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The Extracellular matrix
1. THE EXTRACELLULAR
MATRIX
• What is ECM and where can you find it?
• Building blocks of ECM:
(25% of total protein in your body is collagen)
• More ECM components:
(Laminin, Fibronectin etc. heparan-sulphate proteoglycan)
• ECM functions:
(it is not only to keep cells in place)
• Cell-matrix interactions.
(Integrins)
2. EXTRACELLULAR MATRIX
• In biology, the extracellular matrix (ECM) is a
collection of extracellular molecules secreted by
cells that provide structural and biochemical
support to the surrounding cells.
• The extracellular matrix (ECM) is the non-cellular
component present within all tissues and organs,
and provides not only essential physical
scaffolding for the cellular constituents but also
initiates crucial biochemical and biomechanical
cues that are required for tissue morphogenesis,
differentiation and homeostasis.
3. The importance of the ECM is vividly illustrated by the wide
range of syndromes, which can be anything from minor to severe,
that arise from genetic abnormalities in ECM proteins
(Jarvelainen et al., 2009).
•Complex arrangements of molecules filling in spaces between
the cells.
•Not an amorphous jelly or glue but highly organized structure.
•Mostly found in connective tissues, such as tendon, cartilage,
bone or dermis of the skin.
•Diverse structures created by different amounts and organization
of ECM components
•ECM is a local product for local cells. Cells secrete ECM that is
finally assembled outside the cell.
6. SOME FUNCTIONS OF ECM
• Due to its diverse nature and composition, the
ECM can serve many functions, such as:
Providing support
Segregating tissues from one another, &
Regulating intercellular communication.
The extracellular matrix regulates a cell's
dynamic behavior.
7. Although, fundamentally, the ECM is composed of water,
proteins and polysaccharides, each tissue has an ECM with a
unique composition and topology that is generated during tissue
development through a dynamic and reciprocal, biochemical and
biophysical dialogue between the various cellular components
(e.g. epithelial, fibroblast, adipocyte, endothelial elements) and
the evolving cellular and protein micro-environment.
Indeed, the physical, topological, and biochemical composition of
the ECM is not only tissue-specific, but is also markedly
heterogeneous.
Cell adhesion to the ECM is mediated by ECM receptors, such as
integrins, discoidin domain receptors and syndecans (Harburger and
Calderwood, 2009; Humphries et al., 2006; Leitinger and Hohenester,
2007; Xian et al., 2010). Adhesion mediates cytoskeletal coupling to
the ECM and is involved in cell migration through the ECM
(Schmidt and Friedl, 2010).
8. Moreover, the ECM is a highly dynamic structure that is
constantly being remodeled, either enzymatically or non-enzymatically, and its
molecular components are subjected to a myriad of post-translational
modifications.
Through these physical and biochemical characteristics the ECM generates the
biochemical and mechanical properties of each organ, such as its tensile and
compressive strength and elasticity, and also mediates protection by a buffering
action that maintains extracellular homeostasis and water retention.
In addition, the ECM directs essential morphological organization and
physiological function by binding growth factors (GFs) and interacting with
cell-surface receptors to elicit signal transduction and regulate gene
transcription.
The biochemical and biomechanical, protective & organizational properties of
the ECM in a given tissue can vary tremendously from one tissue to another
(e.g. lungs versus skin versus bone) and even within one tissue (e.g. renal
cortex versus renal medulla), as well as from one physiological state to another
(normal versus cancerous).
9. MAJOR TYPES OF ECM MOLECULES
• Glycosaminoglycans: polysaccharide chains usually
found attached to proteins to form proteoglycans
• Fibrillar proteins such as collagens (mainly structural
role) or fibronectin (adhesive glycoprotein)
10.
11.
12. CONNECTIVE TISSUES
• Tissue that connects, supports, binds, or
separates other tissues or organs, typically
having relatively few cells embedded in an
amorphous matrix, often with collagen or
other fibres, and including cartilaginous, fatty,
and elastic tissues.
13. Connective tissue
Connective tissue (CT) is one of the four types of biological tissue in animal body
that supports, connects or separates different types of tissues and organs.
14. Connective tissue
It develops from the mesoderm. The other three types
are epithelial, muscle, and nervous tissue. Connective tissue is
found in between other tissues everywhere in the body, including
the nervous system. In the central nervous system, the three outer
membranes (the meninges) that envelop the brain and spinal
cord are composed of connective tissue.
All connective tissue consists of three main components: fibers
(elastic and collagenous fibers), ground substance and cells. Not all
authorities include blood or lymph as connective tissue because
they lack the fiber component. All are immersed in the body water.
The cells of connective tissue
include fibroblasts, adipocytes, macrophages, mast
cells and leucocytes.
15. Collagen
Collagen is the most abundant protein in our
bodies, especially type 1 collagen. It's found in
muscles, bones, skin, blood vessels, digestive
system and tendons. It's what helps give our skin
strength and elasticity, along with replacing dead
skin cells.
Tropocollagen molecule: procollagens (red, green,
blue) join to form a triple helical tropocollagen.
16. THE COLLAGEN FAMILY
• Triple helical domain
• Repeated Gly - X - Y amino acid sequence,
where X is often proline and Y hydroxyproline
• 19 different collagen types (+ possibly 4 more)
containing polypeptides encoded by at least 38
genes
Proline hydroxyproline
17. COMMON THEMES IN ECM SYNTHESIS
• Extensive post-translational modification
• Route: ER - Golgi - Secretory vesicles
• During this journey protein are glycosylated or decorated with long GAG
chains
• Amino acid recidues can be modified (in collagens proline ->
hydroxyproline)
Chondroitin sulfate nb R1, R2, R3 Hyaluronan (-4GlcUAβ1-3GlcNAcβ1-)n
Glycosaminoglycans[(GAGs) or mucopolysaccharides[ are long
unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating
unit (except for keratan) consists of an amino sugar (N-acetylglucosamine or N-
acetylgalactosamine) along with a uronic sugar (glucuronic acid or iduronic acid)
or galactose. Glycosaminoglycans are highly polar and attract water. They are therefore
useful to the body as a lubricant or as a shock absorber.
18. Aggrecan (ACAN), also known as cartilage-specific proteoglycan
core protein (CSPCP) or chondroitin sulfate proteoglycan 1, is
a protein that in humans is encoded by the ACAN gene.
This gene is a member of the lectican(chondroitin sulfate
proteoglycan) family. The encoded protein is an integral part of
the extracellular matrix in cartilaginous tissue and it withstands
compression in cartilage.
Aggrecan (ACAN)
19.
20. T cells may recognize these
complexes using their
T cell receptors (TCRs).
These cells process antigens
and present them to T-cells.
Almost all cell types can
serve as some form of APC.
They are found in a variety
of tissue types.
21. Non-collagenous domains
• Triple-helical collagen rods are not the only functional
domains
• Example: Type XVIII collagen that is found in many
tissues is associated with basal lamina.
– Endostatin is a 22kDa polypeptide that is proteolytically
cleaved from the C-terminus of type XVIII collagen
– Endostatin found in blood vessel walls and basement
membranes
– Endostatin is a potent inhibitor of angiogenesis and tumour
growth !!!
– Endostatin is currently being tested in clinical trials
– Similar domains in other collagen family members
22. Collagens in disease
• Inherited diseases with mutations in collagen
genes
• Osteogenesis Imperfecta (Osteogenesis imperfecta (OI), also
known as brittle bone disease, is a group of genetic
disorders that mainly affect the bones. It results in bones
that break easily. The severity may be mild to
severe.[1] Other symptoms may include a blue tinge to
the whites of the eye, short height, loose joints, hearing loss,
breathing problems, and problems with the teeth
• Fibrotic diseases with accumulation of ECM
• Liver Chirrosis
• Lung Fibrosis
23. Collagens in disease
• Osteogenesis Imperfecta - Brittle bone disease (not to
be confused with osteoporosis)
• Variable from mild to embryonic lethal
• Often a point mutation in one of type I collagen genes
can cause disease
• Glycine substitutions to another amino acid more
severe than mutations of X or Y in Gly - X - Y triplet.
• Dominant negative effect of some mutations.
• Predisposing mutations (e.g. Type II collagen in
osteoarthrosis)
24. Collagens in disease
• Fibrotic diseases such as liver cirrhosis are characterized by accumulation
of ECM
• Collagen synthesis is mainly regulated by the level of gene activity.
• Some growth factors such as TGF-b signal to increase collagen synthesis.
• Enzymes in the collagen synthesis are investigated as drug targets to treat
fibrotic diseases
TGFα is upregulated in some human cancers. It is produced
in macrophages, brain cells, and keratinocytes, and
induces epithelial development.
TGFβ exists in three known subtypes in humans, TGFβ1, TGFβ2, and TGFβ3.
These are upregulated in Marfan's syndrome and some human cancers, and
play crucial roles in tissue regeneration, cell differentiation, embryonic
development, and regulation of the immune system. Isoforms of transforming
growth factor-beta (TGF-β1) are also thought to be involved in the
pathogenesis of pre-eclampsia. TGFβ receptors are single
pass serine/threonine kinase receptors.
25. Collagens:
• All collagens contain a repeating Gly-X-Y
sequence and fold into a characteristic triple-
helical structure
• Collagens assemble to fibrils or networks
• Procollagen chains are modified in ER where they
also assemble into a triple helix
• Type I collagen is the most abundant type; it is a
major structural protein of bone, tendon and
dermis
• Mutations in collagen chains can render the fibrils
unstable
26. Fibronectin
• Large extracellular glycoprotein
• Name = fibro + nectere (to bind)
• Multiple domains with different binding sites for other ECM proteins or for
receptors on cell surface
• Present in tissues and in blood plasma
Fibronectin is a high-molecular weight (~440kDa) glycoprotein of
the extracellular matrix that binds to membrane-spanning receptor
proteins called integrins. Similar to integrins, fibronectin binds
extracellular matrix components such as collagen, fibrin, and
heparan sulfate proteoglycans (e.g. syndecans).
27. Fibronectin is essential for embryonic
development
• Gene targeting => complete lack of fibronectin
Embryonic lethal.
• Gross malformations, notochord and somites* missing, heart
malformation
• Problems in cell adhesion, migration and differentiation
• *A somite is a division of the body of an animal or
embryo. Somites are bilaterally paired blocks of paraxial
mesoderm that form along the head-to-tail axis of the
developing embryo in segmented animals.
28. Proteoglycan biosynthesis
• Signal peptide directs the nascent polypeptide to ER
• Protein modifications starts in late ER. GAG side
chains elongation and modification takes place in
Golgi.
• Several specific enzymes to add disaccharide units and
to modify them (e.g. sulphation). For example over 30
enzymes are needed in synthesis of aggrecan, a
cartilage matrix proteoglycan.
29. Syndecans and glypicans
• Syndecans are transmembrane proteins. Four family
members. Short cytoplasmic tail contains highly
conserved sequences that bind to adaptor proteins.
Variable part of syndecan-4 cytoplasmic domain binds
protein kinase C and affect cell signalling
• Glypicans (6 known family members) are lipid
anchored to plasma membrane. GPI=
glycosylphosphatidylinositol.
• Both families: individual family members have distinct
expression patterns: e.g. syndecan-1 in epithelia and
syndecan-3 in neural cells
30. Proteoglycans modulate growth factor
activity
• Matrix associated PG: sequestration
• Membrane-bound PG: presentation
• Certain sugar sequences promote FGF signalling and others
inhibit
• Membrane-bound PGs can be cleaved from cell surface into
matrix
• Sugar chains can be cleaved by heparanase enzymes to
oligosaccharides.
Fibroblast growth factors, or
FGFs, are a family of growth
factors, with members involved
in angiogenesis, wound
healing, embryonic
development and various
endocrine signaling pathways.
31. Heparin binding proteins
• Certain growth factors, especially Fibroblast
Growth Factor family (FGF)
• Enzymes and their inhibitors, e.g. proteases
• Blood coagulation factors
• ECM proteins
• Note: Proteoglycans can bind several
proteins at the same time
32. More functions for proteoglycans:
- syndecan-3 regulates appetite
• Serendipitous discovery in transgenic mice over-
expressing syndecan-1 under a viral promoter =>
maturity-onset obesity.
• Heparan-sulphate sugar chains potentiate signalling in
hypothalamus that induces over-eating.
• In normal mice syndecan-3 is present in hypothalamus
(in addition to other neural tissues).
• Food deprivation induces syndecan-3 expression
several fold and triggers reflex hyperphagia.
33. Aggrecan: Example of Matrix
Proteoglycans
• Aggrecan (ACAN), also
known as cartilage-
specific proteoglycan core
protein (CSPCP) or
chondroitin sulfate
proteoglycan 1, is a
protein that in humans is
encoded by the ACAN
gene. This gene is a
member of the lectican
(chondroitin sulfate
proteoglycan) family.
34.
35.
36. Aggrecan is one of the major macromolecules in the
extracellular matrix of articular cartilage and endows the tissue
with its characteristic water imbibing properties and its ability to
undergo reversible compression.
During normal aging of cartilage, Aggrecan undergoes many
posttranslational modifications to its protein core and to the
number, size, and proportion of the chondroitin sulfate and
keratin sulfate chains that are covalently associated with it.
These events are mostly catabolic in nature and are a
consequence of the relatively long resident time of aggrecan in
the matrix.
However, it is clear that a number of these age-related changes
could only have come about by an altered anabolic response of
the chondrocyte
37. Proteoglycans in human diseases
Proteoglycans are a major component of the animal extracellular matrix, the
"filler" substance existing between cells in an organism. Here they form large
complexes, both to other proteoglycans, to hyaluronan, and to fibrous matrix
proteins, such as collagen. The combination of proteoglycans and collagen
form cartilage, a sturdy tissue that is usually heavily hydrated (mostly due to the
negatively charged sulfates in the glycosaminoglycan chains of the
proteoglycans).[5] They are also involved in binding cations (such
as sodium, potassium and calcium) and water, and also regulating the movement
of molecules through the matrix. Evidence also shows they can affect the activity
and stability of proteins and signalling molecules within the matrix.[citation
needed] Individual functions of proteoglycans can be attributed to either the
protein core or the attached GAG chain. They can also serve as lubricants.
An inability to break down proteoglycans is characteristic of a group of genetic
disorders, called mucopolysaccharidoses. The inactivity of
specific lysosomal enzymes that normally degrade glycosaminoglycans leads to
the accumulation of proteoglycans within cells. This leads to a variety of disease
symptoms, depending upon the type of proteoglycan that is not degraded.
38. Hyaluronic acid
Hyaluronic acid, also called hyaluronan, is an anionic, nonsulfated
glycosaminoglycan distributed widely throughout connective,
epithelial, and neural tissues.
Hyaluronic acid (HA; conjugate base hyaluronate), also
called hyaluronan, is an anionic, nonsulfated glycosamino-
glycan distributed widely throughout connective, epithelial,
and neural tissues. It is unique among glycosaminoglycans in that
it is nonsulfated, forms in the plasma membrane instead of the Golgi
apparatus, and can be very large, with its molecular weight often
reaching the millions.
One of the chief components of the extracellular matrix, hyaluronan
contributes significantly to cell proliferation and migration, and may
also be involved in the progression of some malignant tumors
39.
40. Hyaluronic acid
Hyaluronic Acid is a
glucosaminoglycan consisting of D-
glucuronic acid and N-acetyl-D-
glucosamine disaccharide units
that is a component of connective
tissue, skin, vitreous humour,
umbilical cord, synovial fluid and
the capsule of certain
microorganisms contributing to
adhesion, elasticity, and viscosity
of extracellular substances.
41. CD44*
• Adhesive glycoprotein
• Numerous isoforms from alternative splicing
• Originally found as a ‘homing receptor’ in T-lymphocytes
• Some splice isoforms are suggested to play a role in tumour
metastasis
• Cytoplasmic tail of CD44 binds to ERM proteins (ezrin-radixin-
moiesin family) that can regulate dynamics of actin cytoskeleton
• *Hyaluronic acid (HA), an important component of the extracellular
matrix (ECM), is the principal, but by no means the only, ligand
of CD44. Other CD44 ligands include the ECM components collagen,
fibronectin, laminin, and chondroitin sulfate.
42. Basement membrane
• Also known as basal lamina
• Thin sheet-like network
• Epithelial, endothelial, muscle and Schwann
cells
• Physical support, developmental control,
filtering functions
• Major constituents: laminins, collagen type IV,
perlecan (a proteoglycan)
43. Laminins
• The laminin molecules are named according to their chain
composition. Thus,laminin-511 contains α5, β1, and γ1
chains. ... The trimeric proteins intersect to form a cross-
like structure that can bind to other cell membrane and
extracellular matrix molecules. Molecular composition of
basement membranes is tissue-specific
• Laminins: at least 11 heterotrimers
– Five alternative alpha chains,
– Three alternative beta chains
– Two alternative gamma chains
– For example: in skin in the BM between epidermis and dermis,
Laminin-5 (a3b3g2) is the predominant laminin isoform.
44. Structure and function of laminin: anatomy of a
multidomain glycoprotein
Laminin is a large (900 kDa) mosaic protein composed of many distinct
domains with different structures and functions. Globular and rodlike domains
are arranged in an extended four-armed, cruciform shape that is well suited for
mediating between distant sites on cells and other components of the
extracellular matrix. The alpha-helical coiled-coil domain of the long arm is
involved in the specific assembly of the three chains (A, B1, B2, and possible
variants) of laminin and is the only domain composed of multiple chains.
H-D-Tyr-Ile-Gly-Ser-Arg-OH
Chain, Laminin M
Glycoprotein GP 2
Glycoprotein GP-2
Laminin
Laminin M
Laminin M Chain
M Chain, Laminin
Merosin
45. Laminin
Laminin has active domains for collagen
binding, cell adhesion, heparin binding,
and neurite outgrowth (PHD)
fragment.1Laminin chains are
designated A (molecular weight
400kDa), B1 (molecular weight 210 kDa)
and B2 (molecular weight 200 kDa). The
cohesion between these chains is the
result of many inter- and intrachain
disulfide bonds. Together, they cause
the molecule to look like a crucifix.
Laminin is a ubiquitous non-collagenous
connective tissue glycoprotein that is a
major constituent of basement
membranes.
46. Name Source
Storage
Temp
Target Cells
For
Attachment
Concentration
For Use
Cat. No.
Laminin,
aqueous
solution
from
Engelbreth-
Holm-Swarm
murine
sarcoma
basement
membrane
−20°C
Epithelial
cells,
endothelial
cells, muscle
cells, tumor
cells,
hepatocytes,
Schwannoma
1 - 2 μg/cm2 L2020-1MG
Laminin, liquid
from human
placenta
−70°C
Epithelial
cells,
endothelial
cells, muscle
cells, tumor
cells,
hepatocytes,
Schwannoma
1 - 2 μg/cm2 L6274-.5MG
48. More Basal Lamina Proteins
• Perlecan: a large heparan sulfate proteoglycan.
HS chains bind other BM components and
contribute to filtering functions
• Entactin: interacts with laminin and type IV
collagen
• Nidogen, a laminin binding protein
49. Hemidesmosome: a cell - basement
membrane adhesion site
• In some epithelia: epidermis, bladder, trachea,
breast and amnion
• Shares some ultrastructural features with
desmosomes: both display dense, membrane-
associated cytoplasmic plaques that are connected
to intermediate fialments. But molecular
composition is different.
• Transmembrane glycoproteins connect basement
membrane to intracellular plaque
50. Basal lamina functions
-developmental guidance
• Early embryo: keeps 4 and 8 cell stages together
• Differentiation of epithelial organs; epithelial -
mesenchymal interactions
• Neurite outgrowth: guidance of axon growth by ECM
containing laminin subunits
• (Neurite Outgrowth is a process wherein developing neurons
produce new projections as they grow in response to
guidance cues. ... Dynamic neurite outgrowth during
development results in the formation of a complex neuronal
architecture that results in the establishment of the functional
nervous system and brain)
51. ECM Turnover - MMPs
• Matrix metalloproteinases are enzymes that
cleave components of ECM
• Over 20 different enzymes with different
specifications.
• Common theme: expressed as an inactive pro-
enzyme
• Also other substrates than ECM proteins
• TIMPs = tissue inhibitors of MMPs
52. MMPs - some examples
• “Old names” collagenases, gelatinases and
stromelysin replaced by numbers (e.g. MMP-
1)
• MMP-1 (collagenase-1) cuts triple helical
collagens
• MMP-9, (Gelatinase-B) chops e.g. type IV
collagen and laminins
• MT-MMPs are membrane-bound enzymes
53. MMPs - some functions
• Regulate amount of ECM - degradation and
remodelling
• Cell migration, wound healing, angiogenesis*
• Activate other MMPs
• Release or activate growth factors and other bioactive
molecules
• *Angiogenesis is the formation of new blood vessels. This process involves
the migration, growth, and differentiation of endothelial cells, which line
the inside wall of blood vessels. The process of angiogenesis is controlled
by chemical signals in the body.
• (Angiogenesis is an important process that occurs both during health and disease.
Blood is important in the body as it carries oxygen and nutrients to all the parts of
the body via blood vessels like arteries and brings back the toxins and wastes from
these peripheral organs for purification via veins)
54. MMPs in diseases
• Extensive matrix degradation e.g. in
periodontitis*, rheumatoid arthritis
• Tumour cell invasion and metastasis:
– Carcinoma breaks basement membrane and
invades surrounding stroma.
• MMP inhibitors tested for therapeutic use
*Periodontitis (per-e-o-don-TIE-tis) is a serious gum
infection that damages the soft tissue and destroys the
bone that supports your teeth
55. Integrins
• At least 24 different heterodimers from 9 b subunits and 18 a
subunits.
• Variable pairing: b1 integrin can have 11 different a partners.
• Overlapping ECM binding: e.g. 8 different integrins can bind
fibronectin
• An integrin can bind one or several ECM proteins
Integrins are proteins that function mechanically, by attaching
the cell cytoskeleton to the extracellular matrix (ECM), and
biochemically, by sensing whether adhesion has occurred.
The integrin family of proteins consists of alpha and beta
subtypes, which form transmembrane heterodimers.
57. Integrins and cell behaviour
• Clustering of integrins (“velcro effect” in adhesion)
• Responses to cell adhesion include spreading,
cytoskeletal re-organisation, polarisation, migration,
proliferation, activation of specific genes
• Cell survival: epithelial cells that are detached commit
suicide (this type of apoptosis* is called anoikis).
• Also, inside out signalling: integrins can have inactive
conformation that does not bind matrix unless first
activated.
*the death of cells which occurs as a normal and controlled part of an
organism's growth or development.
Velcro effect: The Velcro Effect is when words and images are associated
with sensation and energy in the body.
58. SUMMARY
• Collagens: Triple helical rod and non-collagenous domains.
Important structural role. Extensive post-translational
modifications
• Fibronectin: adhesive glycoprotein in matrix and plasma
• Proteoglycans: GAG-chains attached to core protein.
• Laminins: major components of basement membranes
• Matrix metalloproteinases degrade and re-model matrix
• Integrins: heterodimeric proteins that mediate cell adhesion
to extracellular matrix.
• Cell-matrix interactions important regulator of cell
behaviour
59. Integrins - variety in functions
Integrins
Basal lamina functions
-developmental guidance
Basal lamina functions
-filter
Basal lamina functions-
structural support
Hemidesmosomes and basement membrane-
molecular composition
Hemidesmosomes and basement membrane-
ultrastructural view
Type IV collagen
Interactions of laminins
Laminins
Basement membrane