Structural Biochemistry/Cell Organelles/Cytoskeleton

The structural biochemistry of the cytoskeleton is very essential to the cell body



The cytoskeleton provides support in a cell. It is a network of protein fibers supporting cell shape and anchoring organelles within the cell. The three main structural components of the cytoskeleton are microtubules (formed by tubulins) , microfilaments (formed by actins) and intermediate filaments. All three components interact with each other non-covalently. Eukaryotic cells contain proteins called intermediate filaments, microfilaments, and microtubules that are collectively termed the cytoskeleton. Also, the cytoskeleton proteins are multifunctional and are also involved in whole-cell movements and movements of substances within the cell.

Actins and Tubulins are abundant cytoskeletal proteins which support diverse cellular processes due to their unique properties of filament-forming proteins. Recent evidences suggest that regulated degradation pathways exist for actin and tubulin collectively maintain the quality control of cytoskeletal proteins, ensuring the appropriate function of microfilaments and microtubules.[1]



Microtubules are polymers of tubulin. They help with cell transport. They also help with the cell shape because it resists compression. It also helps facilitate cell motility. There are two motor proteins that assist organelles to move along the microtubules:

  • Kinesin, which moves things away from the nucleus.
  • Dynein, which moves things towards the nucleus.

Microtubles have a larger diameter than microfilaments and intermediate filaments. The hollow microtubule structure consists of 13 tubulin dimers. They are one alpha-tubulin protein plus one beta-tubulin protein form one tubulin dimer. Microtubules also help movement of substances within the cell and are also involved in powering whole-cell movement by cilia and flagella. The microtubules provide tracks that can move vesicles from one organelle to the next in an efficient, directed fashion. Microtubules also segregate the duplicated chromosomes during mitosis.



Microfilaments are polymers of actin. They help with cell shape also because it bears tension in the cell. It is also involved in cell motility. There are three types of cell shape, which are microvilli, lamellipodia, and filopodia.

  • Microvilli are projections on surface that increase surface area.
  • Lamellipodia are membrane ruffles that help sense the environment and direct movement.
  • Filopodia are like microvilli but are less stable. They also sense the environment. They can turn into lamellipodia.

Microfilaments are formed when individual actin monomers polymerize, in a process fueled by ATP hydrolysis, to form chains of filamentous actin. Microfilaments are dynamic structures, growing and shrinking in a controlled manner. Some microfilaments play a structural role in the cell to maintain cell shape. These structural microfilaments have protein caps at both ends to prevent changes in microfilament length. Other microfilaments have functions that require dynamic changes in length. The microfilaments also mediate cytoplasmic streaming, a mixing of the cytoplasm that aids diffusion.

Intermediate filaments


Intermediate filaments are polymers of keratin. They help with cell shape also because it bears tension in the cell. It is primarily involved in organelle anchorage. The intermediate filaments also consist of various fibrous proteins that have a diameter of about 10 nm. Intermediate filaments often form a meshwork under the cell membrane and, in cells that lack a cell wall, help impart and maintain cell shape. Intermediate filaments are fairly stable and are not thought to undergo acute changes in length the way microfilaments.

Functions of Cytoskeleton


Cytoskeletons are often synthesized based on the cell's needs; however, some protein fibers are permanent. The changeable nature of the cytoskeleton thus contributes to its five important functions.

Cell Shape


The mechanical strength of the cell is due to protein scaffolding in the cytoskeleton. In some cells, protein scaffolding also determines the shape of the cell. Microvilli are supported by cytoskeletal fibers such as microfilaments. Microvilli also increase the surface area of the cell for the absorption of materials.

Internal Organization


The cytoskeletal fibers of the cell help stabilize positions of organelles. However, the interior arrangement of the cell varies based on the cell's needs. The organelles are dynamic and change minute to minute.

Intracellular Transport


The cytoskeleton has the ability to move materials not only in the cell but also within the cytoplasm, thus aiding in the movement of organelles as well. This function is important especially in the nervous system where materials are often transported over long intracellular distances.(3908283293)

Assembly Of Cells Into Tissues


Cells are connected to one another through the linking of the protein fibers of the cytoskeleton as well as the protein fibers in the extracellular space. In the process, materials outside the cell are stabilized as well. The assembly of cells not only contributes to the mechanical strength of the tissue but also allows information to transfer between cells from one to another.


Motor Protein

The cytoskeleton of the cell allows the cell to move. For example, the cytoskeleton of white blood cells allows them to squeeze out of blood vessels. Growing nerve cells are also able to send out extensions that allow them to elongate. The microtubule cytoskeleton of cilia and flagella on the cell membrane allow them to move. In addition, motor proteins using energy from ATP can aid in the movement of cells and intracellular transport by sliding along cytoskeletal fibers. There are three types of motor proteins in the cytoskeleton that include myosins, kinesins and dyneins.Kinesins oftentimes brings vesicles from inside the cell out to the periphery. If the kinesins are mutated, then there is likely to be a neural disorder. KIF1β is one disorder resulting from a mutated kinesin. It causes lack of strengths in arms and legs. Dynein, on the opposite hand, brings the vesicles from the periphery back to the inside of cell. Motor proteins convert energy into movement and those found in the cytoskeleton use stored ATP. Myosins allow for muscle contractions. Kinesins allow for the movement along microtubules as dyneins help with the whiplike motion of the microtubule bundles of cilia and flagella. Many motor proteins consist of two heads (to bind to the cytoskeleton fiber), a neck and a tail region at the end to bind to organelles.

Quality control of cytoskeletal proteins


The evolution of cytoskeletal proteins


The evolution of cytoskeletal proteins required a novel biogenesis machinery. The cytoskeleton in eukaryotes enhances intracellular trafficking and cell division. These functions were once believed to be the distinguish elements of eukaryotes from prokaryotes because bacterial cytoskeleton also composed of proteins that similar to actin and tubulin. The actin-like and tubulin-like proteins in bacteria form filamentous structures which imply in the division of genetic material and maintenance of cell shape.[2] However, actins and tubulins in eukaryotes are distinct from similar prokaryotic proteins in the way that they maintain innovative properties which is critical for eukaryogenesis (the origin of the eukaryotic condition/the evolution of the eukaryotic condition).[3]

Both eukaryotes and bacteria have a cytoskeleton. The bacterial proteins homologous to actin are MreB and ParM, and the bacterial proteins homologous to tubulin are FtsZ. However, actin and tublin differ from MreB, ParM, and FtsZ in the sense that they have properties important for eukaryogenesis.

Actins and tubulins in eukaryotes formed microfilaments and microtubules which unite with their complementary molecular motors (myocin, kinesin, dynein) to be used for phagocytosis. Phagocytosis enables endosymbiosis and also cilia's development which supports mobility and sensory.[4] The inception of actins and tubulins in eukaryotes is an important factor in facilitating their efficient folding and assembly [5][6] which is called cytoskeletal protein biogenesis machinery (CPBM). The CPBM includes molecular chaperones which assist folding of chaperonin containing tailless complex polypeptide-1 (CCT), prefoldin (PFD) - phosducin-like proteins that regulate CCT functions, and five other cofactors. [7][8][9][10][11] In addition, post-translational modifications and proteasomal degradation are required in regulating the function of actins and tubulins which is also unique to eukaryotes.
On the other hand, in prokaryotes, the cytoskeletal protein biogenesis machinery is absent.

Autoregulation of cytoskeletal protein synthesis


Actin and tubulin concentrations are strickly controlled due to their critical effects on cytoskeletal dynamics. In animal cells, tubulin synthesis is regulated by an autoregulatory feedback mechanism that can sense the concentration of tubulin heterodimer in order to regulate the stability of ɑ-tubulin, and β-tubulin mRNAs. [12][13] Similar to animals, the synthesis of tubulin in metazoans is also autoregulated due to its critical influences. Researches have shown that overexpression of β-tubulin in Saccharomyces cerevisiae leads to abnormal microtuble function and slow growth.[14]

Actin overexpression causes by an incompletely characterized feedback mechanism that is sensitive to the concentration of actin monomers is preventable by the presence of the 3' untranslated region of actin mRNA.[15]

Biogenesis of cytoskeletal proteins


The cytoskeleton protein biogenesis machinery is found in eukaryotes but not in prokaryotes. It includes chaperonin containing CCT (complex polypeptide-1) and PFD (prefoldin), phosductin-like proteins, and five cofactors. The chaperonin molecules help actin and tubulin proteins to fold. Challenges to the protein biogenesis machinery include: (1) the high concentrations of actin and tubulin in cells, (2) the tendency of actin and tubulin to self-associate, (3) the inability of actin and tubulin to fold without the help of other molecules, (4) the fact that actin and tubulin compete with one another for the same folding space.

Biogenesis of cytoskeletal proteins faces a lot of difficulty which mainly due to the abundant of actin and tubulin concentrations, self-associate tendency, and the inability in folding independently. In addition, the competing for access to limited folding space is also a challenge to biogenesis of cytoskeletal proteins.[16] Fortunately, the action of specific chaperonin cofactors can account for the regulation of chaperonin-mediated cytoskeletal protein folding.

Eukaryotic cytosolic chaperonin


Eukaryotic cytosolic chaperonin is a unique ability to assist the folding of actin and tubulin. Molecular chaperones can interact with the newly synthesized polypeptide chains to become stable during the folding process form a linear monomer amino acids chain into a more complex and functional protein [17]. There are many types of chaperones that direct the folding of new proteins, refolding of stress-denatured proteins, unfolding of proteins, and transporting proteins [18][19].

Chaperonin, an important family of molecular chaperones, has a barrel-like structure with two multimeric stacked rings of 60kDa (Dalton's atomic mass unit). Chaperonins undergo ATP-dependent conformational changes during its folding cycle: facilitate substrate binding, encapsulation and release[20]. In eukaryotes, the cytosolic chaperonin is the tailless complex polypeptide 1 ring complex (CCT) which is required for viability in yeast and worms.

CCT is crucial for the biogenesis of actin and tubulin. CCT is composed of eight related subunits - ɑ,β,ɣ,ẟ,ɛ,ʝ,ŋ,θ - which show up twice in each oligomer. CCT is closely related to archaeal chaperonin thermosome but quite different from bacterial chaperonin GroEL [21]. CCT is known to have a more specific binding profile than bacterial GroEL [22][23]. As a chaperonin, CCT undergoes ATP-dependent conformational changes during its folding cycle. Due on the copiousness of actin and tubulin, they occupy significant proportion of CCT complexes. Beside actin and tubulin, there are other CCT substrates also have key roles in progression of the cell cycle [24] Researches show that CCT function in vivo is regulated by several dedicated cofactors, including PFD and phosducin-like proteins.

PFD, a jellyfish-shaped molecular chaperone required for stabilization of new cytoskeletal proteins, is a CCT co-chaperone for the biogenesis of actin and tubulin [25][26].

Phosducin-like proteins, regulators of the folding of actin and tubulin in association with CCT, are thioredoxin domain-containing proteins with homology to phosducin, a regulator of retinal G-protein signaling [27].

Tubulin folding cofactors


Scientists believed that actin is released from CCT in a native, assembly-competent state, but cyclase associated protein might interact with and stabilize near-native or unstable forms of actin in close association with the chaperonin [28]. On the other hand, functional tubulin is an obligate ɑ-β heterodimer, and evolved in a folding pathway linked to dimer assembly[29].

Roles of each cofactor:
1. Tubulin cofactor A (TBCA): collects unassembled beta-tubulin
2. Tubulin cofactor D (TBCD): assists beta-tubulin down the assembly pathway
3. Tubulin cofactor B (TBCB): binds to alpha-tubulin after chaperonin release
4. Tubulin cofactor E (TBCE): receives alpha-tubulin from TBCB and processes it further
5. Tubulin cofactor C (TBCC): promotes GTP hydrolysis in beta-tubulin if in the presence of a stable supercomplex (formed by the joining of beta-tubulin-TBCD and alpha-tubulin-TBCE complexes)

a. also facilitates the release of native alpha-beta-tubulin heterodimer

Diseases linked to the cofactors:
It is believed that if TBCB is not degraded properly, a neurological disease called giant axonal neuropathy may result. This disease is related to decreased density of the microtubule of cells. Mutations of TBCE are associated with hypoparathyroidism (a developmental disorder), mental retardation, and facial dysmorphism (HRD).

Prefoldin (PFD) is a CCT co-chaperone required for stabilizing nascent cytoskeletal proteins. It consists of two alpha-type and four beta-type subunits which collectively make its structure resemble the shape of a jellyfish. The six subunits form a cavity shaped like a rectangle that attaches to newly formed actin and tubulin as the chains leave the ribosome. PFD then delivers the actin and tubulin to CCT, probably via a docking and substrate-release mechanism (supported by electron microscopy analysis of PFD-actin complexes). It is also possible that PFD improves the efficiency of actin and tubulin protein folding by navigating partially folded molecules back towards the CCT for more folding. This idea is supported by the fact that yeast cells without PFD were observed to fold actin and tubulin more slowly than wild-type cells. Furthermore, Pfd1 knockout mice showed signs of dysfunction of cytoskeletal proteins, neuronal loss, neuromuscular defects, and defective development of lymphocytes. These Pfd1 knockout mice were only viable for five weeks.

Phosducin-like proteins


Phosducin-like proteins regulate the folding of actin and tubulin. Three such proteins have been termed PhLP1, PhLP2, and PhLP3 as they are CCT-binding proteins. PhLP1 assists in assembling heterotrimeric G-proteins by CCT; this process is regulated by PhLP1 phosphorylation. PhLP2 and PhLP3 are involved in the biogenesis of cytoskeletal proteins. It is believed that there is a specificity of PhLP2 for actin biogenesis and of PhLP3 for tubulin biogenesis. In other words, disruption of PhLP2 function caused severe actin cytoskeletal defects whereas disruption of PhLP3 function alters normal tubulin biogenesis.

When studied in vitro, it is observed that an excess of PhLP2 and PhLP3 prevents actin and tubulin from being folded via the CCT-mediated folding pathway. It is believed that this is due to the reduced activity of CCT ATPase. However, when the study in done on yeast cells, it appears that PhLP2 stimulates actin folding by purified yeast CCT. The researchers of this study concluded that amino acids of mammalian PhPL2 that were not present in yeast PhLP2 are responsible for preventing actin and tubulin from being folded. This also supports the idea that higher eukaryotes evolved with more regulation of cytoskeletal proteins.

Protein biogenesis/quality control pathways


1. Translation

a. Following translation, nascent actin and tubulin undergo folding via:
i. PFD folding pathway.
1. Assisted by PFD and substrate delivery.
ii. PFD-independent pathway.

2. Folding

a. Both PFD and PFD-independent folding pathways lead to formation of CCT (cytosolic chaperonin).
b. CCT-mediated folding follows, leading to:
i. Formation of near-native alpha- and beta-tubulin.
ii. Formation of near-native actin.

3. Assembly, Disassembly, and Polymerization

a. Near-native alpha- and beta-tubulin assembles into a TBCE-TBCD complex.
i. Complex forms folded tubulin heterodimer.
ii. Folded tubulin heterodimer polymerizes into a microtubule.
b. Near-native actin is folded into a folded actin monomer.
i. Folded actin monomer polymerizes into a microfilament.

4. Degradation

a. Free tubulin and free actin not used in polymerization undergo ubiquitylation into proteasome.
i. Proteasome is then degraded.

Post-translational modification (PMT) of cytoskeletal proteins


For actin, post-translational modification is known to affect only folding. Tubulin, on the other hand, is affected by PTM in such a way that allows native proteins to turn on and off activity in a reversible and regulatory manner. Tubulin can be modified in a number of ways such as acetylation, detyrosination, and glutamylation. These tubulin modifications occur on microtubules, and it has been hypothesized (though not well-tested) that the free tubulin heterodimer is the substrate responsible for reversing the modifications. Much study, however, has been dedicated to Tubulin PTMs in general. Tubulin acetylation, for example, has been shown in recent studies to be linked to a human neurodegenerative disease called amyotrophic lateral sclerosis (ALS).

Degradation of cytoskeletal proteins


Part of the quality control process includes the degradation of proteins. Damaged proteins and misfolded proteins that cannot be refolded with the help of chaperones are removed in this process. Unfortunately, the pathway in which the degradation occurs is not as well studied as the biogenesis process.Invalid parameter in <ref> tag

Turn-over rates and steady-state concentrations of all cellular proteins is regulated in the following ways:

1. protein degradation through the ubiquitin-proteasome system (UPS)

2. lysosomal degradation 3. another proteolytic mechanism

“Proteostasis” is the regulatory process in which damaged or incorrectly folded proteins that cannot be repaired by charperones are removed by proteolysis. Not much work has been put into the study of actin and tubulin degradation, but tubulin has been known to rapidly degrade in the presence of microtubule-destabilizing drugs such as colcemid. Such drugs make tubulin more soluble.



Parkin is a ubiquitin-protein ligase important to tubulin degradation. Parkin is mutated in patients with autosomal recessive juvenile Parkinson disease (PD). Its normal function is to interact with HSP70-interacting protein (CHIP) to make stress-denatured proteins undergo ubiquitylation. Parkin is also believed to stimulate ubiquitylation and proteasomal degradation of alpha-tubulin and beta-tubulin. Cells that over-express mutant alpha-synuclein, a toxic inclusion-forming protein in Parkinson disease, reveal increased concentrations of alpha-tubulin and insoluble parkin. These two traits are also observed in patients affected by Lewy body disease.

Cofactor E-like


E-like (COEL), a tubulin folding cofactor, is a protein that destabilizes tubulin. Cells without human COEL contain excess amount of stable microtubules while microtubule disassembly and the degradation of α-tubulin is observed in cells with excess COELs. The degradation of tubulin caused by the presence of COEL is countered by a negative regulator of microtubule called stathmin that isolates tubulins. Overall, COEL is important for three reasons: 1. it can remove misfolded tubulin 2. regulate the concentration of tubulin 3. control tubulin isotype.[30]

Actin Degradation


In metazoan cells, the concentration of tubulin is reduced when CCT or PFD does not function properly. The concentration of actin, however, is not affected significantly. This suggests that the quality control for actin differs from that of tubulin. Relative to the control tubulins, there seems to be less of a need to remove misfolded β-actins. Still, there are incidences when actin degradation is necessary. In the case of ischemic oxidative damage, α-actin specific to the heart is degraded by proteasome. It is also found that α-actins are degraded by lysosomes when muscle contraction in cardiomyocytes is inhibited by the use of drugs. It has been recently observed that TRIM32, an ubiquitin that ubiquitylates α-actin in vitro, when unusually expressed in the human embryonic kidney cells, reduces the concentration of cytoplasmic β-actins. TRIM32 mutations have been found in Bardet-Biedl syndrome and muscular dystrophy, although the actual role TRIM32 plays in these diseases remains unknown.[31]



Reece, Jane (2011). Biology. Pearson. ISBN 978-0-321-55823-7. {{cite book}}: Text "coauthors+ Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson" ignored (help)

Silverthorn, Dee Unglaub. "Compartmentation: Cells and Tissues." Human Physiology. Boston, MA: Pearson Custom Pub., 2007. Print.

  1. Lundin , Victor , Michel Leroux, and Peter Stirling. "Elsevier: Article Locator." | Search through over 10 million science, health, medical journal full text articles and books.., n.d. Web. 17 Nov. 2012. <>.
  2. J. Pogliano The bacterial cytoskeleton Curr. Opin. Cell Biol., 20 (2008), pp. 19–27
  3. M.R. Leroux, F.U. Hartl Protein folding: versatility of the cytosolic chaperonin TRiC/CCT Curr. Biol., 10 (2000), pp. R260–264
  4. T. Cavalier-Smith The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa Int. J. Syst. Evol. Microbiol., 52 (2002), pp. 297–354
  5. S. Bertrand et al. Folding, stability and polymerization properties of FtsZ chimeras with inserted tubulin loops involved in the interaction with the cytosolic chaperonin CCT and in microtubule formation J. Mol. Biol., 346 (2005), pp. 319–330
  6. S. Bertrand et al. Folding, stability and polymerization properties of FtsZ chimeras with inserted tubulin loops involved in the interaction with the cytosolic chaperonin CCT and in microtubule formation J. Mol. Biol., 346 (2005), pp. 319–330
  7. S. Lacefield, F. Solomon A novel step in beta-tubulin folding is important for heterodimer formation in Saccharomyces cerevisiae
  8. P.C. Stirling et al. PhLP3 modulates CCT-mediated actin and tubulin folding via ternary complexes with substrates
  9. P.C. Stirling et al. Functional interaction between phosducin-like protein 2 and cytosolic chaperonin is essential for cytoskeletal protein function and cell cycle progression
  10. E.A. Mccormack et al. Yeast phosducin-like protein 2 acts as a stimulatory co-factor for the folding of actin by the chaperonin CCT via a ternary complex
  11. M. Lopez-Fanarraga et al. Review: postchaperonin tubulin folding cofactors and their role in microtubule dynamics J. Struct. Biol., 135 (2001), pp. 219–229
  12. D.W. Cleveland et al. Unpolymerized tubulin modulates the level of tubulin mRNAs Cell, 25 (1981), pp. 537–546
  13. M.E. Sellin et al. Global regulation of the interphase microtubule system by abundantly expressed Op18/stathmin Mol. Biol. Cell, 19 (2008), pp. 2897–2906
  14. D. Burke et al. Dominant effects of tubulin overexpression in Saccharomyces cerevisiae Mol. Cell. Biol., 9 (1989), pp. 1049–1059
  15. A. Lyubimova et al. Autoregulation of actin synthesis requires the 3′-UTR of actin mRNA and protects cells from actin overproduction J. Cell Biochem., 76 (1999), pp. 1–12
  16. M.R. Leroux, F.U. Hartl Protein folding: versatility of the cytosolic chaperonin TRiC/CCT Curr. Biol., 10 (2000), pp. R260–264
  17. F.U. Hartl, M. Hayer-Hartl Converging concepts of protein folding in vitro and in vivo Nat. Struct. Mol. Biol., 16 (2009), pp. 574–581
  18. F.U. Hartl, M. Hayer-Hartl Converging concepts of protein folding in vitro and in vivo Nat. Struct. Mol. Biol., 16 (2009), pp. 574–581
  19. E.T. Powers et al. Biological and chemical approaches to diseases of proteostasis deficiency Annu. Rev. Biochem., 78 (2009), pp. 959–991
  20. C.R. Booth et al. Mechanism of lid closure in the eukaryotic chaperonin TRiC/CCT Nat. Struct. Mol. Biol., 15 (2008), pp. 746–753
  21. C. Spiess et al. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets Trends Cell Biol., 14 (2004), pp. 598–604
  22. C. Dekker et al. The interaction network of the chaperonin CCT EMBO J., 27 (2008), pp. 1827–1839
  23. A.Y. Yam et al. Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies Nat. Struct. Mol. Biol., 15 (2008), pp. 1255–1262
  24. C. Spiess et al. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets Trends Cell Biol., 14 (2004), pp. 598–604
  25. S. Geissler et al. A novel protein complex promoting formation of functional alpha- and gamma-tubulin EMBO J., 17 (1998), pp. 952–966
  26. J. Martín-Benito et al. Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT EMBO J., 21 (2002), pp. 6377–6386
  27. M. Blaauw et al. Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins EMBO J., 22 (2003), pp. 5047–5057
  28. E.A. McCormack et al. Mutational screen identifies critical amino acid residues of beta-actin mediating interaction between its folding intermediates and eukaryotic cytosolic chaperonin CCT J. Struct. Biol., 135 (2001), pp. 185–197
  29. M. Lopez-Fanarraga et al. Review: postchaperonin tubulin folding cofactors and their role in microtubule dynamics J. Struct. Biol., 135 (2001), pp. 219–229
  30. Lundin, Victor, Loroux, Michel and Stirling, Peter. “Quality Control of Cytoskeletal Proteins and Human Disease” January 2010: 288-295.Retrieved on 20 November 2012.
  31. Lundin, Victor, Loroux, Michel and Stirling, Peter. “Quality Control of Cytoskeletal Proteins and Human Disease” January 2010: 288-295.Retrieved on 20 November 2012.

Berg, Jeremy "Biochemistry", Chapter 35 Molecular Motor. pp1018-1020. Seventh edition. Freeman and Company, 2010.

Slonczewski, Joan L. Microbiology "An Evolving Science." Second Edition.