UNSW Cell Biology

2008 ANAT3231 Lecture 11 Cell Cytoskeleton - Microfilaments

Introduction

This lecture introduces the cell cytoskeleton filament system of microfilaments. Microfilaments are the smallest filament system of the cytoskeleton and have important functions for cellular motility (intra- and whole cell). Note that the role of microfilaments in mitosis (cytokinesis), adhesion (focal adhesions) and muscle (sarcomeres) will not be covered in detail, as this topic is covered elswhere in lecture series. Lecture 11: PDF 2008 lecture11 1 slide/page (view only) (55 pages, 1.7 Mb) PDF 2008 lecture11 4 slides/page (print) (14 pages, 932 Kb) PDF 2008 lecture11 6 slides/page (print) (7 pages, 160 Kb) Online Powerpoint Slide Show Lecture Word Document (not linked yet)

Atomic model of Actin Microfilament Structure     Notes in Preparation (notice removed when complete)

Page Links: Introduction | Objectives | Lecture Audio | Textbooks | Abnormalities | References | Online Textbooks | Web Links | Web Movies | 2007 Lecture Slides | Comments | Acronyms

Objectives

• Understand the role of microfilaments in the cell

• Understand the main components of microfilaments and associated proteins

• Understand the structure and synthesis of microfilaments

• Understand the motor proteins associated with microfilaments

• Brief understanding of the regulation of the microfilament cytoskeleton

• Brief understanding of microfilament dynamics

Lecture Audio

The University has a system for automated recording of lectures called Lectopia (or iLecture).

• No audio recording for this Lecture carried out in the Lab.

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Links: Lectopia Login Page | Cell Biology Podcast Page | Current Course Outline 2008

History

1887 WD Halliburton extracted a protein from muscle that 'coagulated' preparations of myosin,"myosin-ferment."

1942 BF Straub developed a novel technique for extracting muscle protein that allowed him to isolate substantial amounts of actin.

1950 BF Straub reported that actin contains bound ATP

1990 Kabsch solves crystal structure of G-actin

Links:

Motility

• Motility is a fundamental cellular process
• embryonic development
• wound healing, blood vessel growth
• immune responses
• development of tissues
  • "human brain requires the laying down of about 1 million miles of neurites, all proceeding through the crawling motility of growth cones"

Actin

• represents approx 5% of all cell protein
• 10-20% of soluble protein
• small number of key actins
• 100’s of proteins regulate organisation
• Bound to many membrane proteins

(MH - will not cover muscle actin contraction in detail in this lecture)

Links: Electron micrographs of chloroplasts | Chloroplasts | A mitochondrion and chloroplast compared

Microfilaments

• Twisted chain 7 nm diameter
• Compared to Microtubules
• Thinner, more flexible, shorter
• Point in same direction
• Different organisation in different cellular regions

Actin Microfilament Formation

• Globular actin monomer (g actin) polymerise to Filamentous actin (f actin)
• Cells approx G:F is 50:50
• Monomer can add to either (+ or - ) end
• Actin-ATP hydrolysed (ADP) following addition
• Destabilises (like microtubules)

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 and Depolymerization controls filament length

Links:

Actin Types

• 6 Mammalian actin types (isoforms)
• All are 374aa, 43 Kd Protein
• 2 cytoskeletal isoforms in all non-muscle cells
  • Beta cytoskeletal
  • Gamma cytoskeletal
• 4 muscle isoforms in different muscle cells
  • Alpha skeletal (muscle)
  • Alpha cardiac (muscle)
  • Alpha smooth (muscle)
  • Gamma smooth (muscle)

(Human beta gene - 7p22-p12, gamma 17q25)

Actin Protein

• Completely conserved in mammals
• different ratios (beta:gamma) in different cell types
• 4 aa difference between beta and gamma (at N- terminal)
• Highly expressed gene
• Promoter used in gene transfections

Actin Binding Proteins

• Regulate polymerisation and create different structures
• Monomer binding protein
  • Sequester and Release
• Polymer binding proteins
  • Bundling
  • cross-linking
  • Severing
  • contracting

Actin Binding Protein Interactions

Actin Binding Proteins

Actin Cell Cortex

• Layer concentrated directly under cell membrane
• Linked by ABPs and membrane proteins to ECM
• integrins
• Regulates cell shape and motility
• Continuously remodeled

Cortical Actin Filaments

• Cortical Actin Filaments
• organized into 3 types of arrays
• Parallel bundles
  • microspikes and filopodia
  • filaments oriented same polarity
  • closely spaced (10-20 nm apart)
• Contractile bundles
  • stress fibers and contractile ring (mitosis)
  • filaments arranged opposite polarities
  • more loosely spaced (30-60 nm apart)
  • contain the motor protein myosin-II
• Gel-like network of cell cortex
  • filaments arranged loose open array
  • many orthogonal interconnections

Links: The Cell - Model of lamin assembly |

Actin Lamellipodia

Links: Movie - Vesicular Mitochondria | Role of mitochondria in apoptosis |

Cell Movement

• Whole or part of cell
• Amoeba, neutrophil, macrophages
• Neuron processes (axon, dendrites) growth cones
• Common structures
• Contraction
• Intracellular transport

Motile Structures

• Leading/Trailing Edge
• extension/retraction
• Actin nucleation
• Lamellipodia - sheet-like extensions
• Filopodia - thin protrusions (begin as microspikes)
• Integrins anchor to ECM

Adhesive Functions

• Cell signalling
• Modify cell cytoskeleton
• Activate intracellular signalling pathways
• Cell motility
• Note adhesion is covered in detail in earlier Lecture 8

Adhesion Junctions

• Adherens Junctions
  • microfilaments anchor plaque that occurs under membrane of each cell (plaques not as dense)
  • also occur as hemiform
  • Locations – heart muscle, layers covering body organs, digestive tract
  • Cadherin - Transmembrane proteins (E-cadherin) – Links to cadherin in neighboring cell
• Focal Adhesions
  • Adherens (cell-matrix)
  • Integrin – Links to extracellular matrix
  • Adhesive Signalling
  • Actin Signalling
  • Rho – Family of small GTPases organize the actin cytoskeleton
  • Rho, RAC, CDC42
  • Form different actin structures Cell 1995 Apr 7;81(1):53-62
  • Wasp – Wiskott-Aldrich syndrome protein
  • a downstream effector
  • transfers signal from tyrosine kinase receptors and small GTPases to actin cytoskeleton

MF Associated Proteins

• Tropomyosin
  • Reinforces MF
  • Different tropomyosins
  • Muscle/non-muscle
  • Located in different cellular Regions and Structures
  • Phosphorylation
  • Changes location on MF
  • Tropomyosin Isoforms • Many different forms – From different genes – Differential splicing
• Actin-related proteins (Arp2/3)
  • Arp2/3 protein complex
  • control of polymerization – lamellipodia localization
  • human complex has 7 subunits • ARP2, ARP3, ARC41, ARC34, ARC21, ARC20, and ARC16
  • Listeria monocytogenes – Induce actin polymerization by Arp2/3 protein complex at Listeria surface

Actin Motors - Myosin

• Myosins
  • Myosin I • All cells • One head domain – Binds actin –
  • Myosin II • Muscle myosin – Also other cells • Dimer, 2 heads • Bind to each other to form myosin filament – Thick filament
• Movie: Actin - Neuron Vesicle Motility
• Movie: Actin - Neuron Vesicle Motility GFP Actin - Growth Cone Actin Motors- Myosin Myosin Movement
• Muscle Types
  • Skeletal, cardiac – Striated – sarcomeres
  • Cardiac Muscle - Striated
  • Smooth – non-striated
• Contraction
  • sliding of filaments actin against myosin – troponin and tropomyosin
  • contraction of skeletal and cardiac muscle regulated by Ca2+ flux
  • smooth muscle cells and non-muscle cells – contraction same mechanism – contractile units smaller less highly ordered
  • activity and state of assembly controlled by Ca2+ -regulated phosphorylation of a myosin

Microfilament Binding Molecules

• Cytochalasin D
  • Fungal metabolite
  • Binds barbed end – inhibits polymerization and depolymerization
  • Cell permeant
  • Active in low micromolar
• Phalloidin
  • Fungal metabolite
  • Binds and stabilizes F-actin
  • Not cell permeant
  • Fluorescent derivatives are used to stain F-actin in situ and in vitro
• Jasplakinolide
  • Sea sponge metabolite
  • Binds and stabilizes F-actin competitively with phalloidin
  • Causes nucleation
  • Cell permeant
  • Nanomolar Kd for F-actin
• Latrunculin
  • Sea sponge metabolite
  • Binds monomeric actin
  • inhibits polymerization
  • Cell permeant
  • Active at low nanomolar MF Binding Molecules

Movie Slides

• Movie: GFP Actin- Neuron dendrites
• Movie: GFP Actin- Dendritic spines MF/MT
• Movies: Organelle motility in preparations of axoplasm extruded from squid giant axon • Organelle motility in extracts from Xenopus eggs • Movement of phagosomes loaded with latex beads in mouse bone marrow macrophages – http://www.biologie.uni-rostock.de/tierphysiologie/Arbeitsgruppen/Motorenzyme/Movies/main.html

Abnormalities

Actin So essential to cell that diseases due to mutation of cytoskeletal actin rarely seen Cardiac Actin Mutational analysis of the cardiac actin gene in familial and sporadic dilated cardiomyopathy. Am J Med Genet. 1999 Oct 8;86(4):325-7. Tropomyosin Clinical features of hypertrophic cardiomyopathy caused by mutation of a 'hot spot' in the alpha-tropomyosin gene. J. Am. Coll. Cardiol. 29: 635-640, 1997. A mutation in the alpha tropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy. Nature Genet. 9: 75-79, 1995. Myosin Association of unconventional myosin MYO15 mutations with human non-syndromic deafness DFNB3. Science 280: 1447-1451, 1998. Wasp Novel mutations in the Wiskott-Aldrich syndrome protein gene and their effects on transcriptional, translational, and clinical phenotypes. Destrin Hum. Mutat. 14: 54-66, 1999. Aberrant actin cytoskeleton leads to accelerated proliferation of corneal epithelial cells in mice deficient for destrin (actin depolymerizing factor). Hum Mol Genet. 2003 May 1;12(9):1029-37. Filamin Localized mutations in the gene encoding the cytoskeletal protein filamin A cause diverse malformations in humans. Nat Genet. 2003 Apr;33(4):487-91

Genes and Diseases http://www.ncbi.nlm.nih.gov/books/bookres.fcgi/gnd/tocstatic.html OMIM - Online Mendelian Inheritance in Man http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim

Links: NCBI - Genes and Diseases | NCBI - OMIM |

References

There is a complete list of online textbook links at 2008 ANAT3231 Lecture 11 - Microfilaments - References

Search Pubmed Articles "microfilament"

Essential Cell Biology - Chapter 16 p527-542

Molecular Biology of the Cell 4th ed. Chapter 16 p821 Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

The Cell - A Molecular Approach - Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc. ; c2000

Molecular Cell Biology - Chapter 19 Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co. ; c1999

Microfilaement references

Medline (April) Reference Search Results

• Actin 62,901 (08) 58,545 (07) 54,273 (06) 50,096 (05) 46,353 (04)
• Actin Binding Proteins 63,038 (08) 59,067 (07) 54,711 (06) 50,620 (05) 46,945 (04)
• Myosin 30,500 (08) 29,099 (07) 27,683 (06) 26,286 (05) 24,924 (04)

There is a complete list of online textbook links at 2008 ANAT3231 Lecture 11 - Microfilaments - References

Web Links

Web Movies

Molecular Cell Biology

First link is directly to Quicktime move, followed by associated textbook figure with legend. Look at the movie first, then look at the labelled image.

MCB - Photosynthesis | The primary event in photosynthesis

Theriot Lab

Theriot Lab Movie Collection

Shows a collection of educational videos featuring many pathgoens. Some of were featured in the Discovery channel documentry "Intimate Strangers."

Borisy Lab

Borisy Lab Movie Page

Shows a collection of all available supplemental movies for publications

Pollard Lab

Pollard Lab Movie Page

1. Fission yeast mitosis and cytokinesis. 2. Actin filament branching by Arp2/3 complex.

Robinson Lab

The Structural Biology of Cell Movement - Gelsolin Movies

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Acronyms

• ABP - Actin Binding Protein
• ADP - Adenosine Diphosphate
• ARP - Actin-related proteins
• ATP - Adenosine Triphosphate
• DNA - deoxyribonucleic acid
• EM - electron microscopy
• f actin - Filamentous actin polymer
• FL - flourescent
• g actin - Globular actin monomer
• MF - Microfilament
• OMIM - Online Mendelian Inheritance in Man Database
• PDF - Portable Document Format (electronic paper)
• Wasp - Wiskott-Aldrich Syndrome Protein

2007 Lecture Slides

(MH - note that content will not match exactly current lecture structure but has been selected as having similar content)

lecture07 1 slide/page (view only) (55 pages, 1.7 Mb)

lecture07 4 slides/page (print) (14 pages, 932 Kb)

lecture07 outline (print no images) (7 pages, 160 Kb)

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	In 2008 a new way of presenting course content online is being trialled. Please let me know of any difficulties/suggestions or things that work well. Notice also that in some slides I have added annotations in brackets with my initials (MH - )
	Links: Current Course Outline 2008