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
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
• ForminsRapid elongation in presence of formin : 10 µM-1s-1
Formin/profilin : 90 µM-1s-1
Control of assembly Profilin
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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
• 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
Control of assembly Profilin
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
Blanchoin et al. 2014
Actin filament organizations
1a1
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Actin cytoskeleton
Blanchoin et al. 2014
Actin filament organizations
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
Blanchoin et al. 2014
Actin filament organizations
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
Blanchoin et al. 2014
Actin filament organizations
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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Actin cytoskeletonActin filament organizations
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
Blanchoin et al. 2014
1a
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Actin cytoskeletonActin filament organizations
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 )
Blanchoin et al. 2014
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
Blanchoin et al. 2014
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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
• Debranching by Arp2/3 complex
GMF (additional protein) can also induce branch dissociation
1a
Blanchoin et al. 2014
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Actin cytoskeletonDisassembly of actin networks
• Myosin
Induced breakage by faster one end than the other
(eventually buckling)
1a1a
Blanchoin et al. 2014
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Actin cytoskeletonDisassembly of actin networks
ADF/cofilin mechanism
Myosin induced contraction and disassembly
1a1a
Blanchoin et al. 2014
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Actin cytoskeleton
Variety of architectures
Diverse functionality
1a
1a
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Actin cytoskeleton
Blanchoin et al. 2014
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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Actin cytoskeletonFunctions of actin cytoskeleton
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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Actin cytoskeletonFunctions of actin cytoskeleton
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 cytoskeletonFunctions of actin cytoskeleton
Blanchoin et al. 2014
Actin-binding protein composition of the major actin architectures and cellular examples
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Actin cytoskeletonFunctions of actin cytoskeleton
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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Actin cytoskeletonFunctions of actin cytoskeleton
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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Actin cytoskeletonActin cytoskeleton in root hairs
Ketalaar 2013
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Actin cytoskeletonActin cytoskeleton in root hairs
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.
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