Elena A Minina
email: alena.minina@slu.se

Jens Staal

Vanina E Alvarez

John A Berges

Ilana Berman-Frank

Rudi Beyaert

Kay D Bidle

Frédéric Bornancin

Magali Casanova

Juan J Cazzulo

Chang Jae Choi

Nuria S Coll

Vishva M Dixit

Marko Dolinar

Nicolas Fasel

Christiane Funk

Patrick Gallois

Kris Gevaert

Emilio Gutierrez-Beltran

Stephan Hailfinger

Marina Klemenčič

Eugene V Koonin

Daniel Krappmann

Anna Linusson

Maurício F M Machado

Frank Madeo

Lynn A Megeney

Panagiotis N Moschou

Jeremy C Mottram

Thomas Nyström

Heinz D Osiewacz

Christopher M Overall

Kailash C Pandey

Jürgen Ruland

Guy S Salvesen

Yigong Shi

Andrei Smertenko

Simon Stael

Jerry Ståhlberg

María Fernanda Suárez

Margot Thome

Hannele Tuominen

Frank Van Breusegem

Renier A L Van Der Hoorn

Assaf Vardi

Boris Zhivotovsky

Eric Lam

Peter V Bozhkov
email: peter.bozhkov@slu.se

Classification and nomenclature of metacaspases and paracaspases: no more confusion with caspases

Keywords: Caspases, Metacaspases, Paracaspases, Proteases, Classification, Nomenclature Classification

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 current classification of proteases provided by the MEROPS database clusters caspases, meta-and paracaspases to the same family, C14, within the CD clan (https://www.ebi.ac.uk/ merops/). All members of C14 family are annotated to possess aspartate P1 cleavage specificity, and the family is further split into two subfamilies: C14A (caspases) and C14B (meta-and paracaspases).

Importantly, the MEROPS approach of grouping proteases into families or subfamilies is based on statistically significant similarities of the amino acid sequence within the peptidase domain or part thereof, without considering their biochemical properties [START_REF] Rawlings | [END_REF]. Being valuable for high-throughput protease classification, this approach, however, has substantial drawbacks if implemented without further adjustment. Indeed, in contradiction with the MEROPS description, none of the meta-or paracaspases characterized so far cleave after an aspartate residue. Instead, paracaspases are argininespecific (Coornaert et al., 2008;Hachmann et al., 2012;Rebeaud et al., 2008), whereas metacaspases can cleave after either arginine or lysine (Figure S1A; Sundström et al., 2009;Vercammen et al., 2004). Such fundamental differences in the proteolytic specificity between caspases, meta-and paracaspases imply distinct repertoires of new proteoforms that they generate and point to the complex diversification and coevolution of their substrates and downstream pathways. One unfortunate consequence of the current classification is the misuse of caspase-specific probes for studying meta-and paracaspases that is commonly found in the literature and leads to false conclusions.

Apart from substrate specificity, caspases, meta-and paracaspases feature other fundamental differences (Figure S1A). For example, active metacaspases are monomers and their activation usually requires millimolar concentrations of calcium (Hander et al., 2019;[START_REF] Mcluskey | Proc. Natl. Acad. Sci. U. S. A[END_REF]Wong et al., 2012). In contrast, active caspases and paracaspases are calcium-independent dimers (Hachmann et al., 2012;Weismann et al., 2012;[START_REF] Yu | Proc. Natl. Acad. Sci. U. S. A[END_REF]. This indicates that upstream pathways regulating activation of caspases, meta-and paracaspases are likewise different.

In the past two decades we have learned about important differences between caspases, meta-and paracaspases. Thus, simple extrapolation of features typical for caspases to all other members of the C14 family is not justified anymore. Instead caspases, meta-and paracaspases should be separated into three corresponding groups within the family and each group should be properly annotated by providing its key biochemical and structural characteristics. We kindly request curators of MEROPS database to make corresponding changes.

Since structure and substrate specificity of prokaryotic caspase-like proteases named "orthocaspases" remain largely unknown (Klemenčič et al., 2015) we leave their classification and nomenclature open until their structural and biochemical properties have been clarified.

Nomenclature of meta-and paracaspases

The name "caspase" stands for "cysteine-dependent aspartate-specific protease". Thus, the names "metacaspase" and "paracaspase" imply the wrong substrate specificity for these proteases. However, since these names have been used for two decades we propose to keep them, provided that caspases, meta-and paracaspases are recognized as three separate groups within the C14 family.

Based on domain composition and arrangement, meta-and paracaspases are further subdivided into three and two types, respectively (Figure S1A). For the sake of consistency, we propose to maintain a common nomenclature for the different types of meta-and paracaspases using Latin numerals (e.g. type I metacaspases). As for the conserved protein structures, they will be referred to as the p20-like region, the p10-like region, the linker region and the N-terminal pro-domain, matching the nomenclature of caspases (Figure S1A; Alnemri et al., 1996). The p20, p10 and linker regions have been previously defined for the caspase group of the C14 family (Fuentes-Prior and Salvesen, 2004) and can be easily identified in meta-and paracaspase homologs based on a hidden Markov model (HMM) alignment with the C14 peptidase domain (Figure S1B). Notably, although not always clearly stated in the literature, most known members of the C14 family contain the linker region. Furthermore, type II metacaspases are distinguished by a long linker between the p20 and p10 regions and an additional linker within the p10 region (Figure S1A), which are frequently referred to as a single linker.

We suggest to consider the active form of meta-or paracaspases being a monomer if it is a cleaved or intact polypeptide chain derived from a single translational event, and a dimer if it comprises uncut or processed products of two translational events.

We propose to establish a unified nomenclature of meta-and paracaspases in order (i) to facilitate comparison of orthologs from different organisms and (ii) to make it suitable for annotating homologs of species with partially sequenced genomes. Thus, we suggest using simple root symbols such as MCA for metacaspases and PCA for paracaspases. When naming individual family members, these root symbols will be preceded by the abbreviated Latin name of the species and followed by a hyphen, Latin number representing the type and then a small alpha character indicating in alphabetical order the number of the homolog of this type in a given genome (Figure S1C). Proenzymes that require proteolytic processing for activation could be annotated with a prefix "pro-", e.g. pro-AtMCA-Ia for the metacaspase 1 of type I from A. thaliana. Spliceoforms should be indicated by a decimal number (e.g. AtMCA-Ia.1). Please note that these conventions do not consider the letter case, which should conform to gene and protein nomenclature established for a given model organism or taxonomic group.

Importantly, this nomenclature should be used synonymously for meta-and paracaspase homologs with well-established names, e.g. human MALT1/HsPCA-Ia and A. thaliana AtMC1/AtMCA-Ia or AtMC4/AtMCA-IIa. We encourage all researchers to adopt these recommendations. The new classification and unified nomenclature of meta-and paracaspases will facilitate a more comprehensive exchange of relevant findings within the scientific community and help to bridge already existing knowledge with newly discovered homologs, thus promoting mechanistic understanding of these ancient, evolutionarily conserved proteases.

Mol Cell. Author manuscript; available in PMC 2021 March 05.

Acknowledgements

This work was supported by Knut and Alice Wallenberg Foundation. We apologize to colleagues whose work has not been cited due to space limitation.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


Anne Pierres

Anne-Marie Benoliel

Dominique Touchard

Pierre Bongrand

How cells tiptoe on adhesive surfaces before sticking

Keywords: Cell adhesion, membrane undulations, interference reflection microscopy, monocyte, membrane fitting, membrane fluctuations

come

INTRODUCTION

The contact between a cell and a foreign surface often triggers a variety of events such as cell attachment, spreading on the surface and alteration of behavioral patterns relative to survival, differentiation, migration or release of bioactive factors [START_REF] Pierres | Cell fitting to adhesive surfaces : a prerequisite to firm attachment and subsequent events[END_REF]. During the last years, the biologically relevant phenomena occurring after the first minutes following cell-to-surface encounter were subjected to considerable scrutiny. Also, much effort was done to apply biophysical approaches to the analysis of the binding events occurring during the first seconds following contact [START_REF] Lawrence | Leukocytes roll on a selectin at physiologic flow rates : distinction from and prerequisite for adhesion through integrins[END_REF][START_REF] Pierres | Dynamic adhesion of CD8-positive cells to antibody-coated surfaces : the initial step is independent of microfilaments and intracellular domains of cell-binding molecules[END_REF][START_REF] Grabovsky | Subsecond induction of alpha 4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions[END_REF]. However, fewer studies were specifically focussed on the first minutes following cell-to-surface contact and preceding biologically relevant responses such as spreading, lamellipodium formation and migration, or activation of different functions. Interestingly, recent experiments performed with atomic force microscopy at the single cell level [START_REF] Taubenberger | Revealing early steps of α2β1 integrin-mediated adhesion to collagen type I by using singlecell force spectroscopy[END_REF] suggest that cell adhesion to a surface may sequentially involve passive formation of a few receptor-ligand bonds followed a few minutes later by active cooperative events resulting in more than tenfold increase of adhesive strength. Thus, it is warranted to investigate the minute-scale period linking cell-substrate encounter to generation of an active response guided by specific recognition events involving cell membrane receptors and substrate molecular structures.

Numerous electron microscopical studies clearly demonstrated that cell membranes are studded with different membrane folds or protrusions such as microvilli, lamellipodia or filopodia [START_REF] Erikson | Microvilli and blebs as sources of reserve surface membrane during cell spreading[END_REF][START_REF] Grinnell | Initial attachment of baby hamster kidney cells to an apoxy substratum[END_REF][START_REF] Shao | Static and dynamic lengths of neutrophil microvilli[END_REF]. Cell adhesion is essentially mediated by membrane receptors of typically several tens of nanometer length [START_REF] Springer | Adhesion receptors of the immune system[END_REF], this is significantly shorter than the smallest protrusions covering the cell membrane, since microvilli are several tenths of micrometer long [START_REF] Shao | Static and dynamic lengths of neutrophil microvilli[END_REF]. Thus, formation of a substantial contact area on the order of several squared micrometers [START_REF] Cuvelier | The universal dynamics of cell spreading[END_REF] requires nanometer-scale fitting of the cell topography to opposing surfaces. Indeed, while contact formation between a rough cell membrane and a smooth surface obviously requires a minimal smoothing of the cell surface, [START_REF] Mège | Use of cell contour analysis to evaluate the affinity between macrophages and glutaraldehyde-treated erythrocytes[END_REF], intercellular adhesion also requires membrane adaptation [START_REF] Foa | T-cellmediated cytolysis : analysis of killer and target cell deformability and deformation during conjugate formation[END_REF].

While aforementioned requirements were fully illustrated with electron microscopy, little information is currently available on the dynamics of cell membrane at the nanometer scale. It has long been shown that the surface of erythrocytes [START_REF] Brochard | Frequency spectrum of the flicker phenomenon in erythrocytes[END_REF] or deflated lipid vesicles display thermally driven oscillations, and much theoretical work was devoted to account for this phenomenon [START_REF] Helfrich | Undulations, steric interaction and cohesion of fluid membranes[END_REF][START_REF] Lipowsky | Generic interactions of flexible membranes[END_REF]. However, little information is available on the dynamics of nucleated cell membranes at the submicrometer level. Transverse oscillations of up to 20-30 nm amplitude were found on many nucleated cells such as fibroblasts, lymphocytes or monocytes [START_REF] Yu | Local mechanical oscillations of the cell surface within the range of 0.2-30 Hz[END_REF][START_REF] Mittelman | Fast cell membrane displacements in B lymphocytes. Modulation by dihydrocytochalasin B and colchicine[END_REF][START_REF] Zidovska | Brownian motion of nucleated cell envelopes impedes adhesion[END_REF]. Other studies [START_REF] Shao | Static and dynamic lengths of neutrophil microvilli[END_REF] revealed that leukocyte surface microvilli could withstand a pulling force of about 45 nN before forming a thin membrane tether. However, more work is needed to understand the relationship between membrane undulations and the early steps of contact formation and extension between a cell and an adhesive surface.

The purpose of the present work was to provide a real-time description of three-dimensional membrane deformations occurring during the first tens of seconds required to secure cell attachment to adhesive surfaces. We used interference reflection microscopy/reflection interference contrast microscopy [START_REF] Zidovska | Brownian motion of nucleated cell envelopes impedes adhesion[END_REF][START_REF] Curtis | The mechanism of adhesion of cells to glass[END_REF][START_REF] Verschueren | Interference reflection microscopy in cell biology : methodology and applications[END_REF][START_REF] Albersdörfer | Adhesion-induced domain formation by interplay of long-range repulsion and short-range attraction force : a model membrane study[END_REF][START_REF] Pierres | Cell membrane alignment along adhesive surfaces : active or passive mechanism ?[END_REF] to image the initial interaction between human monocytic THP-1 cells and planar fibronectin-coated surfaces. Cell membranes displayed continuous transverse undulations of nanometer amplitude with a lateral correlation on the order of a micrometer. Cells seemed to detect the presence of the surface at about 50 nm separation as revealed by the behaviour of membrane fluctuations. Adhesion was not an immediate phenomenon, rather the cell-to-surface gap decreased by several tens of nanometers during the first few minutes following initial contact. This contact maturation occurred concomitantly with an egress of bulky components of the cell glycocalyx. Observation of cytoskelal elements suggested that actin acted at least partly as a stabilizing structure, and myosin was not detectable in contact areas.

MATERIALS AND METHODS

Cells and transfection : We used the monocytic THP-1 line (23) maintained as previously described [START_REF] Vitte | Beta 1 integrin-mediated adhesion may be initiated by multiple incomplete bonds, thus accounting for the functional importance of receptor clustering[END_REF] in RPMI-1640 medium (Invitrogen) supplemented with 20 mM HEPES buffer, 10 % fetal calf serum (Invitrogen), 2mM L-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin. As previously checked with flow cytometry, these cells expressed typical mononuclear phagocyte markers including CD11b, CD18, CD29, CD32, CD43, CE45, CD64 and class I major histocompatibility complex molecules. Also they were shown to bind fibronectin-coated surfaces with CD49dCD29 (VLA-4) and CD49eCD29 (VLA-5) integrins [START_REF] Vitte | Beta 1 integrin-mediated adhesion may be initiated by multiple incomplete bonds, thus accounting for the functional importance of receptor clustering[END_REF]. THP-1 cells were transfected with pEGFP-actin (Clontech, BD Biosciences) using an AMAXA electroporator, AMAXA solution V and protocol V-01. EGFP-positive cells were selected with G418 selection and limiting dilution plating to isolate clones stably expressing EGFP-actin. For transfected cells 1mg/ml G-418 sulphate (GIBCO, Invitrogen Corporation) was added in culture medium.

Fluorescent labelling and metabolic inhibitors :

Cell overall shape was studied by monitoring cells suspended in culture medium containing 10 µg/ml fluorescein isothiocyanate with confocal microscopy.

In other experiments, cells were labelled with anti-CD43 monoclonal and processed for zenon labelling following the manufacturer (Molecular Probes) procedure. Mouse anti-CD29 (K20) and anti-CD45 were purchased from Beckman Coulter. Cytochalasin D (an inhibitor of microfilament assembly) and butanedione monoxime (a myosin inhibitor) were obtained from Sigma. Intracellular calcium visualization was achieved after labelling cells with Fluo-3-AM (Molecular Probes) as previously described [START_REF] Kaplanski | A novel role for E-and P-selectins : shape control of endothelial cell monolayers[END_REF] Chambers : Custom-made flow chambers (Satim, Evenos, France) were Plexiglas blocks drilled with a rectangular cavity of 17 × 6 × 1 mm 3 and cylindrical ducts for liquid entry and exit. The chamber floor was made of a glass coverslip maintained against the cavity with a steel plate. Coverslips were cleaned by treatment with 98% sulphuric acid for 4 hours, washed overnight with tap running water, then distilled water and dried at 60° C for 30 min. They were then treated with 10 µg/ml fibronectin (Sigma) for 2 hours and rinsed in PBS. Chambers were set on the stage of an Axiovert 135 inverted microscope (Zeiss) bearing a heating stage (TRZ 3700) set at 37°C. Interference reflection microscopy was performed with an antiflex objective (63 X magnification, 1.25 numerical aperture). Illumination was done with a 50 W mercury lamp using a H546 band-pass filter. Images were obtained with an Hamamatsu C4742-95-10 CCD camera yielding 10-bit accuracy. Pixel size was 125×125 nm 2 .

Spreading assays were initiated by injecting 10µl of a concentrated cell suspension (2 × 10 6 cells/ml) into the bottom of treated chambers containing normal culture medium. In a typical experiment, 300 sequential images were recorded with about 1 Hz frequency.

Image processing : Image processing was performed with a custom-made C++ Windows™ compatible software written in the laboratory. This allowed us to perform the following key steps as an improvement of a previously described and validated procedure [START_REF] Pierres | Cell membrane alignment along adhesive surfaces : active or passive mechanism ?[END_REF] -A first step consisted of performing a first order linear correction of brightness with respect to coordinates in order to minimize the illumination differences between the four corners of a 25 × 25 µm 2 area surrounding the cell. The same correction parameters were used throughout each series of 300 images, since they were essentially dependent on the cell position with respect to the microscope field.

-Secondly, the local intensity at each pixel was replaced with the average of intensities measured on the neighbouring 5×5 pixels. This was chosen to reduce the image noise sufficiently to decrease the height measurement error below about 1 nm, which was found necessary to process cell images. Note that the loss of lateral resolution did not result in dramatic loss of information as compared with the maximum resolution yielded with IRM/RICM. -The last step consisted of deriving a relative membrane to substrate distance z from the brightness I with the formula :

z = (λ/4π) Arccosine [(2I -I m -I M )/(I m -I M )] ( 1
)
where λ is the light wavelength, and I m and I M are respectively the minimum and maximum brightness corresponding to contact and (λ/4) separation. The interest of this formula is that estimates may be considered as fairly robust even if underlying assumptions are not fully warranted [START_REF] Pierres | Cell membrane alignment along adhesive surfaces : active or passive mechanism ?[END_REF]. Note also that the ambiguity linked to the arccosine transform is not a problem when we consider only a region close to the contact where the membrane-to-surface distance is expected to remain lower than about 100 nm. This assumption is supported by the absence of concentric fringes usually observed on the rim of molecular contact zones [START_REF] Albersdörfer | Adhesion-induced domain formation by interplay of long-range repulsion and short-range attraction force : a model membrane study[END_REF][START_REF] Pierres | Cell membrane alignment along adhesive surfaces : active or passive mechanism ?[END_REF][START_REF] Simson | Membrane bending modulus and adhesion energy of wild-type and mutant cells of Dictyostelium lacking talin or cortexillins[END_REF]. Parameters I m and I M were determined after examining a set of 300 images on the same microscope field. Corrected values were used for each image in order to account for the fluctuations of illumination intensity of a fixed empty area (the coefficient of variation was close to 1 %) Note that this procedure was not expected to yield absolute distances. However, it must be recognized that there is a necessary arbitrariness in defining a nanometer-scale distance between surfaces coated with macromolecules of various shape.

The results presented in this paper were obtained after examining about 5,000 microscope fields of about 125×125 µm 2 each.

RESULTS

Cells adhere to adhesive surfaces and form growing contacts within less than one minute.

Monocytic THP-1 cells were deposited on fibronectin-coated surfaces in a chamber set on a microscope stage. While these cells were fairly spherical in suspension, they displayed marked flattening at the micrometer scale within seconds following contact (Fig. 1A). Interference reflection microscopy/reflection interference contrast microscopy (IRM) was used to image the details of the cell surface in front of the substratum with nanometer vertical resolution. As shown on Fig. 1B-C, within a few tens of second following contact, a dark area appeared on the IRM image of the cell surface, revealing the formation of a contact zone with surface separation lower than several tens of nanometers. This was previously shown to reflect bona fide adhesion [START_REF] Pierres | Cell membrane alignment along adhesive surfaces : active or passive mechanism ?[END_REF]. Comparing the histograms of images obtained before and after adhesion (Fig. 1D) allowed us to define a threshold intensity (Arrow on Fig. 1D) to define contact zones and measure adhesive areas as was done in previous reports [START_REF] Cuvelier | The universal dynamics of cell spreading[END_REF][START_REF] Albersdörfer | Adhesion-induced domain formation by interplay of long-range repulsion and short-range attraction force : a model membrane study[END_REF][START_REF] Chamaraux | Kinetics of cell spreading[END_REF]. The corresponding separation value obtained with Eq.1 was 34 nm. The kinetics of contact extension was then studied. As shown on Fig. 1E, contact area first increased linearly with respect to time, and the growth rate was weakly dependent on surface concentration of adhesion molecules. This finding is consistent with the concept that contact extension should be essentially driven by cell movements, without any requirement for cooperative interactions between membrane receptors [START_REF] Taubenberger | Revealing early steps of α2β1 integrin-mediated adhesion to collagen type I by using singlecell force spectroscopy[END_REF][START_REF] Garcia | Integrin-fibronectin interactions at the cell-material interface : initial integrin binding and signaling[END_REF].

As exemplified on Fig2A-I, growing contact zones exhibited two important features : i) the contact patches appeared fairly irregular and ii) some isolated patches sometimes appeared and disappeared, demonstrating the need to consider three-dimensional membrane topography in order to account for two-dimensional contact growth.

Cell-substrate contact formation is associated with transverse membrane deformation and fluctuations of contact area superimposed on a continuous decrease of membrane to substrate distance.

IRM images were processed to estimate cell-to-substrate distance within the 100 nm range. Images of cells falling on planar fibronectin-coated surfaces were recorded for periods of 5 minutes with 1Hz frequency. Three sequential color-coded maps of cell-surface distance are displayed on Fig. 3 A-C, corresponding to the two-dimensional representations displayed on Fig. 2 A-C : clearly, contact formation was preceded with important cell surface deformations. These deformations were also displayed on Fig. 4 with a more intuitive 3D representation. Thus, contact initiation and extension were correlated with continuous membrane fluctuations. As shown on Fig. 5, these membrane undulations appeared concomitantly with two phenomena : i) contact area displayed marked fluctuations with about 0.1 Hz frequency, that were superimposed on average growth (Fig. 5A), and ii) during several tens of seconds following cell-to-surface contact, the relative molecular distance between interacting surfaces within previously defined contact zones steadily decreased by at least 30 nm (Fig. 5B). Thus membrane adhesion in a given point is not an all-or-none instantaneous process, but this process involves progressive narrowing of the cell-surface gap.

Transverse membrane fluctuations may be described as a vertical motion of blocks of 0.6µm width and an effective diffusion coefficient of 1-3 nm 2 /s. A conventional display of contact fluctuations is shown on Fig. 6A, emphasizing the concomitant expansion and retraction of contact in different zones. This two-dimensional view is a consequence of three-dimensional deformations of the cell membranes (Fig 6B ) : different regions of the cell membrane facing the substratum displayed upward or downward displacements of 5-10 nm amplitude in different regions of the cell membrane during a 6-second period of time. These overall displacements were associated with membrane fluctuations that were revealed (Fig. 6C) by imaging the standard deviation of z coordinate on each pixel, during the same period of time and using higher image acquisition frequency. Then, to understand the rationale of cell shape control, the following analysis was performed i) we examined the randomness of membrane deformations by studying the time dependence of the squared displacements of individual surface pixels. Indeed, random walks result in a linear dependence of squared displacement on time, whereas a uniform motion would lead to a linear relationship between displacement and time. As exemplified on Fig. 7A, when the mean membrane approach to the surface was subtracted from invidual pixel displacement, the relationship between squared displacement and time was strikingly linear. This finding was obtained on nine separate cells and time intervals ranging between 6 and 80 seconds, immediately after contact, yielding an effective diffusion coefficient equal to half the slope of the curves (since the squared displacement during time t is expected to be equal to 2Dt). This ranged between about 1 and 3 nm 2 per second. Thus, membrane fluctuations separated by a 1-second period of time were not correlated.

Further information was obtained by studying the temporal autocorrelation of membrane shape and fluctuation maps. As exemplified on Fig. 7B, the characteristic time for 50% decrease of correlation was about 5 seconds during contact initiation. However, the onset of adhesion resulted in progressive increase of correlation as a consequence of the formation of fixed adhesive zones (not shown). We also studied the time correlation of displacements (i.e. of the time derivative of membrane maps). No correlation could be found between sequential displacements (Fig. 7B). Together with Fig6A, this is consistent with the view that the characteristic duration of membrane fluctuations was significantly less that 1 second, but a longer correlation was generated by the formation of adhesive patches.

ii) Then we examined the dependence of the amplitude of membrane fluctuations on z coordinate (i.e. distance to the adhesive substrate). Indeed, passive surface undulations of soft vesicles have long been studied [START_REF] Helfrich | Undulations, steric interaction and cohesion of fluid membranes[END_REF][START_REF] Lipowsky | Generic interactions of flexible membranes[END_REF], and the squared amplitude of undulations should be decreased by a neighbouring surface in proportion to the squared distance [START_REF] Netz | Complete unbinding of fluid membranes in the presence of short-ranged forces[END_REF]. The mean correlation coefficient between distance to the fibronectin-coated surface and undulation amplitude calculated on a series of 16 different images was 0.101 (±0.358 SD). The significance of this unexpectedly low correlation was explained by plotting fluctuations (expressed as a standard deviation) versus distance. As exemplified on Fig. 7C, a non-monotonous relationship between local z coordinate and fluctuation amplitude was found. Two domains were apparent : -for higher distances (z>70 nm), the fluctuation strongly increased as a function of distance, in accordance with predictions from the theory of statistical surfaces.

-when z became lower than about 50 nm, fluctuations did no longer decrease concomitantly. Interestingly, during the first tens of seconds following contact, low distance fluctuations increased, supporting the biological relevance of this finding.

iii) Examining deformation and fluctuation maps such as Fig. 6B-C clearly revealed the heterogeneity of surface movement distribution, with a coexistence of more active and quieter membrane regions. The characteristic size of these areas was determined by studying spatial autocorrelation. A typical plot is displayed on Fig. 7D. The mean length for 50 % correlation decay determined on 16 separate cell images was 0.60 µm (0.45µm standard deviation). Accordingly, a comparable correlation length was found on the maps of individual pixel displacements during a fixed period of 2s, suggesting that cell membranes might deform as independent units of nearly micrometer size.

Contact extension is correlated to important changes of cytoplasmic and surface molecules.

In order to obtain more information on the structural basis of observed phenomena, fluorescence microscopy was associated with IRM to study the short term cell structural changes following contact formation First, fluorescent antibodies were made to bind beta 1 integrins, i.e. the adhesion receptors mediating THP-1 cell attachment to fibronectin [START_REF] Vitte | Beta 1 integrin-mediated adhesion may be initiated by multiple incomplete bonds, thus accounting for the functional importance of receptor clustering[END_REF]. High fluorescence levels were observed in cell-surface contact areas (not shown), but more extensive investigations are required to determine whether the surface density of integrins increased during contact maturation.

Second, we labelled CD43, a bulky membrane molecule that is highly abundant on leukocyte membranes and that is supposed to impair adhesive interactions [START_REF] Ardman | CD43 interferes with T-lymphocyte adhesion[END_REF][START_REF] Soler | Leukosialin (CD43) behavior during adhesion of human monocytic THP-1 cells to red blood cells[END_REF]. Interestingly, as exemplified on Fig. 8, decrease of cell-to-substrate distance was associated with a concomitant egress of CD43 molecules.

Third, THP-1 cells were transfected with fluorescent actin and deposited on fibronectincoated surfaces. Contact formation was very similar to processes described above, suggesting that transfection did not markedly alter THP-1 cell properties. The fluorescence distribution was then monitored together with IRM contacts. As exemplified on Fig. 9, in addition to bulk fluorescence, cell displayed localized fluorescence patches that seemed to appear a few tens of seconds before the formation or extension of of contact zones.

Metabolic inhibitors influence contact extension.

While it is well known that soft vesicles can display thermally-driven surface fluctuations and formation of extensive contacts with adhesive surfaces, it was important to determine to what extent cell behaviour was influenced by active processes dependent on biochemical events. This question was addressed as follows. i) Stiffening cell surface with paraformaldehyde abolished contact formation and adhesion. ii) Interestingly, treating cells with moderate concentrations of cytochalasin D (10 µg/ml) resulted in significant decrease of the rate of contact extension, smoothening of the two-dimensional contours of contact surfaces, which make them more alike contact zones formed with artificial vesicles [START_REF] Mège | Use of cell contour analysis to evaluate the affinity between macrophages and glutaraldehyde-treated erythrocytes[END_REF][START_REF] Vitte | Beta 1 integrin-mediated adhesion may be initiated by multiple incomplete bonds, thus accounting for the functional importance of receptor clustering[END_REF], and increase of membrane undulations in membrane zones close to the adhesive surface (Fig. 10) iii) Treating cells with a myosin inhibitor (butanedionemonoxime) to decrease cell motility abolished adhesion and contact formation. iv) Since previous reports clearly demonstrated a role of intracellular calcium changes in cell spreading [START_REF] Kruskal | Spreading of human neutrophils is immediately preceded by a large increase in cytoplasmic free calcium[END_REF][START_REF] Pettit | Release of "caged" cytosolic Ca 2+ triggers rapid spreading of human neutrophils adherent via integrin engagement[END_REF], we investigated whether intracellular calcium level influenced THP-1cell fitting to adhesive surfaces. In preliminary experiments, cells were labelled with fluo-3, a fluorescent calcium probe, before deposition on fibronectin-coated surfaces and monitoring with IRM. While many cells displayed elevated calcium, no clear relationship was found between intracellular calcium and rate of contact growth (not shown).

The requirement for fluid drainage from the cell-surface gap does not seem a limiting parameter for contact extension.

In several situations of physical interest (e.g. aquaplaning) adhesion between surfaces may be hampered by intervening liquid film. The possibility that this might influence contact extension was investigated by monitoring contact extension in highly viscous ficoll solution. However, no significant decrease of contact extension was found when the medium viscosity was increased threefold (not shown).

DISCUSSION

While cell attachment to adhesive surfaces and subsequent deformation and spreading were subjected to considerable scrutiny, little information is available on the mechanisms allowing transverse nanometer-scale adaptation of interacting surfaces. However, in view of the known roughness of cell membranes and length of adhesion molecules, this fitting process seems an obvious requirement to the formation of more than a few adhesive bonds. This point is important since older studies made on the strength of cell adhesion suggested that the minimal number of bonds required to account for experimental properties of cell adhesion was on the order of about at least one thousand [START_REF] Capo | Concanavalin A-mediated thymocyte agglutination : a model for a quantitative study of cell adhesion[END_REF][START_REF] Bongrand | Cell-cell adhesion : parameters and possible mechanisms[END_REF].

Interference reflection microscopy/reflection interference contrast microscopy was demonstrated to be a method of choice to image the fitting of cell membranes to planar surfaces with sufficient vertical resolution [START_REF] Zidovska | Brownian motion of nucleated cell envelopes impedes adhesion[END_REF][START_REF] Curtis | The mechanism of adhesion of cells to glass[END_REF][START_REF] Pierres | Cell membrane alignment along adhesive surfaces : active or passive mechanism ?[END_REF][START_REF] Simson | Membrane bending modulus and adhesion energy of wild-type and mutant cells of Dictyostelium lacking talin or cortexillins[END_REF]. A major advantage of this technique is that it does not require any possibly artifactual treatment. It has been well recognized that the quantitative derivation of membrane-surface distance from intensity measurements relies on several assumptions that are difficult to prove [START_REF] Gingell | Interference reflection microscopy. A quantitative theory for image interpretation and its application to cell-substratum separation measurement[END_REF] and the membrane-surface separation values we obtained may not be considered as absolute values of the distance between lipid bilayers and glass surfaces. However, it must be emphasized that the simple procedure we used to derive these distances is fairly robust and should provide reliable relative values, as acknowledged in previous papers [START_REF] Zidovska | Brownian motion of nucleated cell envelopes impedes adhesion[END_REF][START_REF] Verschueren | Interference reflection microscopy in cell biology : methodology and applications[END_REF][START_REF] Pierres | Cell membrane alignment along adhesive surfaces : active or passive mechanism ?[END_REF]. Indeed, cytoplasmic structures such as the actin cortex might influence image brightness. Although our observations of the kinetics of actin and distance map changes suggest that actin movements were much slower than the membrane fluctuations we described, a reliable proof of the validity of distance determination with IRM/RICM could only be obtained by comparing images obtained with different techniques. We are currently combining total internal reflection fluorescence microscopy and fluorescence labelling to achieve this goal. Several important conclusions may be drawn from our experiments.

First, in addition to the mere confirmation of previous reports demonstrating the occurrence of transverse fluctuations of nucleated cell membranes [START_REF] Yu | Local mechanical oscillations of the cell surface within the range of 0.2-30 Hz[END_REF][START_REF] Mittelman | Fast cell membrane displacements in B lymphocytes. Modulation by dihydrocytochalasin B and colchicine[END_REF][START_REF] Zidovska | Brownian motion of nucleated cell envelopes impedes adhesion[END_REF], our results provide some new information on these fluctuations. The tentative view of these fluctuations as transverse motions of micrometer-size blocks with an effective diffusion coefficient of a few nm 2 /s may be useful to model these movements. Interestingly, this concept of independent motile units is consistent with older reports on the autonomous behaviour of small cytoplasmic fragments obtained from blood neutrophils [START_REF] Dyett | Stimulated cytokineplasts from human polymorphonuclear leukocytes mobilize calcium and polymerize actin. Cytoplasts made in cytochalasin B retain a defect in actin polymerization[END_REF]. The patterns of cell undulations might also account for the recent findings that i) cell spreading on surfaces bearing adhesive structures requires a minimum surface density of anchoring points [START_REF] Massia | An RGD spacing of 440 nm is sufficient for integrin alpha v beta 3 -mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation[END_REF][START_REF] Calvacanti-Adam | Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands[END_REF], and ii) cell behaviour on adhesive surfaces is markedly influenced by topography [START_REF] Dalby | Increasing fibroblast response to materials using nanotopography : morphological and genetic measurements of cell response to 13-nm-high polymer demixed islands[END_REF][START_REF] Mossman | Altered TCR signaling from geometrically repatterned immunological synapses[END_REF].

Second, the dependence of these fluctuations on local distance of the membrane to a surface (Fig. 7B) strongly suggests that cells perceive approaching structures at about 50 nm distance, resulting in increase of oscillations in contrast with an expected behaviour of passive objects [START_REF] Zidovska | Brownian motion of nucleated cell envelopes impedes adhesion[END_REF][START_REF] Netz | Complete unbinding of fluid membranes in the presence of short-ranged forces[END_REF][START_REF] Marx | Helfrich repulsion and dynamical phase separation of multicomponent bilayers[END_REF]. Thus, even the earliest period following cell encounter with a surface might involve active biological functions. Further work is needed to determine the surface molecules or the mechanisms mediating the initial interaction between cells and approaching surfaces.

Third, an important conclusion is that local membrane-to surface adhesion is not an instantaneous process, since the separation gap progressively decreases during the first minute following attachment (Fig. 5B). This phenomenon may be highly significant, since a recent study made on the initial adhesion between natural killer and target cells suggested that the width of the intercellular gap, as estimated with electron microscopy, might influence the outcome of adhesion (i.e. killer cell activation versus inactivation) [START_REF] Mccann | The size of the synaptic cleft and distinct distributions of filamentous actin, ezrin, CD43, and CD45 at activating and inhibitory human NK cell immune synapses[END_REF]. While little information is available on the dynamics of membrane tightening, it is reasonable to speculate that the egress of bulky membrane molecules such as CD43 might be a prerequisite for membrane approach. While the exit of CD43 [START_REF] Soler | Leukosialin (CD43) behavior during adhesion of human monocytic THP-1 cells to red blood cells[END_REF][START_REF] Allenspach | ERM-dependent movement of CD43 defines a novel protein complex distal to the immunological synapse[END_REF] and CD45 [START_REF] Leupin | Exclusion of CD45 from the T-cell receptor signaling area in antigen-stimulated T lymphocytes[END_REF] from adhesion zones formed by immune cells is well demonstrated, the functional consequences remain incompletely understood. Indeed, these molecules may act either by influencing the presence of other signalling molecules, or they may by themselves trigger important biochemical reactions (such as phosphate removal by CD45).

Fourth, there remains to assess the mechanisms responsible for membrane fluctuations. Although the order of magnitude of these deformations is consistent with a passive thermal mechanism [START_REF] Zidovska | Brownian motion of nucleated cell envelopes impedes adhesion[END_REF] as analyzed by Hellfrich ( 14), several possible mechanisms might allow active cell processes to influence these deformations, including i) removal of bulky membrane structures and reduction of repulsive forces, ii) ensuring low enough surface tension and bending rigidity, that are recognized as key parameters of passive deformations, and (iii) generation of active motion. Our findings that i) cytoskeletal impairment with cytochalasin D increased membrane fluctuations in regions closest to the adhesive surface, (ii) increased contact formation was often preceded by local actin concentration would be consistent with the hypothesis that microfilament might act by stabilizing the membrane and adhesive interactions. This interpretation would be in line with the old report [START_REF] Rees | Control of grip and stick in cell adhesion through lateral relationships of membrane glycoproteins[END_REF] that detachment of surface adhesive cells might be triggered by a decrease of membrane associated microfilaments.

In conclusion, cell membrane fitting to adhesive surfaces is an early step of cell adhesion that occurs simultaneously with transverse membrane fluctuations. While strong evidence supports the hypothesis that these events are related by causal links, more work is required to unravel underlying molecular mechanisms. seconds later an extensive contact zone was visible as a dark area with IRM/RICM. Bar length = 2 µm. D: Comparing the brightness histograms of images 1B (broken line) and 1C (full line) allows objective definition of contact zone with a cutoff intensity value (arrow). E: THP-1 cells were deposited on adhesive surfaces treated with different concentrations of fibronectin and the rate of contact growth was determined. Surfaces were derivatized with fibronectin solutions at 0.1 µg/ml (squares), 1 µg/ml (crosses), 10 µg/ml (triangles), 100 µg/ml (circles). Error bars represent standard errors of the mean of 15 to 25 separate cells. A-I: Monocytic THP-1 cells were deposited on a surface treated with 10 µg/ml fibronectin. A microscope field was selected before the onset of the experiment and the cell-surface contact regions (corresponding to less than 34 nm apparent separation between surfaces) were determined at regular intervals of 9 seconds is shown as a black area. Bar length is 2 µm. The distance between a cell and an adhesive surface was derived from IRM images and displayed with coded colours. A: cell membrane immediately after contact (time zero). B and C: images recorded at times 8s and 17s respectively. Bar = 2 µm. A: A representative cell imaged with about 1Hz frequency during contact formation. The squared displacement per pixel was plotted versus time after subtracting the overall displacement (corresponding to decrease of average cell-to-surface distance). The plot is fairly linear. Correlation coefficient is 0.991. B: Lifetime of membrane undulations: the time dependence of correlation between two sequential membrane maps (squares) on a typical cell reveals 50 % decrease of correlation during about 8 seconds. Crosses: displacements of a given membrane point reveal no detectable temporal correlation, suggesting that membrane deformations are driven by random movements with higher that 1 Hz frequency (in line with Fig. 3B). C: Dependence of the amplitude of vertical membrane displacements (as imaged in Fig. 6C) on distance to the adhesive surface. Squares: onset of contact. Crosses: 130 second after contact initiation. D: correlation length of cell contour. Both membrane images (squares) and local displacements during a 2.7 second interval (crosses) display marked spatial correlation with a 50 % decrease at 1 µm separation. Seven control cells (squares) and eight cytochalasin D-treated cells (crosses) were monitored for determination of membrane topographical and fluctuation maps. The standard deviation of membrane height, representing a fluctuation amplitude, was determined on separate groups of point with common distance to the surface. Cytochalasin treatment did not substantially influence fluctuations at 60-80 nm separation, but this seem to increase fluctuations for smaller distances (the difference was significant for 50 nm separation with student's t test).

FIGURE 1 .

 1 FIGURE 1. Imaging contact formation between a cell and an adhesive surface. A: A spherical THP-1 cell deposited on a surface flattens within second as revealed with confocal microscopy in fluorescent medium. White bar length = 25 µm. B: the cell surface first appears fairly bright with interference reflection contrast microscopy. Bar length = 2 µm. C: Seventy-one

Figure 2 .

 2 Figure 2. Typical pattern of growing contact area.A-I: Monocytic THP-1 cells were deposited on a surface treated with 10 µg/ml fibronectin. A microscope field was selected before the onset of the experiment and the cell-surface contact regions (corresponding to less than 34 nm apparent separation between surfaces) were determined at regular intervals of 9 seconds is shown as a black area. Bar length is 2 µm.

Figure 3 .

 3 Figure 3. Quantitative analysis of cell-surface separation.The distance between a cell and an adhesive surface was derived from IRM images and displayed with coded colours. A: cell membrane immediately after contact (time zero). B and C: images recorded at times 8s and 17s respectively. Bar = 2 µm.

Figure 4 .

 4 Figure 4. Three-dimensional image of the topography of the membrane zone facing an adhesive surface. The shape of the cell described on Fig. 3 is shown at times 0, 8, 17, 26, 35 and 64 s respectively on A, B, C, D, E, and F.

Figure 5 .

 5 Figure 5. Quantitative parameters of contact extension and tightening. A:The contact area determined on a single cell is shown, disclosing fluctuations that were not visible on mean curves displayed on Fig.1E. B: The distance between a fixed point of the cell membrane (circle) and the minimum distance between the cell surface and the adhesive plane (crosses are shown), revealing progressive decrease of the distance between surfaces. This representative pattern was found with a similar time-scale on twelve different cells.

Figure 6 .

 6 Figure 6. Imaging cell membrane undulations.A: the contact evolution during a 5.6 second period of time is shown on a representative cell. While the region of stable contact is displayed in yellow, gain and loss of adhesive zone are displayed in red and green. B: The overall vertical membrane displacement is displayed with coded colours. C The amplitude of vertical membrane undulations is displayed as the standard deviation of eight sequential values of z coordinate, revealing the random part of the deformation.

Figure 7 .

 7 Figure 7. Quantitative description of cell membrane transverse fluctuations during contact extension.A: A representative cell imaged with about 1Hz frequency during contact formation. The squared displacement per pixel was plotted versus time after subtracting the overall displacement (corresponding to decrease of average cell-to-surface distance). The plot is fairly linear. Correlation coefficient is 0.991. B: Lifetime of membrane undulations: the time dependence of correlation between two sequential membrane maps (squares) on a typical cell reveals 50 % decrease of correlation during about 8 seconds. Crosses: displacements of a given membrane point reveal no detectable temporal correlation, suggesting that membrane deformations are driven by random movements with higher that 1 Hz frequency (in line with Fig.3B). C: Dependence of the amplitude of vertical membrane displacements (as imaged in Fig.6C) on distance to the adhesive surface. Squares: onset of contact. Crosses: 130 second after contact initiation. D: correlation length of cell

Figure 8 .

 8 Figure 8. Leukosialin redistribution during contact formation.Monocytic THP-1 cells were labelled with fluorescent anti-CD43/leukosialin before deposition on fibronectin-coated surfaces. Fluorescence and IRM images were then recorded at regular intervals. During the first minute, the separation between the cell membrane and the adhesive surface is nearly always higher than 35 nm (A), and CD43 is clearly visible on the whole surface, with local concentration (B -arrow). Sixty seconds later, cell-surface distance decreased by at least 20 nm (C -arrow) and CD43 depletion is clearly visible in the whole cell surface and particularly in the new contact zone (D).

Figure 9 .

 9 Figure 9. Actin redistribution during contact formation. Monocytic THP-1 cells were transfected with GFP before being deposited on fibronectin-coated surfaces and contact regions were analyzed with IRM and fluorescence. Two representative sets of images (A-D and E-H) are shown.A: visible light image of a standard cell (with usual roundish appearance preceding spreading). B and D display contact growth during a 90 second period of time. On the fluorescence image (C), actin appears distributed throughout the whole cell, with local bright patches (arrow) that were associated with and seemed to precede contact. E-H: a similar behaviour was observed several minutes after initial contact, on a markedly polarized cell.

Figure 10 .

 10 Figure 10. Effect of cytochalasin D on membrane fluctuations near a surface.Seven control cells (squares) and eight cytochalasin D-treated cells (crosses) were monitored for determination of membrane topographical and fluctuation maps. The standard deviation of membrane height, representing a fluctuation amplitude, was determined on separate groups of point with common distance to the surface. Cytochalasin treatment did not substantially influence fluctuations at 60-80 nm separation, but this seem to increase fluctuations for smaller distances (the difference was significant for 50 nm separation with student's t test).