducted at room temperature (20 22 C).

  Fig.2Fig.3the entire neuronal cytoplasm, including axons, dendrites and dendritic spines ( , arrows). By contrast, both CHMP2B and Fig.1B

  Fig. 2, b2, c2

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  Fig. 5A investigate the possible effect of CHMP2B mutants on spine dimensions, we determined the mean length of spines and the mean diameter of spine heads for each neuron. None of the CHMP2B constructs modified spine length ( ). Wild-type CHMP2B did not change Fig. 5C,D spine head diameter either. By contrast, mutant proteins strongly affected spine diameter. Expression of CHMP2B induced a 40 intron5

  an enlarged proportion of thin spines. Stubby spines were also reduced, but to a smaller extent, by expression ofCHMP2B. The average diameter of spines in the mushroom spine subset (0.56 m) did not significantly change in cells transfected intron5 μ with the mutants, confirming the consistency of morphotype identification. Taken together, these data indicate that expression of a mutant ESCRT-III subunit linked to a human neurodegenerative disease can strongly affect the density and morphology of dendritic spines, and disrupt the normal pattern of spine development.

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  is difficult, as ) did not mention the amounts of plasmid in their transfection assays). The al. (2007) Lee et al. (2007 non-endosomal location of mutant CHMP2B proteins in our experiments also seem at first sight to be at variance with previously published results from other cell types. However, HA-tagged CHMP2B did accumulate around endosomes when overexpressed in intron5 BHK cells (A. Belly, unpublished data). It should also be noted that even in heterologous cells, a fraction of CHMP2B and CHMP2B intron5 aggregates consistently fail to co-localise with endosomal markers ( ; A.B., unpublished). In transfected 10 Skibinski et al., 2005

Fig. 1 A

 1 Fig. 1 A: primary structure of CHMP2B constructs used in this study. The ESCRT-III domain is the conserved sequence domain common to all members of the Charged Multivesicular body Proteins (CHMP 1 7) superfamily. Positively (blue) and negatively (red) charged alpha helices -(1 to 5) are located along the sequence according to the 3D structure of the closely related CHMP3 protein ( ). MIM: MIT Muziol et al., 2006 (Microtubule Interacting and Transport domain) Interacting Module, required for binding to the SKD1/Vps4 ATPase responsible for dissociation of CHMP polymers. V: a single valine residue replaces the normal C terminus in CHMP2B . The green line in CHMP intron5 10 ⊗ indicates the aberrant C terminus carried by this mutant. Ellipses indicate tags fused to the N terminus (2 9 and 1 10 residues respectively). × × Sequence lengths are those of the untagged proteins. B: subcellular distribution of tagged CHMP2B proteins. Neurones were transfected with the indicated plasmids, processed for immunofluorescence staining with anti-tag antibody, and imaged by confocal microscopy. Two representative cells are shown for each plasmid. Top: CHMP2B diffuses throughout the entire neuron, including the heads of dendritic wt spines (arrows). Middle: CHMP2B forms multiple small clusters in the soma and dendrites, and occasional larger aggregates. Bottom: intron5 CHMP2B has a clustered distribution similar to that of CHMP2B . 10 Δ

Fig. 5 10 Δ

 510 Fig.5effects of FTD-linked CHMP2B mutants on the density and dimensions of spines. All parameters were measured on the same sets of neurones (pooled from 3 independent cultures). Ctrl: control plasmid (n 31 neurones); wt: tagged wild-type CHMP2B (n 33 neurones); i5: CHMP2B = =(n 34 neurones); 10: CHMP2B (n 44 neurones). Measurements were obtained from 700 1500 m dendrite and 600 1200 spines intron5 = Δ

Fig. 9

 9 Fig. 9 effects of CHMP2B depletion on the density and morphology of dendritic spines. All parameters were measured as in and . Ctrl: n Figs. 3 4 = 42 cells; shRNA: n 48 cells; shRNA together with rescuing CHMP2B: n 25 cells. A: the mean density of spines varied as a function of = = CHMP2B depletion (ANOVA: F 3.80, p 0.025) although pairwise differences with control remained below significance ( p 0.021 vs. 2,112 = = * = rescued cells). B: cumulative frequency histogram of spine densities per neurone (normalized in percent), same data as in A. C: CHMP2B depletion had no effect on the mean spine length per neuron. D: cumulative frequency histogram of spine lengths. E: mean spine head diameter per neurone. The CHMP2B shRNA induces a highly significant decrease ( p<0.001) which is rescued by re-expression of CHMP2B * (ns, p 0.19). F: cumulative frequency histogram of spine head diameters. G: normalized distribution of spine head diameters. Note the = superposition of control and rescued curves, and the recovery of larger spines in the rescued profile. H: proportions of basic spine types. " " " " " "The CHMP2B shRNA potently decreases the mushroom spine fraction ( p<0.001) and the effect is suppressed in the rescued cells (ns, p * =

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Ackowledgements:

A. Belly was supported by the Fondation France Alzheimer. G. Bodon was supported by the Minist re de l Enseignement Sup rieur et de la Dournaud for the gift of mCherry, W. Weissenhorn for discussions, J. H. Henley for critical reading of the manuscript, two anonymous reviewers for suggestions, and all members of the lab for support.