Aktin cytoskeleton Seminar PCDU WS15/16 Vera Krieger - 28.10.15

Aktin cytoskeleton

Seminar PCDUWS15/16

Vera Krieger - 28.10.15

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Actin cytoskeleton

https://upload.wikimedia.org/wikipedia/commons/thumb/9/96/La-Jolla-Red-Tide.780.jpg/220px-La-Jolla-Red-Tide.780.jpg

Actin cytoskeleton

Cytoskeleton:

• Microtubule

• Intermediate filament

• Microfilaments or actin filaments

-> Located in cytoplasm of eukaryotic cells

Endothelia cells (MT- green, AF- red, Nuc – blue)

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Actin cytoskeleton

http://www.spring8.or.jp/en/news_publications/press_release/2009/090122_fig/fig4.jpg

Structur of Actin filamentes

Tertiary structures of G-actin and F-actin

G-Actin:

Globular actinCa. 42 kDa ProteinTwo lobes separated by a cleft

Polymers of G-actin subunits -> F-actin

Bound to ATP / ADPMost common: ATP-G-Actin ADP-F-Actin

Actin monomer bind to Mg2+ und Ca 2+

-> affects polymerization dynamicsConcentration increased of free cations – actin filaments stiffer

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Actin cytoskeleton

Blanchoin et al. 2014

Thinnest filaments in cytoskeleton ( 7- 8 nm)

• Thermodynamically limited by nucleation step (dimers and trimers)

• Double-stranded helix (repeating every 37nm)• Right handed, rotate 166° • Polar polymers

0.3/s0.002/s

Pointed end

Barbed endElongates 10 times fasterRate 11.6 µM-1s-1

Thermodynamically unfavorablebut not

above „critical concentration“ (0.1µM)

Building blocks for new actin filament

Structure of Actin filaments

Special : “treadmilling”

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Actin cytoskeleton

http://www.nature.com/nrm/journal/v7/n10/images/nrm2026-f1.jpghttps://upload.wikimedia.org/wikipedia/commons/thumb/7/7b/Profilin_actin_complex.png/223px-Profilin_actin_complex.png

• Profilin- Abundant monomer binding proteinImportant role in actin homeostasisInhibits the spontaneous formation of dimers and trimersDrive actin assembly at barbed endPrevent Polymerization at pointed end -> Polarity in growthActin + profilin need cellular nucleation factor for de novo actin assembly

Control of assembly Profilin

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Actin cytoskeleton



• ForminsRapid elongation in presence of formin : 10 µM-1s-1

Formin/profilin : 90 µM-1s-1


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Actin cytoskeleton



• ForminsRapid elongation in presence of formin : 10 µM-1s-1

Formin/profilin : 90 µM-1s-1

• Arp2/3 complex- complex of 7 proteins:Arp2/3-complex

• Binds on an already existing filaments• Nucleates the formation of daughter filament at 70° angle• Fan-like branched filament network

Acts as actin nucleation machineryLeading-edge of motile cellsClathrin mediated endocytosisMeotic spindle positioningMotility of some bacteria and viruses in host cell cytoplasma


Arp2/3 complex

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Actin cytoskeletonControl of assembly

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Actin cytoskeleton

Polymerization and association with actin regulatory proteins

Variety of architectures

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Actin cytoskeletonActin filament organizations

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Actin cytoskeleton


Actin filament organizations

1a1

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Actin cytoskeleton



Branched actin network:

Involved in cell movementAnd shape changes

Initiated by Arp2/3-complex

Starts with “primer” whose sides interact with Arp2/3-complex

Nucleating-promoting factors (NPFs) activate Arp2/3-complex

1a1a

Array of parallel bundles

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Actin cytoskeleton



Branched actin network:

Involved in cell movementAnd shape changes

Initiated by Arp2/3-complex

Starts with “primer” whose sides interact with Arp2/3-complex

Nucleating-promoting factors (NPFs) activate Arp2/3-complex

Inhibition of branch elongation for force production -> capping proteins (CP)

At small time scales “viscoelastic”e.g. epidermis pinched briefly

At large time scales “viscous”e.g. elbows and knees

1a1a

Array of parallel bundles

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Actin cytoskeleton



1a 1a

Crosslinked actin network:

Actin filament connected trough bridging proteins (EXCLUDING Arp2/3 complex!)

Controlling shape and mechanical integrity

Crosslinking proteins connect already polymerized actin filaments togetherCrosslink distances range from 10 – 160 nmSmall crosslinkers : Fimbrin or fascin – they pack actin into bundles (parallel, anti parallel or mixed polarity)Large crosslinkers.: Filamin or α-actinin – bundles/networkse.g. increased rate of actin assembly prevents bundles formed by α-actinin

Long time force : Time for redistribution Short time force: No time for reorganization of crosslinkers, no resist against load -> network = elastic material, return to shape

1a

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1a

1a

Parallel actin bundles:

Found in filopodia, microvilli and hair cells

Barbed end orientated in the same direction (mainly cell membrane)Crosslinking proteins (α-actinin , fimbrin and fascin) keep close contact filament bundles

Initiation not clear Or Arp2/3 complex required or barbed and elongation enhancement proteins (formins, VASP proteins)

When force applied against:Short stiff bundles stay straightLong have tendency to buckle


1a

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1a1a

Anti parallel actin bundles:

Necessary for cytokinesis and for stress fiber function during establishment of cell/cell and cell-matrix adhesions

Myosin induced

Stabilized with crosslinking proteins (fimbrin and α-actinin)

2 steps : Contraction and myosin induced disassembly

Length of filaments ~ contractile properties ( ~ no of myosin heads/unit )


1a

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Actin cytoskeletonDisassembly of actin networks

Actin disassembling machinery :

• ADF/cofilin

Modifies mechanical properties and nucleotide state of actin monomers in filamentUses fragmentation or severing to break down actin organizationCan increase dissociating rate (25x)

Decreases the persistence length of actin filaments-> actin filaments decorated with ADF/cofilin are more flexible

1a


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Actin disassembling machinery :

• ADF/cofilin

Modifies mechanical properties and nucleotide state of actin monomers in filamentUses fragmentation or severing to break down actin organizationCan increase dissociating rate (25x)

Decreases the persistence length of actin filaments-> actin filaments decorated with ADF/cofilin are more flexible

• Debranching by Arp2/3 complex

GMF (additional protein) can also induce branch dissociation

1a


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• Myosin

Induced breakage by faster one end than the other

(eventually buckling)

1a1a


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ADF/cofilin mechanism

Myosin induced contraction and disassembly

1a1a


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Actin cytoskeleton

Variety of architectures

Diverse functionality

1a

1a

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Actin cytoskeleton


Functions of actin cytoskeleton

Lamellipodia:

Branched and crosslinked networksFilament assembly via Arp2/3 complexArp2/3 complex activated by a specific NPF (WAVE)Sometimes also formins play a role

Many barbed ends growing away from surface

Major engine of cell movement(push cell membrane by polymerizing against it)

Observed in intracellular wound healing systems

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Actin cytoskeletonFunctions of actin cytoskeleton

https://upload.wikimedia.org/wikipedia/en/thumb/f/fa/GrowthCones.jpg/500px-GrowthCones.jpgBlanchoin et al. 2014

Filopodium:

Fingerlike structure at front of cellParallel bundleGrowing end orientated towards membraneContain fascin/fimbrin/formin

Extend beyond the leading edge of lamellipodia

Can form adhesions with substratum, initiating cell contactsSensing the cell environmentTransmitting cell-cell signalsPlay role in fibroblasts (wound healing in vertebrates)

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Stress fibers:

Fingerlike structuresAt front of cellAntiparallel Contractile fibersBundles of unbranched actin filaments of mixed polarity

Contain Non-muscle myosins II

Connect cytoskeleton to the extracellular matrix via focal adhesion sites

Blanchoin et al. 2014https://en.wikipedia.org/wiki/Stress_fiber#/media/File:Stress_fibers.png

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Stress fibers:

Fingerlike structuresAt front of cellAntiparallel Contractile fibersBundles of unbranched actin filaments of mixed polarity

Contain Non-muscle myosins II

Connect cytoskeleton to the extracellular matrix via focal adhesion sites

Adherens junctions:

Characteristic for multicellular organisms with tissue specialization

Blanchoin et al. 2014https://en.wikipedia.org/wiki/Stress_fiber#/media/File:Stress_fibers.png

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Actin cytoskeleton

Blanchoin et al. 2014http://www.actindynamics.org/cms/uploads/images/members/Ellenberg-Lenart/Figure2_1.jpg

Functions of actin cytoskeleton

Cortex:

Coats the plasma membrane at the back and sideCrosslinked networkThin actin shell, contractile (myosin)Underlying the inner face of plasma membraneSeveral hundred of nm thick

Important for cell shape maintenance and changesKeeps membrane proteins on their placeFormation of blebs

The meiotic spindle (shown by microtubules in red and chromosomes in cyan) is transported by an F-actin meshwork (green) to the cell cortex in mouse oocytes.

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Actin-binding protein composition of the major actin architectures and cellular examples

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http://legacy.owensboro.kctcs.edu/gcaplan/anat/images/Image336.gif

Muscle fibers:

Actomyosin myofibrils consistent ofActin and myosin – up to 90% of protein mass

Tropomyosin molecule (40nm)

CapZ (end capping protein)Appears in muscle apparatusPrevents the addition or loss of monomers

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https://upload.wikimedia.org/wikipedia/commons/e/e0/3D-SIM-4_Anaphase_3_color.jpghttp://www.organische-chemie.ch/chemie/2010/apr/krebsg5.JPGVoigt et al. 2004

Cytokinesis:

Arp2/3 complex involved : meotic spindle positioning

Contraction of cells during division

Cell separating by ring of actin, myosin and α- actinin

F: Interphase of cell meristemG: Mitotic cell, with F-actin depleted zone

(asterisk)Scale bar = 10 29µm

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Actin cytoskeletonActin cytoskeleton in root hairs

Ketalaar 2013

Plant actin cytoskeleton:

Backbone for cytoplasmic streamingDelivering of growth materials to expanding surfaces

Root hairs:

Tubular structures emerged from root epidermisWater and nutrient uptake, anchoring root, symbiotic interactionsExpansion by tip growth (pollen tubes, moss protonema)

study of polarized cell expansion

Clear zoneno larger organelles

Subapical cytoplasmic dense regionFor cell expansion needed organelles (Nucleus,

mitochondria, ER, Golgi, endosomes, ribosomes, actin and microtubule cytoskeleton)

Highly vacuolated tube

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Ketalaar 2013

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Ketalaar 2013

Tube away from apex : less turnover

ÞLonger half life of individual filaments

Þ Association with actin bundlers

Þ Increase in bundled filaments

AIP1: Actin interacting protein 1ADF: Actin depolymerizing factorCAP1: Cyclase activating protein

33http://www.frontiersin.org/files/Articles/124351/fpls-05-00786-HTML/image_m/fpls-05-00786-g003.jpg

Based on the functional characterization of ABPs derived mainly from Arabidopsis.

(A) Intracellular localization of several ABPs in the pollen tube

(B) Schematic describing the intracellular localization and function of various ABPs in the pollen tube

Actin cytoskeleton Actin dynamics in the pollen tube

Scale bar = 10 μm.

34http://www.frontiersin.org/files/Articles/124351/fpls-05-00786-HTML/image_m/fpls-05-00786-g003.jpg

Based on the functional characterization of ABPs derived mainly from Arabidopsis.

(A) Intracellular localization of several ABPs in the pollen tube

(B) Schematic describing the intracellular localization and function of various ABPs in the pollen tube

Actin cytoskeleton Actin dynamics in the pollen tube

Scale bar = 10 μm.

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Actin cytoskeleton

Actin cytoskeleton in cells of Arabidopsis seedlings

Voigt et al. 2004

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Actin cytoskeleton

Actin cytoskeleton in cells of Arabidopsis seedlings

Voigt et al. 2004

Plastin-GFP and GFP-mTn expressed in transgenic Arabidopsis show weak and diffuse signals in lateral root cap cells and statocystes

transgenic Arabidopsis

Single optical section trough the root cap:

Columnella cells (stars) and lateral root cap cells (diamonds) showing different actin states

In vivo insights of actin formation

Understanding of assembling and disassembling mechanisms etc.

Thank you for listening !

Documents

Aktin cytoskeleton Seminar PCDU WS15/16 Vera Krieger - 28.10.15