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UNSW Cell Biology

ANAT3231 Lecture 07 Microfilaments

Introduction

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.

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

Page Links: iLecture Audio | Lecture Slides | Lecture Slides Text | NLM Online Textbooks | Search PubMed | Web Links | Movies | OMIM Microfilaments | Related Topics

iLectureAudio

Link to audio presentation of this lecture using iLecture system. Having troubles accessing iLecture Content? iLecture Student Help

2005 Lecture 07 Cytoskeleton 3
2005 Lecture Series

Lecture Slides

Link to lecture slides allows you to open and view the slides in your web browser or download the PDF document for later viewing/printing.

lecture07 1 slide/page (view only) (69 pages, 1.2 Mb)

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See also Lecture Slides Text on this current page

Laboratory 6

The associated Laboratory 07 looks at xxxxxxx.

NLM Online Textbooks

Molecular Biology of the Cell

Molecular Cell Biology

The Cell- A Molecular Approach

Search NLM Online Textbooks- "microfilament"

Molecular Biology of the Cell

Molecular Cell Biology

The Cell- A molecular Approach

Search NLM Online Textbooks- "actin"

Molecular Biology of the Cell

Molecular Cell Biology

The Cell- A molecular Approach

PubMed

Articles

GFP illuminates the cytoskeleton. Ludin B, Matus A, Trends Cell Biol. 1998 Feb;8(2):72-7. uids=9695812

Review Articles

Search NLM PubMed Database

Articles "microfilament" : PubMed Apr 2005 |

Movies

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

Web Links

Actin

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

OMIM- Beta Actin

OMIM- Gamma Actin

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

OMIM- Tropomyosin 1

OMIM- Tropomyosin 3

Arp2/3

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

Myosin

196 entries in the OMIM database.

Related Topics

actin, tropomyosin, cell motility, wound healing, cytokinesis, signal transduction, apoptosis

Lecture Slide Text

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