MSE-2020 L12

The Cytoskeleton Part 1

What is the cytoskeleton?

A dynamic, intricate network of protein filaments involved with various processes

• Dynamicness helps it adapt to cellular needs and respond to external stimuli

What does the cytoskeleton do?

• Provides structural support

• Maintains cell shape

• Enables cellular movement and organisation

What is the cytoskeleton made of?

• Microtubules (tubulin polymers)

• Intermediate filaments

• Microfilaments (actin polymers)

Microtubules

• Cylindrical, tube-like structures composed of protein subunits

• Largest cytoskeleton component, diameter = 25nm

• Exhibit polarity (+/- end)

  • + end is site of rapid growth

  • - end is relatively stable

• Can rapidly assemble and disassemble, which is regulated by GTP hydrolysis

Protofilament and Tubule Formation

• α-tubulin and β-tubulin protein subunits which associate to form heterodimers

• Heterodimers further assemble head-to-tail, forming profilaments

• 13 parallel protofilaments associate to form a microtubule with a hollow core

What do microtubules do?

• Intracellular Transport

• Cell shape maintenance

• Formation of mitotic spindle during cell division

The Mitotic Spindle

• In non-dividing cells, microtubules radiate from microtubule-organising centres (MTOC) near nucleus with minus ends anchored to MTOC and plus ends extending towards cells

• Muring mitosis centrosomes (primary MTOCs) duplicate and move to opposite poles of the cell, with spindle microtubules extending from them

• Some spindle microtubules attach to kinetochores of chromosomes while others interact with microtubules from opposite centrosome

• Motor proteins and microtubule dynamics facilitate chromosome alignment and segregation > genetic material is equally distributed to daughter cells

How do microtubules show dynamic instability?

• Alternating phase of growth (polymerisation) and shrinkage (depolymerisation)

• Regulated by GTP-bound and BDP-bound tubulin at the ends of the microtubules

What does GTP-bound tubulin do?

• Part of microtubule assembly

• Incorporates to growing plus end, and the GTP is hydrolysed to GDP

• Acts as cap at the plus end to promote microtubule growth

What are intermediate filaments?

• Rope-like structures

• Second largest component skeleton, diameter = 10nm

• Named to their size being between microtubules and actin filaments

• Has conserved centrac α-helical rod domains, flanked by non-helical head and tail domains

What are intermediate filaments made of?

Protein types, categorised into types:

• I = acidic keratins

• II = basic keratins

• III = vimentin, desmin, glial fibrillary acidic protein

• IV = neurofilaments

• V = nuclear lamins

• VI = nestin

How are intermediate filaments formed?

1. Proteins assemble into parallel dimers through coiled-coil interactions between α-helical rod domains

2. Dimers further associate in antiparallel manner to form staggered tetramers, aka protofilaments

3. Eight protofilaments come together to form a intermediate filament, with a non-polar and elongated structure

What do intermediate filaments do?

• Distribute mechanical stress evenly, preventing deformation and damage

• Involved in cell-to-cell cell-to-extracellular matrix adhesion

• Regulates cell signalling pathways

• Other cellular processes e.g. cell migration > dynamically reorganise in response to external cues and intracellular signals

Do intermediate filaments show structural polarity?

No, only part of cytoskeleton to not. Not involved with intracellular transport or motor-protein based movements.

Type I: Acidic Keratins

• Expressed in epithelial cells

• For structural and mechanical integrity of epithelial tissues

Type II: Basic Keratins

• aka cytokeratins

• In epithelial cells

• Form heterodimers with Type I keratins, also provide mechanical support

Type III: Vimentin, desmin and GFAP

• Vimentin @ mesenchymal cells e.g. fibroblasts, endothelial cells, leukocytes > cell shape and mechanical resistance

• Desmin @ muscle cells > structural integrity for muscle fibers, connecting myofibrils to sarcolemma and other organelles

• GFAP @ glial cells e.g. astrocytes and Schwann cells > maintain NS structure and function

Type IV: Neurofilaments

• In neurons

• Composed of neurofilament light, medium and heavy (NFL, NFM, NFH) chains

• For radial gorwth of axons and axonal caliber, influences speed of nerve impulse conduction

Type V: Nuclear lamins

• In nuclear lamina, the meshwork underlying inner nuclear membrane

• Composed of

  • A-type lamins: Lamin A, C

  • B-type lamins: Lamin B1, B2

• For nucleus structural support, nuclear envelope assembly/disassembly, nuclear processes e.g. DNA replication and gene expression

Type VI: Nestin

• In neuronal stem cells (+ some progenitor cells @ development)

• Structural scaffold for mitotic cells, helps cell division and differentiation regulation

• Gets replaced by other IL fluids as cells differentiate

Actinfilaments

• aka microfilaments

• thinnest component of cytoskeleton, diameter = 7nm

What are actin microfilaments made of?

Globular actin (G-actin) protein subunits > assemble to helical polymer called filamentous actin (F-actin)

Do actin filaments exhibit polarity?

Yes - have barbed/plus end where subunits are added faster and a pointed/minus end where addition is slower

G-actin

• Globular actin

• Monomeric form of actin, a cell that is important in other cellular processes

• Each G-actin monomer has an ATP or ADP binding site, and two binding sites for interacting with other actin monomers

F-actin

• Filamentous actin

• Linear polymer, formed by head-to-tail assembly of G-actin monomers = flexible and dynamic filament with helical structure

• ATP-bound G-actins are incorporated into growing filament as process needs ATP

How do actin filaments form?

1. Nucleation process - G-actin monomers assmeble into stable nucleus

2. Additional G-actin monomers bound to ATP associate with plus end of filament, extending the length

3. Dynamic turnover of actin filaments = treadmilling, additional G-actin monomers at plus end is balanced by the dissociation of ADP-bound actin subunits from pointed end

4. Regulated by actin binding proteins

What do various ABP’s do to actin filaments?

• Promote nucleation

• Facilitate or inhibit elongation

• Stablisation

• Induce severing and disassembly

What are ABPs?

Actin binding proteins, interact with actin filaments and play essential roles

• Formins

• Arp.2.3 complex

• Profilin

• Cofilin

• Tropomyosin

Formins

Nucleate and elongate unbranched actin filaments by binding to barbed end, promoting actin subunit addition

Profillin

Promotes growth by binding to ATP-bound G-actin, increasing its affinity for barbed end and enhanced filament assembly

Cofilin

Cuts actin filaments, disassembles them by binding ADP-bound actin subunits, enhancing depolymerisation (shrinkage) at the pointed end

Arp2/3

• Actin related proteins 2/3

• Nucleates new actin filaments, creating branched networks by attaching to the sides of pre-existing filaments

Tropomyosin

Stabilises actin filaments, regulating interactions with other proteins e.g. myosin, by binding along filament length

Motor Proteins

Convert chemical energy from ATP hydrolysis into mechanical work > cell movement and force generation

• myosins

• kinesins and dyneins

Myosins

Motor proteins

What is the myosin structure?

• Conserved motor domain = actin binding, ATP hydrolysis

  • actin binding site

  • ATP binding site

  • ATP hydrolysis = conformational changes, enabling myosins to walk along actin filaments

• Variable tail domain = functional specificity by binding against cellular loads and structures

Myosin II

• In muscle contraction

• Molecules assemble into bipolar thick filaments, interacting with actin thin filaments to form sarcomere (muscle contractile unit)

  • Myosin cross-bridge cycle: conformational changes + ATP hydrolysis = myosin heads moving along actin filaments

Non-muscle myosins

• Myosin I, Myosin V

• Involved in cellular processes

• Specialised structures e.g. tall domains facilitate interactions with specific loads and structures

What are non-muscle myosins involved in?

• Vescicle transport

• Cell migration

• Cytokinesis

What are kinesins?

Motor proteins

What is the kinesin structure?

• Conserved motor domain - for movement along microtubules

  • microtubule-binding site

  • ATP-binding site

Kinesin plus-end-directed motors

Move towards plus end of microtubules (most of the kinesin family)

Kinesin minus-end-directed motors

Move towards the minus end of microtubules, e.g. Kinsein-14

Kinesin Stepping Mechanism

• Coordinated action of two motor domains (heads)

• Alternate between microtubule-bound and -unbound states while hydrolysing ATP = hand-over-hand walking motion

Where are kinesins used?

• Mitosis e.g. mitotic spindle formation and positioning

• Chromosome alignment

• Segregation of sister chromatids to daughter cells during anaphase

What are dyneins?

• Family of motor proteins, move along microtubules

• Convert energy from ATP hydrolysis > mechanical work for cellular processes

What type of motors are dyneins?

Minus-end-directed, move towards the minus end of microtubules which is oriented towards cell centre

Can dyneins be further categorised?

• Cytoplasmic dyneins: intracellular transport + mitosis

• Axonemal dyneins: beating cilia and flagella

Why is dynein-mediated transport important?

• For proper positioning and function of organelles

  • Golgi apparatus

  • Endosomes

  • Lysosomes

  • Vesicle transport

What are focal adhesions?

• Specialised multi-protein complexes, connect actin cytoskeleton to extracellular matrix (ECM)

• Facilitates cell adhesion, migration and mechanosensing

Integrins

Family of transmembrane receptors, helps focal adhesion by binding to ECM proteins e.g. laminin, collagen

• connects actin cytoskeleton by adapter proteins

Adapter proteins

• Talin, vinculin, paxillin link integrins to actin cytoskeleton

• Recruits additional signalling and structural proteins = complex focal adhesion network

Focal adhesion kinases (FAK) and Src family kinases

• Initiate downstream signalling cascades that regulate cellular processes

• Become activated during integrin engagement