2010 Lecture 14

Extracellular Matrix 2

Basal Lamina

Introduction

This second lecture on ECM will cover the glycoprotein components of ECM and specialized epithelial ECM. Finally I will discuss some key experiments exploring the role and function of the ECM of epitheilia (basement membrane) and connective tissues. With the epithelial ECM the term "basement membrane" is used with light microscopy and "basal lamina" is used with electron microscopy.

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 end of the lecture. 2009 All Audio Files

Objectives

Understanding of the localisation and origin of extracellular matrix
Understanding of the 3 major components
- fibers, proteoglycans (matrix), adhesive glycoproteins
Broad understanding of structure and function
- fibronectin
- laminin
- basement membrane
Broad understanding of some key extracellular matrix experiments

History

Below are some example historical research finding related to cell junctions from the JCB Archive.

1978 Basal lamina instructs innervation Joshua Sanes and Jack McMahan show that regenerating nerve axons take their cues for new synapse formation from the extracellular matrix (ECM) of muscle cells and not from the muscle cells themselves.

ECM Function

Support for cells
Pattern of ECM regulates
- polarity
- cell division
- adhesion
- motility
Development
- migration
- differentiation
- growth factors

ECM stable and able to be reorganised?

ECM Structure

Glycoproteins
Fibers
- Collagen- main fibers
- Elastin
Hydrated Matrix
- Proteoglycans
- high carbohydrate
Adhesive
- Fibronectin
- Laminin

Cell Adhesion to ECM

Direct linkage to collagen or proteoglycan
insertion of fibers into membrane
covalent attachment to membrane lipid
Linking glycoproteins
- fibronectin
- laminin

Fibronectin

Fibronectin binding domains

Fibronectin Structure

dimer connected at C-terminal
- Mr 550 kDa
- nearly identical subunits composed of types I (F1), II (F2), and III (F3) fibronectin modules
S-S linkages
rigid and flexible domains
fibronectin fibrils have elastic properties and can stretch fibrils up to four-fold their relaxed length.
fibrillogenesis - transformation from the compact (soluble) form to the extended fibrillar (insoluble) form of fibronectin, requires application of mechanical forces generated by cells.

Binding Domains

fibronectin and syndecan binding model

cell binding segment RGDS
- arg-gly-asp-ser
binds integrin receptor in membrane
- then mechanically couples to the actin cytoskeleton
domains bind
- heparin sulphate
- collagen
- hyaluronic acid
- Gangliosides
- fibronectin

Figure 19-54. Coalignment of extracellular fibronectin fibrils and intracellular actin filament bundles

fibronectin stretch

Fibronectin Function

Fibronectin (fl)

soluble protein in blood plasma (200–250 kDa monomer)
- blood clotting process, link to fibrin
insoluble protein in extracellular matrix (ECM)
- ECM fibronectin differs from plasma fibronectin by the presence of additional polypeptide segments and in altering morphology of transformed cells and hemagglutination.

Cell Adhesion

Migration Pathways

blocking fibronectin with antibody
prevents neural crest migration
extension of axons and dendrites
branching

Fetal fibronectin (fFN)

produced by fetal cells and found acting as an adhesive at the interface of the chorion and the decidua ( fetal membrane and uterine lining).
- diagnostic for preterm birth.

Laminin

laminin molecular structure

Laminin Structure

cross-shaped glycoprotein
3 polypeptides a, b1, b2
carbohydrate (13% by weight)
Mr 900K
separate binding domains
- collagen IV
- heparin
- heparin sulphate
- cell binding
- cell specific binding - liver, nerve
- cell surface receptor

Figure 19-57. The structure of laminin

Laminin Function

cell adhesion
migration pathways
stimulates growth of axons
development and regeneration
differentiation
basal laminae
most abundant linking glycoprotein

Integrin- Structure Integrin Function cell membrane receptor for ECM linkers binds RGDS motif 2 subunits alpha (α) and beta (β) transmembrane linked to cell cytoskeleton actin microfilaments via talin and vinculin focal contacts For Review see Integrin signaling revisited. Schwartz MA.Trends Cell Biol 2001 Dec;11(12):466-70

Adhesive Signalling

Integrin and Laminin - Several integrin heterodimers act as laminin receptors on a variety of cell types alpha 1 beta 1 alpha 2 beta 1 alpha 3 beta 1 alpha 6 beta 1 alpha 7 beta 1 alpha 6 beta 4 Microsc Res Tech 2000 Nov 1;51(3):280-301

Integrin and Laminin

Roles of laminin-binding integrins in adhesion-mediated events in vertebrates
embryonic development, cell migration and tumor cell invasiveness, cell proliferation, differentiation and basement membrane assembly
essential role for receptors in maintaining cell polarity and tissue architecture
- Text from: Microsc Res Tech 2000 Nov 1;51(3):280-301

Basement membrane

basement membrane

The epithelial ECM the term "basement membrane" is used with light microscopic observation and "basal lamina" is used with electron microscopy.

The basement membrane is composed of two sublayers.

basal lamina

(about 40–120 nm thick) consists of fine protein filaments embedded in an amorphous matrix.
Membrane proteins of the epithelial cells are anchored in the basal lamina, which is also produced by the epithelial cells.
major component of the basal lamina are two glycoproteins - laminin and (usually type IV) collagen

Figure 19-58. A model of the molecular structure of a basal lamina

reticular lamina

consists of reticular fibres embedded in ground substance.
fibres of the reticular lamina connect the basal lamina with the underlying conective tissue.
components of the reticular lamina are synthesised by cells of the connective tissue underlying the epithelium.

Basal Lamina Experiment

Figure 19-60. Regeneration experiments demonstrating the special character of the junctional basal lamina at a neuromuscular junction

Neuromuscular junction

Basal lamina directs acetylcholinesterase (AChE) accumulation at synaptic sites in regenerating muscle
skeletal muscle damaged such that basal lamina sheaths of the muscle fibers spared
new myofibers develop within sheaths and neuromuscular junctions form at original synaptic sites
regenerated neuromuscular junctions have junctional folds and accumulations of acetylcholine receptors and AChE

Anglister L, McMahan UJ J Cell Biol 1985 Sep;101(3):735-43

Integrin- Function

Activate members of Rho-family of small GTPases
Conversely, Rho- and Ras-family proteins can influence the ability of integrins to bind their ligands
- control of cell motility, and therefore on invasive and metastatic behavior
Integrin binding ECM has effects on cell survival, particularly for cells of epithelial origin
- specific integrins have selective effects on efficiency of signal transduction in cell survival pathways

Text modified from: New aspects of integrin signaling in cancer. Semin Cancer Biol 2000 Dec;10(6):407-14

ECM Reorganisation

Reorganisation can occur through proteolytic degradation changes to ECM proteins (collagen, laminin, and fibronectin). Their activity can be regulated locally by inhibitors.

The proteases form 2 main classes:

Matrix Metalloproteases (MMPs)

dependent upon bound Ca2+ or Zn2+ for activity.
family of enzymes
- MMP-2 (Gelatinase A, 72 kDa type IV collagenase) is the most widely distributed
collagenases can specifically cleave proteins at a small number of sites.
inhibited by tissue inhibitors of metalloproteases (TIMPs).
- MMP-2 appears to be associated with early breast carcinoma and cervical neoplasia

Serine Proteases

have a highly reactive serine in their active site.
inhibited by serpins.
role in metastasis

Links: SOMS Ocular Immunology Group | Expression of MMPs and TIMPs in breast tumours

ECM Scaffold - Tissue Engineering

“Decellularized tissues and organs have been successfully used in a variety of tissue engineering/regenerative medicine applications, …Each of these treatments affect the biochemical composition, tissue ultrastructure, and mechanical behavior of the remaining extracellular matrix (ECM) scaffold, which in turn, affect the host response to the material.”

Reference: Decellularization of tissues and organs. Biomaterials. 2006 Jul;27(19):3675-83. Epub 2006 Mar 7.

References

Textbooks

Essential Cell Biology

Essential Cell Biology Chapter 19 p594-604

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 the Cell 4th ed. - V. Cells in Their Social Context Chapter 19. Cell Junctions, Cell Adhesion, and the Extracellular Matrix
The Extracellular Matrix of Animals
Figure 19-53. The structure of a fibronectin dimer
Figure 19-54. Coalignment of extracellular fibronectin fibrils and intracellular actin filament bundles
Figure 19-57. The structure of laminin
Figure 19-58. A model of the molecular structure of a basal lamina
Figure 19-60. Regeneration experiments demonstrating the special character of the junctional basal lamina at a neuromuscular junction

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 22. Integrating Cells into Tissues
Cell-Matrix Adhesion
Figure 22-22. Structure of fibronectin chains

The Cell- A Molecular Approach

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

The Cell- A Molecular Approach
The Cell - A Molecular Approach - III. Cell Structure and Function Chapter 12. The Cell Surface
The Extracellular Matrix

Essentials of Glycobiology, 2nd ed.

Varki, A.; Cummings, R.D.; Esko, J.D.; Freeze, H.H.; Stanley, P.; Bertozzi, C.R.; Hart, G.W.; Etzler, M.E., editors Plainview (NY): Cold Spring Harbor Laboratory Press; 2008

Search Online Textbooks

"extracellular matrix" Molecular Biology of the Cell | Molecular Cell Biology | The Cell- A molecular Approach | Bookshelf

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

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

"basement membrane" Molecular Biology of the Cell | Molecular Cell Biology | The Cell- A molecular Approach | Bookshelf

"basal lamina" Molecular Biology of the Cell | Molecular Cell Biology | The Cell- A molecular Approach | Bookshelf

Books

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

"extracellular matrix" Entrez all databases
"fibronectin" Entrez all databases
"laminin" Entrez all databases
"basal lamina" Entrez all databases
"basement membrane" Entrez all databases
"matrix metalloprotease" Entrez all databases
"serine proteases" Entrez all databases
- note the spelling differences when carrying out other related ECM searches: UK sulphate, US sulfate ; UK fibre, US fiber

Reviews

Hay ED. The extracellular matrix in development and regeneration. An interview with Elizabeth D. Hay. Int J Dev Biol. 2004;48(8-9):687-94. No abstract available. PMID: 15558460