Fundamentals of Molecular Biology. Part 1: Molecular Cell Biology - A. N. Ogurtsov 2011

Fundamentals of Cell Theory
Cytoskeleton

The Cytoplasm of Eukaryotic Cells is permeated by a three-dimensional network of protein filaments known as the cytoskeleton. Depending on their diameter, these filaments are divided into three groups:

- microfilaments, or Actin filaments (6-8 nm in diameter),

- Intermediate filaments (about 10 nm in diameter),

- microtubules (about 25 nm in diameter).

All of these fibers are polymers composed of subunits of specialized Globular Proteins.

Microfilaments (actin filaments) are composed of actin, the most abundant protein in eukaryotic cells. Actin can exist as a monomer (G-actin, globular actin) or a polymer (F-actin, filamentous actin) (Figure 22).

Class="center">

Figure 22 - Introduction/12.html">Structure of Globular and filamentous actin: a - model of a G-actin monomer; 1 - cleft where ATP binds, with the C- and N-termini located in subdomain I; b - F-actin helix as seen under an Electron microscope; c - model of an actin polymer

G-actin is an asymmetric globular protein (42 kDa) consisting of two domains (each composed of two subdomains). As Ionic strength increases, G-actin reversibly aggregates to form a linear, helically twisted polymer, F-actin. The G-actin molecule carries a tightly bound ATP molecule, which slowly hydrolyzes to ADP upon transition into F-actin; that is, F-actin exhibits ATPase activity.

The amounts of monomers and polymers depend in different ways on the actin concentration in The Cell (Figure 23). As the actin concentration increases, starting from a certain critical value C_c≈ 0.1 μM, F-actin polymerization begins.

Figure 23 - Dependence of monomer and polymer quantities on actin concentration

During the polymerization of G-actin into F-actin, the orientation of all monomers is identical, which gives F-actin a distinct polarity. F-actin fibers have two oppositely charged ends: (+) and (-), which polymerize at different rates because they have different critical concentrations: C⁺_c ≈ 0.1 μM, C^-_c ≈ 0.6 μM. Consequently, during polymerization,

the (+)-end grows 5 to 10 times faster than the (-)-end (Figure 24).

Specialized capping proteins can selectively bind to either end of actin filaments or microtubules, playing a key role in establishing the specific spatial architecture of the cytoskeleton. They ensure the attachment of filaments and microtubules to other cellular structures in the correct orientation, while also providing independent regulation of elongation or disassembly at either end.

For instance, the polymerization process can be inhibited by fungal toxins. Specifically, phalloidin (the toxin of the death cap mushroom) binds to the (-)-end and inhibits depolymerization, whereas cytochalasin (a cytostatic mold toxin) attaches to the (+)-end, blocking polymerization.

The dynamic structure of actin filaments (and microtubules), combined with their vectoral nature where the two ends are non-equivalent, gives rise to a unique property known as treadmilling.

At a critical concentration C^-_c > C_c > C_c⁺, the (+)-end will elongate while the (-)-end shortens, causing G-actin monomers to effectively migrate along the F-actin filament (Figure 24(b)).

Figure 24 - Actin polymerization: a - growth of the (+)-end; b - growth of the (-)-end; c - treadmilling; 1 - capping protein

The treadmilling effect consists in the continuous dissociation of subunits from the lagging end of the filament and their re-addition (reassembly) at the leading edge. As a result, the filament maintains an approximately constant length while "moving" relative to its surroundings. The cell utilizes the treadmilling effect as part of the machinery that drives cell motility.

The Cell Cytoplasm contains over 50 Different types of actin-associated proteins that specifically interact with G-actin and F-actin. These proteins perform various Functions:

- regulating the size of the G-actin pool (profilin),

- influencing the polymerization rate of G-actin (villin),

- stabilizing the ends of F-actin filaments (fragmin, ß-actinin),

- cross-link filaments to each other or to other components (such as villin, a-actinin, spectrin, and MARCKS);

- disrupt the F-actin double helix (gelsolin).

The activity of these proteins is regulated by Ca²⁺ ions and protein Kinases.

Intermediate filaments. The Structural elements of intermediate filaments are proteins belonging to five related families that exhibit a high degree of cell Specificity. Typical representatives of these proteins include cytokeratins, desmin, vimentin, glial fibrillary acidic protein [GFAP], and neurofilaments.

All these proteins share a central basic rod-like dimeric structure known as a coiled-coil domain (Figure 25(a)). Such dimers associate in an antiparallel orientation to form a tetramer. HEAD-to-head aggregation of tetramers yields a protofilament. A bundle of protofilaments forms an intermediate filament.

Unlike microfilaments and microtubules, free monomers of intermediate filaments are rarely found in the cytoplasm. Their polymerization leads to The formation of stable, non-polar polymer molecules.

Microtubules are built from the globular protein tubulin, which is a dimer of a- and ß-subunits (53 and 55 kDa) (Figure 26(a)). The a,ß-heterodimers form linear chains called protofilaments.

Figure 25 - Intermediate filaments: (a) coiled coil; (b) tetramer; (c) intermediate filament: 1 - protofibrils, 2 - tetramer, 3 - protofilament

Thirteen protofilaments form a cyclic complex (Figure 26(b)). These rings then polymerize into a long tube. Like microfilaments, microtubules are dynamic polar structures with (+)- and (-)-ends.

The (-)-end is stabilized by attachment to the centrosome (the microtubule-organizing center, or cell center), whereas the (+)-end is characterized by dynamic instability, allowing it to either slowly grow or rapidly shorten.

Tubulin monomers bind guanosine triphosphate (GTP), which is slowly hydrolyzed to guanosine diphosphate (GDP). Two Types of proteins associate with microtubules: structural proteins, known as MAPs (microtubule-associated proteins), and motor proteins.

Microtubules exhibit an intriguing phenomenon known as dynamic instability. A microtubule elongates as subunits are added to the growing end, and suddenly, this growing end begins to "fray," causing the microtubule to break down into subunits. Due to this property, microtubules are in a state of continuous growth, yet specific stabilizing proteins are required to maintain their structural integrity.

The microtubules of Cilia and flagella have a doublet structure (Figure 26(c)), whereas those of basal bodies and centrioles have a triplet structure.

Figure 26 - Structure of microtubules: (a) tubulin heterodimer; (b) cylindrical polymer; (c) packing of protofilaments; 1 - GTP; 2 - taxol molecule; 3 - GDP; 4 - protofilament; 5-7 - single, double, and triple microtubules

Centrioles, located within the centrosome, act as the cell's "poles" toward which Chromosomes migrate during Cell Division, and are found exclusively in animal cells.

The cytoskeleton performs three main functions.

1. It serves as a mechanical framework that gives the cell its characteristic shape and maintains The connection between The Plasma Membrane and Organelles. This framework is a dynamic structure that constantly reorganizes in response to Changes in external conditions and the cell's physiological state.

2. It acts as a "motor" for cellular motility. Contractile proteins are present not only in Muscle cells but also in other Tissues. Cytoskeletal components direct and coordinate movement, cell division, shape changes during growth, organelle transport, and cytoplasmic streaming.

3. It serves as "tracks" for the Intracellular Transport of organelles and other large molecular complexes.

As an example of cytoskeletal function, Figure 27 shows a cross-section of intestinal epithelial microvilli.

Figure 27 - Diagram of an intestinal epithelial microvillus

Microfilaments composed of F-actin permeate the microvilli, forming cross-linked networks. These microfilaments are held together by actin-binding proteins, the most important of which are fimbrin and villin.

Calmodulin and a Myosin-like ATPase link the peripheral microfilaments to the plasma membrane; another actin-binding protein, fodrin, connects the actin filaments at their base while also anchoring them to the cytoplasmic membrane and the intermediate filament network.

In this case, actin microfilaments primarily perform a static function.

However, actin is most commonly involved in dynamic processes such as Muscle contraction, cell motility, phagocytosis, the formation of plasma membrane microprojections and lamellipodia (cellular extensions), as well as the acrosome reaction during sperm-egg fusion.

The functions of microtubules are illustrated in Figure 28.

Figure 28 - Functions of microtubules in the cell

Microtubules radiate outward in all directions from a structure near The Nucleus known as the centrosome. The (+)-end of a microtubule constantly undergoes cycles of growth and disassembly, whereas the (-)-end is capped by associated proteins within the centriole. The (+)-end can also be stabilized by associated proteins once microtubules reach the plasma membrane, for example.

Microtubules play a key role in maintaining cell shape. They also serve as tracks for organelle transport. Together with motor proteins such as dynein and kinesin, microtubules are capable of mechanical work, including mitochondrial transport, the beating of cilia (Hair-like projections on the epithelium of the Lungs, intestines, and oviducts), and the whipping motion of sperm flagella. Furthermore, microtubules perform vital functions during cell division.

Self-Assessment Questions

1. Define the cell.

2. What is cellular METABOLISM?

3. Define an enzyme.

4. Define a Gene.

5. What topics are studied by molecular cell biology?

6. State the postulates of the Cell Theory.

7. What is cellular totipotency?

8. Describe The structure of a Prokaryotic Cell and compare it with a Eukaryotic Cell.

9. How do eukaryotic cells differ from Prokaryotic Cells?

10. What are the Similarities and differences between the nucleoid and the Cell Nucleus?

11. Define introns. Do eukaryotes have introns?

12. What is the cell Cytosol? How does the cytosol differ from the cytoplasm?

13. What factors limit the size of prokaryotic cells?

14. What is cellular compartmentalization? What are its functions?

15. Under what conditions do cell sizes significantly exceed diffusion limits?

16. What is pinocytosis and how does it differ from phagocytosis?

17. What is phagocytosis and how does it differ from pinocytosis?

18. What are the similarities and differences between Endocytosis and Exocytosis?

19. What is Cell Differentiation and what is its significance?

20. List and describe the main Structural components of a cell.

21. List and describe the main cytoplasmic organelles.

22. Describe the membrane system of Mitochondria.

23. What are mitochondrial cristae?

24. What is the mitochondrial matrix?

25. Which cell organelles contain their own DNA and Ribosomes?

26. What are Chloroplasts and how do they differ from chromoplasts?

27. What are chromoplasts and how do they differ from leucoplasts?

28. What are leucoplasts and how do they differ from chloroplasts?

29. Describe the membrane system of chloroplasts.

30. What is the plastid stroma?

31. What are the similarities and differences between stroma lamellae and thylakoids?

32. What are grana and how do they differ from thylakoids?

33. What is the cell cytoskeleton?

34. What are microfilaments and how do they differ from microtubules?

35. What are microtubules and how do they differ from actin filaments?

36. What is the difference between F-actin and G-actin?

37. During polymerization, which end of an actin filament grows at a faster rate, "(+)" or "(—)"?

38. What is The Role of capping proteins in actin polymerization?

39. Microtubules are polymers of which protein?

40. What is the polymer whose monomer is G-actin?

41. List the primary Functions of the cytoskeleton.