Molecules to cells Flashcards
Signal hypothesis
Blobel - proteins have intrinsic signals that govern their transport + localisation in the cell intracellular postcodes
-> signal sequences made up of specific types of a. acids (can be removed after), often hydrophobic side chains.
70% proteins remain in cytosol
Features of nucleus
Envelope - composed of 2 membranes w/ underlying lamina (protein network), continuous w/ ER
Pores - 30+ proteins act ac gateways, small water sol mols diffuse freely through, larger components (RNA, proteins) actively transported across pore complex
Nuclear localisation signal (NLS)
Target proteins to nucleus, common feature is many +ve a. acids (Arg, Lys), short + can be located anywhere in protein, not removed after.
Protein import into nucleus
- Receptor binds proteins w/ NLS in cytosol
- cytosolic fibrils direct receptor to pore + binds pore proteins, cargo proteins moved into nucleus through gel-like meshwork of fibrils
- pores large so folded proteins can be imported
GTP hydrolysis by RAN
Drives nuclear import
Small GTPase RAN is GTP or GDP bound w/ different localisations
- RAN-GAP (reg1) triggers GTP hydrolysis
- GEF (reg2) promotes exchange of GDP for GTP
High Ran GTP in nucleus displaces cargo protein from receptor - receptor recycled back to cytosol
GTP hydrolysed in cytosol so Ran-GDP dissociates
How is NLS exposed?
Nuclear factor of activated T-cells (NFAT) - family of TFs - stimulated to enter nucleus by calcium (changes their conformation).
-> exposes NLS
Cut and paste experiments used to conclude NLS required + sufficient on its own for nuclear import.
Mitochondria features for protein import
Inner membrane impermeable BUT outer membrane permeable to all mols < 5000 Da.
Targeting sequences normally have high Arg(+) & Ser/Thr (nonpolar)
- located N terminus, 20-80 a.acids long
- cleaved after import
-> can form ampiphilic a-helix w/ 2 different sides
Mechanism of import into mitochondria
- Targeting signal recognised by receptor on outer mem.
- Translocator (TOM) channel moves protein into inter-membrane space.
- Signal binds TIM in inner mem.
- Signal sequence cleaved (protein must be open/unfolded to enter matrix)
-> chaperone protein (Hsp70s) pulls protein into matrix + helps refold it.
Protein targeting to ER
Pancreatic cells have extensive rER, hepatocytes have extensive sER.
ER entry point for proteins destine to other organelles/cells, delivered by vesicular transport
Targeting sequence has 8+ hydrophobic a. acids (leu, val, isoleucine) residue near N terminus.
Proteins enter whilst still being synthesised (co-translational)
Mechanism of import into ER
- SRP binds ER signal sequence as it emerges from ribosome (translation paused)
- SRP binds SRP receptor adjacent to translocator protein (Sec61) on ER mem.
- SRP displaced + released for reuse
- ribosome passes through translocator (translation resumed)
Binding of signal sequence by Sec61 opens channel.
Polypeptide threaded though channel as loop.
Signal sequence cleaved by signal peptidase.
Membrane protein insertion
- ER signal sequence binds Sec61 opening channel
- Hydrophobic stop-transfer seq stops polypeptide movement through channel
- Stop-transfer seq released into bilayer forming transmembrane domain
- Protein inserted into bilayer w/ fixed orientation, N terminus in lumen.
- Signal sequence cleaved
Alternating start/stop transfer sequences generate complex multi pass proteins
Post translational modification in ER
Folding assisted by molecular chaperones.
e.g. BiP is an ATPase that binds exposed hydrophobic residues, Calnexin binds N-glycosylated proteins
Disulphide bonds formation: oxidation of cysteine residue -> increases tertiary structure stability
N-linked glycosylation (N=asparagine): lipid donor dolichol donates oligosaccharide to protein, catalysed by OST, only done in specific consensus sequence
Functions of post-translational modifications
- Assist protein folding
- Creation of manmose-6-phosphate tags act as lysosome sorting signal
- Act as ligand for specific cell-cell recognition events.
Glycocalyx (protective layer) made at ER + golgi - used to coat eukaryotic cells
Quality control in ER
Chaperones bind misfolded proteins + stop them leaving ER.
Unfolded protein response (UPR) occurs when build up of misfolded proteins in ER lumen.
- activates ER sensor protein which activates chaperone genes
SDS-polyacrylamide gel electrophoresis
Sodium dodecyl sulphate (SDS) binds protein to give them negative charge, acts as ionic detergent so will unfold proteins
Polyacrylamide gel is mesh like gel that separates charged proteins by size.
They move towards positive anode faster if they’re smaller
Can use coomassie blue, silver stain, radiolabel or antibodies to visualise proteins.
Membrane lipid synthesis
- Catalysed by enzymes on cytosolic face of ER mem.
- Scramblase transporters transfer phospholipids between leaflets non selectively until equilibrium reached
*far more phosphatidyl serine in inner leaflet
-> flippase transporters in golgi flip specific phospholipids from outer -> inner mem
Membranes + proteins retain orientation during vesicular transport -> lumen domain joins extracellular surface
Vesicle formation
- Protein coat deforms membrane into bud captures cargo.
- Dynamin (GTPase) helps bud pinch off.
- Coat proteins (clathrin in endocytic pathway of plasma mem + golgi) removed.
How is cargo selected and vesicle separated?
Adaptins help clathrin attach to mem forming clathrin-coated pit on cytosolic face.
They also bind cargo receptors which recognise specific sorting signals on cargo proteins, recruiting them into vesicle.
-> clathrin cage causes membrane to invaginate, dynamin assembles ring around neck of bud - GTP hydrolysis changes dynamin conformation contricting neck of bud
How does vesicle uncoating occur?
Coat proteins (e.g. clathrin) removed - requires molecular chaperones + ATP
Mechanism of vesicle docking + fusing
- Rab protein binds vesicle, v-SNARE acts as marker
- specific tethering protein on target organelle binds Rab
- v-SNARE & t-SNARE interact + wrap tightly to allow vesicle docking
- bilayers must be close (1.5nm) to fuse, water must be displaced (energetically unfavourable)
Retention & sorting in ER & golgi
Sorting/trafficking signals vital
ER retention signal: KDEL sequence at C terminus of soluble proteins recognised by KDEL receptor in golgi
-> recruited in COPI vesicles + returned to ER
(BiP & PDI contain KDEL sequences)
- short TM domain (18a. acids) retains proteins in golgi
- addition of Mannose-6-phosphate to N linked glycans of some glycoproteins sends them to lysosome.
Golgi apparatus
cis network - entry, carrying vesicles from ER
trans network - exit, carrying proteins onwards
Vesicular transport model in Golgi
Cisternae static components containing specific enzymes. Vesicles bud + fuse through each cisternae .
- cargo mols present in small transport vesicles (100nm)
Cisternal maturation model
Cisternae matures as it migrates outward through the stack (cis -> trans), resident enzymes carried forward are returned to earlier compartment
- transport large proteins like collagen (300nm), too big for typical vesicles
Golgi function
- protein modification - O linked oligosaccharides added to -OH side chains serine + threonine, N-linked oligosaccharides added in ER can be trimmed + rebuilt at golgi
- Protein sorting - at cis network cargo w/ KDEL signal sorted back to ER, other cargo proceeds onwards, at trans network cargo proteins sorted into transport vesicles.
Unregulated/constitutive exocytosis
Constant stream of transport vesicles from trans Golgi.
- supplies proteins for plasma membrane growth, allows protein secretion
*default, no signal required
Regulated exocytosis
Proteins sorted into secretory vesicles + stored until specific signal received
- only in specialised secretory cells
e.g. release of insulin, increase blood glucose -> insulin secretion by pancreatic B-cells
regulated secretion is rapid
3 forms of endocytosis
- Phagocytosis - e.g. protozoa can take up food, macrophages + neutrophils ingest microorganisms
- cells engulf, form phagolysosome
- M. tuberculosis inhibits mem fusion so multiplies in macrophages - Pinocytosis - non-selective mediated by clathrin coated vesicles, small areas of plasma mem + extracellular fluid internalised.
- Receptor mediated endocytosis - selective uptake of molecules, uses cell surface receptors to capture cargo (increased efficiency 1000 fold)
Endocytosis of low-density lipoproteins (LDL)
- Cholesterol transported in blood as LDL, binds LDL receptors - then internalised by clathrin coated vesicles + fuses w. endosome.
- Endosome has acidic pH -> LDL dissociated from receptor
Many materials taken up by receptor mediated endocytosis:
- lipoproteins
- metabolites
- signalling mols
- virus particles
Endosome & lysosome sfeatures
Endosome - cluster of connected tubules + vesicles, most receptors recycled (LDL) but many degraded (EGF), or moved to different domain of plasma mem (transcytosis)
Lysosomes- around 40 hydrolytic enzymes -> degraded macromolecules, optimal at pH 5so inactive in cytosol
-> acidic pH maintained by proton pump
Sorting enzymes to lysosomes
Enzymes made at ER -> Golgi, modified w/ M-6-P.
M-6-P receptor in trans network sorts + packages into vesicles that deliver them
Autophagy
Damaged organelle engulfed by double membrane formed in cytosol, forms autophagosome that fuses w/ lysosome
Biosynthesis of chylomicrons
- PreChylomicrons assembled in ER from triglycerides + large proteins
- packaged into transport vesicles (PCTV)
- mature into chylomicrons in Golgi
- released by exocytosis + enter capillaries
Chylomicron retention disease (CRD)
PreChylomicrons accumulate in ER + unable to reach Golgi
-> caused by defective ER export: COPII coat responsible for cargo exports fails
What causes COPII to not assemble in CRD?
Sar1 GTPase controls formation of COPII vesicles
- regulatory Sar-GEF activates Sar1p turning it on
- Sar1-GTP initiates assembly of COPII proteins
2 isoforms of Sar1: a & b 90% identical but encoded by different genes.
BUT mutations in Sar1b causes CRD -> no Sar1b made + GTP binding site defective
Sar1a still active + mediates transport of most cargo. Sar1b required for preChylomicron transport .
Symptoms + treatment of CRD
Lipid droplets in cell cytoplasm
- Impaired absorption of fats, cholesterol + fat soluble vitamins
- slow growth + weight gain
- GI + nervous system effects
Treatment is low fat diet to minimise accumulation of intracellular preChylomicrons.
Familiar Hypercholesterolaemia
Autosomal dominant disease - can lead to CHD.
Caused by defect in cholesterol uptake so it accumulates in blood -> atherosclerosis.
6 classes of LDL receptor mutations which disrupt cholesterol uptake:
- class 2 disrupt LDL receptor folding
- class 4 disrupt LDL receptor endocytosis (mutated cytoplasmic tail)
-> adapter proteins also affect endocytic uptake of LDL - class 3 disrupt LDL binding (receptor continues circulating)
Treatments for FH
Inhibit cholesterol synthesis - STATINS inhibit HMG-CoA reductase, stimulates LDL receptor expression + increases its uptake -> heterozygotes w/ one wild type copy.
Inhibit dietary cholesterol absorption - EZETIMIBE acts on intestine
Lysosomal storage diseases
Niemann-Pick type C: 95% cases caused by mutations in mem protein NPC1
Gaucher disease: mutations in lysosomal acid B-glucosidase -> misfolded causing ER retention
-> glycolipids accumulate in lysosome prevent cleavage into glucose + seramide
Potential treatments for lysosomal storage diseases
- Enzyme replacement therapy - injecting synthetic enzymes which are taken up via M6P receptors (expensive
- Substrate reduction therapy - reduce amount of glucosylceramide in lysosomes (miglustat inhibits its synthesis) -> treat Gaucher’s
- Pharmacological chaperones can correct folding -> increase amount of enzyme that escapes ER qual control + reaches lysosome.
Protein targeting diseases
Defective mt targeting:
- point mutation in targeting seq of pyruvate dehydrogenase (PDH), R->P has ring structure which break amphiphilic helix.
- inefficient import of protein reduces PDH in mt -> pyruvate accumulates (converted to lactate, lactic acid build up)
-> inherited congenital lactic acidosis
Defective ER targeting:
- point mutation insulin signal seq, R->C, signal seq does not interact correctly w/ Sec61 so not translocated into ER efficiently.
- mutant insulin diverted into cytosol, hydrophobic signal seq at N terminus + Cys forms toxic aggregates causing B cell death
-> less functional insulin made, diabetes
Mechanism to clear misfolded proteins
Proteins localised incorrectly to cytosol degraded by proteasome int a. acids (proteolysis).
- proteins tagged by ubiquitin
- polyubiquitin chain recognised by proteosome cap (ATPase), unfolds protein
- cytosolic proteasome arranged so active side directed toward inwards cavity
hydrophobic regions/unpaired Cys on misfolded proteins -> prone to aggregation so degraded via autophage using lysosome
ER protein misfolding diseases
Mutations in a. acid seq prevent correct folding.
Chaperones can prevent misfolded proteins leaving via exocytosis so lack of functional protein causes disease
e.g. cystic fibrosis
dF508 mutation (90% patients) - > CFTR cannot fold properly so does not reach plasma mem.
-> no Cl- ions out so thick dehydrated mucus + cilia cannot function
Chaperones remove misfolded CFTR to cytosol (proteolysis)
-> ER-associated degradation (ERAD)
Cystic fibrosis therapies
- express CFTR wild type through gene editing
- drugs to enhance folding + escape from ERAD : correctors (chaperones) w/ potentiators (Trikafta) -> effective but expensive
Diseases due to misfolded protein accumulation in ER + treatment
Aggregation of misfolded proteins -> activates stress sensors that trigger unfolded protein response (UPR).
Increases chaperone expression + inhibits protein synthesis to restore homeostasis
-> if it fails apoptosis triggered
Treatment needs to:
- reduce synthesis of mutant protein
- stimulate degradation of mutant protein
- alter UPR signalling to prevent apoptosis
Microtubule structure + nucleation
Hollow cylinders of 25nm diameter assembled by tubulin heterodimers.
Polar structure: +ve end grows rapidly (B tubulin exposed), -ve end grows slowly if at all.
Nucleation - cells use template of gamma-tubulin + other proteins to speed up polymerisation.
-> gamma rings concentrated on specific structures
e.g. ciliated cells have extra set of MTs in cilia nucleated at basal body
MT dynamics
Each grows + shrinks independently of neighbours - can switch between both (dynamic instability)
- unassembled GDP tubulin cannot polymerise
- GTP tubulin can polymerise
- tubulin is a GTPase (switch + timer)
- protein EB1 preferentially binds GTP-tubulin so marks polymerisation
-> if GTP cap lost then MT will depolymerise (catastrophe)
How can MTs be stabilised or depolymerised?
Stabilised:
- binding MT-associated proteins (MAPs) all along MT
- binding taxol drug
Depolymerised experimentally:
- cells put on ice, MT can depolymerise but not grow
- drugs prevent assembly e.g. nocodazole (binds dimers), colcemid + colchicine (binds MT)
Actin filaments
Components of muscle + found in contractile bundles in many cells.
Also found in non-contractile bundles in many cells (epithelial cells, motile fibroblasts - filopodium + lamellipodium)
Assembled from monomeric actin -> thin flexible 2 stranded helical filaments (7nm diameter)
Hydrolyses ATP after assembly:
Treadmilling effect, polymerise plus end, depolymrise minus end.
-> capping proteins bind -ve end preventing depolymerisation
Actin vs MT depolymerisation
Actin: disassembly from -ve end, uses actin, ATP + Mg2+ for assembly
-> polymerisation altered/prevented by small mols: phalloidin stabilises filaments, cytochalasin caps filament ends, latrunculin binds actin monomers
MTs: disassembly from +ve end, uses tubulin, GTP + Mg2+
MT motor proteins
Kinesin moves towards MT plus ends (plasma mem) - many types: Eg5, kinesin-1
Dynein moves towards MT minus ends (nucleus) - 2 types: cytoplasmic + axonemal.
- cytoplasmic dynein works w/ dynactin complex to move cargo proteins by binding different adaptor proteins.
-> both have 2 heads which bind MT via ATP
What is transported by MT motor proteins?
Facilitate membrane traffic - deliver vesicles + position organelles in cytoplasm
- ER mainly move outward using kinesin, some inward movement by cytoplasmic dynein
- Golgi apparatus transported inwards by cytoplasmic dynein
- Viruses transported to nucleus via cytoplasmic dynein
MTs themselves are cargoes - can cause sliding:
1) anti-parallel sliding (meiotic + mitotic spindles)
2) parallel sliding (cilia + flagella)
Cilia + flagella contain MT structures called axonemes -> axonemal dynein drives ciliary + flagella beating through bending + MT sliding
Actin motor proteins (Myosin I & II) + desmin mutations
Myosin moves along actin filaments via ATP hydrolysis - used by plants, algae + fungi for long distance organelle transport.
Myosin II used in muscle - forms microfibrils in sarcomere.
Cross bridge cycle -> ADP released during power stroke.
Myosin I has 1 head - short distance organelle movement, helps reshape plasma mem by pulling on underlying actin filaments
Desmin mutations -> muscular dystrophy + cardiac myopathy
- desmin IFs form scaffold that stabilises muscle Z discs, maintain organisation + connect cell-cell junctions
Actin organisation + dynamics at leading edge of lamellipodia
Arp 2/3 complex binds side of existing actin filament + nucleates assembly of new actin, prevents disassembly at minus end
-> branching, main nucleator of actin in lamellipodia
Polymerisation pushes membrane forward. Distribution of Arps controls actin dynamics.
Actin disassembles at rear of lamellipodium, when Arp not bound.
Actin organisation + dynamics at leading edge of filopodia
Formins are actin-nucleating proteins. Attach to plasma membrane + add actin monomers to plus end of filaments -> filopodia.
Filaments do not slide back - anchored by interactions w/ other filaments via cross-linking proteins.
Filopodia guide migrating cells by probing new environment + establish new contacts w. surrounding ECM
Mechanism of animal cell migration
- Cell pushes out protrusions at leading edge of cell
- Protrusions adhere to surface via focal contacts (w/ integrins)
- Rear of cell pulled forward - using myosin II
Intermediate filaments
Cytoplasmic:
- keratin in epithelial cells
- vimentin + desmin in connective tissue, muscle + glial cells
- neurofilaments in nerve cells
Nuclear:
- nuclear lamins in all nucleated animal cells, underlie envelope - mutations lead to many diseases inc. progeria (premature ageing)
IF properties
10nm diameter, do not bind nucleotides, strong 8-stranded flexible helix, stable.
- symmetrical + not polarised
Some disassemble (phosphorylation) during cell division + reassemble in telophase
-> provide strength + protection against stretching
e.g. keratin + desmin in adjacent cells linked by desmosome provide strength across epithelial sheet.
Plectins link IFs, actin, Mts + desmosomes
- mutations causes severe disease
What needs to happen before mitosis
Interphase - cells increase size, DNA replicates in S phase
Cohesin rings added S phase, hold sister chromatids together until anaphase.
Centrosomes focus for MT polymerisation - triggered by Cdk (phosphorylate key proteins)
Mitotic spindle structures (MT types)
Interpolar - grow from form one pole + meet those from other pole forming anti-parallel interactions
Kinetochore - find + attaches kinetochores on chromatids -> must undergo coordinated assembly + disassembly
Astral - highly dynamic, crucial in anaphase, bind cell mem.
How is mitotic spindle set up?
Nucleation of MTs by centrosomes increases -> more dynamic
- dynamics increased due to MAPs inactivated when phosphorylated by M-Cdk
SO more chance MTs growing from centrosome contact each other or chromosomes
Effects of phosphorylation by M-Cdk
Phosphorylation activates:
Condensin - chromosomes condense
MT catastrophe proteins - MTs more dynamic
Phosphorylation inactivates:
MAPs - MTs more dynamic
Nuclear lamins - nuclear envelope disassembles
Prophase
Condensins compact DNA - chromosomes condense (phosphorylation by M-Cdk)
Interpolar MTs start to form + create anti-parallel interactions -> overlap drives pole separation
Kinesin Eg5 cross links anti-parallel MTs + pushes centrosomes apart.
-> stabilises MTs + indices chromosome separation
Prometaphase
Nuclear envelop disassembles (not in yeasts), lamina also disassembles in animal cells (M-Cdk substrate)
Golgi apparatus fragments - secretion + endocytosis stops
More MTs nucleated so more dynamic -> more chance MTs contacting chromosomes.
Kinetochore attaches chromosomes to + end of MTs by assembling onto centrosome region in prophase - dynamic linker holds onto MT.
- can move both directions using MT motors
- MTs attached grow + shrink in co-ordinated way creating tension
Metaphase/spindle assembly checkpoint
Transition to anaphase blocked (apopostosis) if: MTs depolymerised (nocodazole), stabilised (taxol), spindle not assembled properly, or single kinetochore not attached.
If unaligned or kinetochores unattached - stop signal generated by SAC, anaphase delayed
When attached, chromosomes align as SACs removed from KTs by cytoplasmic dynein.
Entering anaphase early -> aneuploid daughters (incorrect number of chromosomes)
Metaphase to anaphase transition
Once chroms aligned SAC is off, APC/C is active -> separase activated triggering changes in M-Cdk + sister chromosomes come apart as cohesin cleaved.
APC/C triggers proteolysis of specific protein
- covalently attaches ubiquitin tag (proteasome degradation)
- degrades Cyclin B subunit of M-Cdk & securin (separase inhibitor)
Anaphase
A - chromosomes pulled poleward as kinetochore MTs shorten, sister chromatids stay attached to depolymerising MTs.
B - kinesin Eg5 cross link anti-parallel MTs, interpolar MTs continue growing by addition of tubulin dimers at + ends.
Dynein anchored at cell cortex pulls on astral MTs, hauls spindle towards plasma mem.
Telophase
Golgi starts to reassemble. Secretion + endocytosis start.
Nuclear envelope + lamins reassemble.
Lamins are M-Cdk substrates but M-Cdk inactive so nuclear pore proteins + lamins dephosphorylated, fusion of nuclear envelope vesicles continue.
Cytokinesis
In animals:
Contractile ring formed from myosin II + actin -> cleavage furrow. Recruited + activated by central spindle so nucleus not split.
Rings get smaller as actin + myosin v. dynamic.
- integrins phosphorylated, weakening grip on ECM
In plant/algal cells:
No centrosome or dynein, has very broad spindle poles & organised by - end.
Golgi derived vesicles/MTs gradually build up in centre of cell -> new cell wall (not actin + myosin
Epithelia
Cell in sheets, can be rolled to create tubules.
- serve as barriers, can be cuboidal, columnar, squamous or stratified in structure.
Usually polarised - free surface faces air/liquid, basal side connects to basal lamina.
Epithelial cell junctions
Tight junctions from seal between cells stopping diffusion across sheet.
- in vertebrates, formed from occludin + claudin proteins
- lipids in mem an diffuse freely but not mem proteins.
Apical + basal membranes have different protein compositions.
- epithelia functionally polarised
-> polarised transport of nutrients e.g. glucose + a. acids in intestine
Transcytosis - polarised transport of proteins across epithelium e.g. megalin transports from collecting duct to gut
Adherens junction
Adherens + desmosomes link cytoskeleton to neighbours, both use cadherins. But link to different filaments.
Adherens: cadherins link to actin filaments -> network across epithelium (adhesion belt).
Actin network in apical region can be contractile due to myosin II presence (vital form sheet movement)
Desmosomes
Link IFs together across epithelial sheet -> great tensile strength.
IFs have rope like structure so protect against stretching.
Keratin filaments connect to clathrin linker proteins
Abundant in cardiac muscle - linked to desmin in adjacent muscle cells.
- mutation of which cause muscular dystrophy + cardiac myopathy
Hemidesmosomes
Anchors cytoskeleton of epithelial cells to basal lamina using integrins.
Have keratin + plaque of linker proteins like desmosomes BUT integrin binds laminin in basal lamina .
IFs inside cell are cytokeratin linker proteins
HOWEVER, integrins in focal adhesions in fibroblast link to actin filaments
Cadherins
Family that mediate mechanical attachment between epithelial cells.
Transmembrane proteins can bind to identical cadherin in next cell - interaction requires Ca2+.
Used in adherens junctions + desmosomes
Importance of cell-cell interactions
- Cadherins define which cells can interact w/ each other - epithelial cells express E-cadherin, muscle cells M-cadherin (specificity). Cancer cells do not express specific cadherins - M instead of E makes cells highly motile, expression of ECM proteases helps their escape through basal lamina.
- Neighbouring cells can be directly connected by channels
a) Gap junctions (6 connexons) from cytosolic channel allowing direct transfer of inorganic ions.
b) Plasmodesmata in plants allow transfer of proteins + mRNA
Role of ECM
Found in large quantities in connective tissues - specific protein composition determines mechanical properties of tissue.
Great variation:
- tough + flexible (skin, tendon)
- hard + dense (bone)
- shock absorbing (cartilage)
- soft + transparent (in eye)
Collagen
Fibrous protein in connective tissue. 40 different types, ~90% collagen is collagen I.
Collagen I key component of bone. Osteoblasts deposit ECM in bone - arranged in oriented fibres + calcium phosphate fills gaps.
laminin + collagen IV main components of basal lamina
Synthesis & organisation of collagen
Sythesised by osteoblasts (bone) and fibroblasts (skin + tendon)
Trimerisation of collagen polypeptide chain in ER needs ascorbic acid (vit C) - helical structure. Packed into fibrils which are packed into fibres.
Secreted as a precursor (procollagen), cant assemble into fibrils until cleaved by protease outside cells.
- transported through golgi by cisternal maturation + rearranged using integrins in focal contacts.
Genetic conditions of ECM
- abnormally stretchy skin
- brittle bone disease
- skeletal abnormalities
Changes in ECM stiffness can alter gene expression .
- involves SUN-KASH complexes which link filaments to nuclear lamins.
Role of integrins
Attach cells to ECM.
Transmembrane proteins in plasma membrane - link ECM to cytoskeleton.
- focal adhesions in migrating/collagen secreting cells
- hemidesmosomes in epithelial monolayers
How do protrusions adhere to surface during cell migration?
- Focal contacts contain integrins.
- Contractile actin bundles attach to focal contacts
Fibroblasts also attach to ECM via integrins in focal contacts, but need linker protein.
- fibronectin in focal adhesions
- laminin in basal lamina
How can mitosis change cell morphology?
Cell become rounded + less well attached while dividing, actin + myosin drastically rearranged.
Integrins phosphorylated + weaken their grip on ECM.
What is in between collagen fibres in ECM?
Gels of polysaccharide + protein fill space & resist compression
Glycosaminoglycan (GAGs) - negatively charged polysaccharides, very hydrophilic, huge volume relative to mass
Proteoglycans - extracellular secreted proteins w/ covalently linked GAGs
Cartilage is tough + resists compression
- GAGs generate swelling pressure as they bind water mols
-> pressure resisted by collagen fibres
Proteoglycan vs hyaluronan synthesis
Proteoglycan:
- protein made in ER where glycosylation starts -> completed in golgi
- delivered to plasma mem by constitutive secretion
Hyaluronan (only made of carbs):
- hyaluronan synthase at plasma mem, extruded directly int extracellular space
- has high molecular weight + unbranched
Plant cell wall
Give strength has it has no IFs.
Primary laid down 1st - relatively thin so allows growth.
Secondary composition determines its properties:
- hard + thick in wood (cross linked lignin)
- thin + flexible in waxy leaves
Cellulose fibres
Polysaccharide of D-glucose, long fibres containing 16 strands held by H bonds.
Interwoven w/ other polysaccharides - pectin forms compression resistant gel, lignin meshwork for wood.
Cellulose orientation determines axis of cell growth.
Synthesised at cell membrane (like hyaluronan) using cellulose synthase - made in ER + transported via golgi.
Cellulose movement not driven by MT motor proteins BUT powered by cellulose synthesis from synthase complex - assembles them into microfibrils.
Type of signals between cells
- Signals generated by cell-cell contact
Signals are TM proteins which interact w/ adjacent cells e.g. immune system - Signals generated by free diffusion of ligands (long or short distance)
- small hydrophobic mols can cross plasma membrane + bind intracellular receptors e.g. steroids
-> steroid hormone receptors have binding site for steroid hormone + DNA sequence. Forms a dimer. e.g. cortisol binds nuclear receptor protein + activates transcription. - large hydrophilic mols bind surface receptors, can be hormone or growth factors
e.g. EGF short distance in local paracrine signalling, NTs in local neuronal signalling.
Vs remote endocrine cells produce hormones that act over whole body via bloodstream e.g. insulin + adrenaline.
Specificity through cell receptors
1 signal -> 1 response OR 1 signal -> 2 responses.
Often multiple target cells w/ same receptor type e.g. salivary gland cell + heart muscle cell both have mAChRs (different response to activation)
-> response depends on how cell interprets signal
Many signalling pathways can be activated simultaneously as can receive signals from many sources.
GPCR mechanism
When signal binds, GPCR/GEF takes up GDP from G protein + releases GTP
-> binds a subunit + activates trimeric complex causing it to dissociate
Acts as molecular switch .
Conformational change of GPCR induces G protein activation.
GTP hydrolysis turns G protein pathway off.
Principles of signal transduction
Amplification - signal cascade results in strong activation in very short time
Termination of signalling causes receptor degradation or recycling.
cAMP 2nd messenger pathway
adenylyl cyclase converts ATP -> cAMP, when G protein activated (Gs) + dissociates
- activates PKA (has serine-threonine residues) which phosphorylates substrates.
cAMP rapidly degraded by phosphodiesterase
PKA activation + roe in gene transcription
Has 2 regulatory subunits w/ 2 receptor sites each that bind cAMP causing dissociation of complex
-> 2 catalytic subunits released
It is a serine-threonine kinase
Active PKA translocated to nucleus, phosphorylates specific TFs -> increased /suppressed transcription target genes.
DAG & IP3 2nd messenger pathway
Dissociation of trimeric G protein causes phospholipase C to cleave PIP2 -> DAG + IP3
*phosphorylated form of phosphatidyl inositol
- can also be activated by GPCRs, RTKs + are present in cytoplasm
DAG -> PKC translocation from cytosol to membrane
IP3 binds ER receptor -> Ca2+ release which binds C2 domain on PKC activating it
PKC structure: DAG binds C1, ATP binds C3, substrate binds C4
Ca2+ independent PKCs
Novel PKC is group B + PL sensitive, does not have C2 domain.
Atypical PKC is group C + PL insensitive, does not have C1 + missing part of C2.
Enzyme coupled receptors
Phosphorylation is molecular switch, occurs via protein kinases.
Catalyses covalent modification of serine, threonine + tyrosine residues (-OH).
It is reversible , inactivated by protein phosphatases (transduction is dynamic)
Different protein kinase cascades (e.g. MAPK kinase cascade)
Mechanism of activation in RTKs + inactivation
e.g. EGF, HGF, insulin
Intracellular tyrosine kinase domain can be activated by dimerisation - signal mol in form of dimer.
Kinase domains in contact after signal binding causes dimerisation -> activated kinases phosphorylate each other on multiple tyrosines.
Phospho-tyrosine residues have specific binding sites allowing downstream signalling.
Inactivation -> phosphate removal by tyrosine phosphatases, receptor endocytosis + degradation in lysosome.
RTK mediated RAS activation
RAS is monomeric G protein - downstream molecular switch of RTK in plasma membrane
- controlled by GTP binding/hydrolysis
RTK to RAS activation via GRB2/SOS - SOS is a GEF, no direct binding of RTK & RAS.
RTK signalling through MAPK (via RAS)
activated RAS activates MAPK3 -> MAPK2 -> MAPK
MAPK then changes protein activity or gene expression through phosphorylation
Each kinase able to phosphorylate multiple substrates
RTKs signalling through PI3K
PI3K is heterodimeric lipid kinase
- reuglatory subunit has SH2 domain
- catalytic subunit has ATP domain + dual specificity so phosphorylates associated p85 reg subunits
series of phosphorylation leads to PDK1 & AKT activation
-> nuclear translocation Foxo3a (TF), phosphorylated by AKT activating it
Chromosome cycles
Distribution of chromosomes must be co-ordinated w/ cell division.
Embryonic cell: cycles can be simple S -> M oscillators, to generate as much mass as possible.
Somatic cell: gap phases (G1, G2) which separate alternating S & M phases.
Cell cycle
Cdk-cyclin complexes drive various cell cycle phases.
- Cdk levels remain constant
- Cyclin levels rise + fall
Cdk inactive w/o cyclin as T loop occludes active site (no ES complexes).
-> when cyclin bound, conformational change pulls T loop away from active site
G1: cyclin D + Cdk4/6
G1/S: cyclin E + Cdk2
S: cyclin A + Cdk2
M: cyclin B + Cdk1
Checkpoint throughout cell cycle
1) Entering S phase
2) Entering mitosis (is DNA damaged/replicated?)
3) Pull chromosomes apart (are they properly attached?)
-> ensures cell + chromosome cycles coupled
Regulating checkpoints
- Switch off G2/M DNA checkpoint - CdK1 activation blocked + DNA repair mechanisms induced
- Activate Cdk1 to enter mitosis, Cdc25 (removes inhibitory phosphates) + Wee1 control inactive -> active Cdk1, feedback loops used so spike in Cdk1 activity in mitosis
- Silencing SAC - SAC blocks APC activation until chromosomes correctly aligned + kinetochores attached. When attached, SAC silenced, APC activated, Cyclin B degraded -> mitotic exit.
Securin also degraded by APC -> chromatid separation (anaphase onset) - Inactivating Cdk1 - cyclin B ubiquinated (directionality), E3 is anaphase promoting complex (APC) -> active when cyclin B cleaved.
Erythropoietin (EPO)
Secreted cytokine that stimulates rbc production (used for doping).
How is cell proliferation controlled?
EPO activates JAK/STAT pathway leads to proliferation.
Intestine, skin, embryo + stomach tissues highly proliferative.
Extrinsic factors play large role:
- mitogens stimulate proliferation by driving cell cycle
- GF stimulate growth
- Survival factors suppress apoptosis
- Differentiation factors control cell lineage
-> myostatin exerts inhibitory effect - blocks cell cycle progression via TGF(B) pathway
Belgian blue cow has myostatin mutation + is jacked
How do mitogens trigger cell entry?
Beyond restriction point (R), cells no longer need mitogens to continue cycle - handover from Cdk4/6 to Cdk2 (driven by cyclin E rise)
-> rise cyclin D in early G1 causes rise in cyclin E late G1
Mitogen activates Cyclin D transcription through MAP kinase pathway (RTKs -> Ras GTPase -> MAP kinase)
Cyclin D binds Cdk4/6 to make active complex
- many signalling pathways in different tissues feed into Cyclin D formation
How does cyclin D induce cyclin E formation?
Cdk4/6-cyclin D phosphorylates Rb protein which releases E2F (moves into nucleus)
E2F drives cyclin E transcription. Cyclin E then activates Cdk2 -> cell proliferation.
Regulated by +ve feedback: Cdk2 phosphorylates Rb + E2F drives its own synthesis
-> surge in cyclin E drives G1/S transition
Proliferation in tissues
Embryonic stem cells rapid cycles give ~ 200 cells
Small intestine: villi tubes regenerated by underlying tissues are (1 stays, 1 migrates upwards), 3-6 days
Proliferation restricted to base of crypt - proliferation only takes place in certain range
e.g. Paneth cells secrete Wnt (signalling pathway for division), division only at villi base.
Cdks in causing cancerous growth
Cdk2 activity drives S phase entry, Cdk4 activity drives progression beyond R.
Activating mutation would causes cell proliferation in absence of mitogen (uncontrolled)
Cdk4/6 inhibitors (PALOMA-2 trial 2016) can suppress autonomous proliferation.
How does cancer develop + its hallmarks?
Most solid tumours derived from epithelial cells - carcinomas.
early/intermeditae/late adenomas -> carcinomas
Clonal expansion increases accumulation of mutations in same cells
Hallmarks include:
- evading growth suppressors
- sustaining proliferative signalling
- resisting cell death
- genome instability + mutation
Cancer manifests as deregulated tissue homeostasis but is caused by mutations in oncogenes + tumour suppressor genes.#
Viral oncogenes
Rous discovered 1st oncogene - 1964 RSV transforms cells so they hyper-proliferate (abnormal). Has RNA genome.
RSV had extra 4th gene, v-src - expression responsible for tranformation.
Src is cellular gene - codes for non-receptor kinase
Oncogenic mutation examples
Ras GTPase: mutation of gly->val at pos 12 locks RAs in active GTP bound form. Constant downstream signalling even in absence of mitogens.
- only needs 1 mutated Ras allele
-> frequent mutation in causing variety of cancer types
Gene amplification mutations: MYC, ERB2
Chromosome rearrangement mutations: Bcl-2, Bcr-Abl.
Tumour suppressor gene cancers
Eye cancer (retinoblastoma) has 2 types:
1) Sporadic - one eye, no family history, low risk other tumours.
2) Familial - both eyes, family history, high risk of other tumours, already inherited 1 mutation.
- both copies of Rb mutated for complete loss of function (higher risk), cannot inhibit E2F so division driven to S phase w/o mitogens
Neurofibramatosis - familial cancer syndrome due to mutations in NF1 tumour suppressor gene.
- NF1 needed to switch Ras to inactive form (Ras remains constantly active)
- both alleles must be mutated to lose NF1 function
Wnt signalling pathway
Wnt binds cell surface receptor, disrupts complex so B catenin not degraded - can enter nucleus + stimulate transcription of cyclin D.
Wnt pathway mutation is obligate step in colon cancer:
1) APC tumour suppressor function lost
2) B catenin oncogene constantly activated
In cancerous tissue:
B catenin still on despite Wnt being out of range (too far up villi).
Cyclin D still on, proliferation maintained.
Dont differentiate + stay in same location.
Ovarian cancers
p53 normally gets ubiquitinated + destroyed
~95% have p53 mutation
Can be induced by stress -> acts as TF for p21 -> Cdk2
Apoptosis vs necrosis
Apoptosis is programmed cell death - important in normal tissue physiology e.g. immune system, NS, development.
- cells have highly condensed DNA + large vacuoles when undergoing apoptosis
Necrosis is spilled cellular contents due to damage which causes inflammation
Upstream events in intrinsic apoptosis pathway
Fight to control pore:
pro death factors open pore vs pro-survival factors keep pore closed.
Apoptotic stimulus changes balance of factors in favour of pro death.
- can also stimulate p53 which can inhibit pro-survival factors
e.g. Bcl-xl inhibitor can suppress pro-survival function, sensitises taxol induced apoptosis
Dowsntream events of intrinsic apoptosis pathway
Cytochrome C released from mt into cytosol. Membrane asymmetry lost (internal marker externalised) + integrity lost (DNA dye enters cell)
-> cyt C release cases apoptosome assembly + caspase activation
-> susbtrates proteolyzed + DNA cleaved by CAD (formed after ICAD substrates degraded)
