This lecture continues to look at the cell cytoskeleton, covering in detail the 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) and adhesion (focal adhesions) will not be covered in detail, as this topic is covered elswhere in lecture series.
The key concepts are: microfilaments, actin, associated proteins, tropomyosin, actin regulation, contraction, muscle actins, myosin. The lecture slides and textbook alone contain enough information as an introduction to the subject for this level of study. If you are interested in further reading, I have also included below links to more detailed textbooks with further information and images. Please note this additional information is not necessarily examinable, but may be useful if you have not previously studied biology. The link below to lecture slides allows you to open and view the slides in your web browser or download the PDF document for later viewing/printing.
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
Page Links: iLecture Audio | Lecture Slides | Lecture Slides Text | NLM Online Textbooks | Search PubMed | Web Links | Movies | OMIM Microfilaments | Related Topics
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Molecular Biology of the Cell
Molecular Cell Biology
The Cell- A Molecular Approach
Search NLM Online Textbooks- "microfilament"
Search NLM Online Textbooks- "actin"
GFP illuminates the cytoskeleton. Ludin B, Matus A, Trends Cell Biol. 1998 Feb;8(2):72-7. uids=9695812
Search NLM PubMed Database
Articles "microfilament" : PubMed Apr 2005 |
Shows a collection of educational videos featuring many pathgoens. Some of were featured in the Discovery channel documentry "Intimate Strangers."
Shows a collection of all available supplemental movies for publications
1. Fission yeast mitosis and cytokinesis. 2. Actin filament branching by Arp2/3 complex.
The Structural Biology of Cell Movement - Gelsolin Movies
Actin is the major component of the thin filaments of muscle cells and of the cytoskeletal system of nonmuscle cells. The amino acid sequences of muscle and cytoskeletal actin isoforms are very similar and well conserved in evolution.
Search OMIM database: OMIM entries for Actin
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
The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of 7 subunits.
OMIM- ACTIN-RELATED PROTEIN 2/3 COMPLEX
196 entries in the OMIM database.
actin, tropomyosin, cell motility, wound healing, cytokinesis, signal transduction, apoptosis
Note that the text without the slide context and associated images may not make sense and is insufficient to understand all key issues.
ANAT3231 - Cell Biology Lecture 7 School of Medical Sciences The University of New South Wales Dr Mark Hill Cell Biology Laboratory Room G20 Wallace Wurth Building Email: m.hill@unsw.edu.au UNSW Copyright Notice
Lecture Overview
Microfilaments
Structure, function and regulation
Actin
Motility
Adhesion, focal adhesions
Actin binding proteins, myosin motors
Muscle actins
Microfilament diseases
UNSW Cell Biology http://cellbiology.med.unsw.edu.au/units/science/lecture0505.htm
Cytoskeleton
Cytoskeleton Filaments
Actin Dynamics
Introduction Motility- fundamental cellular process embryonic development wound healing 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 100's of proteins regulate organisation Bound to many membrane proteins
Microfilament References Medline (April) References Actin 50,096 (05) 46,353 (04) 271 (05) 193 (04) (review, 1 year) Actin Binding Proteins 50,620 (05) 46,945 (04) Myosin 26,286 (05) 24,924 (04) Textbooks Essential Cell Biology Ch16 p527-542 Molecular Biology of Cell Ch16 p821 Molecular Cell Biology Ch19
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 43 Kd Protein 2 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
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 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 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 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/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
Cell motility
Note adhesion is covered in detail in later Lecture
Adhesion Junctions
Adherens Junctions 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
Adhesion Junctions Adherens (cell-cell) cadherin (E-cadherin) Links to cadherin in neighboring cell Adherens (cell-matrix)
Integrin Links to extracellular matrix
Focal Adhesions
Adhesive Signalling
Actin Signaling
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 Regions
Structures Phosphorylation Changes location on MF
Tropomyosin Isoforms 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
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
Actin- Neuron Vesicle Motility
Movie: Actin- Neuron Vesicle Motility
GFP Actin- Growth Cone
Actin Motors- Myosin Myosin Movement
Muscle Types
Skeletal, cardiac
Striated sarcomeres
Smooth non-striated
Muscle- Skeletal
Muscle- Cardiac
Muscle Smooth
Skeletal Muscle
Muscle Contraction sliding of filaments- actin against myosin troponin and tropomyosin allow contraction of skeletal and cardiac muscle to be regulated by Ca2+ flux smooth muscle cells and nonmuscle cells contraction the same mechanism contractile units smaller and less highly ordered both activity and state of assembly are controlled by Ca2+ -regulated phosphorylation of a myosin
MF 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
Cytochalasin D Fungal metabolite Binds barbed end inhibits polymerization and depolymerization Cell permeant Active in low micromolar
Latrunculin Sea sponge metabolite Binds monomeric actin inhibits polymerization Cell permeant Active at low nanomolar MF Binding Molecules
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
Movie: GFP Actin- Listeria
A Selection of MF Diseases 1
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.
A Selection of MF Diseases 2
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
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
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