B. The Cytoskeletal System and Adhesion
• Red = Actin Microfilaments
• Green = Tubulin Microtubules
Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings
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3 Fibrous Components and Motors
• Thin Filaments: Actin family of proteins
• Microtubules: Tubulin family of proteins
• Intermediate Filaments: Very cell‐specific family of proteins
• The first two have molecular motors
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1. Actin Microfilaments
• Chains of few to many 42kDa monomers
• Found in all eukaryotic cells (except nematode worm sperm)
• Highly Conserved: Genes vary <20% from algae to humans; human primary protein sequence <5%
• Three main isoforms: alpha, beta, gamma
• Alpha is muscle actin, beta and gamma are found in nearly all cells
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Figure 16-12 Molecular Biology of the Cell (© Garland Science 2008)
ATP Binding
ADP Binding
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Figure 16-74c,d Molecular Biology of the Cell (© Garland Science 2008)
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Figure 16-47 Molecular Biology of the Cell (© Garland Science 2008)
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Fig. 6‐26
Microvillus
Plasma membrane
Microfilaments (actin filaments)
Intermediate filaments
0.25 µm
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Figure 16-69a Molecular Biology of the Cell (© Garland Science 2008)
Actin tracks allow specific movement between two points
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Figure 17-49a Molecular Biology of the Cell (© Garland Science 2008)
Remember.....
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The Key Structural Elements
1. 70 Å filaments (f‐actin) ‐ polymers of globular monomers (g‐actin) formed as double helix
2. Structural polarity –sequences and structures unequally directed towards each of the ends
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Figure 16-7 Molecular Biology of the Cell (© Garland Science 2008)
“Dynamic instability”
a. Can form, crosslink, breakdown and reform rapidly
b. The cell can build new structures as its needs change
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2. Tubulin Microtubules
• Large hollow tubes of many 110kDa dimers
• Three main isoforms: alpha, beta, gamma
• Found in all eukaryotic cells
• Alpha and beta found in microtubules, gamma found in microtubule organizing center (MTOC)
• Highly Conserved: Primary sequences vary <10% from algae to humans
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Figure 16-11 Molecular Biology of the Cell (© Garland Science 2008)
GTP Binding GDP Binding
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Figure 16-30c Molecular Biology of the Cell (© Garland Science 2008)
Large‐scale, long distance movement of cellular components, including organelles
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Fig. 6‐22Centrosome
Microtubule
Centrioles
0.25 µm
Longitudinal section of one centriole
Microtubules Cross sectionof the other centriole
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Figure 16-30b Molecular Biology of the Cell (© Garland Science 2008)
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Figure 16-85c Molecular Biology of the Cell (© Garland Science 2008)
Projections of asters to move the chromosomes in cell division
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Figure 16-104 Molecular Biology of the Cell (© Garland Science 2008)
Directed transport along the neuronal axon
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Cilia andflagella
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Give direction to plant cell division and growth and dictate sites of plant wall synthesis
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Plant microtubules have no obvious MTOC
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The Key Structural Elements
1. These are also polar – unequally directed towards each end
a. most: (–) end toward nucleus and (+) end to cell membrane
2. 250 Å filaments formed as a hollow bundle of 13 protofilaments
a. protofilaments are long polymers of tubulin dimers
b. dimers are one alpha and one beta globular monomers
c. gamma tubulin makes up the MTOC: centriole or basal body
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Figure 16-16c Molecular Biology of the Cell (© Garland Science 2008)
Like actin, microtubules can be changed as the cells needs change
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3. Intermediate Filaments
• Rope‐like filament structures formed from 8 protofilaments twisted together
• Lamins found in nucleus, cytosolic IF’s found in some cells and not others
• IF’s found only in some animals, including vertebrates, nematodes, molluscs
• Plants and single‐celled organisms have no IF’s
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Figure 16-19 Molecular Biology of the Cell (© Garland Science 2008)
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Figure 16-20 Molecular Biology of the Cell (© Garland Science 2008)
Keratinsin the cytosol
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Lamins in the Nucleus
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Sarcomere Support
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The Key Structural Elements
1. Apolar – very stable, not structurally nor functionally oriented towards one end or the other
2. No motors
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4. Bacterial Homologs and Analogs
• Homologs are genes and proteins that share common sequence
• Analogs are proteins that share common function
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Figure 16-26 Molecular Biology of the Cell (© Garland Science 2008)
Actin homologs: MreB and Mbl
Helical proteins give cell its shape by distributing themselvesthroughout the cell and directing sites of cell wall synthesis.
Mbl+ Mbl‐
Analogous to plant tubulin!
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Figure 16-27a Molecular Biology of the Cell (© Garland Science 2008)
Actin homolog: ParM
Analogousto tubulin!
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Figure 16-25a Molecular Biology of the Cell (© Garland Science 2008)
Tubulin homolog: FtsZ
As a bacterium divides: after the two chromosome copieshave been segregated, the Z‐ring forms and then contractsto split the daughter cells. Two rings reform in the daughters.
This is analogous to actin!
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Figure 16-28 Molecular Biology of the Cell (© Garland Science 2008)
Intermediate filament homolog: Crescentin
Creates the curved shape by forming a cortical band
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b. Molecular motors allow filament‐based intracellular transport
The three basic types
1. Myosins: move along microfilaments towards the positive end (mostly), Some myosin gene products move toward negative end
2. Kinesins: move along microtubules towards the positive end (away from the MTOC)
3. Dyneins: move along microtubules towards the negative end (toward the MTOC)
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Figure 16-54a Molecular Biology of the Cell (© Garland Science 2008)
Globular heads on fibrous tails:
a. Heads have ATPase activity, dictate filament track, direction and speed
b. Tails have “cargo” binding and myosin thick filament organizing activity
Molecular Motors are ATPases that Couple Hydrolysis with Movement
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Kinesins and Myosins are Evolutionarily Related
1. Both have single head (type I ) and double head (type II) designs
2. Little sequence similarity but remarkable similarities in tertiary structures
Dyneins are Something Else
1. They are an ancient family of proteins found in all eukaryotes
a. Cytosolic dyneins move most structures, ciliar dyneinsmove ciliaand flagella
2. Generally similar structure to the others – can have trimer heads
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Figure 16-67 Molecular Biology of the Cell (© Garland Science 2008)
The motorcargo complexgrabs onto
meshwork and transmembrane
proteins.
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“Power Stroke” mechanism to couple movement to ATP metabolism
1. At the end of one cycle the motor is bound to actin at 45o
2. ATP binding Filament Release
3. ATP Hydrolysis Conformational Relaxation to 90o
4. Release of Pi Filament Binding
5. Release of ADP Conformational Stroke to 45o
6. The shift in the motor back to 45o moves the cargo forward
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2. Regulation of Cell Function via Control of the Cytoskeleton
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a. Actin filaments are assembled and disassembled rapidly and thus are able to help regulate long‐term activities, change and movement
Assembly of specific actin structures and cellular functions is dependent on the expression and activity of microfilament associated proteins (MFAP’s) in specific cellular locations
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Figure 16-10 Molecular Biology of the Cell (© Garland Science 2008)
Actin fiber assembly is spontaneous if only actin and ATP are present and the bonds between subunits are non‐covalent
Nucleation is the rate‐limiting step in assembly and the first control
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Figure 16-34c Molecular Biology of the Cell (© Garland Science 2008)
To make a dense actin network, or gel, the cell must express and activate ARP complexes at that site
Activating factor can followinternal or external signals
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Figure 16-36 Molecular Biology of the Cell (© Garland Science 2008)
Formin facilitates nucleation and causes rapid extension
Expressed with ARP complexes, leads to very rapid assemblage and branching
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Thymosin and Profilin Regulate Speed
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Figure 16-43 Molecular Biology of the Cell (© Garland Science 2008)
Caps mean slow growth and stabilized fibers
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Figure 16-49a Molecular Biology of the Cell (© Garland Science 2008)
Lateral association of double helical fibers by bundling proteins
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Figure 16-51 Molecular Biology of the Cell (© Garland Science 2008)
Stabilization of gel structures
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Figure 16-50a Molecular Biology of the Cell (© Garland Science 2008)
Microvilliassemblage
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Many changing cell functions are regulated by actin fiber reorganization, which requires coordinated disassembly of existing fibers followed by nucleation and polymerization at a new location
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Rapid disassembly of fibers
Severing Depolymerization
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Combine disassembly with localized assembly
Profilin promotes ATP exchange and ARP nucleation
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Figure 16-90 Molecular Biology of the Cell (© Garland Science 2008)
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Lamellipodia
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Assembly of the contractile ring followed by severing is required in cytokinesis
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Figure 13-46 Molecular Biology of the Cell (© Garland Science 2008)
Pseudopod‐Driven Phagocytosis in Neutrophils
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Figure 13-47a Molecular Biology of the Cell (© Garland Science 2008)
Actin polymerizationis required to extendpseudopods
Actin depolymerization isrequired to seal off thephagosome
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Regulation of the active actin structure is associated with known Rho‐family signaling pathways
stressfibers
microspikesor
filopodialamellipodia
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Figure 16-98b Molecular Biology of the Cell (© Garland Science 2008)
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Figure 16-98a Molecular Biology of the Cell (© Garland Science 2008)
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b. Microtubules are also assembled and disassembled rapidly and are used to regulate movement and positioning of materials, including the cell
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Figure 16-30a Molecular Biology of the Cell (© Garland Science 2008)
γ‐Tubulin ring complex (γ‐TuRC) nucleates assembly from the MTOC and remains associated with the minus end
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Figure 16-30b Molecular Biology of the Cell (© Garland Science 2008)
A high concentration of free tubulinsubunits is required for assembly and elongation
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Figure 16-41 Molecular Biology of the Cell (© Garland Science 2008)
MAP2, Tau and Plus‐end tracking proteins (TIPs) bind and stabilize microtubules
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Figure 16-45a Molecular Biology of the Cell (© Garland Science 2008)
Green = MT
Red = +Tipprotein
Long‐termstable
structures
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Expression of stathmin protein will bind free tubulinsubunits and inhibit assembly and elongation
Each stathmin protein binds two dimers
Phosphorylation of stathmin will inactivate it
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Microtubule Severing
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Figure 16-44 Molecular Biology of the Cell (© Garland Science 2008)
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Disassembly and Reassembly in Mitosis
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Figure 16-19 Molecular Biology of the Cell (© Garland Science 2008)
c. Intermediate filaments are the most stable element in the cytoskeleton but their structure can be regulated to facilitate specific cell activities
Remember....
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The aminoterminal head and carboxyterminaltail appear to be key points of regulatory control
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Glycosylation and phosphorylation of intermediate filaments.
Hyder C L et al. J Cell Sci 2011;124:1363-1372
©2011 by The Company of Biologists Ltd
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Disassembly of nucleus in mitosis
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Nuclear Envelope Fragmentation
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CELLULARADHESION
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Figure 19-46 Molecular Biology of the Cell (© Garland Science 2008)
Hemidesmosomes bind intermediate filaments to anchor proteins to integrins to ECM fibrous proteins
Very stable
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Figure 19‐45 Molecular Biology of the Cell (© Garland Science 2008)
Focal adhesions bind microfilaments to anchor proteins to integrins to ECM fibrous proteins
Can be stable or used for migration
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Figure 19-48b Molecular Biology of the Cell (© Garland Science 2008)
Internal binding and external binding are linked
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Figure 19‐17a Molecular Biology of the Cell (© Garland Science 2008)
Very stable
Desmosomes bind intermediate filaments to anchor proteins to their own cadherinswhich bind to the adjacent cell’s cadherins
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Adherens junctions bind microfilaments to anchor proteins to their own cadherinswhich bind to the adjacent cell’s cadherins
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Figure 19‐7 Molecular Biology of the Cell (© Garland Science 2008)
A fairly large protein family, all have homophillicbinding and are Ca++‐dependent
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Figure 19-24 Molecular Biology of the Cell (© Garland Science 2008)
Tight Junctions can block molecules assmall as ions from passing between cells
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Figure 19-26a Molecular Biology of the Cell (© Garland Science 2008)
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When there is an infection or inflammation, endothelial cells will express selectins on their surface that specifically bind WBC.
WBC also express integrins that bind strongly, stop their rolling and aid in escape
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Figure 19‐20 Molecular Biology of the Cell (© Garland Science 2008)
NCAM is foundon a variety ofcell types and mediates homo‐philic binding.
Ig superfamily cell adhesion molecules
ICAM is foundon endothelialcells and bindsheterophilicallyto integrins onwhite blood cells.
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This is a large gene family
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