Aktin cytoskeleton
Seminar PCDU
WS15/16
Vera Krieger - 28.10.15
Actin cytoskeleton
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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https://upload.wikimedia.org/wikipedia/commons/thumb/9/96/La-Jolla-Red-Tide.780.jpg/220px-La-Jolla-Red-Tide.780.jpg
Actin cytoskeleton
Structur of Actin filamentes
G-Actin:
Globular actin
Ca. 42 kDa Protein
Two lobes separated by a cleft
Polymers of G-actin subunits -> F-actin
Bound to ATP / ADP
Most common: ATP-G-Actin
ADP-F-Actin
Actin monomer bind to Mg2+ und Ca 2+
-> affects polymerization dynamics
Concentration increased of free cations
– actin filaments stiffer
Tertiary structures of G-actin and F-actin
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http://www.spring8.or.jp/en/news_publications/press_release/2009/090122_fig/fig4.jpg
Actin cytoskeleton
Structure of Actin filaments
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/s
0.002/s
Building blocks for new actin filament
Pointed end
Special : “treadmilling”
Blanchoin et al. 2014
Barbed end
Elongates 10 times faster
Rate 11.6 µM-1s-1
Thermodynamically unfavorable
but not
above „critical concentration“ (0.1µM)
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Actin cytoskeleton
Control of assembly
Profilin
• Profilin
- Abundant monomer binding protein
Important role in actin homeostasis
Inhibits the spontaneous formation of dimers and trimers
Drive actin assembly at barbed end
Prevent Polymerization at pointed end
-> Polarity in growth
Actin + profilin need cellular nucleation factor for de novo actin assembly
http://www.nature.com/nrm/journal/v7/n10/images/nrm2026-f1.jpg
https://upload.wikimedia.org/wikipedia/commons/thumb/7/7b/Profilin_actin_complex.png/223px-Profilin_actin_complex.png
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Actin cytoskeleton
Control of assembly
Profilin
• Profilin
- Abundant monomer binding protein
Important role in actin homeostasis
Inhibits the spontaneous formation of dimers and trimers
Drive actin assembly at barbed end
Prevent Polymerization at pointed end
-> Polarity in growth
Actin + profilin need cellular nucleation factor for de novo actin assembly
• Formins
Rapid elongation in presence of formin : 10 µM-1s-1
Formin/profilin : 90 µM-1s-1
http://www.nature.com/nrm/journal/v7/n10/images/nrm2026-f1.jpg
https://upload.wikimedia.org/wikipedia/commons/thumb/7/7b/Profilin_actin_complex.png/223px-Profilin_actin_complex.png
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Actin cytoskeleton
Control of assembly
Profilin
• Profilin
- Abundant monomer binding protein
Important role in actin homeostasis
Inhibits the spontaneous formation of dimers and trimers
Drive actin assembly at barbed end
Prevent Polymerization at pointed end
-> Polarity in growth
Actin + profilin need cellular nucleation factor for de novo actin assembly
• Formins
Rapid 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 machinery
Leading-edge of motile cells
Clathrin mediated endocytosis
Meotic spindle positioning
Motility of some bacteria and viruses in host cell cytoplasma
Arp2/3 complex
http://www.nature.com/nrm/journal/v7/n10/images/nrm2026-f1.jpg
https://upload.wikimedia.org/wikipedia/commons/thumb/7/7b/Profilin_actin_complex.png/223px-Profilin_actin_complex.png
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Actin cytoskeleton
Control of assembly
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Actin cytoskeleton
Polymerization and association with actin regulatory proteins
Variety of architectures
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Actin cytoskeleton
Actin filament organizations
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Actin cytoskeleton
Actin filament organizations
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Blanchoin et al. 2014
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Actin cytoskeleton
Actin filament organizations
Branched actin network:
Involved in cell movement
And 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
Array of parallel bundles
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Blanchoin et al. 2014
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Actin cytoskeleton
Actin filament organizations
Branched actin network:
Involved in cell movement
And 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)
Array of parallel bundles
At small time scales “viscoelastic”
e.g. epidermis pinched briefly
At large time scales “viscous”
e.g. elbows and knees
Blanchoin et al. 2014
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Actin cytoskeleton
Actin filament organizations
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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 together
Crosslink distances range from 10 – 160 nm
Small crosslinkers : Fimbrin or fascin – they pack actin into bundles (parallel, anti parallel or mixed polarity)
Large crosslinkers.: Filamin or α-actinin – bundles/networks
e.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
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Blanchoin et al. 2014
Actin cytoskeleton
Actin filament organizations
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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 straight
Long have tendency to buckle
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Blanchoin et al. 2014
Actin cytoskeleton
Actin filament organizations
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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 )
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Blanchoin et al. 2014
Actin cytoskeleton
Disassembly of actin networks
Actin disassembling machinery :
• ADF/cofilin
Modifies mechanical properties and nucleotide state of actin
monomers in filament
Uses fragmentation or severing to break down actin
organization
Can increase dissociating rate (25x)
Decreases the persistence length of actin filaments
-> actin filaments decorated with ADF/cofilin are more flexible
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Blanchoin et al. 2014
Actin cytoskeleton
Disassembly of actin networks
Actin disassembling machinery :
• ADF/cofilin
Modifies mechanical properties and nucleotide state of actin
monomers in filament
Uses fragmentation or severing to break down actin
organization
Can 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
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Blanchoin et al. 2014
Actin cytoskeleton
Disassembly of actin networks
• Myosin
Induced breakage
by faster one end than the other
(eventually buckling)
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Blanchoin et al. 2014
Actin cytoskeleton
Disassembly of actin networks
ADF/cofilin mechanism
Myosin induced contraction and
disassembly
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Blanchoin et al. 2014
Actin cytoskeleton
Variety of architectures
Diverse functionality
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Actin cytoskeleton
Functions of actin cytoskeleton
Lamellipodia:
Branched and crosslinked networks
Filament assembly via Arp2/3 complex
Arp2/3 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
Blanchoin et al. 2014
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Actin cytoskeleton
Functions of actin cytoskeleton
Filopodium:
Fingerlike structure at front of cell
Parallel bundle
Growing end orientated towards membrane
Contain fascin/fimbrin/formin
Extend beyond the leading edge of lamellipodia
Can form adhesions with substratum, initiating cell contacts
Sensing the cell environment
Transmitting cell-cell signals
Play role in fibroblasts (wound healing in vertebrates)
https://upload.wikimedia.org/wikipedia/en/thumb/f/fa/GrowthCones.jpg/500px-GrowthCones.jpg
Blanchoin et al. 2014
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Actin cytoskeleton
Functions of actin cytoskeleton
Stress fibers:
Fingerlike structures
At front of cell
Antiparallel Contractile fibers
Bundles 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. 2014
https://en.wikipedia.org/wiki/Stress_fiber#/media/File:Stress_fibers.png
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Actin cytoskeleton
Functions of actin cytoskeleton
Stress fibers:
Fingerlike structures
At front of cell
Antiparallel Contractile fibers
Bundles 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. 2014
https://en.wikipedia.org/wiki/Stress_fiber#/media/File:Stress_fibers.png
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Actin cytoskeleton
Functions of actin cytoskeleton
Cortex:
Coats the plasma membrane at the back and side
Crosslinked network
Thin actin shell, contractile (myosin)
Underlying the inner face of plasma membrane
Several hundred of nm thick
Important for cell shape maintenance and changes
Keeps membrane proteins on their place
Formation 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.
Blanchoin et al. 2014
http://www.actindynamics.org/cms/uploads/images/members/Ellenberg-Lenart/Figure2_1.jpg
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Actin cytoskeleton
Functions of actin cytoskeleton
Actin-binding protein composition of the major actin architectures and cellular examples
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Blanchoin et al. 2014
Actin cytoskeleton
Functions of actin cytoskeleton
Muscle fibers:
Actomyosin myofibrils consistent of
Actin and myosin
– up to 90% of protein mass
Tropomyosin molecule (40nm)
CapZ (end capping protein)
Appears in muscle apparatus
Prevents the addition or loss of monomers
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http://legacy.owensboro.kctcs.edu/gcaplan/anat/images/Image336.gif
Actin cytoskeleton
Functions of actin cytoskeleton
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 meristem
G: Mitotic cell, with F-actin depleted zone
(asterisk)
Scale bar = 10 29µm
https://upload.wikimedia.org/wikipedia/commons/e/e0/3D-SIM-4_Anaphase_3_color.jpg
http://www.organische-chemie.ch/chemie/2010/apr/krebsg5.JPG
Voigt et al. 2004
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Actin cytoskeleton
Actin cytoskeleton in root hairs
Plant actin cytoskeleton:
Highly vacuolated tube
Backbone for cytoplasmic streaming
Delivering of growth materials to expanding surfaces
Clear zone
no larger organelles
Root hairs:
Tubular structures emerged from root epidermis
Water and nutrient uptake, anchoring root, symbiotic
interactions
Expansion by tip growth (pollen tubes, moss protonema)
study of polarized cell expansion
Subapical cytoplasmic dense region
For cell expansion needed organelles (Nucleus,
mitochondria, ER, Golgi, endosomes, ribosomes,
actin and microtubule cytoskeleton)
Ketalaar 2013
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Actin cytoskeleton
Actin cytoskeleton in root hairs
Ketalaar 2013
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Actin cytoskeleton
Actin cytoskeleton in root hairs
AIP1: Actin interacting protein 1
ADF: Actin depolymerizing factor
CAP1: Cyclase activating protein
Tube away from apex : less turnover
Longer half life of individual filaments
 Association with actin bundlers
 Increase in bundled filaments
Ketalaar 2013
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Actin cytoskeleton
Actin dynamics in the pollen tube
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
Scale bar = 10 μm.
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http://www.frontiersin.org/files/Articles/124351/fpls-05-00786-HTML/image_m/fpls-05-00786-g003.jpg
Actin cytoskeleton
Actin dynamics in the pollen tube
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
Scale bar = 10 μm.
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http://www.frontiersin.org/files/Articles/124351/fpls-05-00786-HTML/image_m/fpls-05-00786-g003.jpg
Actin cytoskeleton
Actin cytoskeleton in cells of Arabidopsis seedlings
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Voigt et al. 2004
Actin cytoskeleton
Actin cytoskeleton in cells of Arabidopsis seedlings
Plastin-GFP and GFP-mTn expressed in transgenic Arabidopsis show weak and diffuse signals in lateral root cap cells and statocystes
Single optical section trough
the root cap:
In vivo insights of
actin formation
Columnella cells (stars) and
lateral root cap cells
(diamonds) showing different
actin states
Understanding of
assembling and
disassembling
mechanisms etc.
transgenic Arabidopsis
Voigt et al. 2004
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Thank you for listening !
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