Cell Shape Is Reinforced By
Nature. Author manuscript; available in PMC 2010 Jul 28.
Published in last edited course every bit:
PMCID: PMC2851742
NIHMSID: NIHMS183197
Cell mechanics and the cytoskeleton
Daniel A. Fletcher
aneBioengineering and Biophysics, University of California, Berkeley, California 94720, The states
2Physical Biosciences, Lawrence Berkeley National Laboratory, Berkeley, California 94720, U.s.a.
R. Dyche Mullins
3Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143, Us
Abstract
The ability of a eukaryotic cell to resist deformation, to transport intracellular cargo and to change shape during move depends on the cytoskeleton, an interconnected network of filamentous polymers and regulatory proteins. Contempo piece of work has demonstrated that both internal and external physical forces can act through the cytoskeleton to bear on local mechanical properties and cellular behaviour. Attention is now focused on how cytoskeletal networks generate, transmit and respond to mechanical signals over both short and long timescales. An important insight emerging from this piece of work is that long-lived cytoskeletal structures may act as epigenetic determinants of cell shape, function and fate.
In a 1960 lecture, cell and developmental biologist Paul A. Weiss encouraged his audience to call back of the cell every bit an integrated whole "lest our necessary and highly successful preoccupation with cell fragments and fractions obscure the fact that the cell is not simply an inert playground for a few almighty masterminding molecules, but is a system, a hierarchically ordered organization, of mutually interdependent species of molecules, molecular groupings, and supramolecular entities; and that life, through cell life, depends on the society of their interactions"1.
This argument may exist more than relevant today than it was 50 years agone. Despite tremendous progress, fundamental gaps remain between our understanding of individual molecules and our understanding of how these molecules part collectively to form living cells. The sequencing of genomes outpaces characterization of the cellular components they encode and far exceeds our power to reassemble these components into the types of complex system that can provide mechanistic insight into cellular behaviour. An even more than difficult chore is to connect the behaviour of cells in culture with that of more circuitous living tissues and organisms.
Ever since muscle fibres were beginning examined under rudimentary microscopes in the seventeenth century, researchers have been motivated to understand how the process of self-organization generates dynamic, robust and elaborate structures that organize and 'breathing' cells. The biological importance of establishing social club over various length scales and timescales, also as the challenges of understanding how systems of self-organizing molecules carry out cellular functions, is perhaps best illustrated by studies of the cytoskeleton.
The cytoskeleton carries out 3 broad functions: information technology spatially organizes the contents of the cell; it connects the cell physically and biochemically to the external environment; and it generates coordinated forces that enable the prison cell to movement and change shape. To achieve these functions, the cytoskeleton integrates the activity of a multitude of cytoplasmic proteins and organelles. Despite the connotations of the word 'skeleton', the cytoskeleton is non a fixed structure whose function can be understood in isolation. Rather, it is a dynamic and adaptive structure whose component polymers and regulatory proteins are in abiding flux.
Many basic building blocks of the cytoskeleton accept been identified and characterized extensively in vitro, and researchers are now using advanced light microscopy to determine, with great spatial and temporal precision, the locations and dynamics of these cytoskeletal proteins during processes such every bit prison cell division and motility. For instance, more than 150 proteins have and then far been found to incorporate binding domains for the protein actin, which polymerizes to course one of the key cytoskeletal filaments in cells2. One set of actin regulators forms a macromolecular ensemble called the WAVE complex that promotes assembly of actinfilament networks at the leading edge of motile cells3. High-resolution light microscopy of rapidly crawling leukocytes revealed that the Wave complex forms highly coherent travelling waves whose motility correlates with cell protrusion4.
Such observations in living cells can stimulate the formation of detailed hypotheses for how molecules collaborate to grade functional cytoskeletal structures, but to test these hypotheses definitively, the components must exist isolated from cells and purified. Remarkably, experiments that combine a minor number of purified proteins have demonstrated that many circuitous cytoskeletal structures observed in cells can be reconstituted in vitro from purified components. For case, only 3 proteins are required to actively runway and send cargo on the growing cease of microtubules, which are formed by the polymerization of subunits consisting of αβ-tubulin heterodimers and are another key cytoskeletal filament in cells5. Although the list of proteins associated with the cytoskeleton continues to grow, the ultimate goal remains — agreement how the interactions of the individual molecules of the cytoskeleton give ascension to the big-scale cellular behaviours that depend on them.
In this Review, we discuss contempo progress towards an integrated understanding of the cytoskeleton. In detail, nosotros focus on the mechanics of cytoskeletal networks and the roles that mechanics have in many cell biological processes. Rather than focusing on i cellular procedure or cytoskeletal filament, we place a set of bones concepts and link them to work in several cytoskeleton-related fields. We begin with a brief introduction to the major polymers that constitute the cytoskeleton so shift focus from molecules to more than complex structures, emphasizing iii concepts that repeat Weiss's 1960 claiming to view cells every bit an integrated whole. The start concept is that long-range guild arises from the regulated self-assembly of components guided by spatial cues and physical constraints. The second is that beyond simply limerick, it is the architecture of the cytoskeleton that controls the physical properties of the cell. And the third is that cytoskeletal links to the external microenvironment can mediate both curt and long timescale changes in cellular behaviour. We finish by discussing the intriguing and nether-appreciated question of whether long-lived cytoskeletal structures can function as a cellular 'retentivity' that integrates by interactions with the mechanical microenvironment and influences future cellular behaviour.
Cytoskeletal building blocks
The proteins that make upwards the cytoskeleton accept many similarities to LEGO, the popular children'southward toy. Both consist of many copies of a few key pieces that fit together to form larger objects. Both can exist assembled into a wide range of structures with diverse properties that depend on how the pieces are assembled. And both can be disassembled and reassembled into unlike shapes according to changing needs. But simply the cytoskeleton fulfils all of these functions through self-assembly.
At that place are iii main types of cytoskeletal polymer: actin filaments, microtubules and a group of polymers known collectively as intermediate filaments. Together, these polymers control the shape and mechanics of eukaryotic cells (Fig. 1). All iii are organized into networks that resist deformation but can reorganize in response to externally applied forces, and they have important roles in arranging and maintaining the integrity of intracellular compartments. The polymerization and depolymerization of actin filaments and microtubules generate directed forces that drive changes in prison cell shape and, together with molecular motors that move along the actin filaments and microtubules, guide the organization of cellular components. The compages of the networks that are formed by cytoskeletal polymers is controlled past several classes of regulatory protein: nucleation-promoting factors, which initiate filament formation; capping proteins, which cease filament growth; polymerases, which promote faster or more sustained filament growth; depolymerizing factors and severing factors, which disassemble filaments; and crosslinkers and stabilizing proteins, which organize and reinforce higher-lodge network structures. Mechanical forces from inside or outside the cell can affect the activity of these regulatory factors and, in turn, the local system of filaments in the networks. The well-nigh important differences between the three main cytoskeletal polymers — the differences that distinguish the architecture and function of the networks they class — are their mechanical stiffness, the dynamics of their associates, their polarity, and the blazon of molecular motors with which they associate.
Elements of the cytoskeleton
The cytoskeleton of eukaryotic cells provides structure and organization, resists and transmits stresses, and drives shape modify and movement. a, Neurons are specialized eukaryotic cells that extend long processes to form connections in the nervous organisation. Like other eukaryotic cells, neurons have a cytoskeleton that consists of iii main polymers: microtubules (green), intermediate filaments (purple) and actin filaments (ruddy). b, A fluorescence micrograph of the neuronal growth cone, which migrates in response to chemical cues during the development of the nervous system, is shown. Microtubules (green) emanate from the axon, and actin-filament networks (crimson) course sheet-like structures and filopodial protrusions at the leading edge. Scale bar, 20 μm. (Image reproduced, with permission, from ref. 82.) c, The neuronal axon is a long membrane-bounded extension, in which neurofilaments (a class of intermediate filament in neurons) course a structural matrix that embeds microtubules, which send materials from the cell torso to the axon terminals at the synapse. d, The growth cone contains dendritic actinfilament networks and parallel actin-filament filopodia. due east, Microtubules consist of thirteen protofilaments of tubulin dimers arranged in a hollow tube. f, Neurofilaments take flexible polymer arms that repel neighbouring neurofilaments and determine the radius of the axon. g, Actin filaments are arranged into networks. These networks can have many architectures, including the branched structures depicted here, which are formed by the Arp2/3 circuitous (blue). The diameters of microtubules, intermediate filaments and actin filaments are within a gene of three of each other; the diagrams in e, f and g are drawn approximately to scale. Just the relative flexibilities of these polymers differ markedly, as indicated by their persistence lengths: from least to most flexible, microtubules (5,000 μm), actin filaments (thirteen.5 μm) and intermediate filaments (0.5 μm).
Microtubules are the stiffest of the three polymers and have the most complex assembly and disassembly dynamics. The persistence length of microtubules, a measure out of filament flexibility that increases with stiffness, is so large (~5 mm) that single microtubules tin can form tracks that are well-nigh linear and span the length of a typical beast cell, although microtubules are known to buckle under the compressive loads in cellsvi. During interphase, the part of the cell cycle during which cells fix for partition, many cells have advantage of this stiffness past assembling radial arrays of microtubules that role every bit central hubs and 'highways' for intracellular traffic. During mitosis, the part of the cell cycle during which cells separate chromosomes into 2 identical sets, the microtubule cytoskeleton rearranges itself into a high-fidelity Dna-segregating machine called the mitotic spindle. The ability of the mitotic spindle to find and marshal chromosomes depends, in part, on the complex associates dynamics of private microtubules7. A microtubule tin switch between 2 states: stably growing and rapidly shrinking8. This 'dynamic instability' enables the microtubule cytoskeleton to reorganize rapidly and allows individual microtubules to search the cellular space quickly9, upwardly to 1,000-fold faster than a polymer that is sensitive only to changes in the cellular concentration of its constituent subunits or to the actions of regulatory proteins.
Actin filaments are much less rigid than microtubules. But the presence of high concentrations of crosslinkers that demark to actin filaments promotes the assembly of highly organized, stiff structures, including isotropic networks, bundled networks and branched networks. Bundles of aligned filaments support filopodial protrusions, which are involved in chemotaxis (directed movement along a chemic gradient) and cell–cell communication. By contrast, networks of highly branched filaments support the leading edge of most motile cells and generate the forces involved in changes in prison cell shape such as those that occur during phagocytosis. Unlike microtubules, actin filaments do not switch between discrete states of polymerization and depolymerization; instead, they elongate steadily in the presence of nucleotide-spring monomers. This steady elongation is well suited to producing the sustained forces that are required to advance the leading edge of a migrating cellten. Also unlike the microtubule cytoskeleton, the compages of which is often adamant by one or two primal organizing centres, the actin cytoskeleton is continually assembled and disassembled in response to the local activity of signalling systems. For example, protrusive, branched actin-filament networks, such as those in crawling leukocytes, are assembled at the leading border of the cell in response to signals downstream of prison cell-surface receptors that guide chemotaxis11. Similarly, the assembly of contractile actin-filament bundles known every bit stress fibres, such as those in adherent fibroblasts, is triggered locally when jail cell-surface adhesion receptors called integrins engage their ligands12. And, in the final stages of endocytosis, one of the processes by which cells take up extracellular molecules, signals from the invaginating plasma membrane trigger actin filaments to assemble locally, helping this region of the membrane to become internalized equally an endocytic vesicle. In addition to the network dynamics discussed here, more complex dynamics tin occur when actin filaments interact with disassembly factors such as members of the cofilin family or with polymerases such as members of the formin family.
Both actin filaments and microtubules are polarized polymers, meaning that their subunits are structurally asymmetrical at the molecular level. Equally a result of this structural polarity, both types of polymer function as suitable tracks for molecular motors that move preferentially in one direction. For microtubules, the motors are members of the dynein or kinesin families, whereas for actin filaments, they are members of the large family unit of myosin proteins. These molecular motors have essential roles in organizing the microtubule and actin cytoskeletons. Microtubule-associated motors are crucial for the assembly of the microtubule assortment, in interphase, and the mitotic spindle. These motors besides carry cargo between intracellular compartments along microtubule tracks. Some actin networks, such as the branched networks that underlie the leading border of motile cells, seem to assemble without the help of motor proteins, whereas others, including the contractile array at the rear of a motile cell, require myosin motor activity for their formation and function. Myosin motors also human activity on the bundles of aligned actin filaments in stress fibres, enabling the cells to contract, and sense, their external environment.
Intermediate filaments are the least strong of the three types of cytoskeletal polymer, and they resist tensile forces much more finer than compressive forces. They can be crosslinked to each other, as well as to actin filaments and microtubules, by proteins chosen plectins13, and some intermediate-filament structures may be organized mainly through interactions with microtubules or actin filaments. Many prison cell types get together intermediate filaments in response to mechanical stresses, for instance airway epithelial cells, in which keratin intermediate filaments form a network that helps cells to resist shear stress14. Ane form of widely expressed intermediate filament, consisting of polymerized nuclear lamins, contributes to the mechanical integrity of the eukaryotic nucleus, and phosphorylation of nuclear lamins past cyclin-dependent kinases helps trigger nuclear-envelope breakdown at the start of mitosisxv. Unlike microtubules and actin filaments, intermediate filaments are not polarized and cannot back up directional movement of molecular motors.
Long-range club from curt-range interactions
The cytoskeleton establishes long-range society in the cytoplasm, helping to turn seemingly chaotic collections of molecules into highly organized living cells. Spatial and temporal information from signalling systems, too as pre-existing cellular 'landmarks' such as the 'bud scar' left after division of budding yeast, can affect the associates and function of cytoskeletal structures, but much of the architecture of these structures emerges from simple brusk-range interactions between cytoskeletal proteins. The long-range order that is generated by the cytoskeleton typically refers to cellular dimensions (tens of micrometres), which are large compared with molecular dimensions (a few nanometres).
The mode that cytoskeletal structures form is studied in vivo by genetically eliminating, reducing or increasing the expression of a protein through knockout, knockdown or overexpression experiments, respectively, and is demonstrated in vitro past reconstituting cytoskeletal filament networks from purified proteins. Radially symmetrical arrays of microtubules similar to those establish in interphase cells, for example, can spontaneously assemble from mixtures of microtubules and motorsxvi. The mitotic spindle, which is more complex, has yet to be reconstituted from purified cellular components, simply Heald and colleagues establish that extracts from Xenopus laevis ova undergoing meiosis tin robustly assemble bipolar spindles around micrometre-sized polystyrene particles coated with plasmid Dna17. The formation of such structures shows that spindles tin can cocky-get together in vitro in the absence of both centrosomes (the microtubule-organizing centre in animal cells) and kinetochores (the site on chromosomes to which spindle microtubules adhere to pull the chromosomes apart).
Long-range order of actin-filament networks is created past the activeness of actin-binding proteins and nucleation-promoting factors. One example of how a gear up of elementary rules can result in an extended structure is the formation of branched actin networks (Fig. 2). The Arp2/three complex (which consists of seven proteins, including actin-related protein ii (Arp2) and Arp3) binds to actin and initiates the formation of new actin filaments from the sides of pre-existing filaments, thereby generating highly branched actin filaments that form entangled 'dendritic' networks18. Nucleation-promoting factors activate this Arp2/3-complex-mediated branching. These factors are typically only establish associated with membranes, and they specify the front (or leading edge) of a jail cell, ensuring that the nucleation of new filaments in a dendritic actin-filament network occurs but from filaments growing towards the membrane19 , 20. The growth of all filaments is somewhen stopped by a capping poly peptide, which prevents the improver of more actin monomers21. Taken together, the repeated steps of growth, branching and capping lead to the formation of micrometre-scale, protrusive, branched actin-filament networks, which are of import for crawling motility. A process of disassembly so disrupts the network and recycles the actin subunits for subsequent use22.
Edifice cytoskeletal structures
Long-range order of the cytoskeleton is generated by simple rules for network assembly and disassembly. a, A fluorescence micrograph of a fish keratocyte is shown (with the nucleus in blue). Motile cells such as these grade branched actin-filament networks (scarlet) at their leading edge, and these branched networks generate protrusions. Together with coordinated adhesions to a surface (indicated by vinculin, green) and myosin-driven retraction, the protrusions lead to directed movement. Scale bar, fifteen μm. (Paradigm courtesy of K. van Duijn, Univ. California, Berkeley.) b, At that place are three bones steps involved in the assembly of protrusive, branched actin-filament networks: filament elongation; nucleation and crosslinking of new filaments from filaments close to the membrane; and capping of filaments. Disassembly of the network involves a separate set up of proteins that severs the filaments and recycles the subunits. c, The branching of actin filaments tin be reconstituted in vitro with soluble proteins, generating various branched structures such as those in these fluorescence micrographs of labelled actin (white). (Images courtesy of O. Alike, Univ. California, San Francisco.)
Crosslinkers also affect the structural organization of cytoskeletal networks, particularly as a result of their geometry and binding kinetics. For example, the crosslinker fascin preferentially stabilizes parallel bundles of filaments such as those in filopodia, owing to the rigid coupling between the filament-binding sites on fascin. By contrast, actin crosslinkers such equally α-actinin, in which the filament-binding sites rotate much more freely, tin can stabilize either orthogonal gels (such equally those institute in non-aligned actin-filament networks, which back up the plasma membrane of cells) or parallel bundles. In this case, the compages of the network is determined by the kinetics of the interaction. If the dissociation rate of the crosslinker from the actin filaments is high, so filaments are aligned into bundles. If the dissociation charge per unit is low, then filaments are stabilized in a more randomly ordered country23.
Some cytoskeletal structures tin can span distances much larger than that of the typical cell. 'Cytonemes' and 'membrane nanotubes', which contain actin filaments and are essentially specialized filopodia, can abound to lengths of millimetres and have been shown to mediate cell–prison cell signalling across sea-urchin blastulae and Drosophila melanogaster fly imaginal discs24. The mechanisms that enable filopodium-like structures to grow to such extreme lengths may involve cooperative interactions between the elastic properties of the actin filaments and the plasma membranes, stabilizing the protrusions against buckling afterward a membrane tube has formed25.
Network compages and mechanics
Although distinct in their backdrop and the types of network they form, the polymers of the cytoskeleton are intricately linked together. The organisation of these links and the resultant architecture of the cytoskeletal networks has a central role in transmitting compressive and tensile stresses and in sensing the mechanical microenvironment26. Structures formed from microtubules, actin filaments or intermediate filaments interact with each other and other cellular structures either nonspecifically (through steric interactions and entanglement) or specifically (through proteins that link one filament type to another). For case, the actin nucleation-promoting factor WHAMM binds not but to actin merely also to microtubules and membranes27, and the GTPase Rac1 is activated by the growth of microtubules, which in plough stimulates the polymerization of actin in lamellipodial protrusions28. This interconnectivity creates continuous mechanical coupling through the cytoskeleton, providing a means for internal or external forces and fluctuations to be distributed throughout the cell. But, despite their interconnectivity, cytoskeletal networks take typically been investigated by studying the component polymers individually to understand their contributions to prison cell mechanics.
The mechanical response of gels formed from purified cytoskeletal filaments that have been reconstituted in vitro provides insight into the properties of these polymers in cells. Actin-filament networks have been of considerable interest because of the variety of structures they form and because they function every bit model semi-flexible polymers29. As such, their elasticity can arise from ii sources: entropic elasticity, which results from a reduction in configurations available to thermally fluctuating filaments, such as when they are stretched; and enthalpic elasticity, which is due to changes in spacing of the molecules that make up the filaments, such as when they are bent, even in the absenteeism of thermal fluctuations. The importance of these two rubberband contributions seems to depend on the architecture of the network.
When shear stresses are applied to actin-filament networks, as well as to networks of intermediate filaments or extracellular-matrix filaments such as collagen and fibrin, the networks stiffen and resist boosted deformation, as a result of filament entanglement (in which the displacement of i filament is impeded by some other filament) and the entropic elasticity of private filaments30. When a rigid crosslinker such equally scruin is added to randomly organized actin filaments and shear stress is applied, the magnitude of the rubberband modulus (a measure out of the resistance of the network to deformation) increases significantly, and the network retains the stress-stiffening behaviour attributed to the entropic elasticity of private filaments31 , 32. When the more than flexible crosslinker filamin A is added to randomly organized actin filaments together with the molecular motor myosin, the rigidity of the network increases to more than that of an entangled filament network, and the network stiffens nonlinearly equally though it were subject to external stress33. These studies demonstrate the importance of the entropic elasticity of filaments in the mechanical backdrop of networks without specific filament orientation.
By contrast, in networks with highly organized architectures, the bending of actin filaments, rather than their entropic stretching, can dominate the rubberband properties. When branched actin-filament networks, such as those growing at the leading edge of crawling cells, are exposed to compressive forces, the network shows nonlinear stress stiffening, followed by stress softening at high stresses34. Interestingly, the softening behaviour is completely reversible, every bit would be expected from the buckling of actin filaments oriented towards the load, suggesting that filaments bearing a compressive load can exist of import contributors to network elasticity. Although the bending and stretching of filaments has been the focus of many actin-filament network studies, the mechanical properties of crosslinked networks must also depend on the properties of the crosslinkers themselves. In one written report of crosslinker length, variation in the spacing between actin-binding domains significantly afflicted both the elastic modulus and the network architecture, with short crosslinker lengths resulting in high stiffness and arranged filament arrangements35. In general, the highly nonlinear behaviour of filament stretching and bending, together with the organizational constraints imposed by crosslinkers and nucleation-promoting factors, indicates the importance of network architecture in determining the mechanical behaviour of the cytoskeleton.
In whole cells, the actin cytoskeleton has a wide variety of architectures that are associated with specific functional structures (Fig. 3). By using in vitro reconstitutions such every bit those described above, researchers are starting time to identify the fundamental molecular determinants of filament social club, just how these actin-filament structures are linked to other cellular systems remains an important area of investigation. In detail, the deformation of cytoskeletal networks in response to mechanical load is coupled to changes in plasma-membrane tension and displacement of fluid in the cytoplasm. For case, Herant and colleagues observed pregnant increases in plasma-membrane tension as neutrophils engulfed antibiotic-coated beads past using phagocytosis, which is an actin-driven process36. Furthermore, the resistance to menstruation through dense cytoskeletal networks, known as poroelasticity, can dull the flow of fluid to the point at which it takes several seconds for stress to propagate across an individual cell37, in contrast to times of the order of microseconds for direct mechanical coupling through tensed filaments38.
Form meets function
The cytoskeleton forms structures that have a wide variety of architectures and are associated with different types of cellular strength. Shown are four structures generated by actin filaments and the stresses typically encountered past these structures (red arrows, compression; green arrows, tension). a, Branched actin-filament networks push against the plasma membrane and external barriers every bit they generate protrusions, thereby encountering an in force of compression. b, Filaments bundled into filopodia also generate protrusive forces as they extend from the cell body, encountering like compressive force. In this case, the linker molecule is the bundling poly peptide fascin. c, Cortical networks (that is, non-aligned networks), such as this i involving filamin equally a crosslinker, form below the plasma membrane and deport tension loads in multiple directions. d, Stress fibres form from bundled actin filaments, shown here associated with filaments of myosin, and generate tension confronting prison cell adhesions to the extracellular matrix. The illustrations are based on micrographs from refs 83 - 86 (a–d, respectively).
The broad range of cytoskeletal architectures and mechanisms for stress transmission has presented considerable challenges to the evolution of a complete model of the cytoskeleton. Appropriately, different theoretical frameworks have been developed to capture diverse aspects of the commonage behaviour of filament networks and the cytoplasm. For example, filament hydrodynamics and molecular motors are included in a theory of active polar gels39. Some models have also attempted to describe the complex viscosity of cytoskeletal networks, which, at the molecular scale, partly arises from the binding kinetics of crosslinkers. The range of relaxation timescales that has been observed for cells at temporal frequencies (measured in hertz) has led to the suggestion that the cytoskeleton behaves as a glassy fabric that transitions between several kinetically trapped states40. What is needed now are more process-level models that connect the cytoskeleton with the plasma membrane or other physical purlieus atmospheric condition and provide an understanding of cellular behaviours that depend on membrane–cytoskeleton interactions, as in a recent study describing shape variations in motile cells41.
What is the value of determining the cytoskeletal architecture and mechanical backdrop of a cell? In short, this information tin provide insight into where forces are acting. Stress practical to a cell is distributed broadly by the cytoskeleton, just the magnitude of transmitted stress to a particular location depends on network mechanics and architecture and tin take marked furnishings on cellular processes, from private filament polymerization up to unabridged network reorganization. Growing microtubules that encounter resistance decrease their growth rate exponentially equally the strength increases42 and have a greater likelihood of complete disassembly of the polymer. The polymerization of a small number of actin filaments is similarly limited by force43, but filaments growing as function of a dendritic actin-filament network bear differently from predictions based on the single-filament model of forcefulness dependence. When reconstituted on the end of an atomic-strength-microscope cantilever, dendritic networks that feel increasing loads abound at a constant velocity over a wide range of forces, suggesting that the network adapts to the increasing load by increasing local filament density through a force-sensing chemical element in the network44. This constant-velocity behaviour of actin-filament networks under increasing load was also observed in measurements of lamellipodial protrusions in crawling cells45. The complication of network growth, in contrast to that of single filaments, highlights the importance of thinking nigh the collective behaviour of cytoskeletal structures rather than only private filaments. This challenge becomes even greater when considering how the cytoskeleton interacts with external signals.
Sensing the mechanical microenvironment
Cells are intricately connected to the external environment through their cytoskeleton. Whether in directly contact with neighbouring cells or with a dumbo meshwork of polymers known equally the extracellular matrix, cells receive external signals that guide circuitous behaviours such equally motility and, in some cases, differentiation (for case from stalk cells into cells of a specific lineage). Whereas the contribution of chemical signals has long been understood, physical signals have simply recently been widely recognized to be pervasive and powerful. The observation that physical properties of the microenvironment tin can affect jail cell shape and behaviour dates to the 1920s, when studies showed that mesenchymal cells embedded in clots of diverse stiffnesses had different shapes (run across ref. 46 for a review). More recent studies have shown that the tension generated past a contracting cytoskeleton can be used to sense the mechanical properties of the extracellular matrix, which in turn have been shown to affect cytoskeletal organization and jail cell behaviour47, although whether stiffness or force is the most important point remains a subject of debate48.
Of particular interest is how cell–extracellular matrix and cell–jail cell interactions can lead to long-lived changes in cellular system in tissues and cell behaviour. Several studies accept highlighted the importance of physical cues in the arrangement of tissues during development. For case, Thery and colleagues constitute that the orientation of the mitotic spindle in dividing cells, and hence the location of the division plane and the spatial arrangement of the daughter cells, is affected by the spatial distribution of extracellular matrix proteins49. Using microcontact printing to define patterns of extracellular matrix proteins to which cells adhere, they institute that the cells divided with predictable orientations controlled by cortical contacts with the extracellular matrix. In improver to the pattern of adhesion sites, the mechanical backdrop of cells themselves also contribute to tissue arrangement. As one case, in gastrulating zebrafish embryos, actin- and myosin-dependent plasma-membrane tension and differential adhesion among cells drives the sorting of germ-layer progenitor cellsl. Cell proliferation tin even be affected when external forces are applied to tissue (Fig. four). When a tumour spheroid, formed from murine mammary carcinoma cells grown in a cluster within an agarose gel, is exposed to compressive loads during growth, the cells proliferate more slowly at regions of high stress and undergo programmed cell death when exposed to sufficiently high stress51.
Force and shape
a, Unmarried cells can exert big contractile forces that touch on their shape. An osteosarcoma cell attached betwixt the cantilever of an atomic forcefulness microscope and a surface can exert contractile forces (ruddy arrow) of more than 100 nN (depicted diagramatically from left panel to eye console and in a fluorescence micrograph, right panel). Actin-filament structures (white), including contractile stress fibres spanning the upper and lower surfaces, are generated in the contracting osteosarcoma jail cell (right). Scale bar, 10 μm. (Image reproduced, with permission, from ref. 87.) b, Mechanical stress on cancer cells in three-dimensional tumour spheroids changes their growth. There are fewer proliferating cells (green) in tumour spheroids (cherry) at the regions of highest compressive stress (red arrows) than at the regions of low stress (white arrows). The image on the right is an overlay of the centre and left images. Calibration bar, 50 μm. (Images reproduced, with permission, from ref. 51.)
When the 'normal' mechanical properties of tissue are disrupted, the effects tin exist considerable. In epithelial cell layers, altered stiffness of the supporting tissue disrupts morphogenesis and drives the epithelial cells towards a cancerous phenotype52. In fact, the stiffness of the substrate seems to be of import for stalk cells to differentiate properly. A substrate with a stiffness that emulates normal tissue tin part equally a developmental cue that directs stalk cells to differentiate into cells of specific lineages, including mesenchymal stem cells53 and neural stem cells54. How substrate stiffness, besides as growth factors and matrix properties, affect stem-prison cell differentiation is reviewed in more detail in ref. 55.
Emulating the native backdrop of a cell'southward surroundings is an important consideration that is oftentimes overlooked when studying cells ex vivo. For example, the traditional method of culturing cells on stiff substrates, which has been used for decades, tin can itself bulldoze changes in the mechanical backdrop and gene-expression profiles of the cells. Primary human foreskin epithelial cells cultured in plastic dishes were plant to increase in stiffness with passaging, with cells beingness twofold to fourfold stiffer afterward eight passages than were cells passaged fewer than three times56. Similarly, human being epithelial breast carcinoma (MCF7) cells were constitute to stiffen with increasing passage number when cultured on glass coverslips57 and endometrial adenocarcinoma cells cultured in plastic dishes expressed more α-actin equally a function of passage number, as they moved towards a stromal phenotype58. In each of these cases, the cells were grown on substrates with markedly different mechanical properties from native tissue, and long-lived changes in cytoskeletal properties and cytoskeletal organization were observed.
As more examples of cells responding to mechanical cues through the cytoskeleton are establish, the questions of how, what and where concrete inputs are sensed is becoming fundamental. There is substantial evidence implicating stress-induced changes in focal adhesions and adherens junctions26, and several molecules have been identified as specific mediators of mechanical inputs. For example, mesenchymal stem cells differentiate into cells of various lineages depending on the substrate stiffness, and this elasticity-sensitive lineage-specific differentiation is blocked past inhibiting the protein non-muscle myosin53. A force-induced conformational change in p130Cas (also known as BCAR1), a scaffolding protein that is involved in focal adhesions, causes it to be more hands phosphorylated past Src59. And sensitivity to the elasticity of the extracellular matrix during angiogenesis is mediated by the Rho inhibitor p190RhoGAP (also known as GRLF1), through its effect on ii antagonistic transcription factorssixty.
Although changes in gene expression may exist the end indicate of mechanosensing, the process tin take days for cells in civilisation, and it is unclear how information about physical interactions with the mechanical micro environment is stored. Are heritable changes in gene expression in mechanically perturbed cells due simply to changes in chromatin construction and organization or other familiar epigenetic mechanisms? The heritability of changes that ascend from mechanical interactions — and that are mediated by the cytoskeleton — raises the question of whether the organization and reorganization of long-lived cytoskeletal structures themselves might have a function in recording the prison cell's mechanical 'history'.
Cytoskeletal epigenetics
The idea that cellular structures tin can exist passed on and influence the behaviour of subsequent generations of cells is not new. In the 1930s, embryologists recognized that regional differences in the molecular limerick of ova caused girl cells to inherit dissimilar surface molecules61. In the 1960s, Paramecium aurelia cells that were genetically identical to wild-type cells were plant to pass on alterations in the orientation of their cilia for hundreds of generations62 , 63. Internal structures of the cytoskeleton can likewise persist afterwards cells divide. Daughter 3T3 fibroblast cells have similar actin-filament stress-fibre system and motile behaviour64 , 65, and adjacent epidermal 'siamese twin' cells in Calpodes ethlius caterpillars take the same number of actin-filament bundles66. The emergence of principal cilia from sister cells was recently plant to depend on centriole historic period, with sister cells that inherit the onetime centriole growing a primary cilium before sister cells that inherit the new centriole, which was formed earlier jail cell sectionalization67.
As a wait at any dish of cultured cells volition ostend, genetically identical cells tin can have markedly different cytoskeletal structures, presumably as a result of random events also equally slight differences in external conditions. Endothelial cells grown in vitro under similar weather condition, for example, contain stress fibres that are oriented randomly. Merely, if those cells are exposed to shear stress from fluid flowing above them, they respond by elongating and orienting their stress fibres in the direction of the flow. If the shear stress is removed, then the variability in stress-fibre orientation returns, simply it does and then slowly. Interestingly, if the elongated cells are discrete from the surface, then the elongated shape persists68. In this way, the cytoskeleton can be a record of a cell's past mechanical interactions. Given the interconnectedness of the cytoskeleton and its role in the transduction of mechanical signals from the external microenvironment, too as its function as a scaffold for many reactions69, the ability of cytoskeletal structures to tape the past may event in the cytoskeleton profoundly affecting the cell'due south future and even the future of the cell's progeny70.
Given that cytoskeletal structures are often highly dynamic, with specific factors that promote disassembly and recycling of the cytoskeletal building blocks competing with factors that assemble and stabilize them, is it possible for mechanical inputs to be recorded? During endocytosis, actin-filament networks can get together around clathrin-coated pits and readapt the invaginated membrane in less than 15 seconds71. But, when the timescales for assembling and stabilizing a cytoskeletal construction are longer than those for disassembling and recycling it, the result can be a persistent construction that affects the behaviour of a cell over a longer timescale than the initial signal. In essence, the system shows hysteresis, a common characteristic of magnetic, electrical and elastic properties of materials, in which there is a lag betwixt awarding or removal of a stimulus, such as a force, and its effect. In biological systems, this hysteresis seems to involve agile, energy consuming processes. In one example, a growing actin-filament network that had been reconstituted in vitro was exposed to a weak compressive force during growth. When the compressive strength on the network was gradually increased and and then apace reduced to the previous level of strength, the velocity of network growth increased and persisted at a rate that was significantly higher than the original velocity at that force44. It is probable that this increase in velocity resulted from an increase in actin-filament density caused past the transient increase in load, with the system showing hysteresis. In contrast to molecular motors, for which the human relationship between force and velocity is immediately reversible, the observation that there is more ane growth velocity for a given force suggests that actin-filament network growth depends on history. The cytoskeletal construction and the process by which information technology is congenital tin can record mechanical interactions, whereas a unmarried filament could non.
If mechanical interactions with the external environment can change the cytoskeleton in a lasting manner, what are the implications? To the extent that the cytoskeleton is intricately involved both mechanically and biochemically in cellular processes such as cell division and move, long-lived cytoskeletal structures could create variability in jail cell behaviour and may guide variation towards certain phenotypes. This behaviour could be as transient equally the mirror-prototype movements of sis cells65 or every bit permanent as alterations in cell fate53. Although it is plausible, the hypothesis that specific cytoskeletal structures are necessary and sufficient determinants of cell behaviour has but just begun to be explored in item. Farther research at the level of tissues, cells and reconstituted cytoskeletal structures is needed to understand when and how the cytoskeletal history can markedly influence a cell'southward future.
Future research on the cytoskeleton
With the experimental techniques at present available for studying the cytoskeleton, ranging from super-resolution imaging of molecular organization to directly physical manipulation of cellular structures and processes, there are many opportunities to probe the link betwixt concrete strength and cell behaviour. To guide experiments, new models are needed to search for the mechanisms and molecules that link cell mechanics and the cytoskeleton to cellular controlling. Computational simulations will become increasingly important as a way of testing the properties of hierarchical ordered systems. If successful, this effort to follow forces, whether internally generated or externally imposed, from the mechanical input to the phenotypic output could have a profound impact on our agreement of how normal and diseased cells behave.
Because of the cardinal part of the cytoskeleton in cell structure and intracellular organization, perturbations in the architecture of any of the 3 primary cytoskeletal networks tin can upshot in marked pathologies. For example, if the mitotic spindle does not function properly, dividing cells cannot sectionalisation the genetic material as between the daughter cells. Chromosomal instability is associated either with a loss of the checkpoint that ensures that all chromosomes are correctly attached to microtubules in the mitotic spindle or with the presence of likewise many microtubule-organizing centres at the fourth dimension of jail cell division72. And mutations in the genes encoding intermediate filament proteins are associated with many diseases in humans (see ref. 73 for a review), including a predisposition to liver illness in the case of some keratins, amyotrophic lateral sclerosis (also known equally Lou Gehrig's disease) in the case of a neuronal course of intermediate filament called neurofilaments, and progeria (a hereditary form of premature ageing) in the case of improperly assembled nuclear lamins.
The in vitro reconstitution of purified proteins will continue to be a powerful tool for identifying the conditions that are necessary and sufficient for a cytoskeletal process and could guide the search for therapeutic drug targets and candidate drug molecules. For example, a decrease in the activity of cardiac-muscle myosin proteins is associated with heart failure, and myosin motors are at present important targets for cardiovascular drug therapy. Reconstitution experiments that involve just a few proteins take the potential to uncover circuitous physical behaviour and long-range structural organisation (Fig. 5). Like cells, reconstituted cytoskeletal polymers are sensitive to the physical boundary conditions imposed past their environment. Circumscribed cytoskeletal polymers within lipid vesicles affects both their dynamics and their organization, equally has been shown for microtubules74 and actin filaments75, and this can be used to test the effect of forces on the compages and function of the cytoskeleton. Every bit experiments with purified proteins increment in complexity and in fidelity to cellular processes, new methods volition be needed so that non merely cytoskeletal structures but as well integral membrane proteins and metabolic processes can be reconstituted76.
Learning by building
The reconstitution of cytoskeletal structures is a fundamental method for understanding how functional behaviour emerges from discrete components. In this instance, actin filaments were nucleated from chaplet coated with the nucleation-promoting factor ActA (not shown) and were then crosslinked by fascin inside a unilamellar lipid vesicle. a, Purified proteins were loaded into a vesicle by a microfluidic encapsulation technique that allows the dynamics of filament associates to be observed immediately subsequently encapsulation88. b, The micrograph (left) shows fluorescently labelled actin filaments (white) that have polymerized within the vesicle and take assembled into a fascin-crosslinked network. Calibration bar, 5 μm. The diagram (right) is a schematic depiction of the actin-filament network present in the inset box of the micrograph. (Prototype courtesy of D. Richmond, S. Hansen and Thousand. Zanic, Physiology Course, Marine Biological Laboratory, Woods Pigsty, Massachusetts.)
Until not long agone, eukaryotic cells were idea to exist distinguished from bacteria and archaea by the presence of a cytoskeleton. But the discovery of cytoskeletal polymers even in comparatively simple cells of modest size and genome are revealing the cardinal importance of internal arrangement for cell function. Now, clues to the origin of the eukaryotic cytoskeleton and the polymers that establish it are emerging from the study of leaner. Filamentous proteins with homology to actin filaments, microtubules and intermediate filaments take been identified and shown to have a function in organizing the bacterial cytoplasm. Rod-shaped bacteria such every bit Escherichia coli require an actin-similar polymer formed from MreB to be assembled in order to ascertain their shape77. To abound into its curved shape, Caulobacter crescentus (also known equally Caulobacter vibrioides) requires an additional cytoskeletal component, an intermediate-filament-like poly peptide called crescentin78. Other polymers have a role in organizing the DNA in bacteria. ParM, for example, is an actin-like protein that forms polymers required for segregating type II plasmids during prison cell sectionalisation79. Reconstituting ParM-dependent DNA segregation in vitro has revealed a machinery by which large, slowly diffusing cargo can be moved speedily through the bacterial cytoplasmfourscore. More than 35 actin-like proteins have been identified in bacteria, but well-nigh remain to be characterized81.
In the lecture quoted at the beginning of this Review, Weiss also stated: "Life is a dynamic process. Logically, the elements of a procedure can be merely elementary processes, and not elementary particles or any other static units. Prison cell life, appropriately, tin can never be divers in terms of a static inventory of compounds, notwithstanding detailed, merely only in terms of their interactions"1. The cytoskeleton is a manifestation of those elementary interactions, exemplifying the rich behaviour that can emerge in hierarchically organized systems. The progress over the by 50 years indicates that the commonage properties of the cytoskeleton, including the compages of the networks and their mechanical history, are essential for understanding the push and pull of cellular behaviour.
Acknowledgements
We thank O. Chaudhuri, D. Richmond, V. Risca and other members of the Fletcher laboratory for discussion and assistance with this Review. We also benefited from interactions with the researchers and students in the 2009 Physiology course at the Marine Biological Laboratory, Wood Hole, Massachusetts. Work in our laboratories is supported by R01 grants from the National Institutes of Health (NIH) and by the Cell Propulsion Lab, an NIH Nanomedicine Development Middle. We repent to those colleagues whose piece of work could not be cited considering of infinite constraints.
Footnotes
The authors declare no competing fiscal interests.
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Cell Shape Is Reinforced By,
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