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Cell biology 2014 (revised 11/2-14). Lecture 8 & 9:. The cytoskeleton Function, design and regulation. Chapter 16 965-1020 1026-1050 A lot of reading! Focus on general principles and topics highlighted in the lecture synopsis .

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The cytoskeleton function design and regulation

Cell biology 2014 (revised 11/2-14)

Lecture 8 & 9:

The cytoskeleton

Function, design and regulation

Chapter 16

965-1020

1026-1050

A lot of reading!

Focus on general principles

and topics highlighted in

the lecture synopsis

Cell Biology interactive  media  ”video” or ”animation”


The cytoskeleton function design and regulation

Classical cytoskeletons

Microtubules

Actin filaments

(Microfilaments)

Intermediate

filaments


The cytoskeleton function design and regulation

1. Why do we need a cytoskeleton?

Cell containing

cytoskeleton

Cell without

cytoskeleton

  • Establishment of cellular

  • shape and intracellular

  • organization

ER

Golgi

1

  • Resistance against

  • mechanical stress

1


The cytoskeleton function design and regulation

2. Why do we need a cytoskeleton?

Cell containing

cytoskeleton

Cell without

cytoskeleton

  • Cellularappendages

  • Cell locomotion

  • Genomic and

  • cellular division


The cytoskeleton function design and regulation

Principle architecture of cytoskeletal filaments

Actin

filaments

Intermediate

filaments

Microtubules

7 nm

10 nm

25 nm

Tubulin

heterodimer

Actin

Subunits:

A family of coiled-coil proteins


The cytoskeleton function design and regulation

Intermediate filaments – structure and function

Non-polar filaments

Cytosol: support of cell layers ( tensile stress )

Nucleus: supporting the nuclear envelope

amphipathic a-helical monomers

+

Tetramer of coiled-coil dimers

Cell adhesion

(desmosome)

animation16.4

-Intermediate _filament


The cytoskeleton function design and regulation

Tissue specific intermediate filaments

Intermediate filaments can be composed of either:

Homodimers or heterodimers

- Intermediate filament super-family: >60 genes in mammals

Cytosolic

Nuclear

Protein:

Cyto-

keratins

Vimentin,

Desmin

Neuro-

filaments

Lamins

Lining of the nuclear membrane of all cells

Location:

Epithelia

Neurons

Cells in

connective-

and muscle

tissue


The cytoskeleton function design and regulation

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

Intermediate filaments in epidermis

Keratin:

1 + 10

5 + 14

ECM

(Basal lamina)


The cytoskeleton function design and regulation

Actin filaments – structure and function

Structure

- Polar filaments composed of actin

Functions

- Linking the interior to the exterior ( )

- Contraction ( )

- Spreading & protrusions

 cell shape

- Locomotion

- Contractile ring during cell division

ECM

Video 01.1-keratocyte_dance

Video 22.7 –neurite_outgrowth


The cytoskeleton function design and regulation

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Actin filaments are dynamic in migrating cells

Rapid assembly and disassembly is central to a variety of functions, such as cell remodeling and locomotion

Stimuli

Time


The cytoskeleton function design and regulation

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Subunit interactions within actin filaments

A polymer with only longitudinal subunit interactions

 uniform (and poor) polymer stability

+

+

Protofilament

(proto = a prefix meaning the “earliest”)

A polymer with both longitudinal and lateral subunit interactions

 stability within the polymer but dynamic ends

Internal stability

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Dynamic ends


The cytoskeleton function design and regulation

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Fanta

Fanta

Fanta

Fanta

Nucleation of actin filaments

Spontaneous nucleation is slow because the initial interactions are unstable (low degree of cooperativity)

Spontaneous nucleation

Spatially regulated nucleation factors  local nucleation

Fanta

Fanta

Fanta

Fanta

Nucleation

factor


The cytoskeleton function design and regulation

Fanta

Fanta

Fanta

Fanta

Fanta

Fanta

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Fanta

Fanta

Fanta

Fanta

Fanta

Fanta

Fanta

Fanta

Fanta

Fanta

Fanta

Fanta

Control of actin filament nucleation

No availabile

nucleation factor

Inactive

nucleation factor

No (specific) nucleation

No (specific) nucleation

Localized activation

of nucleation

factors

Global activation

of nucleation

factors

Local

nucleation

Global

nucleation


The cytoskeleton function design and regulation

Actin nucleation factors

Arp 2/3

Formin

+ end

Arp 2/3 may also

bind pre-existing

filaments to create

branching

+ end

- end

- end


The cytoskeleton function design and regulation

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Concept of the critical free concentration

The monomer ( = soluble subunit) concentration ( = [Free]) at steady state is referred to as the critical concentration

0

100%

Steady state

Elongation

Monomer concentration

[Free subunits]

% Subunits in filament

(% Bound)

Spontaneous

nucleation

0 %

60

Time (minutes)


The cytoskeleton function design and regulation

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Dynamics at different free concentrations

[Monomer] = [Free subunits]

[Monomers] > critical concentration, e.g. 3 nM

Net polymerization

[Monomers] = critical concentration, e.g. 2 nM

No net effect on

polymer length

[Monomers] < critical concentration, e.g. 1 nM

Net depolymerization


The cytoskeleton function design and regulation

Nucleotide turnover in cytoskeletal subunits

Actin subunits bind ATP

Tubulin heterodimers bind GTP

The subunit changes its conformation upon nucleotide hydrolysis

Subunit bound to a nucleoside

triphosphate

Coca Cola

Nucleotide hydrolysis

Subunit bound to a nucleoside

diphosphate

Coca Cola

A nucleoside is the portion of a nucleotide that doesn't include the phosphate groups


The cytoskeleton function design and regulation

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

Coca Cola

I. ATP fueled actin treadmilling

”Low” critical

concentration

(e.g. 2 nM)

”High” critical

concentration

(e.g. 10 nM)

- end

+ end

Treadmilling occurs when [Monomers] (i.e. [Free subunits]) is

between the critical concentrations at the two ends (2  10 nM)

ADP

ATP

During treadmilling the filament length remains constant, while subunits are added at the (+) end and dissociate from the (-) end


The cytoskeleton function design and regulation

II. ATP fueled actin treadmilling

ADP

P

P

:interaction strength

+

-

ADP

ADP

ADP

ADP

ATP

ADP

ATP

P

P

ADP

ADP

ADP

ATP

[ADP] << [ATP]

ADP

ATP

ATP

ATP

Time

-

+

= Fluorescent actin

Treadmilling  actin subunits "move" towards the (-) end


The cytoskeleton function design and regulation

Treadmilling requires actin severing

[G-actin] = [Monomer] = [Free]

Arp2/3 stabilizes the (-) end

-

+

Polymerization ceases due to low [G-actin]

A severing protein – ADF/Cofilin –

binds to ADP-actin containing filament

Polymerization at the (+) end can resume

and the filament will treadmill, which will

facilitate continuous growth at the (+) end

ADP/ATP exchange

Significance ?!

 slide 10 & 52


The cytoskeleton function design and regulation

Microtubules

Structure

- Hollow polar tubes of

tubulin protofilaments

Tubulin heterodimer

b-tubulin

a-tubulin

Protofilament

Microtubule

Function

Exert both pushing and pulling forces

Structural support and railroad tracks, which establish intracellular organization

Locomotion by cellular appendages (cilia and flagella)

Segregation of chromosomes during mitosis


The cytoskeleton function design and regulation

Pushing and pulling by microtubules during mitosis

Interphase (G2)

Prophase

Telophase/

cytokinesis

Prometaphase

Anaphase

Metaphase

Astral-

video 13.2 –

biosy_secret_path

Video 17.7 –

mitotic_spindle

Overlap-

Kinetochore MT


The cytoskeleton function design and regulation

The centrosome – the site for microtubule nucleation

The centrosome contains ~100 g-tubulin ring complexes,

which act as nucleation sites for microtubule assembly

Centriole pair

g-Tubulin Ring

Complex

Minus-end

All subunits are encoded for by the genome, but assembly requires an inherited copy as a template

Plus-end


The cytoskeleton function design and regulation

-

-

-

-

-

+

+

+

+

+

Different microtubules arrangements

+

+

+

-

  • “Most” cell types

+

+

+

+

  • Columnar epithelial cells

  • (small intestine)

-

+

-

-

+

+

-

-

+

+

-

  • Neurons

+


The cytoskeleton function design and regulation

GTP

GTP

GTP

GTP

GTP

GTP

GTP

GTP

GTP

GTP

Pi

GTP hydrolysis at the E-site of the tubulin heterodimer

E-site

E-site

E-site = Exchangeable site

b

a

GTP

GDP

b

a

Catalytic

loop

Catalytic

loop


The cytoskeleton function design and regulation

Proteins that control microtubule dynamics

Stabilization by Microtubule Associated Proteins (MAPs)  Multivalent binding along the polymer

Destabilization by catastrophe promoters  Peeling of proto-filaments at the end


The cytoskeleton function design and regulation

GTP-tubulin

GDP-tubulin

MT dynamics – catastrophe

GTP cap (E-site exposed)

Catastrophe promoting protein

+ end

(delay in GTP

hydrolysis)

  • end

  • (nucleated at

  • the centrosome)

The + end

is “capped” by

GTP-tubulin

Catastrophe,

followed by

depolymerization

Peeling of proto-filament


The cytoskeleton function design and regulation

GTP-tubulin

GDP-tubulin

MT dynamics – rescue

Paus

Depolymerization

Regain of GTP cap

through re-initiated

polymerization

: Rescue promoting protein

video 16.1- MT_instability


The cytoskeleton function design and regulation

Dynamic instability – stochastic switches

[GDP] << [GTP]

GTP

GDP

+ end

Polymerization

Depolymerization

Catastrophe

Rescue

  • end

  • (nucleated at

  • the centrosom)

: Rescue promoting protein

: Catastrophe promoting protein

Dynamic instability ”search and capture” of a variety of structures


The cytoskeleton function design and regulation

P

Cdk

Cdk

M

M

P

P

Cell cycle regulation of microtubule dynamics

Interphase

Mitosis (active Cdk/M)

P

P

Few and long microtubules:

- Few nucleation events

- Slow dynamics

Many and short microtubules:

- Many nucleation events (5x)

- Rapid dynamics (10x)

video 16.5- microtubule_dynamics

Note- visualization by fusion to a fluorescent protein (EB1-GFP& aTub-GFP)


The cytoskeleton function design and regulation

Capture of kinetochores by microtubules

MTs continuously searches the cellular space...

1

...and are stabilized at the

kinetochores of chromosomes

2

*

*

*

*

*

*

*

*

*

*

Finally, both kinetochores are captured by MTs from opposite centrosomes.

*The sister chromatid pair is positioned at the cellular equator by the polar ejection force generated by MTs (* )

3

Generation of pulling force


The cytoskeleton function design and regulation

Unidirectional transport on polar polymers

Candy

Check-out

Polarity in a queue at the supermarket

Motor proteins (unidirectional movement)

+ end

- end


The cytoskeleton function design and regulation

A

B

A

A

B

A

B

B

A

B

Movement of MT dependent motor proteins

Dynein

Kinesin

- end

+ end

Head-over-head walking (an ATP dependent process)

1.

2.

3.

4.

5.

animation 16.7- kinesin


The cytoskeleton function design and regulation

+

+

MT dependentpushingforcesduringmitosis

Kinesin dimer

Kinesin dependent pushing forces via anti-parallel MTs are required for:

Prophase

Anaphase (B)


The cytoskeleton function design and regulation

Control of division plane in epithelia

Incorrect

Correct


The cytoskeleton function design and regulation

Control of division plane by astral microtubules

Dynamic (astral) microtubules are stabilized by tip binding proteins ( ) at specific sites at the cell cortex

1.

Membrane anchored

dynein ( ) pulls at astral microtubules

-

2.

1.

+

2.

Basal lamina(ECM)

Pulling forces specify the correct division plane

3.

3.


The cytoskeleton function design and regulation

Cell polarization by localized MT stabilization

A non-polarized cell in which MTs search the intracellular space

Stabilization of MTs that encounters localized tip-binding proteins ( )

Reorientation of the MT- system

by membrane anchored dyneins ( )

A polarized cell: stabilized MTs serve as

rail tracks that transport membrane vesicles

and actin regulatory proteins to MT (+) ends


The cytoskeleton function design and regulation

MT-dependent trafficking in the cell

+

Kinesin

Dynein

Virus

ER

+

-

-

Vesicle

Axon

Golgi

Lysosome

Synapse

Endocytosis

Mitochondrion

Exocytosis

video 16.6- organelle_movement


The cytoskeleton function design and regulation

Cellular appendages built of microtubules

Cilia

Flagella

  • - 5 -10 mm appendages projecting

  • from cell surfaces

  • - Capable of movement

  • - Moves fluids over

  • the cell surface

- In essence a cilia, but longer (100-200 mm)

- Only one per cell

- Move the cell in a

wavelike fashion


The cytoskeleton function design and regulation

Arrangement of microtubules in cilia and flagella

Flagella

Axoneme

Cilia

Basal

body

Axoneme: the part of a cilia or flagella that bends back and fourth


The cytoskeleton function design and regulation

The beating of a cilia

The beating of cilia is dependent on MT bending forces

Power

Stroke

(energi

input)

Axoneme

Basal

body

Recovery

Stroke

(back to default)


The cytoskeleton function design and regulation

1.

2.

3.

Dynein dependent MT bending in cilia and flagella

Nexin, holds the MTs together

Anchorage to

dynein tail

Bending of MTs upon

dynein movement


The cytoskeleton function design and regulation

+

Head

ADP

+Pi

-

Myosin: a family of (+) end-directed actin motors

Myosin bound to actin filament

ATP binding dissociates myosin

ATP

ATP is rapidly hydrolyzed, which cause

a simultaneous conformational change

ADP

+Pi

Following ATP hydrolysis, myosin

binds an actin subunit

Binding to actin causes the release

of ADP + Pi. This results in the

conformational change termed

the “power stroke”

ADP

+Pi

Video 16.9 –crawling_actin


The cytoskeleton function design and regulation

+

-

-

-

-

+

“Non-muscular” myosin family members

A large family of related (+) end directed motors.

Example of functions:

+

Monomeric myosin

Transport(short range)

Non-muscular myosin II

Contraction

(movement towards the +ends

of two anti-parallel actin filaments)

+

Cargo


The cytoskeleton function design and regulation

Lumen

Lumen

Muscles – a brief overview

  • Skeletal muscle, fused myoblast that forms a multinucleated cell

  • (fast but non-persistent)

  • Cardiac muscle cells

  • (persistent)

  • Smooth muscle cells

  • i) surrounds hollow organs – intestines and blood vessels

  • ii) Arrectorpili muscles attached to hair follicles

  • (slow and very persistent)

video 16.11- beating_heart


The cytoskeleton function design and regulation

Principle of skeletal muscle contraction

When stimulated to contract, the heads of the bipolar myosin filament walk along actin in repeated cycles of attachment and detachment  contraction of the sarcomere unit

Actin

Myosin

Actin

Myosin

Contraction

+

+

+

+

Sarcomere

Sarcomere

  • - The actin and myosin filaments remain the same length

  • - The sarcomere length shortens because the actin and myosin filaments slide relative each other

animation 16.8- myosin (compare with picture 43)


The cytoskeleton function design and regulation

Tropomyosin

Tropomyosin

Tropomyosin

Tropomyosin

Tropomyosin

Tropomyosin

Tropomyosin

Tropomyosin

Regulation of skeletal muscle contraction

  • Tropomyosin binds along the actin filament:

  •  No contact between actin and myosin filaments

Contraction is initiated by an increase of cytosolic Ca2+:

Troponin mediated translocation of tropomyosin

Ca2+

animation 16.10- muscle_contraction


The cytoskeleton function design and regulation

-

-

-

-

Higher-order architecture of actin filaments

Actin filaments (in non-muscle cells) may associate into bundles or networks via different cross-linking proteins

Anti-parallel bundles

allowing access to

myosin II

Sparse 3D

network

Tight parallel bundles

+

+

a-actinin

a-actinin

Fimbrin

Fimbrin

+

+

Filamin

short and thin fibers

Long and thick fibers


The cytoskeleton function design and regulation

Cell migration requires locally acting GTP switches

Rho

Rac

Cdc42

GTP

GTP

GTP

-

-

+

-

-

+

Stress fibers

(contraction)

Actin web

(tread milling)

Actin bundles

(protrusions)

+

Rho/Rac/Cdc42 are GTP switches (similar to Ras)

+


The cytoskeleton function design and regulation

An integrated view of actin dependent migration

Actin structure:

Stress fibers

(contractile)

Lamellipodia

(pseudopodia)

Filopodia

(microspikes)

+

-

-

-

+

Chemoattractant (e.g. PDGF)

+

+

-

Rho family member:

Rho

Rac

Cdc42

video 23.9- wound_healing


The cytoskeleton function design and regulation

Fanta

Fanta

Actin nucleation and bundling by Cdc42

Chemotactic signal

Cdc42

GEF

Cdc42

GDP

GTP

Cdc42

Arp 2/3

Fanta

Fanta

Fimbrin

Actin nucleation

Tight parallel

actin bundles

video 10.1- membrane_fluidity


The cytoskeleton function design and regulation

P

P

P

Fanta

Fanta

Rac dependent lamellipodiaformation

Chemotactic signal

(PI3-K dependent – see slide 56)

Rac

GTP

GDP

3

Rac

Rac

GEF

ADF/Cofilin

Fanta

Fanta

Stable actin

meshwork

Filamin

Actin nucleation

and branching

ADF/Cofilin dependent severing  treadmilling


The cytoskeleton function design and regulation

Stable filaments

2.

ADF/Cofilin

1.

Actin

nucleation

3.

4.

Rho dependent stress fiber formation

Internal (localization dependent) signals

GTP

Rho

Formin

- end

a-actinin

a-actinin

Myosin II

- end

Anti-parallel

actin bundles

Contraction


The cytoskeleton function design and regulation

Summary of cell motility

1.

2.

3.

4.

Contraction and

translocation

Protrusion

+

+

-

Chemoattractant

+

-

-

+

-

ECM

ECM attachment at the leading edge (focal adhesions)

Detachment at trailing end

Video 01.2 –crawling_amoeba


The cytoskeleton function design and regulation

1

2

3

Role of actin for neutrophil migration

NeutrophilshavefMet-Leu-Phe receptors

Bacteria, releasing

peptidescontaining

N-formyl-Methionine

The activated receptor provides the direction of pseudopodia formation

Neutrophil engulf bacteria through phagocytosis

video 15.2chemotaxis

video 16.2-neutrophile_chase


The cytoskeleton function design and regulation

P

P

P

P

P

g

Neutrophil chemotaxis

fMet-Leu-Phe receptor

N-formylatedbacterial protein

GPCR

GTP

GDP

b

a

a

+

b

3

3

g

PI-3 Kinase

Rac

GEF

Phosphatidyl-

inositol

PI-3 Kinase

Rac

GTP

Arp 2/3

Stable actin

meshwork

zzz

Filamin

GDP

Rac

Rac GEF

Actin nucleation

and branching


The cytoskeleton function design and regulation

g

II. Regulation of hetero-trimeric G-proteins

No ligand (default state)

P.M.

GDP

a

b

g

Ligand binding causes a conformational change

P.M.

GTP

GDP

b

a

a

+

b

g

GTP

GDP

The G-protein is recruited to the receptor, which acts as a GEF  the a-subunit exchanges GDP for GTP

 dissociation of an active a-subunit


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