cytoskeleton introduction - cellbiology · pdf file15/04/2015 3 functions of the cytoskeleton...

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ANAT3231 Cytoskeleton Introduction

31st March 2015

COMMONWEALTH OF AUSTRALIA

Copyright Regulations 1969

WARNING

This material has been reproduced and communicated to you by and on behalf of the University of New South Wales pursuant to

Part VB of the Copyright Act 1968 (the Act).

The material in this communication may be subject to copyright under this Act. Any further reproduction of communication of

this material by you may be the subject of copyright protection under the Act.

Do not remove this notice

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Three filament systems: microtubules, microfilaments (actin filaments) and intermediate filaments

Image: The world of the Cell

Filament dimensions

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Functions of the cytoskeleton

• Functions based upon the filaments physical properties

• each filament system has different properties

• Integral strength

• Cell shape

• Motility

• inside the cell

• whole cell

• motor proteins associated with 2 filament systems

• Signal transduction

Note - the Extracellular Matrix has a similar structural role outside of the cell

Functions of the cytoskeleton

MBoC Fig 16-102

Signaling during neutrophil polarization Cytoskeleton driven force generation moving cells forward

MBoC Fig 16-86

Polarization of a cytotoxic T cell after target-cell recognition

MBoC Fig 16-103 Alberts et al. (2008)

Complex morphological changes during development

MBoC Fig 16-106

Alberts et al. (2008)

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Structure of the cytoskeleton

• Network of filamentous proteins

• filaments formed from a few proteins

• monomer protein forms polymer filaments

• Located in nucleus and cytoplasmic compartments

• not within organelles

• Location based upon cellular function

• Named on basis of physical size

Organization of the cytoskeleton

• Cytoplasmic

• cortical meshwork under plasma membrane

• three dimensional meshwork through

cytoplasm

• Nuclear

• cortical meshwork under nuclear envelope

• Assembly

• some spontaneous

• assembly sites

• Dynamic

• variable stability

• high to low stability

• stability can be altered by associated proteins

and signals

• drugs can alter stability

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Organization of the cytoskeleton

MBoC Fig 16-47


Microfilaments

• Twisted chain 7 nm diameter

• Most abundant protein in cells (5% of all cell protein)

• Actin 43 Kd

• Motility

• Adhesion, focal adhesions

• Actin binding proteins

• Myosin motors

• Muscle actins

MBoC Fig 16-47


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Intermediate Filaments

• Different cell types, different intermediate filaments

• all eukaryotes nuclear cytoskeleton the same

• Resist stresses applied externally to the cell

• Cytoplasmic

• Anastomosed network

• Flexible intracellular scaffolding

• 10-nanometer diameter

• Cross-linking proteins allow interactions with other cytoskeletal networks

• Intermediate filament associated proteins (IFAPs)

• coordinate interactions between intermediate filaments and other

cytoskeletal elements and organelles

• Human disorders

• mutations weaken structural framework

• increase the risk of cell rupture

Microtubules

• 25 nm diameter, 14 nm internal channel

• Tubulin

• Cytoplasmic

• All cells contain

• Same core structure

• Same motors Dynein (-) and Kinesin (+)

• Different associated proteins

• Dynamic

• Continuous remodelling

• Movement

• Intracellular > cellular

• Cell division mitotic spindle

• Specialized structures

• centrosome, basal bodies, Spindle pole

• Cell processes - cilia (9+2)

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Polar versus non-polar


Image: MBoC Figure 16-11

Microtubules

Microfilaments


No

n-p

olar

Po

lar


Polar filament structures allow directed transport

Albus et al. (2013)

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Alberts et al. (2008) Hirokawa et al. (2010)


Kneussel and Wagner (2013)

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• Polarized mRNA localization in the yeast bud tip

MBoC Fig 16-69


Prokaryotic Cytoskeleton Filaments

FtsZ ring

• Microtubule homolog

• Dynamic and exchanges subunits with the cytoplasmic pool

• Assembles into a ring at the future site of bacterial septum in cell division

MreB

• Microfilament (actin) homolog

• Dynamic and exchanges subunits with the cytoplasmic pool

• Assembles into helix-like structures

• Thought to spatially restrict cell growth activities during cell elongation

Crescentin

• Intermediate filament homolog

• Form stable filamentous structures

• Continuously incorporate subunits along their length

• Grow in a nonpolar fashion

• Stably anchored to the cell envelope

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How do we generate the diversity of the cytoskeleton?

• Large number of actin- and

microtubule associated

proteins


• Large number of actin- and

microtubule associated

proteins



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• Many proteins of the cytoskeleton are generated by alternative splicing

• Some examples are:

• lamins (nuclear intermediate filaments)

• tau (mictotubule-associated proteins)

• tropomyosins (actin-associated proteins)


(Blencowe, 2006)


Many proteins of the cytoskeleton are generated by alternative splicing

• The lamin protein family

Peter and Stick (2012)

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• The tau protein family

Wang and Liu (2008)


• The tropomyosin protein family TPM1

TPM2

TPM3

TPM4


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Abnormal Cytoskeleton

• Many mutations associated with human diseases

• Toxins can affect organization


References

Website:

http://php.med.unsw.edu.au/cellbiology/index.php?title=Cytoskeleton_Introduction

Articles:

• Alberts et al. (2008) Molecular Biology of the Cell, 5th Edition. • Albus et al. (2012) Cell length sensing for neuronal growth control. Trends Cell Biol, 23: 305-310. • Blencowe (2006) Alternative Splicing: New Insights from Global Analyses. Cell, 126: 37-47. • Hirokawa et al. (2010) Molecular Motors in Neurons: Transport Mechanisms and Roles in Brain

Function, Development, and Disease. Neuron, 18: 610-638. • Kneusel and Wagner (2013) Myosin motors at neuronal synapses: drivers of membrane transport

and actin dynamics. Nat Rev Neursci, 14: 233-247. • Peter and Strick (2012) Evolution of the lamin protein family. Nucleus, 3: 44-59. • Wang and Liu (2008) Microtubule-associated protein tau in development, degeneration and

protection of neurons. Progr Neurobiol, 85: 148-175.

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ANAT3231 Filament Dynamics and Research Methods to study the Cytoskeleton

2nd April 2015

Concepts of regulation of filament dynamics

[1] Microtubules [2] Microfilaments

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Microtubules

T. Wittmann [Nikon Small World, 2003]

• Cell organizing role

• Cytoskeleton – Largest fibre

– 25 nm diameter

– cytoplasmic

• All cells contain – Same core structure

– Same motors

– Different associated proteins

• Dynamic – Continuous remodelling

• Movement – Intracellular => cellular

– Cell division

Microtubules (EM)

Electron Microscope images transverse/longitudinal sections


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MT Structure

• Long hollow tubes – 25 nm diameter

• Radiate from forming structure – Centrosome

– Spindle pole

– Basal Body


• Polarized – (+) plus and (-) minus ends

• Formed from Tubulin – 55 kD protein


MT Structure


• dimers polymerize to form microtubules

• 13 linear protofilaments

– head-to-tail arrays of tubulin dimers

– arranged in parallel

– assembled around hollow core


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Microtubule Polarity and Treadmilling

– dynamic behavior when tubulin bound to GDP continually lost from minus end

– replaced by the addition of tubulin bound to GTP to plus end of same microtubule

– GTP hydrolysis also results in dynamic instability

• individual microtubules alternate between cycles of growth and shrinkage

- Visualisation of Treadmilling: Fluorescence speckle microscopy of mitotic spindle

(Keatin and Borisy, (2000); http://download.cell.com/biophysj/mmcs/journals/0006-3495/PIIS0006349503745640.mmc2.mov)

GTP-Cap of microtubules – Dynamic instability

Image: MBoC Figure 16-16c


http://download.cell.com/biophysj/mmcs/journals/0006-3495/PIIS0006349503745640.mmc2.mov

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• Addition of tubulin adds GTP to end of protofilament – grows in linear conformation

readily packed into MT wall

– becoming stabilized

• Hydrolysis of GTP – changes subunits conformation

– force protofilament a curved shape

– less able to pack into the MT wall

– protofilaments with GDP-containing subunits forced linear conformation by lateral bonds within MT wall, mainly in stable cap of GTP-containing subunits

Image: MBoC Figure 16-6a



Image: MBoC Figure 16-6b

• Addition of tubulin adds GTP to end of protofilament – grows in linear conformation

readily packed into MT wall

– becoming stabilized

• Hydrolysis of GTP – changes subunits conformation

– force protofilament a curved shape

– less able to pack into the MT wall

– protofilaments with GDP-containing subunits forced linear conformation by lateral bonds within MT wall, mainly in stable cap of GTP-containing subunits



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Microtubules originate from a microtubule organising centre


• slow-growing minus end of MT embedded in centrosome matrix surrounding a pair of centrioles

• matrix determines number of MTs in a cell

– By nucleating growth of new MTs


The diversity of microtubule-associated proteins

Image: MBoC Panel 16-3 Alberts et al. (2008)

http://www.nature.com/srep/2013/130508/srep01681/extref/srep01681-s3.avi

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Microfilaments

• Twisted chain 7 nm diameter

• Compared to MT – Thinner, more flexible, shorter

• Point in same direction

• Different organisation in different cellular regions

MBoC Figure16-49


Actin Types

• 6 Mammalian actin types (isoforms) – All are 43 Kd Protein

• 2 cytoskeletal isoforms in all non-muscle cells – Beta (b) 7p22-p12

– Gamma (g) 17q25

• 4 muscle isoforms in different muscle cells – Alpha (a) skeletal

– Alpha (a) cardiac

– Alpha (a) smooth

– Gamma (g) smooth

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Actin Protein

• Conserved in mammals

• Different ratios (b:g) in different cell types

• 374aa, 43 kD protein

• 4 aa difference between beta and gamma – at N- terminal

• Highly expressed gene – Promoter used in gene transfections

Actin Microfilament Formation

• Globular actin monomer (G-actin) polymerise to Filamentous actin (F-actin)

– Cells approx. 50:50

– Monomer can add to either (+ or - ) end

• Faster at + end

• Actin-ATP hydrolyzed (ADP) following addition

– Destabilizes (like MT)


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Nucleation/Elongation

• Nucleation – Two actin molecules bind weakly

– addition of a third (trimer) stabilizes the complex

– forms a "nucleation site”

• Elongation – Additional actin molecules form a long helical polymer

• Initial period of growth

• Then equilibrium phase reached

• Dynamic Equilibrium • Elongation ><Depolymerization controls filament length

Actin Binding Proteins

• Regulate polymerisation and create different structures

– Monomer binding protein • Sequester

• release

– Polymer binding proteins • Bundling

• cross-linking

• Severing

• contracting

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Actin Binding Proteins

Image: MBoC Figure 16-79 Alberts et al. (2008)

(Revenu et al. 2004)

Actin filament dynamics

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Actin filament dynamics

(Baum et al. 2006)

How can we study the structure and function of the cytoskeleton?

3 Major Functions

• Spatial organization of the cellular contents • Physical and biochemical link to the external environment • Generation of forces for cell movement and reshaping

3 Levels at which to study the cytoskeleton

• In vitro • In vivo

• Intra vital

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Detailed imaging using a diverse range of imaging technologies

T. Wittmann [Nikon Small World, 2003] http://www.nhlbi.nih.gov/research/intramural/researchers/core/electron-microscopy-core/media-gallery

Electron microscopy Light microscopy

Reconstitute in vitro

Studying filament assembly outside of the cell

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Structure of microfilaments

Detailed imaging using a diverse range of imaging technologies

Structure of microtubules

Mandelkow et al. (1991)

In vitro analysis of filament assembly

Microtubules

Microtubule assembly assay

(Eidenmueller et al., 2000)

Pyrene polymerisation assay

Microfilaments

(Gupton et al., 2007)

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Combined information from immunofluorescence and immunogold-EM analysis

Schaefer et al. (2002)

Analysis of filament dynamics using fluorescent speckle microscopy

Schaefer et al. (2008)

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(Danuser and Waterman-Storer, 2006)

Analysis of filament dynamics using fluorescent speckle microscopy

(Svitkina, 2009)

Correlative phase-contrast and EM

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References

Articles:

• Baum et al. (2006) Regulation of apicomplexan actin-based motility. Nat Rev Microbiol, 4: 621-628. • Danuser and Waterman Storer (2006) Quantitative Fluorescent Speckle Microscopy of Cytoskeleton

Dynamics. Annu Rev Biophys Biomol Struct, 35:361–87. • Eidenmueller (2000) Structural and Functional Implications of Tau Hyperphosphorylation: Information • from Phosphorylation-Mimicking Mutated Tau Proteins. Biochemistry, 39, 13166-13175. • Gupton et al. (2007) mDia2 regulates actin and focal adhesion dynamics and organization in the

lamella for efficient epithelial cell migration. J Cell Sci, 120: 3475-3487 • Mandelkow et al. (1991) Microtubule Dynamics and Microtubule Caps: A Time-resolved Cryo-Electron

Microscopy Study. J Cell Biol, 114: 1991977-991. • Revenu et al. (2004) The co-workers of actin filaments: from cell structures to signals. Nat Rev Mol

Cell Biol, 5: 1-12. • Schaefer et al. (2002) Filopodia and actin arcs guide the assembly and transport of two populations of

microtubules with unique dynamic parameters in neuronal growth cones. J Cell Biol, 158: 139-152. • Schaefer et al. (2008) Coordination of Actin Filament and Microtubule Dynamics during Neurite

Outgrowth. Dev Cell, 15: 146–162 • Svitkina (2009) Imaging Cytoskeleton Components by Electron Microscopy. Methods Mol Biol. 2009 ;

586: 187–206.

ANAT3231 Mechanistic insights into the regulation of the cytoskeleton and how the cytoskeleton regulates biological functions

14th April 2015

Intermediate filaments &

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COMMONWEALTH OF AUSTRALIA

Copyright Regulations 1969

WARNING

This material has been reproduced and communicated to you by and on behalf of the University of New South Wales pursuant to

Part VB of the Copyright Act 1968 (the Act).

The material in this communication may be subject to copyright under this Act. Any further reproduction of communication of

this material by you may be the subject of copyright protection under the Act.

Do not remove this notice


Physical Characteristics

- 10 nm diameter

- Named by size relative to other cytoskeletal filaments

- intermediate filaments have no structural polarity

- Monomer - central α-helical domain

- Dimer - 2 monomers form parallel coiled coil

- Tetramer - pair of parallel dimers associates in an antiparallel staggered fashion

- tetramer is the soluble subunit (analogous to MT αβ-tubulin dimer, or MF actin

monomer)

- Provide rope-like resistance to mechanical stress

- In muscle: link Z discs of adjacent myofibrils

- Organization can be altered by phosphorylation

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Intermediate Filaments - Structure

(Hermann et al., 2007)


(Tang, 2008)

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ULF

(Hermann et al., 2007)

Type I (n = 28) Acidic keratins (pI < 5.7) 40–64 kDa

K9-28 (epithelia) K31-40 (hair/nail)

Type II (n = 26) Basic keratins (pI ≥ 6.0) 53–67 kDa

K1-8, K71-80 (epithelia) K81-86 (hair/nail)

Keratins form heterodimers that assemble into heteropolymeric keratin filaments

Intermediate Filament - Types

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Differential expression of Keratins

Dey et al. (2014)

Type III Desmin (cardiac, skeletal and smooth muscle) Vimentin (widespread distribution: leukocytes, blood vessels, endothelial, some epithelial and mesenchymal cells) 56 kDa Peripherin (neurons) 57 kDa Glial fibrillary acidic protein (GFAP) (astrocytes/glia) 50 kDa

Type III intermediate filament proteins can form both homo- and heteropolymeric filaments


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Type IV Neurofilament Low NF-L (neurons) 62 kDa Neurofilament Medium NF-M (neurons) 110 kDa Neurofilament High NF-H (neurons) 130 kDa

Neurofilaments form heteropolymers

α-internexin (CNS neurons) Synemins (muscle) Syncoilin (muscle) Nestin (stem cell marker) 220 kDa

Type V Lamin A/C (ubiquitous) 62–72 kDa Lamin B1/2 (ubiquitous) 65–68 kDa

Davidson and Lammerding (2014)


Type IV Neurofilament Low NF-L (neurons) 62 kDa Neurofilament Medium NF-M (neurons) 110 kDa Neurofilament High NF-H (neurons) 130 kDa

Neurofilaments form heteropolymers

α-internexin (CNS neurons) Synemins (muscle) Syncoilin (muscle) Nestin (stem cell marker) 220 kDa

Type V Lamin A/C (ubiquitous) 62–72 kDa Lamin B1/2 (ubiquitous) 65–68 kDa

Orphan Phakinin (lens) Filensin (lens)

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Intermediate Filament Associated Protein (IFAP)

Cross-link intermediate filaments with one another

forming a bundle (also called a tonofilament) or a network

IFAPs

Plectin 500 kDa Striated muscle, epithelia Nuclear envelop

Syncoilin 64 kDa Striated muscle

Nesprin-3 117 kDa Kerotinocytes

Paranemin 280 kDa

Desmuslin 230 kDa

Intermediate Filaments - Function

(Kottke et al. , 2006)

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Vikstrom et al. (1992)

Intermediate Filaments - Dynamics

rhodamine-vimentin networks in 3T3 cells

Pre-bleach Bleach 13.5 min after bleach


(Moch et al – 2013)

FRAP in AK13-1 cells producing fluorescent HK13

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Windoffer et al. (2002)

Stably expressed fluorescent desmosomal cadherin desmocollin 2a (Dsc2a) chimeras

Intermediate Filaments - Function

- Desmin interacts with nebulin linking intermediate filament network

and sarcomeres at the Z-discs

(Koniecny et al., 2008)

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Intermediate Filaments Impact on dynamic cellular processes

Chung et al. (2013)

Chung et al. (2013)

Intermediate Filaments Impact on dynamic cellular processes

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(Huber et al., 2015)

Interplay between Microtubules, the Actin Cytoskeleton and Intermediate Filaments

(Baum et al. 2006)

How is Filament Dynamics and Stability Regulated?

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Rho Rho GDP- GTP-

GAP

GEF

Rac Rac GTP- GDP-

GAP

GEF

ROCK PAK

Cofilin Cofilin -P

LIMK

Slingshot

Actin depolymerisation

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Gunning et al. (2005)

(Ridley, 2010)

Different signalling pathways lead to different spatial

organisation of the actin cytoskeleton

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(Ligeti et al., 2012)

Selected Signal Transduction Pathways Acting on the Actin Cytoskeleton

Receptors

Kinases

GEFs and GAPs

Small GTPases

Kinases and effector proteins

Actin filament assembly and

disassembly

(Kuehn and Geyer, 2014)

Spatial Regulation of Actin Filament Nucleation

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Microtubules and the Actin Cytoskeleton in Cell Migration

(Blanchoin et al., 2014)

Regulation of Cell Polarity by the Actin Cytoskeleton

(Mack and Georgiou, 2012)

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(Priya and Yap, 2015)


(Priya and Yap, 2015)


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(Mack and Georgiou, 2012)



(Stiess and Bradke, 2011)

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Microtubules and the Actin Cytoskeleton in Neuronal Cell Morphogenesis

(Akhmanova and Hoogenraad, 2015)

Microtubules and the Actin Cytoskeleton Neurite Branching

(Spillane and Gallo, 2014)

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(Spillane and Gallo, 2014)

Microtubules and the Actin Cytoskeleton Neurite Branching

(Lamprecht, 2011)

Regulation of a Dynamic Actin Cytoskeleton at the Neuronal Synapse

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(Hanley, 2014)

Regulation of a Dynamic Actin Cytoskeleton at the Neuronal Synapse

AMPA receptor subunits

Endosomal system

The Cytoskeleton in the T-cell Response

Angus and Griffiths (2013)

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(Chircop, 2014)

Regulation of the Actin Cytoskeleton during Cell Division

Regulation of the Actin Cytoskeleton during Cell Division

(Chircop, 2014)

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(Gruenbaum and Medalia – 2015)

The Actin Cytoskeleton in Mechanotransduction

The Actin Cytoskeleton in Mechanotransduction

(Osmanagic-Myers et al., 2015)

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Abnormal Cytoskeleton

• Many mutations associated with human diseases

• Toxins can affect organization

The Cytoskeleton in Disease

Goetz and Ittner (2008)

APP = Apolipoprotein

http://www.ahaf.org

http://med.kuleuven.be

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(Suchowerska and Fath, 2014)

(Toivola et al., 2015)


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Learning outcomes

After the Cytoskeleton lecture, you should be able to

• Describe the structure of the three cytoskeletal filament systems in eukaryotic cells, microtubules, intermediate filaments and microfilaments

• Identify the key regulatory mechanisms of the dynamics of these systems • Acquired an understanding of research methodologies to study the

function of the cytoskeleton • To name key cellular processes that are regulated by the cytoskeleton and

to be able to explain the functional role of the cytoskeleton in these processes and how these are disrupted in disease

References

Articles:

• Akhmanova and Hoogenraad (2015) Curr Biol, 25: R162-171. • Alberts et al. (2008) Molecular Biology of the Cell, 5th Edition. • Angus and Griffiths (2013) Curr Opin Cell Biol, 25: 85–91. • Baum et al. (2006) Nat Rev Microbiol, 4: 621-628. • Blanchoin et al. (2014) Phyysiol Rev, 94: 235-263. • Chircop (2014) Small GTPases, 5: e29770—1 - e29770—14. • Chung et al. (2013) Curr Opin Cell Biol, 25: 600–612. • Davidson and Lammerding (2014) Trends Cell Biol, 24: 247–256. • Dey et al. (2014) Diagnost Cytopath, DOI: 10.1002/dc.23132. • Goetz and Ittner (2008) Nat Rev Neurosci, 9: 532-544. • Gunning et al. (2005) Trends Cell Bio, 15: 333-341. • Gruenbaum and Medalia (2015) Curr Opin Cell Biol, 32:7–12. • Hanley (2014) Front Neurosci, 8: 1-8. • Herrmann et al. (2007) Nature Reviews - Mol Cell Biol, 8: 562-573. • Huber et al. (2015) Curr Opin Cell Biol, 32: 39–47. • Koniecny et al. (2008) J Cell Biol, 181, 667-681. • Kottke et al. (2006) J Cell Sci, 119, 797-806. • Kuehn and Geyer (2014) Small GTPases, 5: 1-15. • Lamprecht (2011) Progr Neurobiol, 117: 1–19. • Ligeti et al. (2012) Physiol Rev, 92: 237–272. • Mack and Georgiou (2012) Small GTPases, 5: 1-16. • Moch et al. (2013) Proc Nat Acad Sci USA, 110: 10664-10669. • Osmanagic-Myers et al. (2015) Genes & Dev 29:225–237. • Priya and Yap (2015) Curr Topics Dev Biol, 112: 65-102. • Revenu et al. (2004) Nat Rev Mol Cell Biol, 5: 1-5. • Ridley (2006) Trends Cell Biol, 16: 522-529. • Spillane and Gallo (2014) Small GTPases, 5, e279741-9. • Stiess and Bradke (2011) Dev Neurobiol, DOI 10.1002/dneu.20849 • Suchowerska and Fath (2014) Front Biol, 9: 5-17 • Tang (2008) Am J Physiol Cell Physiol, 2008: C869–C878. • Vikstrom et al. (1992) J Cell Biol, 118: 121–129. • Windoffer et al. (2002) J Cell Sci, 115: 1717-1732.

Book:

• Alberts et al. (2008) Molecular Biology of the Cell, 5th Edition.

cytoskeleton introduction - cellbiology · pdf file15/04/2015 3 functions of the cytoskeleton...

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