sign in sign up
<<
Home , Prokaryotic cytoskeleton

Course - PGPathshala-Biophysics Paper 11 - Cellular and molecular Biophysics Module 14 - and molecular motors Content Writer: Dr. Karthikeyan Pethusamy, AIIMS, NEW DELHI. Objectives:

 To understand the role of cytoskeleton & molecular motors  To differentiate the three types of cytoskeletal elements  To enlist the drugs acting on cytoskeletal elements and their uses  To understand the pathological basis of disorders associated with cytoskeletal elements  To understand the interaction between cytoskeleton and molecular motor Introduction: The cytoskeleton is a cellular structure that helps cells maintain their shape and internal organization. It also provides mechanical support that enables cells to carry out essential functions like - division, anchorage and movement. In this module, we will discuss the cytoskeleton. Cytoskeleton is not a single component. It is made up of network of filamentous proteins. In certain cells, cytoskeletal proteins constitute 80% of total cellular proteins. Cytoskeleton consists of three major class of elements namely , intermediate filaments and . Out of these, microfilaments are the thinnest, microtubules are the largest and intermediate filaments are intermediate-sized. 1. Microfilaments is formed by family of proteins in all eukaryotic cells. Apha, beta, and gamma are the three major actin isoforms. Alpha actin is found in muscles and is involved in muscle contraction. Beta and gamma are found in various cells. Microfilaments are composed of two intertwined strands of double helical polymers that are arranged head to tail. Each strand is made up of multiple actin monomers. Microfilaments are dynamic structures which undergo rapid assembly and disassembly. ATP binding promotes the addition of actin monomers. Polymerisation of G-actin (globular actin) produces F-actin (filamentous actin). This polymerisation requires cations like magnesium.

Schematic figure illustrating the polymerisation of Globular actin to Fibrous actin and the requirement of ATP.

The end in which rapid association of actin monomers takes place is known as plus (+) end and the end in which rapid dissociation of actin takes place is known as minus (-) end. Thus, actin fiber has polarity. Constantly deconstruction and reassembly of microfilaments is known as “dynamic instability.” 1.1. Actin fold ATP binding domain of actin is known as actin fold. This domain is also present in the glycolytic enzyme hexokinase and the chaperone hsc70. Role of microfilaments: 1. Actin- complex is involved in muscle contraction 2. Microfilaments help the cells to change their shape. of phagocytes is due to pseudopodia (false feet) formation. Rapid polymerisation of actin filaments towards the one end of the cell forms these pseudopodia. 3. In plants, cytoplasmic streaming (movement of inside the cell towards the optimal area for photosynthesis) is mediated by the current of actin flow. 4. Contractile ring that forms at the end of mitosis is made up of actin-myosin complex. Constriction of this ring produces two new daughter cells. 1.2. Actin associated proteins Actin cross-linking proteins help in the assembly of actin filaments into stable network and bundles. Some of the actin associated proteins are given in the table below.

Actin associated proteins Protein Location Fimbrin Microvilli, Stereocilia α Filopodia, Lamellipodia, Stress fibers, Adhesion plaques Cortical network of cells, i.e. specialized layer of cytoplasmic protein on the inner face of the plasma membrane of the cell periphery Muscle cortical network Filopodia, Pseudopodia, Stress fibers Fascin Filopodia, Lamellipodia, Stress fibers, Microvilli, Acrosomal process Villin Microvilli in intestinal and renal brush border

2. Microtubules Microtubules are the largest cytoskeletal element made up of proteins. There are two types of namely alpha and beta. Tubulin heterodimers organise into a hollow shape to form microtubules. The hollow shape provides the mechanical strength to bear the shear stress exerted upon the microtubules.

Structure of a . The ring shape depicts a microtubule in cross-section, showing the 13 protofilaments surrounding a hollow center. By Thomas Splettstoesser (www.scistyle.com) - Own work (rendered with Maxon Cinema 4D), CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=41014850

Multiphoton fluorescence image of HeLa cells with cytoskeletal microtubules (magenta) and DNA (cyan). Appreciate the microtubules spreading across each and every corner of the cell.

By National Institutes of Health (NIH) (National Institutes of Health (NIH)) [Public domain], via Wikimedia Commons Microtubules form a dense network across all part of the cells not only providing structure but also providing a pathway for transport of various intracellular cargo across the cell. For examples, vesicles that bud off from move along the microtubule network with the help of other proteins including molecular motors (discussed later). Simply speaking, molecular motor proteins walk along the microtubule rail road carrying their cargo.

Kinesin, a molecular motor protein moving along the microtubules.

In addition to the maintenance of shape of cell and transport, microtubules play important role in the separation of during . Microtubules and other proteins attach to the of each to form mitotic spindle. Shortening of the spindle pulls the chromosomes apart. Non-disjunction of chromosomes leads to aneuploidy (e.g. Down Syndrome).

Image of the mitotic spindle in a human cell showing microtubules in green, chromosomes (DNA) in blue, and in red. By Afunguy at English Wikipedia [Public domain], via Wikimedia Commons

Microtubules are not important for the movement inside the cell but also for the movement of entire cell. Alpha and beta tubulins are coded by two different genes and are highly conserved throughout the eukaryotic kingdom. Cilia are short bundles that beat in a wave like motion to move cells are move the fluid around the cells. Flagella are long-bundles of microtubules that helps in unidirectional movement of cell. In humans, the only flagellated cell is the spermiocyte. Cilia are present in many cells of the body and serve roles in motility and sensory function. 2.1 Microtubules in cancer and metastasis Cancer cells divide rapidly and are able to metastasise to distant places. As we have discussed above, microtubules are important in the formation of mitotic spindles. Thus, microtubule inhibitors are used in the therapy of cancer.

 Vinca alkaloids (vincristine and vinblastine) and Taxanes (Paclitaxel) – Anti-cancer agents

 Colchicine is used to cause metaphase arrest for karyotyping and to prevent chemotaxis in acute gouty arthritis

Mechanism of action of Vinca alkaloids and taxanes are different

2.2. Beta actin and tubulin genes as control Beta actin and tubulin genes are expressed almost in every kind of cell at all time (constitutive expression). This is why beta actin and tubulin proteins are used in western blot as controls for the relative quantification of proteins. 3. Intermediate filaments As the name suggests, intermediate filaments are of intermediate size between microtubules and microfilaments. There are many group of proteins that make up intermediate filaments. Unlike other cytoskeletal elements, intermediate filaments are less dynamic and are made of at least 40 different

subunit proteins. This stability makes them excellent as anchors for that don’t need to move around e.g. nucleus. , one type of intermediate filaments constitutes the nuclear lamina which is found inside the nuclear membrane. Different subunits of intermediate filaments are classified in the following manner.

Subtype Protein composition Tissue distribution Type I Acidic Epithelia Type II Basic Keratin Mesenchymal cells Muscle cells Glial cells, astrocytes, Glial fibrillary acidic protein Type III stellate liver cells Diverse neuronal cells Diverse neuronal cells – Low, Type IV Medium and high Neurons

Type V Lamins – A, B and C Nuclear Lamina Phakinin Unclassified Lens Filensin

Cultured epithelial cells Keratin is stained red and DNA (nucleus) is stained green. Look at how the keratin filaments are surrounding cells.

Compared to microfilaments and microtubules, intermediate filaments are unique in the following ways. 1. Intermediate filaments are less dynamic 2. They do not contain polarity. 3. They are tissue-specific, e.g. Neurofilament is present in neurons. Tissue specific nature of intermediate filaments is utilised in immunohistochemistry. As we have seen, keratin is a marker of epithelial cells and vimentin is a marker of mesenchymal cells. Presence of keratin pearls (shown in image) is a marker of epithelial malignancies. Disappearance of keratin and appearance of vimentin is a marker of epithelial to mesenchymal transition (EMT).

H&E staining of carcinoma. Look at the whorled pattern of keratin. This is known as Keratin pearls.

3.1. Diseases due to defective intermediate filaments 1. Epidermolysis bullosa simplex (bullous formation after a trivial trauma) – due to mutation 2. Hutchinson-Gilford (Progeria) causes premature aging, involves mutations affect A protein 3. Amyotrophic lateral sclerosis (ALS) progressive loss of motor neurons, muscle atrophy and paralysis. – defect in (NF-L, NF-H) 4. Comparison of three classes of cytoskeletal elements: Microfilaments Intermediate filaments Microtubules Size (nm) 5-7 8-10 23-25 Subunit proteins Actins (alpha, beta and Lamins, , Tubulins (alpha and gamma) Vimentin, Desmin, beta) Neurofilaments Structure Beaded structure α helical rods that Hollow tubules. assemble into rope like filaments. Requirement of ATP hydrolysis for Not needed nucleotide polymerisation of G-actin hydrolysis to F-actin

5. Role of cytoskeleton  Giving shape & strength to the cell  Anchorage  Intracellular transport  Muscle contraction  Beating of cilia  Amoeboid movement of phagocytic cells

6. Prokaryotic Cytoskeleton

It was initially believed that cytoskeleton is unique to . Recently eukaryotic cytoskeletal protein analogues have been found in also. The following are the prokaryotic cytoskeletal elements

FtsZ ring microtubule homolog MreB microfilament (actin) homolog homolog

7. Molecular motors Molecular motors are protein that convert chemical energy to mechanical work. Most of the molecular motors utilise the free energy of hydrolysis of ATP. 7.1. Classes of molecular motors

Class Example Cytoskeletal Myosin, , , motors Rotary motors FoF1-ATP synthase Nucleic acid DNA polymerase, RNA polymerase, Helicase, motors Topoisomerases

7.2. Cytoskeletal Motors Cytoskeletal Molecular motors are the vehicles carrying the cargo. They run upon the cytoskeletal framework. For example, Myosin is the motor, actin filaments are the tracks on which myosin moves, and ATP is the fuel that powers movement. So, myosin is an ATPase.

Cytoskeletal Fuel Molecular motor Function railroad used

Myosin Actin ATP Muscle contraction

Dynamin Actin GTP Receptor mediated endocytosis

Anterograde transport (e.g. Kinesin Tubulin ATP neuronal transport)

Retrograde Dynein Tubulin ATP transport (e.g. cilia)

All those motor proteins that move on actin filaments belong to the myosin superfamily. The motor proteins that move on microtubules are members of either the kinesin superfamily or the dynein family. Even though myosin and kinesin walk along different cytoskeletal rail road, they share a common structural core. This suggest that myosin and kinesin are derived from a common ancestral gene. 7.3. Functions of molecular motors:

 Muscle Contraction due to sliding of myosin II and actin filaments. Atin, myosin, and are involved in this.

 Motor Proteins Mediate the Intracellular Transport of Membrane-enclosed Organelles.

Eukaryotic showing the 9+2 arrangement of microtubules. Dynein arms are shown in blue.

By en:User:Smartse - File:.JPG and Figure 19.28 on page 819 of "Molecular , 4th edition, Lodish and Berk" ISBN 0-7167-3706-X, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=9423365 7.4. Dynein arm defect leads to Primary ciliary dyskinesia/Kartagener syndrome Primary ciliary dyskinesia (PCD), also called immotile ciliary syndrome or Kartagener syndrome is an autosomal recessive disorder due to dynein arm defect. Dynein is important for the proper function of cilia and flagella. Thus, dynein arm defect leads to respiratory infections, chronic sinusitis. We have already seen that the only flagellated cell in human body is the sperm. Patients of immotile ciliary syndrome are infertile because of the inability of sperm to move (azoospermia).

A – Normal cilia B – Cilia of Kartagener’s syndrome

By Filip em - Own work, CC BY 2.5, https://commons.wikimedia.org/w/index.php?curid=3838971

8. Summary

 The cytoskeleton is a cellular structure that helps cells maintain their shape and internal organization.

 Cytoskeleton consists of three major class of protein elements namely microfilaments, intermediate filaments and microtubules.

 Microfilament is formed by actin family of proteins

 Microtubules are the largest cytoskeletal element made up of tubulin proteins.

 Microtubule inhibitors are used in the therapy of cancer.

 Intermediate filaments are less dynamic and are made of at least 40 different subunit proteins.

 Cytoskeletal Molecular motors are the vehicles carrying the cargo. They run upon the cytoskeletal framework.

 Myosin is the motor protein that move along actin filaments using the energy of ATP hydrolysis while Kinesin is the motor protein that move along tubulin using the energy of ATP hydrolysis

 Dynamin requires free energy of hydrolysis of GTP.

 Primary ciliary dyskinesia (PCD), also called immotile ciliary syndrome or Kartagener syndrome is an autosomal recessive disorder due to dynein arm defect.