L2: Actin organisation in vivo

Why are nucleation factors (nucleators) required for nucleation of actin-filaments

• kinetically unfavvourable

• need to suppress the lag phase

What ultimately dictates the spatial distribution and type of strucutres made of actin filaments

• localisation

• mode of activation

• mechanism

characterising these nnucleators

Three main classes of actin nucleators

1. ARP2/3 complex

2. Spire

3. Formins

1. ARP2/3 complex

• similar to actin monomer + end ‘actin related protein’

• mimics and actin dimer or trimer

What they do

1. can initiate a new actin filament

2. can branch off of an existing filament→ generate Y branched actin networks

   • stays at the branch point

3. cannot form filaments on their own

4. Filaments continue to grow at the + end, while the - end is capped by Arp2/3 complex

1. What activates the ARP2/3 complex in vivo

• Associated with WASP (Wiskott-Aldrich Syndrome Protein)

  • family proteins

  • Nucleation-promoting factors

  • name→ genetic sex linked disorder

    • migration of actin function and tendency to malignancies

How it works

1. Signals

2. WASP released from an auto inhibitory conformation

3. Open conformation permits binding and activation to Arp2/3

2. Spire protein

• may promote linear association of 4 actin subunits

  • to act as scaffold for polyermisation into an unbranched filament

• may also cap the pointed end of the filament

3. Formins

• Promote nucleation of unbranched filaments

  • dimer stabilises an actin dimer/trimer to facilitate the nucleation event

  • remain associated with growing barbed ends of filaments

    • through sequential binding and release interactions

3. How do formins work→ e.g Diaphanous (Dia)-related formins

Act as dimers

• Contain FH1 and FH2 signature domains

• Adopt an open, active conformation upon recruitment by GTP-bound Rho-like GTPases

• Act as dimers and nucleate from G-actin only→ producing linear filaments

How it works

• FH1 domains recruit profilin-actin complexes (by polyproline sequences)

  • can increase the rate of elongation

  • i.e brings actin monomers to close proximity

• FH2→ processively tracks the growing barbed end of actin filament

  • Protecting ti from capping proteins

  • i.e actively does the polymerisation

    • by causing a ring conformation that allows nucleation that be elongated

  • Stablise actin oliogmers to induce nucleation and maintain a leaky cap

note: this also involves APC

What is meant by the leaky cap on the barbed end

• Formins act as as leaky caps at the + end

  • cap the end but still permit polymer elongation by allowing monomers to add on

What is APC

• Adenomatous polyposis coli

• nucleation promoting factor

  • reamins at the base of where actin starts

• collaborates with Formin

How APC and formin work together→ Rocket Launcher Model

• Formin at the + end→ walks along to stay on the + end

• APC→ remains at the base

  • helps aggregate the monomers and keep monomers together

  • Starts off the elongation

How was this visualised

Single-molecule formin association to the barbed end

1. followed in reconstitution experiments by total internal reflection fluoresence microscopy (TIRFM)

2. high resolution cryo-EM showed actin filaments bound to formins

What is the actin filament turnover regulated by

1. Profilin

2. Cofilin (ADF→ Actin-Depolymerising factor)

How does the actin filament turnover work

Profilin (polymerisation)

1. Bind exclusively G actin

2. exchanges ADP→ ATP

3. allowing recycling of D subunits back into T subunits

4. T subunits delivered to + end as a profilin-actin complex

5. Bind to + end and flatten→ into D form?

6. Decrease Affinity for Profilin

7. Profilin released

Cofilin (De-polymerisation of older filaments)

1. Old filaments→ are in D form

2. Target D subunits (due to higher affinity) (in - end)

3. binds to the filament to drop D subunits and causes stress→ fragmentation (severing)

4. Accelerates turnover of filaments

5. Cofilin phosphorylated→ releases D subunits

6. once D monomers released→ can be re used again by the action of profilin

7. Cofilin also blocks polyermisation at the - end

   • by inhibiting spontaneous ADP>ATP exchange on G-actin

What does binding to PIP2 or to the formin FH1 domain help do

• modulates locally profilin delivery of actin monomers

• to sites of polymerisation near the plasma membrane

What are motor proteins

• Mechanochemical enzymes

• move uni-directionally along a cytoskeletal track

• by coupling ATP hydrlysis with specific conformational changes

The direction of the movement is dictate by

1. the motor properties

2. polarity of the track

Features of motor proteins

1. Motor domain/ head region

   • has ATP and track binding sites

   • hydrolyses atp

   • cycles between sates in which binds and releases filament→ causes walking

   • The structure of the motor domain dictates the

     • Choice of cytoskeletal filament

     • Direction of movement

2. Tail Region

   • interacts with a ‘cargo’

   • determines the specific biological function of the motor protein

     • depending on what cargo it binds to

One type of motor protein→ actin based

Myosins

• Superfamily→ 18 members

• First found myosin 2 (in muscles) but found to be in all cells

What they do:

• Use energy fromATP hydrlysis to move along actin filaments

Role they play:

• carry organelles along actin tracks

• cause adjacent actin filaments to slide pas each other in contractile bundles

In which direction do myosins move

• Move towards the + end

  • (except type VI)

Example 1: Myosin II→ structure

• elongated protein

• two heavy chains

  1. globular head at N-terminus→ force generating machinery

  2. coiled coil tail→ mediates heavy chain dimerization

     • bundles itself with the tails of other myosin molecules

• two essential light chains

  • bind close to the head domain

What does the bundling of the myosin tails do

• generates bipolar ‘thick filaments’

• with several hundreed myosin heads

• orientated in opposite directions at the two ends of the thick filaments

• allows contractile acitivty due to directionality

Role of myosin II

1. contractile activity in muscle and non-muscles cells

2. cytokinesis→ (as component of actomyosin ring)

3. cell migration→ Forward translocation of the cell body

How does the fibrous tail help with contraction

• formation of filaments with heads pointing away from centre

• a bipolar strucutre than can drive contraction by promoting the sliding of actin filaments past each other

• as the heads both move towards polar ends

Example 2: Myosin V what is it involved in

• vesicular transport

How does Myosin V work

• continuous hand-over-hand fashion along filament

• processive motor that travels long distances without detaching from the track

• must make sure that it is coordinated movement so that there is always one head atached→ ensures it does not fall off

  • ATP cycle in a continuous manner

• only lets go when at destination

Myosin V compared to Myosin II

• Myosin II spends a major fraction of its cycle in a detached state

  • around 300 motor heads for rapid sliding of actin filaments as the muscle is signalled to contract

Example 2.1→ Myosin VIIa where present

• stereocillia

• in hair cells of the cochlea

• mutants→ responsible for deafness in humans

How to do video imaging of walking myosin V

High-speed atomic force microscopy

• single-molecule fluoresence miscroscopy (e.g using TIRFM- total internal reflection fluorescence microscopy)

  • observe individual fluorescent spots (from fluorophore attached to a protein)

    • not the proteins themselves

Compared to other methods (e.g EM or X-ray crystallography)

• show only a static view

• high-speed atomic (AFM)→ direct observation of the structure and dynamics of biomolecules simultaneously

Example of imaging myosin V by AFM

1. mica surface sully covered with biotin-containing lipid bilayers

2. Streptavidin molecules (green circles) deposited on the substrate

3. biotinylated actin filaments immobilised on the bilayer surface through streptavidin molcules

4. M5-HMM (a tail-truncated Myosin V) deposited on the lipid bilayers

Overall: Made is possible to directly visulaise myosin V molecues walking along actin tracks and to corroborate the ‘hand-over-hand’ model

Actin polymerisation-Driven Motility: why is cell migration important

• needed for

  • development→ growth cones

  • repair→ fibroblasts to repair wounds, osteoclasts to reach sites for bone remodelling

  • defence processes→ WBC

  • spread of tumour cells→ metastasis

• almost all cell locomotion occurs by actin-driven crawling

  • except sperm cells

Principles of cell movement

1. protrude a front

2. attach it to its substrate

3. retreat its rear

All driven by actin cytoskeleton

What does the cell need to be for movement and how

Cell must be polarised→ so it can persistently extend projections on one edge and reduce its net protrusive activity elsewhere

How→ Barbed + end features

• Lamellipodium→ branched

• Filopodium→ Bundled

Process of how a cell moves

1. Protruding either

   • thin leaflet of cytoplasm→ lamellipodium

   • Finger-like projection→ filopodium

     • (based on polymerization of an actin filament network

2. Actin and myosin make stress fibres

   • contractile pundles ending at focal contacts

3. Retraction of the rear of the cell driven by myosin II

4. Reorgnaisation of the actin cytoskeleton during crawling is coupled to cel adhesion to the underlying substratum

Adhesion is achieved by

• linkage of parts of the actin cytoskeleton to transmembrane receptors

• for extracellular matrix (integrins)

• at specialized focal contacts

• these must be dissolved from the back→ to ensure continued in the forward direction

Note: when observing movement and then mitosis

• shows that during mitosis→ movement stops

• suggsts→ the two processes clash

This hints to how in meiosis and the making of oocyte→ how it must be seriously regulated

• ensure not moving at the same time as meiosis or embryo

The cells are polarised but what directs the movement?

1. Chemotaxis

   • direction signalled by a gradient of a diffusible chemical

2. Non-diffusible cells

Examples of chemotaxis

1. Neutrophil in hot pursuit of bacterium

2. Dictyostelium discoideum responding to cAMP→ leads to aggregation for development of fruiting bodies

3. Neutrophile migration towards the site of a wound in a zebra fish larva

Direction of cell migration by non-diffusible cues

• from the extracellular matrix

example:

• nerve projections are generated by ‘crawling’ of growth cones

Outstanding question

Chemotaxis is good for short distances

• but how does it guide cell migration over long distances

  • e.g during embyronic development

  • e.g spreading metatstic cancer cells

• and complex paths branched paths

Does chemotaxis allow for this?

• gradients over long distances would be too shallow for guidance

What is the proposed explanation as to how cells can do this then?

Self-generated gradient

• cells may break down attractant

  • by cell surface enzymes or depleted by receptor-ligand endocytosis

• remains sufficiently steep near the cell to sustain long-distance migration

Why are self-generated gradients advantageous

1. steeper→ made in vicinity of cells

2. Work over long distances→ remade locally as cells migrate

3. Follow complex paths> generated by interactions between cells and environment

This has been proven by

• modelling and simulations

• factoing into attractant depletion and diffusion

What did it predict

• self- constructed chemoattractant gradeints may allow cells to navigate complex pathas

How was this tested

Microfluidic mazes of variable complexity

• Dictostelium cells led by cAMP self-generated gradient

• cancer cells directed by LPA (lysophosphatidic acid)

• could solve mazes→ sense upcoming junctions, accurately choose live channels over dead ends and identify optimum paths as shown in the figure

Add more info on the trakcs pls

Now that we have the signals, how do they get tansduced to direct movement→ what they use

Rho-like GTPases transduce signals to promote the polarised organisation of specific actin structures

• Rho, Rac and Cdc42→ members of the Ras superfamily of small GTPases

• 21kDa proteins

• with weak intrinsic GTPase activity

• act as molecular switches to signal transduction at the plasma membrane

  • by cycling between GTP and GDP bound forms

Why is GTPase acitivity significant for this

• Small GTPases are central in transducing external signals or internal cues to effector ABPs

  • to impart directionality to cytoskeletal structures

  • Also underlie the basis of pattern formation and embryonic development asymmetric partition of fate determinants or organelles in polarised asymmetric divisions

How do Rho-like GTPases transduce signals to promote the polarised organisation of specific actin structures

Directional migration arises from the asymmetrical activation of small GTPases at the cell surface according to the extternal single gradent

1. When GTP-bound→ Induce distinct membrane protursions:

2. Rac activate Arp2/3

   • causes Lamellipodia (branched F-actin networks)

3. Cdc42 activate Arp2/3

   • causes filopodia (linear bundles)

4. Rho promtes contraction by activating actin organisation by formins and myosin II

What happens when signal is received and Rho GTP is made

MLC→ two regulatory light chains of MLC that polymirses into thick filaments associating with actin filaments

1. RhoGTP

2. Activates Rho kinase (ROCK)

3. ROCK phosphorylates both MLC and MLC phosphatase (inactivating it)

4. MLC phosphorylation= myosin II filaments can slide along actin filaments and exert contractile force

Therefore: ROCK drives contraction both by activating MLC phosphoylation and inhibiting its deposphylation

Forms stress fibres

How is focal-adhesion developed and maintained

• linked to myosin-II induced contractility

  • stimulated by Rho

• With traction at focal contacts to transport the cell body forawrd

Therefore: directional movement is coupled to polaried adhesion-site turnover as well

Overall what do the Rho-like GTPases do

1. Lamellipodia

2. Filopodia

3. Stress fibres and focal adhesion

Extracellular signals that locally activate Rho, Rac and Cdc42 as plasma membrane trigger what

1. highly polarised F-actin nucleation

   • therefore→ promoting the ‘leading edge’

2. Branched (Arp2/3) and linear formins F-actin polyers aim their + ends towawrd the leading edge

3. further back in the cell→ as actin filaments age, cofilin cataluses depolymerisation

4. results in continuous treadmilling of actin subunits from front to back coupled to progression forward of the leading edge as shown in the model (below)

5. docal contacts also follow an asymmetric pattern

Model: Polarised actin-driven protrusion in crawling cells: the actin network at the leading edge of motile cells may consist of

• a branched array of F-actin pusing the plasma membrnae

• with + ends either facing or reaching tangentially the mmebrane

• - ends making Y-junctions with other filaments linked by Arp2/3 complex

MODEL: What happens when stimulants bind to the plasma membrnae

1. bind to receptors activating signalling pathways via small GTPases

2. activate WASP and related proteins by freeing autoinhibition

3. activates Arp2/3 complex

4. Arp2/3 initiates a branch on the side of an existing filament

5. new filaments grow rapidly at the + end, fed by profilin-bound actin

6. Capping proteins bind to the growing ends, terminating elongation

7. Small GTPases also activate formins to form filopodia

8. As filaments age→ cofilin binds to ADP-actin subunits and severs or depolymerises the ADP filaments

9. Profilin re-entes the cycle at this point→ promoting ADP-ATP exchange

10. Rho family GTPases also activate p21-activated protein kinase PAK

11. stimulates LIM kinase to phosphorylate and inhibit cofilin

Strong support to this model comes from

• biochemical and

• EM studies

what was shown:

• ability of Arp2/3 complex to promote actin filament formation by generating branch points on existing actin filaments

  • demonstrated in vitro asays

• acitivty has been correlated with EM images of cytoskeleton

  • show a richly branched actin network underlying the protruding plasma membrane

Protrusive activity may also play an important role in

• sampling gradients for guidance

However→ certain aspects of this dendritic network model have been challenged by studies based on

• electron tomography

and

• alternative fixation protocols

What has been questioned

The importance of Arp2/3-dependent nucleation in promoting the network (relative to alternative actin nucleators such as formins)

• controversy hinged on the existence and density of branch points in the network

• and relative stability of branched strucutres

• as later studies detected fewer branches and indicated instead the the presence of long linear fibres

Overall consensus

• actin filaments grow at the lemellipodium tip

• forming a treadmilling network

• with filaments polymerising at the front and under continuous turnover toward the rear of the lamellipodium

Contrasting views regarding the mechanistc impact of filament severing by cofilin on the dynamics of the actin network

1. View 1→ indirect contribution to treadmilling by increase in actin monomer pool

   • broought about by depolymerisation

2. view 2→ cofilin stimulates network formation through its severing activity that may give rise to short filaments

   • providing novel sites for initiation of Arp2/3-dependent actin branches

   • it follows that the detailed molecular action of the various players in setting up this strucutre is far from understood

Actin organisation in cells→ key concepts

1. ABPs control actin organisation, dynamics and turnover. Additional ABPs can form bundles or networks of F-actin and link them to membranes. These higher order structures generate a variety of cell membrane protrusions and impart cell shape.

2. Nucleation is temporally and spatially promoted by local signals that cause the recruitment and activation of the Arp2/3 complex (branched F-actin) or formins (linear structures).

3. Cofilin stimulates actin turnover and targets ADP-rich filaments (aged). Profilin promotes ADP/ATP exchange and delivers ATP-actin to sites of polymerisation. Actin bound to profilin can only be added to the barbed/(+) end of a filament. Additional sequestering, severing and capping proteins contribute to overall control of actin polymerisation.

4. Myosin II is the prototypic actin-based motor protein involved in contractile structures. Other family members play a variety of roles including vesicular transport along actin tracks. Most myosins travel to the (+) barbed end.

5. ABPs are under the control of signal transducers (Rho, Rac, Cdc42) linking actin organisation to internal cues or external signals. These pathways can induce cell polarity to support a variety of cellular processes. Among them we have discussed the generation of directional movement in crawling cells by stimulation of actin organisation and turnover at the leading edge.