7-11: Connective Tissues

Question 1

What actually is the ECM?

Answer 1

The Extracellular Matrix is a dynamic, multiprotein polymer, which makes up the majority of our organs and tissues

Question 2

Name 2 key (general) functions of the ECM

Answer 2

1. Organisation: Cells within organised tissue compartments have different functions, and are ORGANISED BY THE ECM
2. Tissue Strength: Cells are linked to the ECM to resist mechanical forces of tension + compression

Question 3

What is the significance of collagen (and its different types) in the body?

Answer 3

It is the most abundant protein in the body and plays a key role in holding tissues together

There are 28 types (though more than 80% is Col1), which form different types of ECM structure and determine its properties

Question 4

What causes Scurvy?

Answer 4

A lack of vitamin C in the diet:

Vitamin C is a cofactor for the enzymes Proline Hydroxlase and Lysyl Hydroxylase, both of which form intermolecular H bonds between collagen chains

Lack of Vitamin C hinders the formation of stable collagen fibres

Question 5

State the steps of Fibrillar Collagen Formation

Answer 5

1. Pro-alpha collagen chain synthesised as a single polypeptide and co-translationally imported into ER
2. Modifications of amino acids (especially Hydroxylation of Pro and Lys, and Glycosylation of Hydroxylysines)
3. Three Pro-alpha chains assemble into a triple helix - driven by C-terminal, non-collagenous domains that lack the Gly-X-Y repeat
4. Collagen enters a secretory vesicle; N and C domains are cleaved to leave just the triple-helical collagen molecule
5. Collagen triple-helices self-assemble into fibrils outside the cell

Question 6

What is the Repeating Sequence found in collagen, and what is the significance of this repeat for the function of collagen?

Answer 6

A repeating, Proline-rich sequence, defined by the Gly-X-Y repeat (either Gly-Pro-Y or Gly-X-Hydroxyproline)

• Proline has a cyclical side chain which restricts rotation in the polypeptide backbone
• Glycine is very flexible (as its R group is just H)
• Glycine wraps around, Pro forces it into a STABLE HELIX STRUCTURE

Question 7

What is the Basement Membrane?

Answer 7

The BM is a specialised form of ECM, containing Col4, that organises cell layers in metazoans

Question 8

Give 5 examples of tissues where an important basement membrane separates different cell layers

Answer 8

1. Kidney (GBM)
2. Muscles
3. Blood Vessels (Muscle/Epithelium)
4. Epithelium (either at Lumen or External Surface)
5. Neuromuscular Synapse

Question 9

Describe how the structure of Col4 differs from Col1, and the consequences of this for the polymers it forms

Answer 9

• In Col4, the N and C domains (i.e. non-collagenous regions) are NOT cleaved
• C domain drives triple-helix formation via end-end interactions, and drives hexamer (“dimer of trimer”) formation
• N domain cross-links 4 triple-helices to form tetramers
• Interruptions within the Gly-X-Y repeats of the coiled-coil allows increased flexibility and sites for cross-linking

Overall, these interactions drive self-assembly of Col4 into a 2D mat, rather than a 3D network like Col1

Question 10

How many heterotrimers of Col4 are there (and what drives this specificity)?

Answer 10

Only 3 heterotrimers form in practice:
a1a1a2
a3a4a5
a5a5a6

Even though 6 genes for Type 4 Collagen gives 56 theoretical combinations

The specificity of NC1 interactions drives specificity of trimer and hexamer formation

Also specificity BETWEEN trimers too (i.e. a1a1a2 will ONLY tetramerise with a1a1a2)

Question 11

Where are the different isoforms of Col4 found

Answer 11

Most BMs contain a1a1a2, including Bowman’s Capsule

The GBM also contains a1a1a2 in development, but in adults forms a THICK BM containing a3a4a5 to act as a filtration barrier between endothelial cells and podocytes

Not sure for a5a5a6

Question 12

What is Alport’s Syndrome and what causes it?

Answer 12

An X-linked Nephritis disease due to mutations in COL4A5 (or also COL4A3/4)

Can’t properly form a3a4a5 collagen, so a1a1a2 still expressed in GBM in adults, which forms less inter/intra-chain crosslinks and is thus less protected from physical pressure and proteolysis

-> Gradual degradation of GBM leads to disease

Question 13

What factors can affect the severity and onset of Alport’s Syndrome?

Answer 13

• Much more severe in Hemizygous males than heterozygous females (because X-linked)
• Most severe disease caused by large gene rearrangements, nonsense mutations and splice-site-induced truncations
• Later onset disease caused by Gly-X-Y missense mutations, or conserved Cys residues that affect crosslinking

Question 14

How are knock-out or knock-in mice created for transgenic model organism experiments?

Answer 14

Introduce DNA into embryonic stem cells, inject these ESCs into an embryo (-> hybrid embryo), implant this embryo back into a mouse, SOME tissues in the offspring will have the transgene
Identify which offspring have the mutation in their germ cells -> breed these

(Also these days CRISPR allows more specific editing, e.g. mimic 1-AA mutations)

Question 15

What happens if we knock out COL4A3 in a mouse, and what is the use of this?

Answer 15

Progressive kidney failure, or “glomerulopathy” (as no longer correct Col4 isoform in GBM) - phenocopies Alport’s in humans

Can use these mice to test treatments, e.g., ACE inhibitors

Question 16

Are ACE inhibitors and Gene Therapy effective in treating mice that phenocopy Alport’s syndrome?

Answer 16

They seem to extend the lifespan, as they reduce blood pressure on the GBM, so it takes longer for collagen to wear down -> this could extend the time to find a suitable organ donor

There is also some evidence that Gene Therapy to rescue COL4A3 in mice causes repair and return to function of the defective GBM

Question 17

What is Laminin?

Answer 17

Laminin is a high-MW glycoprotein with a cruciform structure, and is the second most abundant BM protein

Question 18

Describe the structure of Laminin

Answer 18

Laminin is made up of three chains assembled into a cruciform structure

The long arm is a coiled-coil a-helix of the three chains, consisting of a 7-AA repeated sequence - these 7 include hydrophobic, hydrophilic, positive and negative AAs, allowing non-covalent interactions

The three N-terminal globular domains interact with other laminins, and with accessory proteins such as nidogen and perlecan - this promotes polymerisation into a 2D network (self assembly like collagen)

The C-terminal globular domain consists of 5 LG (laminin G-like) domains that interact with cell surface receptors: LG1-3 interact with integrins, while LG4-5 interact with Dystroglycan and Heparin

Small note: while electrostatic and hydrophobic interactions stabilise the coiled-coil, each end is stabilised by DISULPHIDE CROSS-LINKS

Question 19

How many laminin trimers are there (and how does this compare to Col4)?

Answer 19

There are 15 heterotrimeric combinations of Laminin

A bit like Col4, there are 11 laminin genes, but interaction specificity means only 15 combinations

However, there is MORE tissue-specific variation than in col4 (as Col4 is almost all in the BM whereas laminins are found more widely)

SOME laminins are ubiquitous and essential (e.g., a1ß1y1), while others are tissue specific (e.g. a2 KO = MD, a3 KO = blistering of stratified epithelium)

Question 20

What happens when the Laminin ß2 isoform is lost?

Answer 20

PIERSON SYNDROME - a rare lethal condition similar to the loss of GBM Col4 isoforms

Laminin11 (a5ß2y1) is expressed in GBM, eye, and synaptic BM, resulting in phenotype of nephrotic syndrome, eye abnormalities, AND muscular hypotonia

Question 21

Which Laminin isoform is linked to a form of Epidermolysis Bullosa, and how/why?

Answer 21

Laminin 5 (a3ß3y2) - it links the epidermis to the dermis:

Lam5 links integrins (a6ß4) in the epidermis to the underlying Col4 in the BM, which is anchored by Col7 to the Col1 network in the dermis

Mutations in any of LAMA3, LAMB3 or LAMC2 cause Junctional Epidermolysis Bullosa (JEB), either Herlitz-type (lethal due to complete loss) or Non-Herlitz type (milder due to limited Lam5 function)

Question 22

Can Gene Therapy be used to treat Non-Herlitz JEB?

Answer 22

YES - and here is how/why:

• A modified retrovirus is used to deliver a functional copy of (for example) LAMB3 to the patient’s UNaffected keratinocytes
• Grow them in culture, then graft this transgenic epidermis onto the patient
• Eventually the holoclones (containing proliferative stem cells) will make up the majority of the patient’s skin

Question 23

What are the limitations of Gene Therapy for treating BM-related conditions

Answer 23

Limitations on gene size when using retroviruses to deliver (e.g., laminin gene is small enough to use, but dystrophin is NOT)

Also potential issues when randomly altering the genome of a cell that is being grafted back onto the patient

Question 24

What do the cytoplasmic domains of integrins interact with?

Answer 24

MOST (e.g., in adherens junctions and FAs) interact with actin

BUT a6ß4 in hemidesmosomes link to Keratin Intermediate Filaments

Question 25

Describe the tissue-specific distribution and effects of tissue-specific deletion of key ß integrin isoforms

Answer 25

ß4 = HEMIDESMOSOMES IN STRATIFIED EPITHELIUM (between epidermis and BM) - deletion leads to detachment of epidermis from BM, like Lam5 deletions ß1 = Widely expressed - deletion is lethal ß2+3 = Blood cells - deletion affects clotting + inflammation

Question 26

If integrin ß4 is deleted, why does the epidermis lift off as 1 sheet instead of simply falling apart?

Answer 26

Because ß4 deletion results in loss of hemidesmosomes, but not cell-cell adhesions (AJs) that link to IFs

Question 27

What is PA-JEB and what causes it?

Answer 27

[Junctional Epidermolysis Bullosa with Pyloric Atresia] is a rare, fatal, AutRecessive condition caused by the loss of a6ß4 integrin It causes neonatal blistering and GI obstruction through loss of GI, Genitourinary, and Respiratory Epithelium

Question 28

Why is the loss of integrin (e.g., in PA-JEB) more severe than the loss of Lam5 (in Herlitz or non-Herlitz JEB)?

Answer 28

Integrin ß4 is more widely expressed in tissues, and interacts with multiple Laminins Laminin 5 is more specific in its integrin interactions, so its deletion has less widespread effects

Question 29

Does loss of integrin ß4 gene also result in loss of a6 integrin expression?

Answer 29

YES - similar to Laminins and Col4, integrin interactions are specific

Question 30

Why can BM formation still be seen when integrin ß is lost?

Answer 30

Lam 5 can be seen forming a continuous membrane, as the BM still forms, but just can't attach to the layer underneath due to the lack of ß4

Question 31

What is the result of a6 integrin deletion?

Answer 31

Same effect as loss of ß4 (in mice and humans)

Question 32

What is EB Simplex and what is it caused by?

Answer 32

It is a milder form of EB, usually restricted to blisters on regions subject to mechanical stress Often due to mutations in components of Keratin5/14 IFs... OR in the cytoplasmic tails of integrin ß4 -> Cells are weakened internally, but the BM is still intact; therefore, cells can rip off but leave others still attached to BM (Can also be caused by mutations in any of the adaptor proteins linking cytoplasmic domain of ß4 with IFs)

Question 33

Describe how the structure of muscles allows contractile sarcomeres to transmit force

Answer 33

- Multinucleate muscle cells contain multiple contractile fibres, each of which is made up of repeating sarcomeres - Around a muscle cell is the PM (sarcolema), and around THAT is the Basement Membrane - When sarcomeres contract, most of the force is transmitted through cell surface receptors to the BM - Muscles attach to tendons (at myotendinous junctions), which then attach to bone, allowing muscles to move the skeleton

Question 34

Describe the specific composition of the BM found in SKELETAL muscle

Answer 34

It contains specific laminin isoforms, mainly Laminin2 (a2b1y1) At neuromuscular junctions specifically, laminins a4b2y1 and a5b2y1 are found (spatial specialisation)

Question 35

What fundamentally causes all muscular dystrophies

Answer 35

Defects in muscle attachment to the basement membrane

Question 36

What are the TWO main ways that the skeletal muscle attaches to the BM?

Answer 36

1. The Dystroglycan Complex 2. ECM-Laminin(a2b1y1)-Integrins

Question 37

Describe the form of Muscle-BM attachment that relies on integrins

Answer 37

The ß1 integrin subunit (unlike ß4 in epidermis) links LamininA2 with the Actin Cytoskeleton of the muscle cell ECM-LamininA2-Integrinß1-Actin

Question 38

Describe the form of muscle-BM attachment that relies on a very large complex

Answer 38

The Dystroglycan complex: ECM - LamininA2 - alpha-Dystroglycan Complex (incl. Sarcoglycan) - Dystrophin - Actin

Question 39

Describe the genetic cause and severity of Musculary Dystrophy

Answer 39

MDs can be caused by disruption of ANY PART of the linkage between BM and cytoskeleton (laminin/integrin - congenital; dystropglycan; dystrophin - Duchenne/Becker; Collagen - Bethlem myopathy) MDs have a range of severity depending on whether there is NO expression or PARTIAL expression E.g., among Dystrophin mutations, Duchenne (no expression) results in death by mid-30s, whereas Becker (reduced expression) can still have a normal lifespan

Question 40

Describe Congenital Muscular Dystrophy - causes, most common cause, severity and symptoms

Answer 40

Caused by a range of mutations affecting the Muscle BM 50% have LamininA2 chain mutations (because laminin a2b1y1), but can also be caused by integrin mutations Complete loss of expression leads to a severe phenotype, whereas partially functional/truncated LamA2 (e.g., due to splice site mutations) show a less severe phenotype [NOTE THE SIMILARITY WITH EB] Symptoms include progressive degeneration of muscles, weakness, PNS and CNS defects

Question 41

Describe how integrin mutations can cause Muscular Dystrophy

Answer 41

Integrin a7b1 is the main laminin-binding integrin on the muscle cell surface Several splice variants are known to exist (A, B and C) - disruption or wrong variant can disrupt link between laminin and costamere (and thus laminin with actomyosin), causing CMD Loss of a7 subunit results in a progressive form of MD (more tissue specific than ß1 so not lethal)

Question 42

What is a costamere?

Answer 42

The protein complex on the cytoplasmic side of a sarcolema (including vinculin, a-actinin, talin, filamin) which links integrins to actin They allow force transmission between neighbouring muscle cells, and between muscle cells and bone

Question 43

What is the most common form of Muscular Dystrophy, how does it progress, and what is the underlying cause?

Answer 43

Duchenne's - caused by complete loss of Dystrophin (This is independent of the a7b1 integrin-mediated attachment of muscles to BM) Symptoms (e.g., muscle wasting, poor balance, cardiomyopathy) first show when child starts walking - wheelchair by teens, death in early 20s

Question 44

What happens if both integrins a7b1 AND dystrophin are knocked out?

Answer 44

The MD phenotype is much more severe than either knockout alone (one linkage can partially compensate for loss of the other, just not completely)

Question 45

Describe Becker Muscular Dystrophy and how it differs from Duchenne's

Answer 45

Caused by *partially* functional Dystrophin Less severe: symptoms first show in teens/20s (difficulty walking, climbing stairs, etc.) Death after 40, though may even have a normal lifespan

Question 46

Is there a cure or therapy for Duchenne Muscular Dystrophy?

Answer 46

There is no known cure, but there is potential for gene therapy. A retroviral approach (as used for Laminin in Non-Herlitz JEB) is NOT possible, as the Dystrophin gene is too big However, using CRISPR (specifically, NHEJ), a reading frame shift in exon 50 can be restored by inserting an extra base pair, thus removing the truncation Alternatively, can use splice sites to skip exon 50 completely (shorter but still functional dystrophin) - use CRISPR to induce random INDELS, some of which will result in functional protein Practically, use AAV and a muscle-specific promoter to deliver CRISPR-Cas9 to cells

Question 47

What is the preferred animal model for human DMD and why?

Answer 47

DOG - a mutation in exon51 in dogs is very similar to common human mutations in exon51 Better than mouse model, as the Mdx mouse has a mutation in exon23 introducing a stop codon - different kind of mutation from that which is commonly found in humans

Question 48

How widely is Dystropglycan expressed?

Answer 48

UBIQUITOUSLY (KO in mice is lethal, and no known human mutations, probably because they are also lethal) However, unclear what its roles are outside of muscle in mice

Question 49

Describe the structure of Dyrstroglycan

Answer 49

It is made as a single (extremely large) polypeptide, then is cleaved into alpha and ß alpha contains N-terminal, Muscin and C-terminal domains, while ß-DG contains the transmembrane domain a-Dystroglycan is heavily glycolsylated (this varies in different tissues)

Question 50

How does Dystroglycan connect to the ECM?

Answer 50

LG4 and LG5 domains of Laminin(a2b2y2) bind to O-glycosylated moieties on dystroglycan Having the RIGHT sugar modifications is key, as removing N-linked does not affect laminin binding, but removing O-linked DOES.

Question 51

What is FCMD and what is the underlying cause?

Answer 51

Fukuyama Congenital Muscular Dystrophy - autosomal recessive form of MD found mainly in Japan, involves severe muscle degeneration and brain abnormalities Caused by a 3kb transposon insertion into the 3'-UTR of the Fukutin gene, resulting in reduced mRNA expression of Fukutin FUKUTIN is involved in Glycosylation of alpha-dystroglycan Therefore, FCMD is associated with aberrant a-DG glycosylation

Question 52

Describe the significance of modification of alpha-dystroglycan as it passes through the ER and Golgi - how can this go wrong?

Answer 52

It MUST BE glycosylated by several enzymes as it passes through - defects in many of them are associated with forms of Muscular Dystrophy: Mutations in POMT1/2, POMGnT1, FKRP, LARGE, etc.

Question 53

What cellular process explains the degenerative component of many Muscular Dystrophies (also why does this occur and how can we demonstrate it)?

Answer 53

Defective control of APOPTOSIS: Normally, when Laminin binds to either receptor (DG or a7-integrin) it promotes activation of Akt, which suppresses pro-apoptotic Bcl2s If this binding cannot occur, less suppression of pro-apoptotic Bcl2 proteins, MORE APOPTOSIS Knocking down Bax, OR overexpressing Bcl-2 in muscles, partially rescues muscle phenotype of Laminin2 deficient mice - it doesn't rescue muscle weakness, but it DOES protect against the degenerative component

Question 54

What is Young's Modulus?

Answer 54

Young's Modulus (E) is a measure of Stiffness or Elasticity (calculated as Stress/Strain)

Question 55

Give some examples of cell behaviours that can be controlled/altered by substrate stiffness

Answer 55

Morphology (flatten), Contractility (increases), Proliferation (increases), Apoptosis (decreases), Differentiation (stiff->stiff, soft->soft), Movement (Durotaxis)

Question 56

Give some examples of key mechanotransduction pathways

Answer 56

1. Focal Adhesions and Cytoskeletal Remodelling 2. Mechanosensitive Ion Channels (e.g., TRPV4) 3. Force transmission to the nucleus + remodelling 4. Mechanosensitive Translocation of TFs

Question 57

Describe the role of Focal Adhesions in Mechanotransduction

Answer 57

Cells can "feel" stiffness by deforming their surroundings They need mechanisms of force generation (actomyosin), transmission (cytoskeleton) and mechanosensing (conversion into signals) Key proteins involved are integrins, actin, myosins, talin (-> MAPK and RhoA)

Question 58

Describe the role of mechanical linkage to the nucleus in mechanotransduction

Answer 58

The LINC Complex (Nesprins and SUN proteins) links the cytoskeleton to the nuclear lamina This allows extracellular force to be transduced to a conformational change in chromatin, affecting gene expression

Question 59

Describe the role of mechanosensitive translocation of TFs in mechanotransduction

Answer 59

Certain transcription factors (e.g., YAP1, which drives osteogenic differentiation) translocate to the nucleus in response to tissue stiffness This kind of transduction allows mechanical regulation of genetic programmes

Question 60

What is fibrosis?

Answer 60

EXCESSIVE MATRIX (due to dysregulation of feedback and loss of homeostasis), making tissues STIFFER so mechanical properties are NO LONGER MATCHED TO FUNCTION

Question 61

What do fibroblasts do?

Answer 61

They are necessary for wound healing - they migrate towards sites of healing and synthesise ECM molecules such as collagen

Question 62

How can fibroblasts be "activated" and what does this mean?

Answer 62

They are activated by mechanical stimulation (e.g., stiff tissue) to become proto-myofibroblasts, then by Transforming Growth Factor ß1 (TGFß1) to become MYOfibroblasts Myofibroblasts are more contractile and secrete more ECM

Question 63

Give two (mentioned) examples of fibrotic diseases

Answer 63

Chronic Obstructive Pulmonary Disease (COPD) and Atherosclerosis

Question 64

How does a Homeostatic response become a Fibrotic Response?

Answer 64

Scarring is part of the healthy tissue response to injury, but dysregulation can lead to TOO MUCH SCARRING, which can disrupt function

Question 65

What is IPF and what are its main symptoms (at the organ and the body level)?

Answer 65

Idiopathic Pulmonary Fibrosis - i.e. pulmonary fibrosis of unknown cause "Honeycomb" areas of fibrosis within lung; enlarged, damaged bronchioles; distorted alveoli Shortness of breath, chronic dry cough, finger clubbing

Question 66

Describe simply how IPF develops

Answer 66

Inflammation and thickening of alveoli walls [DYSREGULATION] -> Loss of gas exchange, Organ Failure

Question 67

What is meant by the "Matrisome" and the "Matrisome Project"?

Answer 67

Matrisome - the complete set of proteins that compose (core matrisome) or associate with the ECM Matrisome project - attempt to build a database of all ECM components using *in silico* predictions and mass spectrometry

Question 68

How are tissues analysed to determine the matrisome?

Answer 68

1. Disrupt and Solubilise as much protein as possible from a tissue 2. Digest the protein into fragments with trypsin 3. Separate by hydrophobicity (LC column) 4. First round of mass spec quantifies the peaks (how much protein is at each peak) 5. Second round of mass spec fragmentises the peaks (to reveal identities)

Question 69

What are the main components of the Core Matrisome?

Answer 69

1. Collagens (e.g., Type 1) - structural component of all CT 2. Proteoglycans (e.g., pelecan, biglycan, lumican) - bind to water, salts and soluble factors; fill space and provide lubrication 3. Glycoproteins (e.g., fibronectin, laminins) - ECM assembly, cell adhesion, signalling, etc.

Question 70

How constant/ubiquitous are the matrisome proteins?

Answer 70

They can vary both spatially and temporally, AND with disease (e.g., healthy vs early cancer vs metastasis) However, there are many common proteins in all situations

Question 71

What 4 main features of the matrix determine its mechanical properties?

Answer 71

1. Identities of matrix proteins 2. Concentration 3. Orientation/Assembly 4. Crosslinks between proteins binds them together and increases tissue stiffness

Question 72

How does concentration affect mechanical properties of the ECM?

Answer 72

Many matrix proteins are biological polymers, and MORE POLYMER = MORE STIFFNESS (a bit like jelly)

Question 73

How does orientation/assembly affect mechanical properties of the matrix?

Answer 73

Collagen fibres are strongest along their length (e.g., in tendons) - orientation determines tissue strength

Question 74

According to the more modern model, what are some of the main cell types found in a tumour?

Answer 74

Cancer Cells (CC) Cancer Stem Cells (CSC) Cancer-Associated Fibroblast (CAF) Immune Inflammatory Cells (ICs) Endothelial Cells (EC) Perictyes (PC)

Question 75

Which Cancer Hallmarks are most strongly influenced by mechanical signals?

Answer 75

- Sustaining Proliferative Signalling - Resisting Apoptosis - Cell movement (Activating Invasion and Metastasis)

Question 76

Give examples of some key Matrix-Regulated pathways that are linked to cancer hallmarks

Answer 76

1. FAK - limits sensitivity to Growth Inhibitors and Apoptotic Signals 2. MAPK and ERK - promote cell Proliferation 3. Vascular Endothelial Growth Factor - induces Angiogenesis 4. Rho/ROCK and Rac - drive invasion and metastasis

Question 77

What is the most significant measurable risk factor for breast cancer apart from age (and explain this factor)?

Answer 77

High Mammographic Density (due to altered matrix) Increased fibrillar collagen and fibre organisation - stiffer tissue!

Question 78

What are some features of the CANCER ECM?

Answer 78

1. Increased matrix production (by fibroblasts) 2. Increased crosslinking (e.g., by Lysyl Oxidase, LOX) 3. Reorganisation of the matrix (by proteases)

Question 79

Describe the role of Fibroblasts in Cancer (CAFs)

Answer 79

Solid tumour formation can be considered a fibrotic process - dysregulation of collagen synthesis by fibroblasts

Question 80

What are the two main limiting factors in metastasis?

Answer 80

Ability to degrade the matrix, and ability to squeeze through small gaps

Question 81

Describe how Squeezing through Gaps is a limiting factor in metastasis and the key protein that affects this?

Answer 81

The nucleus is the largest and stiffest organelle, so is harder to deform LAMIN proteins in the nuclear envelope (especially Lamin-A) determine nuclear stiffness Lamin-A knockdown can make tumours spread more rapidly (but this is a complex role and depends on the type of cancer)

Question 82

Describe the role of crosslinking enzymes and proteases in tumour formation, and whether this understanding has therapeutic potential

Answer 82

Extracellular enzymes can remodel the matrix: Crosslinking enzymes (e.g., Transglutimase and Lysyl Oxidase/LOX) can stiffen the ECM Matrix Metalloproteases (MMPs) degrade a range of ECM substrates, regulated by TIMPs Also ADAMTs cleave collagen and proteoglycans Both of these groups have a very complex role in cancer, so their therapeutic potential is unclear - some MMPs can actually have anti-tumorigenic effects