Talk:Extracellular Matrix 2

From CellBiology

2017

An organizing function of basement membranes in the developing nervous system

Mech Dev. 2014 Aug;133:1-10. doi: 10.1016/j.mod.2014.07.003. Epub 2014 Jul 22.

Halfter W, Yip J

Abstract

The basement membranes (BMs) of the nervous system include (a) the pial BM that surrounds the entire CNS, (b) the BMs that outline the vascular system of the CNS and PNS and (c) the BMs that are associated with Schwann cells. We previously found that isolated BMs are bi-functionally organized, whereby the two surfaces have different compositional, biomechanical and cell adhesion properties. To find out whether the bi-functional nature of BMs has an instructive function in organizing the tissue architecture of the developing nervous system, segments of human BMs were inserted into (a) the parasomitic mesoderm of chick embryos, intersecting with the pathways of axons and neural crest cells, or (b) into the midline of the embryonic chick spinal cord. The implanted BMs integrated into the embryonic tissues within 24h and were impenetrable to growing axons and migrating neural crests cells. Host axons and neural crest cells contacted the epithelial side but avoided the stromal side of the implanted BM. When the BMs were inserted into the spinal cord, neurons, glia cells and axons assembled at the epithelial side of the implanted BMs, while a connective tissue layer formed at the stromal side, resembling the tissue architecture of the spinal cord at the pial surface. Since the spinal cord is a-vascular at the time of BM implantation, we propose that the bi-functional nature of BMs has the function of segregating epithelial and connective cells into two adjacent compartments and participates in establishing the tissue architecture at the pial surface of the CNS. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved. KEYWORDS: Basement membrane; Collagen IV; Extracellular matrix; Extracellular matrix proteins; Laminin


PMID 25058486 DOI: 10.1016/j.mod.2014.07.003


Proteoglycans

MBoC - Proteoglycans in the extracellular matrix of rat cartilage MBoC - Examples of a small (decorin) and a large (aggrecan) proteoglycan found in the extracellular matrix

  • consist of protein (~5%) and polysaccharide chain (~95%)
  • form a gel to embed the fibril network
  • Golgi apparatus - GAG disaccharides are added to protein cores to form proteoglycans
  • 10% by weight but fill most of space
  • unbranched polysaccharide chains
  • disaccharide subunits
  • amino sugar

Glycosaminoglycans (Gags)

Five types

  • Hyaluronan (or hyaluronic acid) main glycosaminoglycan in connective tissue
  • high molecular weight (~ MW 1,000,000 )
  • length of about 2.5 µm hyaluronan
  • "backbone" for the assembly of other glycosaminoglycans
    • Hyaluronan is also a major component of the synovial fluid, which fills joint cavities, and the vitreous body of the eye.
  • Other 4 major glycosaminoglycans
    • chondroitin sulphate, dermatan sulphate, keratan sulphate and heparan sulphate (UK sulphate, US sulfate)
    • attach through core and link proteins to hyaluronic acid backbone

Proteoglycan Function

  • trap water
  • resistant to compression
  • return to original shape
  • occupy space
  • link to collagen fibers
  • form network
    • in bone combined with calcium hydroxyapatite, calcium carbonate

Development

  • produce a “cell-free” space
  • for cell proliferation and migration into

Adult

  • in areas of compression
  • tissues, joints

hyaluronan-binding proteins

Hyaluronan Synthesis

  • differs from other GAG synthesis
    • synthesized at plasma membranes
    • nascent chains directly extruded into ECM

Cell adhesion

embryonic migration

Proteoglycan- Disease

  • Mucopolysaccharidosis type I (MPS I) - Hurler disease
    • deficiency of alpha-L iduronidase => lysosomal storage disease, associated with an altered elastic matrix
    • excess heparan sulphate and dermatan sulphate
  • Cancer development
    • altered types and kinds of proteoglycans formed by cells
    • normal cells -> malignant
  • Arthritis
    • Cartilage breakdown (cartilage erosion)
    • chondrocytes elicit a catabolic response which exceeds anabolism of new matrix molecules
    • Degrade proteoglycan (aggrecan)
    • Also a mouse model generates antibodies to proteogycan


This next section will be covered in the second lecture.

Fibronectin

Fibronectin binding domains

Fibronectin Structure

  • dimer connected at C-terminus
    • 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


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




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

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.


2015 Course Content

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cytoskeleton Introduction | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Cytoskeleton - Intermediate Filaments | Cell Mitochondria | Cell Junctions | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | Development | 2015 Revision


Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Cell Knockout Methods | Cytoskeleton Exercise | Immunochemistry | Project Work | Confocal Microscopy | Tissue Culture | Stem Cells Lab | Microarray Visit

2015 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7

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