Fundamentals of Cell Biology

Chapter 5: The Cytoskeleton and Cellular Architecture

• Chapter foci: – Cytoskeletal proteins form a skeleton inside the

cell– Intermediate filaments provide the cell with

mechanical strength– Microtubules are associated with cellular

trafficking– Actin is responsible for large-scale movements– Eukaryotic cytoskeletal proteins evolved from

early prokaryotes

Chapter Summary: The Big Picture (1)

Chapter Summary: The Big Picture (2)

• Section topics:– The cytoskeleton is represented by three

functional classes of proteins– Intermediate filaments are the strongest, most

stable elements of the cytoskeleton– Microtubules organize movement inside a cell– Actin filaments control the movement of cells– Eukaryotic cytoskeletal proteins arose from

prokaryotic ancestors

The cytoskeleton is represented by three functional classes of proteins

• Key Concepts:– The cytoskeleton is a complex mixture of 3 different types of

proteins that are responsible for providing mechanical strength to cells and supporting movement of cellular contents.

– The most visible form of cytoskeletal proteins are long filaments found in the cytosol, but these proteins also form smaller shapes that are equally important for cellular function.

– The structural differences between the 3 protein types underscores their 4 different functions in cells.

Cytoskeleton

• occupies large portion of cytosol and appears to link organelles to each other and to plasma membrane

• 3 elements: IFs

MTs

Actin• Elements do not form

mixed polymers

Figure 05.01: The cytoskeleton forms an interconnected network of filaments in the

cytosol of animal cells.

IFs are the strongest, most stable elements of the cytoskeleton

• Key Concepts (1):– Intermediate filaments are highly stable polymers

that have great mechanical strength.– Intermediate filament polymers are composed of

tetramers of individual intermediate filament proteins.

– Several different genes encode intermediate filament proteins, and their expression is often cell- and tissue-specific.

IFs are the strongest, most stable elements of the cytoskeleton

• Key Concepts (2):– Intermediate filament assembly and disassembly

are controlled by posttranslational modification of individual intermediate filament proteins.

– Specialized intermediate-filament-containing structures protect the nucleus, support strong adhesion by epithelial cells, and provide muscle cells with great mechanical strength.

IFs provide mechanical strength to cells

Figure 05.02: Two types of intermediate filaments.

Figure 05.03: Intermediate filaments have the most mechanical strength of the

cytoskeletal proteins.

IFs are formed from a family of related proteins

The primary building block of IFs is a filamentous subunit

• α-helices in the central rod domain

Figure 05.04: The central rod domain of intermediate filament proteins forms an alpha helix. The head (and tail) regions form globular shapes.

IF subunits form coiled-coil dimers

Figure 05.05: A model for intermediate filament assembly. The coiled coil formed by the dimer formed the structural basis for the

strength of intermediate filaments.

Heterodimers overlap to form filamentous tetramers

• Coiled-coils align to form antiparallel staggered structures

Assembly of a mature IF from tetramers occurs in 3 stages

Figure 05.05: A model for intermediate filament assembly. The coiled coil formed by the dimer formed the structural basis for the strength of intermediate filaments.

Posttranslational modifications control the shape of intermediate filaments

• Chemical modification of IF controls their shape and function– Phosphorylation-dephosphorylation– Glycosylation– Farnesylation– Transglutamination of head and tail domains

IFs form specialized structures

Keratins in epithelium Costameres

Figure 05.06: Keratin expression patterns vary in different epithelial tissues.

Figure 05.07: Costameres link the contractile apparatus of muscle cells to the plasma

membrane and extracellular matrix.

Microtubules (MT) organize movement inside a cell

• Key Concepts (1):– MTs are hollow, tube-shaped polymers

comprised of proteins called tubulins.– MTs serve as “roads” or tracks that guide the

intracellular movement of cellular contents.– MT formation is initiated at specific sties in the

cytosol called MT-organizing centers. The basic building block of a MT is a dimer of two different tubulin proteins.

MTs organize movement inside a cell

• Key Concepts (2):– MTs have structural polarity, which determines the direction of

the molecular transport they support. This polarity is caused by the binding orientation of the proteins in the tubulin dimer.

– The stability of MTs is determined, at least in part, by the type of guanine nucleotides bound by the tubulin dimers within it.

– Dynamic instability is caused by the rapid growth and shrinkage of MTs at one end, which permits cells to rapidly reorganize their MTs.

MTs organize movement inside a cell

• Key Concepts (3):– MT-binding proteins play numerous roles in

controlling the location, stability, and function of microtubules.

– Dyneins and kinesins are the motor proteins that use ATP energy to transport molecular “cargo” along MTs.

– Cilia and flagella are specialized MT-based structures responsible for motility in some cells.

MT cytoskeleton is a network of "roads" for molecules "pass to and fro"

MT assembly begins at a MT-organizing center (MTOC)

Figure 05.08: The distribution of microtubules in a human epithelial cell. The microtubules

are stained green and the DNA is stained red.

The MTOC contains the gamma tubulin ring complex (γTuRC) that nucleates

MT formation• Centrioles• Pericentriolar material• gamma (γ ) tubulin  

Figure 05.09: The structure

and location of the centrosome.

The primary building block of MTs is an alpha-beta tubulin dimer

• α - and β -tubulin bind together to form stable dimer

• If purified α-β  tubulin dimers bound to GTP are concentrated enough (critical concentration), they spontaneously form MTs  

Figure 05.10: A three dimensional model

of the dimer formed by α- and β-tubulin.

Figure 05.11: In vitro assembly of microtubules is spontaneous and GTP-dependent. The graph

represents the turbidity of a solution of α-β tubulin dimers

over time.

MTs are hollow "tubes" composed of 13 protofilaments

• Polymers of dimers sheet composed of 13 protofilaments folds into a tube

• GTP binding and hydrolysis regulate MT polymerization and disassembly

Figure 05.12: A simple model of microtubule assembly.

The growth and shrinkage of MTs is called dynamic instability

• Some microtubules rapidly grow and shrink in cells = dynamic instability

• Elongation is at the

+ end by GTP-bound dimers

Figure 05.13: Growth and shrinkage of microtubules in a living cell. The microtubules have been tagged with a fluorescent

molecule, and recorded by video over time.

Figure 05.14: The growth of microtubules begins at the gamma tubulin ring and continues as long as the plus end contains

GTP-bound tubulin dimers.

Catastrophe?

• What happens when the supply of GTP-bound tubulin dimers runs out?

1) MT depolymerizes at the + end

OR

2) Capping proteins prevent depolymerization Figure 05.15: Two fates of the plus

ends of microtubules.

Some MTs exhibit treadmilling

• In cases where neither end of MT is stabilized, tubulin dimers are added to the + end and lost from the - end

• Overall length of these MTs remains fairly constant, but the dimers are always in flux

Figure 05.16: Treadmilling in microtubules.

Benefits of dynamic instability

Allows cells to have – flexibility with

trafficking during cell movement

– ability to exert force by bonding with cargo molecules

Figure 05.17: Microtubules exert enough force to move cargo by dynamic instability.

Figure 05.18: Longitudinal and lateral bonds make microtubules strong.

MT-associated proteins regulate the stability and function of MTs

• “MAPs” = capping proteins, rescue-associated proteins, and proteins that govern the motion

• motor protein = special type of MAP that transports organelles/vesicles– Dyneins and kinesins

Motors

Figure 05.19: The structure of dynein and kinesis, the two most

common motor proteins that bind to microtubules.

Figure 05.20: How a microtubule motor protein moves along a microtubule.

Cilia and Flagella

Axoneme

Sliding dynein = whip movement

Figure 05.23: The structure of an axoneme.

Figure 05.24: The coordinated motion of a cilium and a flagellum.

• Key Concepts (1):– Actin filaments are thin polymers of actin proteins.– Actin filaments are responsible for large-scale

changes in cell shape, including most cell movement.– Actin filament polymerization is initiated at numerous

sites in the cytosol by actin-nucleating proteins.– Actin filaments have structural polarity, which

determines the direction that force is exerted on them by myosin motor proteins.

Actin filaments control the movement of cells

• Key Concepts (2):– The stability of actin filaments is deteremined by

the type of adenine nucleotides bound by the actin proteins within them.

– Actin-binding proteins play numerous roles in controlling the location, stability, and function of actin filaments.

– Cell migration is a complex process, requiring assembly and disassembly of different types of actin filament networks.

Actin filaments control the movement of cells

The building block of actin filaments is the actin monomer

• Smallest diameter of cytoskeletal filaments – 7nm “microfilament”

• Great tensile strength• Structural polarity

+ end – barbed end - end – pointed end

• Often bound to myosin

Figure 05.25: The general structure of an

actin filament. The lateral and longitudinal

bonds holding actin monomers together

are indicated at right.

Figure 05.26: An electron micrograph of an actin

filament partially coated with mysoin proteins.

Actin found in wide variety of locations and configurations

Figure 05.27: A number of different actin filament-based structures in cells.

ATP binding/hydrolysis regulate actin filament polymerization and disassembly

• + ATP = polymerization• ATPADP = depolymerization

Figure 05.28: The structure of an actin monomer. A

ribbon model, derived from a crystalized form of the

protein.

Actin polymerization occurs in 3 stages

Figure 05.29: The three stages of actin filament assembly in vitro.

Actin filaments have structural polarity

• Actin filaments undergo treadmilling

Figure 05.30: Treadmilling in actin filaments. Note the similarity of this

treadmilling with that shown for microtubules in Figure 5-16.

6 classes of proteins bind to actin to control its polymerization/organization

1. Monomer-binding proteins regulate actin polymerization

2. Nucleating proteins regulate actin polymerization

Figure 05.31: The structure and

function of profilin, an actin

monomer-binding protein.

Figure 05.32: ARP2/3 nucleates the formation of a new actin

filament off the side of an existing filament.

6 classes of proteins bind to actin to control its polymerization/organization

3. Capping proteins affect the length and stability of actin filaments

4&5. Severing and depolymerizing proteins control actin filament disassembly

6. Cross-linking proteins organize actin filaments into bundles and networks

Figure 05.34: Three forms of crosslinked actin filaments created by different

crosslinking proteins.

Cell Migration

• Actin-binding motor proteins exert force on actin filaments to induce cell movement

• Cell migration is a complex, dynamic reorganization of an entire cell

• Migrating cells produce three characteristic forms of actin filaments: filopodia, lamellopodia, and contractile filaments

Filopodia

Figure 05.35: Different forms of actin in stationary and migrating cells.

Myosins are a family of actin-binding motor proteins

• myosins = multisubunit proteins organized into 3 structural domains– Motor– Regulatory– Tail Figure 05.36: Myosin proteins contain three

funtional domains

Contractile cycle

• Myosins move towards one end of the actin filaments– myosin V crawls

towards the - end, all other myosins crawl towards the + end

– Allows for movement of cell Figure 05.37: The contractile cycle of myosin.

Striated muscle contraction is a well-studied example of cell movement

Figure 05.38: The anatomy of a skeletal muscle. The sarcomere contains actin and myosin arranged in parallel bundles.

Eukkaryotic cytoskeletal proteins arose from prokaryotic ancestors

• Modern prokaryotic cells express a number of cytoskeletal proteins that are homologous to eukaryotic cytoskeletal proteins and behave similarly– Vimentin (IF)– FtsZ (MT)– MreB and ParM (actin)

• Shared properties seem to include protection of DNA, compartmentalization and motility.