2009 Lecture 12

Adherens Junction

Cytoskeleton - Microfilaments

This lecture introduces the smallest of the three cytoskeleton filament systems, microfilaments.

This lecture will be presented by a guest expert lecturer Prof Peter Gunning.

With more than 150 papers in microfilament research

The guest lecturer will provide their own notes for this lecture.

Recent Tropomyosin review - Gunning PW, Schevzov G, Kee AJ, Hardeman EC. Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends Cell Biol. 2005 Jun;15(6):333-41. Review. PMID: 15953552 PDF version

Objectives

• Introduction to microfilaments
• Understand microfilament functions
• Understand microfilament structure
• Understand microfilament associated proteins
• Understand microfilament motors
• Brief understanding of other microfilament cellular roles

Actin Filament Model

Myosin Microtubule Actin Collagen

Lecture Slides

These are links to PDF versions of the powerpoint slides used in the 2009 lecture.

1 slide/page viewing only‎ | 4 slides/page printing‎ | 6 slides/page printing‎

Lecture Audio

The University has a system for automated recording of lectures called Lectopia. Lectopia requires login using your student number and unipass. I will be adding the link to each iLecture Audio following the Lecture. Due to the automated recording method, most lectures begin 4-5 minutes into MP3 recordings and occasionally stop before the lecture does. 2009 All Audio Files

Lecture 12: Cytoskeleton Microfilaments Lecture Date: 22-04-2009 Lecture Time: 10:00 Venue: BioMed E Speaker: Peter Gunning

Actin filament nucleation and elongation

History

• Actin in non-muscle cells Howard Holtzer’s group uses heavy meromyosin as a probe to find actin filaments in non-muscle cells.
• Actin in locomotion Ken Yamada, Brian Spooner and Norman Wessels use the newly discovered drug cytochalasin B to show that actin filaments drive cell locomotion.
• Actin pushes in bizarre places Lewis Tilney discovers that actin polymerization is a means of force generation. His studies use unorthodox systems: the acrosomal reaction in both starfish and sea cucumber sperm and the cell-to-cell motility of the Listeria monocytogenes bacterium.
• Powered by gel Thomas Stossel and John Hartwig nab the very first actin-binding protein, find that it spurs actin fibers in vitro to coalesce into a mesh, and tie this process to what happens in vivo during phagocytosis.
• Actin and microtubules interact via MAP A viscometer allows Linda Griffith and Tom Pollard to demonstrate that actin and microtubules interact via MAPs.

Actin

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

Microfilaments

• Twisted chain 7 nm diameter
• Compared to MT
• 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 50:50
• Monomer can add to either (+ or - ) end
• Faster at + end
• Actin-ATP hydrolysed (ADP) following addition
• Destabilises (like MT)

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 Types

• 6 Mammalian actin types (isoforms)
• All are 43 Kd Protein
• 2 cytoskeletal isoforms in all non-muscle cells
  • Beta (β) 7p22-p12
  • Gamma (γ) 17q25
• 4 muscle isoforms in different muscle cells
  • Alpha (α) skeletal
  • Alpha (α) cardiac
  • Alpha (α) smooth
  • Gamma (γ) smooth 2p13.1

Actin Protein

• Conserved in mammals
• Different ratios (β:γ) 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

Accessory proteins in same intracellular process are shown in same colour Adapted from D. Botstein et al., in The Molecular and Cellular Biology of the Yeast Saccharomyces [J.R. Broach, J.R. Pringle, E.W. Jones, eds.], Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1991.

Actin Binding Proteins

• Regulate polymerisation and create different structures
• Monomer binding protein
  • Sequester
  • release
• Polymer binding proteins
  • Bundling
  • cross-linking
  • Severing
  • contracting
• Actin Binding Protein Interactions

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

• 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 are arranged opposite polarities
  • more loosely spaced (30-60 nm apart)
  • contain the motor protein myosin-II

• Gel-like network of cell cortex
  • filaments arranged relatively loose open array
  • many orthogonal interconnections

Movie: Actin Lamellipodia Movie: GFP Actin Lamellipodia and Bundles

Cell Movement

• Whole or part of cell
  • Amoeba, neutrophil, macrophages
• Neuron processes
  • axon, dendrites
• Common structures
  • Contraction
  • Intracellular transport
• Motile Structures
  • Leading/Trailing Edge
  • extension/retraction
  • Actin nucleation

Lamellipodia

Sheet-like extensions

Filopodia

Thin protrusions

Integrins anchor to ECM

Cell Migration Movie: Adhesion and Motility

Adhesive Functions

Cell signalling

Modify cell cytoskeleton

Activate intracellular signalling pathways

Adhesion Junctions

Note adhesion is covered in another Lecture

• microfilaments anchor the plaque that occurs under the membrane of each cell.
• plaques not as dense
• also occur as hemiform

Adherens Junctions

• heart muscle, layers covering body organs, digestive tract.
• transmembrane proteins
• Cadherin

• Adherens (cell-cell)
  • cadherin (E-cadherin)
  • Links to cadherin in neighboring cell
• Adherens (cell-matrix)
  • Integrin
  • Links to extracellular matrix

Focal Adhesions

• Adhesive Signaling through actin polymerisation

Signaling pathway

• • Activation cell surface receptor
  • Rho family GTPases activated
  • WASP family proteins (WASp, N-WASP, and Scar/WAVE)
  • stimulate Arp2/3 complex to nucleate actin filaments
  • filaments grow at a fixed 70 degrees angle from side of pre-existing actin filaments
  • new filaments push the membrane forward, grow at their barbed ends

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

Tropomyosins are ubiquitous proteins of 35 to 45 kD associated with the actin filaments of myofibrils and stress fibers. In vertebrates, 4 known tropomyosin genes code for diverse isoforms that are expressed in a tissue-specific manner and regulated by an alternative splicing mechanism

• Reinforces MF
• Different tropomyosins
• Muscle/non-muscle
• Regions, Structures
• Phosphorylation
  • Changes location on MF

• Tropomyosin Isoforms
  • From different genes
  • Differential splicing

Links: OMIM- Tropomyosin 1 OMIM- Tropomyosin 3

Actin-related proteins (Arp2/3)

The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells.

• 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

Links: OMIM- Arp2/3 protein complex

Actin Motors

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 GFP Actin - Growth Cone Actin Motors- Myosin Myosin Movement

Muscle

• Striated
  • Skeletal, cardiac
  • sarcomeres
• Non-striated
  • Smooth

Muscle 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

Microfilament Diseases

• 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

From the JCB Archive

• Actin in non-muscle cells Howard Holtzer’s group uses heavy meromyosin as a probe to find actin filaments in non-muscle cells.
• Actin in locomotion Ken Yamada, Brian Spooner and Norman Wessels use the newly discovered drug cytochalasin B to show that actin filaments drive cell locomotion.
• Actin pushes in bizarre places Lewis Tilney discovers that actin polymerization is a means of force generation. His studies use unorthodox systems: the acrosomal reaction in both starfish and sea cucumber sperm and the cell-to-cell motility of the Listeria monocytogenes bacterium.
• Powered by gel Thomas Stossel and John Hartwig nab the very first actin-binding protein, find that it spurs actin fibers in vitro to coalesce into a mesh, and tie this process to what happens in vivo during phagocytosis.
• Actin and microtubules interact via MAP A viscometer allows Linda Griffith and Tom Pollard to demonstrate that actin and microtubules interact via MAPs.

References

Textbooks

Essential Cell Biology

• Chapter 16 p527-542

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

• Molecular Biology of Cell Chapter 16 p821
• Figure 16-7. The structures of an actin monomer and actin filament
• Figure 16-38. Actin arrays in a cell
• Table 16-2. Drugs That Affect Actin Filaments and Microtubules

Molecular Cell Biology

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

• Molecular Cell Biology Chapter 19
• The Actin Cytoskeleton

The Cell- A Molecular Approach

Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000

• Actin Structure and Organization

Search Online Textbooks

• "microfilament" Molecular Biology of the Cell | Molecular Cell Biology | The Cell- A molecular Approach | Bookshelf

• "actin" Molecular Biology of the Cell | Molecular Cell Biology | The Cell- A molecular Approach | Bookshelf

Books

• Cell Junctions: Adhesion, Development, and Disease‎ by Susan E. La Flamme, Andrew Kowalczyk

PubMed

• PubMed is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. PubMed

• PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. PMC

• Online Mendelian Inheritance in Man (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM

• Entrez is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others Entrez

Search Pubmed

• "microfilament" Entrez all databases
• "actin" Entrez all databases
• "tropomyosin" Entrez all databases

Reviews

• Gunning P. Emerging issues for tropomyosin structure, regulation, function and pathology. Adv Exp Med Biol. 2008;644:293-8. Review. PMID: 19209830

Movies

• Theriot Lab - Listeria

• Robinson Lab
• Pollard Lab - Yeast

• UNSW Cell Biology Microfilament Lecture 2008

Work Area

• Medline (April 2008) References
  • 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)

• Motility- 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”

2009 Course Content

Lectures

Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton 1 Intermediate Filaments | Cytoskeleton 2 Microtubules | Cytoskeleton 3 Microfilaments | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells | Stem Cells | Development | Revision

Laboratories

Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Tissue Culture 1 | Tissue Culture 2 | Microarray Lab visit

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G