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New technologies in scanning probe microscopy for studying molecular interactions in cells
Petri P. Lehenkari, Guillaume T. Charras, Stephen A. Nesbitt and Mike A. Horton
Atomic force microscopy (AFM) is a specialised form of scanning probe microscopy,
which was invented by Binnig and colleagues in 1986. Since then, AFM has been
increasingly used to study biomedical problems. Because of its high resolution,
AFM has been used to examine the topography or shape of surfaces, such as during
the molecular imaging of proteins. This, combined with the ability to operate
under known force regimes, makes AFM technology particularly useful for measuring
intermolecular bond forces and assessing the mechanical properties of biological
materials. Many of the constraints (e.g. complex instrumentation, slow acquisition
speeds and poor vertical range) that previously limited the use of AFM in cell
biology are now beginning to be resolved. Technological advances will enable
AFM to challenge both confocal laser scanning microscopy and scanning electron
microscopy as a method for carrying out three-dimensional imaging. Its use as
both a precise micro-manipulator and a measurement tool will probably result
in many novel and exciting applications in the future. In this article, we have
reviewed some of the current biological applications of AFM, and illustrated
these applications using studies of the cell biology of bone and integrin-mediated
adhesion.
Expert Reviews in Molecular Medicine © Cambridge University Press ISSN 1462-3994
Biochemical assays such as enzyme-linked immunosorbent assays and surface plasmon resonance analysis are extremely efficient and widely used to measure many of the chemical interactions upon which physiological phenomena in living organisms are based. This is especially so for those reactions that are not appreciably influenced by isolation from their normal surroundings. However, to function optimally, many biomolecules must be embedded in their natural surroundings, for example within a matrix or cell membrane. Such biomolecules must be analysed using even more specialised techniques that operate at the level of individual cells and target specific receptor proteins. Here, these receptors often need to be activated, either by their anchoring to the cytoskeleton or associated membrane proteins, or through interactions with intracellular signalling molecules. An example that typifies these requirements is the integrin family of cell-adhesion receptors (Ref. 1). Thus, integrin receptors are coupled ‘mechanically’ to the cytoskeleton and are functionally dependent upon associated signal-transduction processes, which are considered in more detail later in this review.
Previously, molecular interactions at the level of the cell membrane have been investigated using various micro-manipulative techniques, such as the surface-force apparatus (Ref. 2), pipette suction (Ref. 3), laser tweezers (Ref. 4), magnetic beads or hydrodynamic drag (Ref. 5). Several methods have also been developed to measure more specifically the affinity between receptors and their ligands. However, these have been limited either to the analysis of protein–protein interactions analysed with radioactively labelled probes and by surface plasmon resonance (Ref. 6), or to the characterisation of average ‘affinities’ in bulk cell-binding assays. All of the micro-manipulative techniques are based on the use of a physical probe that is ‘functionalised’ with a molecule that acts as a ligand, which binds to a cell-surface receptor. A classical example of such a micro-manipulative approach is the use of laser tweezers to study the molecular basis of muscle contraction, during which myosin molecules move along actin filaments (Refs 7, 8); the size and direction of the forces imparted by this ‘molecular motor’ have been characterised in detail.
‘May
the force be with you!’
Atomic force
microscopy (AFM) is a specialised form of scanning probe microscopy that enables
the acquisition of high-resolution topographical images of intact cells; furthermore,
it allows the measurement of single molecular events under physiological conditions
(Ref. 9). AFM was devised by Binnig and colleagues in 1986
(Ref. 10) for the examination of material surfaces, and was
subsequently applied to the life sciences by Marti and colleagues (Ref. 11).
Today, AFM is used widely in biology, and a summary of the range of the applications
of AFM in cell biology is provided in Table 1 (tab001mhu).
Intriguingly, AFM is increasingly being used as an ultra-precise micro-manipulator
rather than an imaging tool, because it can apply precisely regulated, small
forces. Indeed, as regards its imaging capabilities for cell-biological work,
the rate at which it captures images is rather disappointing (usually requiring
several minutes per image); thus, AFM faces ‘competition’ from confocal
laser scanning microscopy and scanning electron microscopy, both of which can
accumulate data faster. However, owing to the method by which surface topography
is determined, AFM has many other uses, including: (1) the measurement of the
binding force between a molecule and its ligand, (2) the determination of the
elastic properties of cells, (3) the evaluation of the friction coefficient
of materials and (4) the high-resolution imaging of isolated proteins and structures.
Importantly, AFM can be performed on living samples such as cells and tissues.
In this review, our aim was to outline some of the different uses of AFM in the life sciences, specifically focusing on its uses as a nanoscale micro-manipulator and a detector of molecular interactions. As an example, we have also described current views of the mechanics involved in integrin–ligand recognition, and considered the potential applications of AFM in bone cell biology (Ref. 12).
Principles
of AFM
The measurement
of the topography of a sample using AFM involves a micro-fabricated cantilever
(usually 100–200 µm
long) with a very small tip (which has a contact area of only a few square nanometers)
being raster scanned above the surface of the sample (i.e. the microscope tip
is moved progressively backwards and forwards across the surface; see Fig. 1;
fig001mhu). A piezo-electric crystal is used to
raise or lower the cantilever, to maintain a constant bending of the cantilever.
A laser beam is reflected from the top of the cantilever towards a photo-detector,
which detects any bending of the cantilever, thus enabling the actual position
of the cantilever to be back-calculated. As a result, the atomic force microscope
records images of surface topography under a constant applied force (in the
low nN range), which is optimised to produce maximal resolution without damaging
the surface of the sample.
Many different imaging modes have been developed for AFM, and these have been reviewed by Hansma and colleagues (Ref. 13). Contact-mode imaging involves the whole surface being scanned with the tip of the cantilever in constant contact with the surface. Using the contact mode, it is also possible to measure the torque applied on the cantilever as it is scanned sideways over the surface. Differences in torque equate to differences in the friction of the surface and variation in material properties (Refs 14, 15). An alternative non-contact, or ‘tapping’, mode involves the cantilever being rapidly oscillated while it is slowly lowered towards the surface. When the cantilever comes close to the surface, the amplitude of the oscillations is dampened and the surface can be detected. This mode is much less damaging to the samples (Ref. 16). During a third mode of operation, ‘force–distance measurement’, the cantilever is slowly lowered towards the surface and its deflection is constantly recorded. This mode yields a curve showing the bending of the cantilever as a function of the distance travelled (see Fig. 2; fig002mhu). Using the spring constant of the cantilever, it is then possible to calculate the force needed to create that deflection. This final mode is the basis for the measurement of the material properties of cells and the binding forces between ligands and receptors.
Applications
of AFM
Topography measurements
AFM was originally designed for the study of surface topography. Initially,
the technique yields a height map, which is generated from height measurements
taken at multiple positions along the cell surface. From this map, it is possible
to construct a three-dimensional profile of the cell surface (Fig. 1b; fig001mhu).
The resolution is proportional to the deformation caused by the force used for
imaging. When an atomic force microscope is used in contact mode on surfaces
that are harder than the probe, the lateral resolution of the microscope is
at the atomic level. However, for surfaces that are ‘softer’ than
the tip of the atomic force microscope, the resolution obtained is lower than
the ‘atomic scale’, owing to indentation of the tip along the probed
surface. Indeed, on materials as soft as living cells, the resolution can be
as low as in the order of 500 nm. In comparison, a resolution of 350 nm is achievable
using confocal laser scanning microscopy, which is, in turn, much lower than
that achievable using scanning electron microscopy (i.e. ~1 nm). The resolution
achievable using AFM can be enhanced by operating the atomic force microscope
in non-contact mode. The estimated resolution in tapping mode is in the order
of a few tens of nanometers, which is better than that of confocal laser scanning
microscopy (Ref. 16). However, non-contact imaging is difficult
to operate in liquids and particularly so in the case of living cells in an
aqueous phase; this is due to the confounding effects of oscillations of the
object being imaged and the surrounding fluid that are induced by movement of
the cantilever.
Topography measurements yield the three-dimensional aspect of the examined surface with a vertical resolution at the nanometer level (Fig. 1b; fig001mhu). Samples can be either fixed (i.e. using conventional histological fixatives) or unfixed. However, as shown in Figure 3 (fig003mhu), fixed cells present striking morphological differences when compared with live cells, as has been reported by several investigators (Refs 17, 18). In contact mode, because the softer parts of the cytoskeleton inside the cell are indented more than the harder parts of the cell by the tip of the atomic force microscope, this mode of imaging can also reveal components of the underlying cytoskeleton, thereby providing structural information about the interior of the cell. Thus, sub-surface biological processes such as vesicle trafficking, mitosis or cytoskeletal rearrangement can be evaluated under ambient conditions (Refs 19, 20), and the imaging of these processes enhanced in live cells because they are ‘softer’ than the ‘more rigid’ fixed cells. Furthermore, AFM is beginning to be used to measure other physiological responses. For example, Schneider and colleagues (Refs 21, 22) have used AFM to measure the aldosterone-induced volume increase in live endothelial cells, whereas Spudich and Braunstain used it to follow the process of basophil de-granulation (Ref. 23).
Even though AFM can be used to acquire images of surface topography, similar images can be obtained by using confocal laser scanning microscopy. This can be achieved by labelling cells with fluorescent markers (e.g. tagged antibodies or lectins) that bind to structures that are found solely on the cell surface, such as the avb3 integrin (i.e. the vitronectin receptor) on osteoclasts (Refs 24, 25, 26). Similar confocal techniques that utilise membrane-excluded dyes (e.g. fluorescein-linked dextrans, which have a high molecular weight) have been developed to reveal surface topography (Ref. 27). Using confocal laser scanning microscopy, a series of confocal cross-sectional images (separated by a minimum thickness of 350 nm) are collected vertically through a cell. The stack of images is then post-processed (using a computer and commercially available software) to recreate a three-dimensional rendered image (Fig. 4; fig004mhu). The resolution and surface detail obtained using AFM and optical (confocal) techniques are comparable.
Measurements of binding
forces
Since its invention, AFM has proven of value for quantifying the binding
forces of interactions between proteins, between cells, between receptors and
ligands (either on cells or plated onto mica), or between cells and their substrates
(Table 2; tab002mhu). Receptor–ligand binding
forces in the range of 15–250 pN have been reported for protein interactions,
and up to 220 nN for total cellular binding forces (Refs 28,
29, 30, 31, 32,
33, 34, 35, 36,
37, 38, 39). Using the
atomic force microscope, it is possible to evaluate directly the binding force
between a single receptor molecule and its ligand (Refs 40,
41). To achieve such a measurement, the tip of the atomic
force microscope is ‘functionalised’ by adding a specific interactive
molecule, such as an antibody or protein ligand. Two basic techniques have been
developed to achieve this. One relies on the passive chemi-sorption of a solution
of the ‘coating’ molecule onto the tip of the microscope, often using
polyethylene glycol as a linker or adsorbent (Ref. 42). The
other technique uses covalent coupling (using various chemistries) to link the
‘coating’ molecule directly onto the tip (Ref. 39).
Force–distance curves are then recorded in different locations until an
adhesion event occurs, which is easily recognised by the shape of the force–distance
curve. The shape and interpretation of a typical force–distance measurement
are shown in Figure 2 (fig002mhu).
The measurement of the binding forces of cellular components depends on the ligand being either adsorbed onto atomically smooth, cleaved mica, or present as a natural component of the cell membrane. However, it is inherently difficult to carry out measurements on living cells for three reasons: (1) the target molecule is easily lost from the cell membrane, (2) the target molecule may be present at too low a density to make a successful interaction likely or (3) the tip of the atomic force microscope may become contaminated by cellular debris during the analysis (Ref. 43). Recently, our group has reported the first measurements of binding forces between an arginine–glycine–asparagine (arg–gly–asp or RGD) peptide and integrin, its cognate receptor, on living cells (Ref. 42). Details of this application of AFM are described later in this review.
Measuring the material
properties of a cell
The intracellular cytoskeleton gives a cell its physical integrity but the
cytoskeleton also adapts to the external environment and the activity levels
of the cell. However, the exact role of each element of the cytoskeleton is
still unclear. By measuring the material properties of a cell (e.g. stiffness,
plasticity and visco-elasticity) and determining the effects of induced cytoskeletal
changes on them, it is possible to gain insight into the particular role of
each cytoskeletal element. Moreover, by analysing the slopes of force–distance
curves that are recorded during indentation with the probe of an atomic force
microscope (Fig. 5a and 5b; fig005mhu),
the elastic modulus (i.e. the stiffness) of a cell can be determined at a particular
location, using the theory of indentation (Ref. 44). Thus, AFM can determine
the cellular elasticity of tissues (as reviewed by Radmacher in Ref. 45).
Applying these principles (Fig. 5; fig005mhu),
such information has been accumulated during raster scanning across a cell,
and a three-dimensional map of information about elastic properties and height
of structures generated (Fig. 5c, 5d and 5e; fig005mhu).
Common materials, such as steel or bone, have elastic moduli of 200 000 kPa and 10 000 kPa, respectively, whereas living cells are more compliant, with stiffness values in the range of 1–150 kPa. A summary of the reported elastic moduli of different cell types and cell structures is presented in Table 3 (tab003mhu). As expected, different constituents and regions of a cell have very different stiffness values. The effects of cytoskeleton-disrupting drugs on cell stiffness have been studied extensively (Ref. 46; Table 4; tab004mhu). In particular, it was found that disrupting the F-actin network with cytochalasin D reduced cell stiffness. In contrast, glutaraldehyde (a common histological fixative) increased cell stiffness by up to threefold, by increasing the degree of crosslinking between the F-actin fibres and the rest of the cytoskeletal network. Together, these studies show that F-actin filaments participate in the maintenance of cellular elasticity. In focal-adhesion plaques, the F-actin cytoskeleton is linked to integrin receptors on the cell surface by vinculin. Recently, Goldmann and colleagues (Ref. 47) reported that the elastic modulus of vinculin-deficient cells was lower than that of wild-type cells; when vinculin expression was reinstated by gene transfer of the gene encoding vinculin, an elastic modulus close to that of the wild-type cells was attained. These results point to a role for vinculin in stabilising focal adhesions and transferring mechanical stresses to the cytoskeletal network. Banes and colleagues have extensively reviewed the field of transduction of mechanical signals through the cytoskeleton (Ref. 48). Other material properties such as the visco-elasticity and the plasticity of cells have also been measured. Visco-elasticity measurements are determined by indenting (deforming) a cell, and following its recovery over a period of time (Ref. 46). Plasticity measurements are, in contrast, determined by measuring the permanent deformations that are inflicted on a cell as a result of a similar indention (for a review of the basic biomechanical measurements see Ref. 49).
Using the atomic force
microscope as a micro-manipulator
Recently, there have been several reports of experiments that fall under
neither of the previous categories in that they are not aimed at surface measurement,
but instead these studies use the atomic force microscope as either a micro-manipulator
or a micro-detector. Domke and colleagues used AFM to map the mechanical ‘pulse’
(i.e. contraction and relaxation) of cardiomyocytes in culture (Ref. 50).
Thie and colleagues, using similar technology, examined the adhesive forces
between a trophoblast (i.e. an implanting embryo) and the uterine epithelium
using whole trophoblast cells, instead of isolated molecules, bound to the tips
of atomic force microscopes (Ref. 28). Adhesion forces of
~3 nN were recorded between the cells, which is an order of magnitude higher
than reported adhesion forces between receptors and ligands. Recently, Sagvolden
and colleagues developed a new use for AFM; they used it to quantify the adhesion
forces of cells to a substrate (Ref. 51). This new application
is of interest in the field of bioengineering, particularly in orthopaedic and
vascular surgery. For example, such data might be useful in the selection of
suitable biomaterials for tissue reconstruction. In the future, we are likely
to see an increase in the number of similar studies, and their findings used
in the development of new therapies.
Integrin–RGD recognition:
an example of intermolecular-force measurement in cells using AFM
Members of the integrin family of cell-adhesion receptors play key roles
in cell–matrix recognition (Ref. 52). They are found
in virtually all types of cells (Ref. 52) as transmembrane
receptors, consisting of two non-covalently linked subunits that require the
presence of divalent cations for optimal function (Refs 53,
54). In cells that adhere to extracellular-matrix substrates,
integrins are concentrated at the underside of the cells in focal-adhesion plaques
that are associated with intracellular linker proteins, such as vinculin with
both the F-actin cytoskeleton and signalling molecules (Ref. 55).
Many integrins recognise peptides that contain the RGD consensus sequence. This
sequence is found in several ligands including cell-surface and extracellular-matrix
proteins; these proteins include non-collagenous proteins that are found in
the bone matrix, such as osteopontin and vitronectin (Ref. 56).
The affinity of the integrin receptor for a given ligand depends not only on
the integrin type, but also on the conformational changes that follow receptor
activation. Such conformational changes are triggered by alterations in the
extracellular microenvironment of the integrin receptor, and also by interactions
between: (1) the cytoplasmic part of the receptor, (2) the intracellular signalling
molecules and (3) the cytoskeleton (Ref. 57). Knowledge of
the biological properties of integrins suggests that the reliable evaluation
of their affinity for ligands depends on the properties of the intact cells
that are being used (Ref. 42). It is likely that this is also
true for many other systems of receptors on the cell-surface membrane.
RGD–integrin binding forces, measured using AFM in live osteoclasts and osteoblasts (bone cells), appear to depend not only on the integrin type and its activation status, but also on the spatial conformation of the sequence of the peptide or protein containing the RGD sequence (Ref. 42). Thus, the forces observed for a linear RGD peptide are lower than those for echistatin, a snake-venom protein that contains RGD in a different conformation and is a potent antagonist of integrins. This dependence on conformation is most likely because the more favourable orientation between the ligand and the integrin receptor results in shorter interaction ranges for the molecules, and thus higher interaction forces between RGD and the binding site. Surprisingly, the dynamic range of RGD-binding forces is small compared with affinity measurements that are based on the inhibition of receptor–ligand interactions (see Table 2; tab002mhu). For example, the binding affinity of a linear RGD peptide and echistatin differs by a factor of ~1 x 105, whereas the binding forces that are measured using AFM vary by only approximately twofold (Ref. 42). The relationship between these two values is unclear, and it is probable that fundamental differences exist between the parameters measured. AFM can be used to determine binding force and binding probability separately, whereas affinity measurements combine both (Ref. 30).
AFM also provides further insights into the nature of intermolecular interactions. During the evaluation of intermolecular interactions, the ‘pulling velocity’ (i.e. the rate at which the tip of the atomic force microscope is removed from the object under analysis during a force–distance measurement) is critical, as shown by Fritz and colleagues, using isolated proteins (Ref. 38), and by our group, using intact cells (Ref. 42). Thus, the measured binding force (e.g. that for the biotin–avidin interaction) varies with the pull-out speed (Ref. 58); higher speeds yield higher binding forces. There are several possible explanations for this phenomenon. The first explanation is based on the occurrence of different adhesive forces within the binding pocket of the receptor that rupture separately; thus, the observed force consists of a series of sub-binding forces, which are added together to yield a higher force at high pulling speeds. The separation into different forces can be explained in two different ways: (1) the bound molecules can undergo different orientations during the pull-out step from the receptor molecule, and the initial binding energy can then be absorbed by both of the molecules and finally transformed into conformational changes; or (2) a series of forces could be explained by the existence of physically separate binding and acceptor domains within the interacting molecules. A second, more simplified explanation is based on the gradual increase in distance and the proportional decrease in force during a slow pull-out. This model does not assume any conformational changes in the molecules, but rather is based on linear binding fields between the molecules, which get weaker the further they are separated. A third explanation considers the properties of the atomic force microscope itself, rather than intermolecular forces. Thus, different pull-out speeds change the recognition capabilities of the probe of the microscope, owing to changes in either the visco-elasticity of the ‘vehicle’ (i.e. the culture fluid) or the absorption of energy by the probe itself. Which of the three proposed explanations is true awaits confirmation. Meanwhile, the pull-out speeds and cantilever mechanics must be carefully considered when ‘absolute’ force values are cited.
Research
in progress and outstanding research questions
Intriguingly, the use of AFM in biology in the future might be based not so
much on its atomic-scale imaging capabilities, but rather on the opportunity
it offers to apply and measure forces between biological entities, such as molecules,
cells or biomaterials. As a consequence, the atomic force microscope is being
used increasingly as an ultra-precise micro-manipulator rather than an imaging
tool. This capability has already been used in some extremely interesting preliminary
studies that have focused not only on intermolecular forces but also on intramolecular
bonds, such as unfolding force measurements of the muscle protein titin or DNA
(Refs 59, 60). As instrumentation for AFM
improves and a wider range of equipment becomes commercially available, it is
likely that cell biologists will routinely use AFM to measure molecular interaction
forces.
Acknowledgements
and funding
P.P. Lehenkari
and G.T. Charras contributed equally to this paper. This work was supported
by grants to M.A. Horton from the Wellcome Trust and Monsanto Searle, Inc. P.P.
Lehenkari was in receipt of fellowships from both the EMBO and the Finnish Academy
of Science, and G.T. Charras was funded by a COSAT award from Johnson and Johnson.
We also thank our peer reviewers, Manfred Radmacher (Department of Physics,
Ludwig Maximilians University, Munich, Germany), Kalervo Väänänen
(Department of Anatomy, University of Turku, Finland) and Nigel Loveridge (Department
of Medicine, University of Cambridge, UK), for their helpful comments during
the production of this article.
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Further
reading, resources and contacts Manufacturers
of AFM instrumentation Burleigh Instruments Inc., New York, USA. Digital Instruments, California, USA. Molecular Imaging Corporation, Arizona, USA. OMICRON, GmbH, Germany. ThermoMicroscopes/Park Scientific Instruments, California, USA. TopoMetrix, California, USA. WITec GmbH, Germany. Academic research
groups Applied Optics Group in the Department of Applied Physics at the University of Twente, The Netherlands. Blaine’s Scanning Probe Microscopy Home Page. This site provides a long list of useful links. Hansma Research Group in the Department of Physics at the University of California Santa Barbara, USA. Heckl’s Nano Science Research Group at the University of Munchen, Germany. Hermann Gaub’s Group at the University of Munchen, Germany. Institute for Biophysics at the University of Linz, Austria. Jan Hoh’s Laboratory in the Department of Physiology at the Johns Hopkins University School of Medicine in Baltimore, Maryland, USA. John Cross’s website. This site provides a useful introduction to scanning probe microscopy (or ‘imaging surfaces on a fine scale’). Laboratory of Biophysics and Surface Analysis in the School of Pharmaceutical Sciences at the University of Nottingham, UK. Lindsay Laboratory at the Arizona State University, USA. Nottingham Nanoscience Group at the University of Nottingham, UK. Phil Haydon’s Research Group at the Laboratory of Cellular Signalling, Iowa State University, USA. Scanning Probe Microscopy Group in the Department of Physics at the University of Bristol, UK. Scanning Probe Microscopy Group at the Institute of Food Research, Norwich, UK. Scanning Probe Microscopy Laboratory at the North Carolina State University, USA. The Local Probes Group in the European Molecular Biology Laboratory, Heidelberg, Germany. This site is produced by Stephan Altmann and contains links through to many useful sites. Zhifeng Shao’s Research Group at the Department of Molecular Physics and Biological Physics, University of Virginia, USA.
Nanotechnology
General microscopy Confocal laser
scanning microscopy Leica Microsystems Heidelberg GmbH, Germany. This site is dedicated to confocal laser scanning microscopy. Microscopy
index sites Microscopy links. This site is compiled by Bio-Rad Laboratories, Inc., and aims to provide ‘a definitive links page for microscopy-related issues’.
Molecular Expressions Microscopy Primer. This site was produced by Michael W. Davidson (of Mortimer Abramowitz, Olympus America Inc., and the Florida State University). Computation NIH Image. This site provides information on NIH Image, a public domain image-processing and analysis program for the Macintosh, which was developed at the Research Services Branch (RSB) of the National Institute of Mental Health (NIMH), part of the National Institutes of Health (NIH). The BioImage Homepage. This site describes a European initiative for a new database of multidimensional biological images. |
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Features
associated with this article Figures Figure 2. How the
atomic force microscope generates force–distance curves for a cell
receptor and ligand Figure 3. Examples
of results of scanning cells with an atomic force microscope in contact
mode in liquid Figure 4. Confocal
laser scanning microscopy of fixed and stained cells Figure 5. Examining
the material properties of a cell using atomic force microscopy Tables Table 2. Examples
of the interactive forces between specific molecules Table 3. Elastic
properties of different cell types and cell structures Table 4. Effects
of cytoskeleton-disrupting drugs on the material properties of cells Movie |