Roles Core Primaty Cilia

Question 1

Primary cilia

Answer 1

Ligand receptors on cilia
Activating pathways
Regulation of pathways

One per cell
Microtubule based
Most cell assemble - myeloid doesn’t
From basal body (modification of mother centriole)
Assembly takes outside cell cycle
250nm width
Similar structure to motile multiple cilia

Cillopathies

Question 2

Primary cilia action

Answer 2

Left, right, up, down flow

Mutagenesis screen: cystic kidneys, mutated genes looks like a gene of trafficking flagella (cilia protein)
Polycystines - no cilium so no recruitment of mechano sensitive ion channels to surface of epithelium so don’t respond to flow

Question 3

Hedgehog pathway

Answer 3

Cilia use scaffold, place to be regulated
Know how components are recruited and more insight into pathway due to cilia
More and more pathways are being discovered to use cilia for signalling

Question 4

Skeletal ciliopathies

Answer 4

Eyes - retinal dystrophy (non motile cilia)
Ears - hearing loss (non motile)
Heart - congenital heart defects (both types)
Dysfunction in motile/non motile cilia cause cilliopathies that encompass most human organ systems

Question 5

Disrupted endochondral ossification

Answer 5

Rib cages stunted in growth
Narrow thorax

Question 6

Endochondral ossification

Answer 6

Axolotl - can regrow limbs by switching on and off

Question 7

Growth plate

Answer 7

Skeletal templating
1) zone of reserved cartilage - small stem cell population (renew or change)
2) zone of cell proliferation (makes a lot of chrondrocytes)
3) zone of cell hypertrophy (massive grow)
4) zone of calcification (become osteoblast)
5) zone of bone deposition (death of chrondrocytes, bones then deposited by osteoblasts forming bone)

Makes different matrixes at each stage and then break down to make new

Question 8

Primary cilia hypothesis

Answer 8

Cilia or ciliary machinery act go recieve/transducer external cues eg growth factors, inflammatory cytokines (some elicited by biophysical changes eg pH, force)
Turn up or Dow the response to changes
Factors often mechanically activated/regulated themselves
Cilium can act as mechanostat or rheostat that modifies integrated response to signals (changes receptor resistance) and force accordingly to achieve cullular output desired

Question 9

Mice model of osteoporosis/osteoarthritis

Answer 9

Never get rid of growth plate tho unlike humans
Snip- destabilised- wear at cartilage so model (DMM) medial goes first coz more force

Question 10

Delete IFT88 = loss of calcified cartilage

Answer 10

Delete gene of interest in cartilage
Flank with loks-p sites (cute sites), cute gene out when exposed to CreER, only activated within cells making agrecal

Specific and inducible - adults so induce later on

Progressive atrophy (of calcifying cartilage) after deletion of cilia

6 months - spontaneous loss of cartilage and osteophyte formation- looked like OA

Question 11

Mechanoadaptation

Answer 11

Cartilage during adolescence- shape skeleton later
But don’t know much about adolescence cartilage

But by adding running wheels save the cartilage
More OA than should be so change environmentally to change it eg exercise

Cilia acting as mechanosensitive brake
Release brake deactivate developmental program hedgehog is meant to be running in development and cartilage turns to broke again
Cartilage > bone > no cartilage to protect bone

Question 12

Growth plate mechanics

Answer 12

950kPA - Articulate cartilage
20-100kPA - cartilage
11,500,000kPA - compact bone

Secondary ossification centre - spread load across limb

Spot loading would kill hypertrophic cell

Question 13

But does the load distribute evenly?

Answer 13

Still a little bit of spot loading

Question 14

What happens when we delete a “mecganothtesholder” (IFT88) in the growth plate as it’s closing?

Answer 14

Didn’t close growth plate, fusion wasn’t happening
Didn’t make the bone
Made on one side not the other

A-bi lateral effect
Forces greater on outsides, IFT88 removed and not ossifying on the outsides = cilia are a MECHANODAMPENER
Off loaded by cutting nerve- looks normal
Contra lateral looks like removed gene
Wheel exercised - acute response of adolescent mice - more plastic so can adapt quickly

KO increased bad phenotype if wheel exercises, naive decreased bone, off loaded normal - mechanodependent over genetic phenotype

INTERPRETATION - bones made quicker outside due to force but cilia says no we’ll do it as the same pace as the middle

Question 15

Hypertrophic niche

Answer 15

Greater force, osmotic flux, cilia - control response
Genetics

Question 16

Matrix related cause loss of ossification

Answer 16

Not making correct signal
Osteoclasts aren’t there in KO’s
Vessels don’t arrive - signal VEGF not there
Tumour uses VEGF to metastasise, also use cilia for it

Question 17

Problem = maturation of hypertrophic niche before trans differentiation

Question 18

Modelling chrondrocytes osseous mechano-stability/plasticity

Answer 18

Make gels, buoyancy to cast gradieysns of growth factors
Click chemistry
Helparin methacryalayl with stem cells - bone growth
Can ask for skin graft stem cells of patient to create replica of their mutation - tissue engineering