The structural properties of the cytoskeletal polymers
- Intermediate Filaments
|Fig. 1: Structural Components of an Eukaryotic Cell. This image of an epithelial cell taken by Steve Rogers, UIUC Fluorescence Gallery, (used with permission) shows fluorescent staining of some of the structural components of a cell. Larger view.|
Eukaryotic cells are both very small and very complex a typical eukaryotic cell spans ~20 µm with a 6-8 µm nucleus. Their constituents and associated signaling pathways have fascinated molecular biologists for a long time. The interior of the eukaryotic cell is a complex material made of carbohydrates, lipids, nucleic acids and proteins, as seen in this Web site offering a virtual tour of the cell.
|Fig. 2 Cytoskeletal Polymers. This image shows a schematic of the individual structures of the three cytoskeletal polymers actin (left), microtubules (center) and intermediate filaments (right). Although actin and microtubule monomers assemble to form one type of filament, different cells have different types of intermediate filaments such as keratin, vimentin, desmin and neurofilaments. The interaction of the cytoskeletal polymer assemblies results in a composite network that spans the entire interior of the cell, as seen in Figure 1. Together, the cytoskeletal polymers perform crucial cell functions such as organelle transport, signal transduction, cell motility and cell division.(Larger view) (Drawings by author.)|
A cytoskeleton spans the entire interior of the cell. It is a network of three distinct proteinaceous polymers: actin, microtubules and intermediate filaments . These polymers inside the cell have been imaged in detail in recent years, as shown in Fig. 1 and the other images of this [University of Illinois, Urbana-Champaign] fluorescence gallery. The actin cytoskeleton, a meshwork of short semi-flexible actin filaments, is found predominantly just beneath the cell membrane (Fig. 1 and Fig. 2), with its concentration decreasing towards the cell interior. Microtubules are rigid rod-like polymers that originate from the centrosome located close to the nucleus and extend towards the actin network at the cell periphery (Fig. 1 and Fig. 2). Intermediate filaments are flexible coil-like polymers, acting like molecular cables that connect the nucleus to the plasma membrane and that create a fibrous network throughout the cell interior (Fig. 2).
These three cytoskeletal polymers are connected in vivo via several accessory proteins, such as crosslinking-, binding- and motor-proteins, to form a compound or composite material that is the main determinant of the cell's structural or elastic properties, assumed here to imply the passive deformation response of the cell to an applied force . The composite cytoskeleton's elastic properties allow the cell to respond to (show a deformation to) a wide range of forces, as it is subjected to in the body. For example, while a cell moving in the body is soft or deformable enough to squeeze through surrounding tissue, it can also resist high stresses such as an osmotic pressure. Thus, this composite material inside the cell endows it with unique and versatile structural properties, which cannot be obtained by any one of the polymers alone.
These cytoskeletal structural properties are relied on by the cell in its functioning. The elastic properties of the actin cytoskeleton, for instance, are critical during cell movement. When they are altered due to molecular changes in the actin cytoskeleton, as in a cancer cell , so are its movement and functioning . This tight coupling between the functioning of the cell and its structural response leads us to expect that understanding the cell's structural properties will shed further light on its functioning as well. Hence, in recent years, several cell deformation techniques such as Atomic Force Microscopy (AFM), Micropipette Aspiration, Microplates, Optical Tweezers and Optical Stretcher have been developed to better understand the relation between the cell's deformation response to an external force and the structural properties of the individual cytoskeletal polymers (see cells being deformed by the opposing laser beams of the Optical Stretcher). This article describes the current state of knowledge, after these investigations, regarding the role of each of the cytoskeletal polymers in determining the overall structural properties of the cell.
There is ample experimental and theoretical evidence that demonstrates that of the three cytoskeletal polymers, actin networks dominate the cell's structural response, of which some important observations are discussed below.
|Fig. 3: Crosslinked Actin Network Structure of the three dimensional network formed by the actin crosslinking protein filamin, adapted from . An actin mesh or network crosslinked by filamin is structurally strong. Filamin (yellow) is a homodimeric protein, 160 nm in length. Hence, it is very flexible and can tether two actin filaments (red) with each of its arms even at large angles. (Drawings by author.) (Larger view)|
Another experiment has studied the deformation of fibroblasts (rat embryo cells) by probing them with glass needles . By simultaneously injecting the cell with green fluorescent protein that enables the visualization of cytoskeletal components, their role in cell shape and deformation has been observed. The experiment shows that the elastic response of the cell can almost exclusively be attributed to the actin cytoskeleton. The microtubules and cell interior show a more fluid-like behavior, while the nucleus also shows an elastic response.
In vitro rheology experiments on actin networks have also been performed to study their elastic properties. Rheology is a common technique used to study the elasticity of reconstituted polymer networks and yields the network's modulus, a measure of its elasticity, as a function of time or frequency (in Hz). When the elastic properties modulus of the polymer network varies with time, the material is termed viscoelastic. In terms of frequency, the rheology experiments on actin networks show that the network displays an elastic behavior at low frequencies between 0.02 and 100 Hz and a viscoelastic behavior at frequencies above 100 Hz.
Rheology experiments on crosslinked actin networks reveal the crucial role of crosslinkers such as filamin (Fig. 3) or α-actinin in determining the network's structural properties a network that is crosslinked has a much higher elastic modulus than one that is not. Theoretical estimates of the structural strength of crosslinked actin networks show that the actin cytoskeleton contributes significantly to cell elasticity when both the actin and crosslinker are concentrated into a specific region of the cell (note that most of the actin in vivo is concentrated in the leading edge of a moving cell or in the actin cortex beneath the cell membrane of suspended cells) . In this case, the estimated elastic modulus of the actin network, calculated using the theory of crosslinked polymers with in vivo actin and crosslinker concentrations, is close to the moduli values of the cell obtained from whole cell deformation experiments such as the Optical Stretcher ; the elastic moduli of cells range from 100s of Pa to 1000s of Pa depending on the cell type and measurement technique used, while the estimated elastic modulus of crosslinked actin networks with in vivo concentrations is also 100s of Pa.
|Fig. 4: Stress-Strain Characteristics of the Cytoskeletal Components (actin, microtubules and intermediate filaments) from rheology experiments. Stress is a measure of the force exerted on the polymer network, while the strain is a measure of the deformation of the polymer network. Note that vimentin is a type of intermediate filament; fibrin is a biopolymer whose structural response was investigated in this study as a comparison. (Courtesy Rockefeller University Press . Used with permission.) (Larger view)|
However, experiments on whole cells reveal that the structural strength of the in vivo actin network is necessary but not sufficient to explain cell elasticity . In addition, from in vitro rheology experiments, actin filament networks are seen to break at strains > 20% (Fig. 4), while the cell can withstand higher strains (strain is a measure of deformation, and its simple definition is the ratio of the change in length to the original length of the material).
Thus, the above observations demonstrate that while actin networks clearly play a key role in the structural response of cells, the actin cytoskeleton alone cannot adequately explain the cell's entire structural response, especially at large strains.
In vitro rheology experiments show that intermediate filament networks, on the other hand, can withstand strains up to 80% without rupture (Fig. 4). At small strains, the linear elastic modulus of the intermediate filament network is estimated to be very low (~10-5 Pa). However, intermediate filament networks display strain hardening, which means that their modulus increases significantly at higher strains. This occurs because as the slack in the flexible intermediate filaments gets pulled out at higher strains, they become stiffer and stiffer. Eventually, when the slack is completely pulled out, they behave like rigid rods. This process of straightening out the individual filaments leads to a large increase in the network stiffness (modulus) . Thus, although intermediate filament networks play an insignificant role at small deformations, experiments show that they play a key role in the cell's structural response at large deformations .
The exact structural contribution of microtubules to cell strength is not yet clear but existing experimental evidence suggests that they do not play a major role. As mentioned earlier, experiments involving drugs to depolymerize microtubules show that cell properties such as elasticity are not significantly affected by the absence of microtubules. In fact, other studies on neutrophils (white blood cells) show that cells without microtubules can still perform normal and crucial functions such as motility , while cells cannot move without actin. Analyzing the deformation of a computational cell-like structural model created with actin and with and without microtubules shows quantitatively that microtubules do not greatly impact cell elasticity relative to actin . However, although microtubules alone may not contribute much structurally, their presence could stabilize the other structural elements in the cell. They could also play a role in transmitting forces to other interior structural elements in the cell, such as the nucleus.
Although not one of the cytoskeletal polymers, the nucleus is also a structural element of the cell. Some studies show that its modulus is ~10 times higher than that of the cytoskeleton, and is of the order of 5000 Pa . This implies that the cytoskeleton, rather than the nucleus, is what is primarily deformed, unless the cell is subjected to very high forces.
Experimental studies, in conjunction with cell modeling, show that the actin cytoskeleton is the main determinant of the structural response of cells. However, the other cytoskeletal polymers are also required to adequately explain the cell's overall structural response, and to maintain cell integrity. The structural properties of each cytoskeletal polymer are ideally suited to its function and location in the cell. Since the actin network is located near the cell periphery, it has elastic properties that are suited to withstand forces exerted on the cell from the outside. At low strains, the linear modulus of the actin network is high and it can respond to the applied force and offer elastic resistance. However, at high strains that cause the actin network to yield or break, intermediate filament networks likely take over due to their strain hardening properties, preventing cell lysis or rupture even at high strains. The microtubules aid these other structural elements but may not play a crucial structural role by themselves. Together, these cytoskeletal polymers create a composite material that not only has optimal structural properties for the functioning of the cell but also has structural properties that are unrivaled by any man-made (synthetic) polymer.
I gratefully acknowledge the continued support of my thesis advisor Josef Kaes. I also thank Jochen Guck, Falk Wottawah, Stefan Schinkinger, Bryan Lincoln, Timo Betz, Allen Ehrlicher and Bjoern Stuhrmann of the Kaes lab and Melanie Thein of the Laboratory of Cell and Computational Biology. I also wish to thank my postdoctoral advisor Alex Mogilner for his interest and support of this work.
B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, Molecular Biology of the Cell, Fourth Edition, Garland Science, New York, 2002.
J. Guck, S. Schinkinger, B. Lincoln, F. Wottawah, S. Ebert, M. Romeyke, D. Lenz, H. M. Erickson, R. Ananthakrishnan, D. Mitchell, J. Kas, S. Ulvick, and C. Bilby, Biophys. J., 88(5), 3689-98, 2005.
C. Rotsch, K. Jacobson, J. Condeelis, and M. Radmacher, Ultramicroscopy, 86(1-2), 97-106, 2001.
C. Rotsch, and M.Radmacher, Biophys J., 78(1), 520-35, 2000.
S. R. Heidemann, S. Kaech, R. E. Buxbaum, and A. Matus, J Cell Biol., 145(1),109-22, 1999.
R. Ananthakrishnan, PhD Thesis, University of Texas at Austin, 2003; J. Guck, R. Ananthakrishnan, H. Mahmood, T. J. Moon, C. C. Cunningham, and J. Kas, Biophys J., 81(2):767-84, 2001.
R. E. Mahaffy, C. K. Shih, F. C. MacKintosh, and J. Kas, Phys. Rev. Lett., 85(4), 880-3, 2000.
S. Yamada, D. Wirtz, and S. C. Kuo, Biophys J., 78(4), 1736-47, 2000.
P. A. Janmey, U. Euteneuer, P. Traub, and M. Schliwa, J. Cell Biol.,113(1), 155-160, 1991.
C. Storm, J. J. Pastore, F. C. MacKintosh, T. C. Lubensky, and P. A. Janmey, Nature, 435(7039), 191-4, 2005.
N. Wang, and D. Stamenovic, Am. J. Physiol. Cell Physiol., 279(1), C188-94, 2000.
P. Hofman, L. d'Andrea, E. Guzman, E. Selva, G. Le Negrate, D. F. Far, E. Lemichez, P. Boquet, and B. Rossi, Eur. Cytokine. Netw., 10(2), 227-36, 1999.
R. Ananthakrishnan, J. Guck, F. Wottawah, S. Schinkinger, B. Lincoln, M. Romeyke, and J. Kas, Current Science, 88(9), 1434-1440, 2005.
N. Caille, O. Thoumine, Y. Tardy, and J. J. Meister, J Biomech., 35(2), 177-87, 2002.
One of the classic and well-known cell biology text books. The site contains several illustrative animations and videos for each chapter. Molecular Cell Biology, 5e by Harvey Lodish, Arnold Berk, Paul Matsudaira, Chris A. Kaiser, Monty Krieger, Matthew P. Scott, Lawrence Zipursky, and James Darnell
A simple cytoskeleton tutorial.
A more detailed description of the cytoskeletal filaments and their functions in the cell.
A nice background to the cytoskeleton.
A very detailed power point presentation on the cytoskeleton. The first slide says that the aim of the presentation is to explain "the structural properties of the proteins that make up the cytoskeleton, how are the filaments assembled, localisation and function of the cytoskeleton in cells and some methods for studying the cytoskeleton." The level of this presentation may be more appropriate for college students taking cell biology.
Desprat N, Richert A, Simeon J, Asnacios A. Creep function of a single
living cell. Biophys
A recent and elegant paper on deforming individual cells with parallel plates in order to understand the relation between the deformed cell shape and the mechanical or structural properties of the cell.
Haga H, Sasaki S, Kawabata K, Ito E, Ushiki T, Sambongi T. Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cytoskeleton.
Ultramicroscopy. 2000 Feb;82(1-4):253-8.
Using the technique of Atomic Force Microscopy, the authors study the contribution of the individual cytoskeletal elements to cell elasticity in fibroblast cells. They find that the elastic properties of the cells are controlled by the actin cytoskeleton as well as intermediate filament network, but are not significantly governed by microtubules.
Stamenovic D, Coughlin MF. A quantitative model of cellular elasticity based on tensegrity. J Biomech Eng. 2000 Feb;122(1):39-43.
This paper attempts to create a simple model of the cell to study its steady-state elastic response. The cell-model incorporates the actin filaments and microtubules - which are modeled as inter-connected elastic cables and struts respectively - and predicts the lower and upper bound for the elasticity of cells.
Mahaffy RE, Park S, Gerde E, Kas J, Shih CK. Quantitative analysis of the
viscoelastic properties of thin regions of fibroblasts using atomic force
This paper uses the technique of Atomic Force Microscopy to study the elastic properties of cells. This as well as previous work of the authors shows that the visco-elastic properties of cells are similar to that of in vitro actin networks, implying that they contribute significantly to the cell's structural response.
Gardel ML, Shin JH, MacKintosh FC, Mahadevan L, Matsudaira P, Weitz DA.
Elastic behavior of cross-linked and bundled actin networks. Science.
2004 May 28;304(5675):1301-5.
An elegant experimental-based paper that studies the elastic properties of in vitro crosslinked actin networks using the technique of rheology. Such a detailed study that predicts the elastic range of an actin network in vitro also has implications for its elastic behavior in vivo.