Organelle Interactions and coordination of cellular function Flashcards
(10 cards)
Neuron mt transport, kinesin KIF5, damaged mt and tissue-specific morphology
Neuron mt transport: Mt (+other organelles) transported long-distance in axons by motors to provide ATP where highest demand, mainly for synaptic transmission+ maintenance/synaptic vesicle pool. Some mt stationary, some move both directions. Kinesins drive mvmt to axon terminal, dyneins to soma.
Kinesin KIF5 complexes w/ adaptor Trak (Milton)+ mt outer mem Miro-> mt transport. Transient Ca2+ spikes@ synaptic terminals-> conf change in Miro, kinesin-adaptor complex dissociates, mt mvmt stops. Kinesin activity inhibited @ synapse by syntaphilin binding through increase in Ca-> mt become stably located @terminal. Dynein also binds trak/Miro to allow retrograde mvmt. High glucose reduces mt mvmt in neurons, driven by sugar mods on Milton/trak adaptors due to increased OGT enzyme activity (transfer N-acetyl glucosamine GlcNAc) to protein Sers. Damaged Mt transported back-> soma on dyneins or as part of autophagosomes.
Tissue-specific morphology reflects functional demands. Cardiomyocyte mt numerous, full of cristae+ globular. Lymphocyte mt few+tubular.
mt fission and fusion, internal mt remodelling
Mt fission: ER contact establishes pre-fission site drives initial constriction/ mt mem by actin binding+ actin polymerising proteins on opposite sides of ER-mt contact. When mt region <~150nm diameter, Drp1 rings wrap around, drive further constriction. Dnm2 complex assembles around neck, finalises fission. Timelapse imaging of ER+ mt show mt division machinery Drp1 localised to where ER tubule crosses over mt constriction- ER-mt contact important for Drp1 recruitment.
Mt fusion: outer mt mem fusion driven by mfn1/2 complexes, inner mem by OPA1 (these can be uncoupled). Dynamin involved here as well as fission. Dnm2, Drp1, Mfn1+Opa1 all have GTPase domains
Internal mt remodelling: cristae house protein complexes for ATP synth, can remodel content, org+ density in mt. Stress conditions (nutrient starvation, HBSS media)-> mt fuse+ increase #cristae to increase ATP synth. Individual cristae vary in mem potential, so form independent bioenergetic units.
Communications via contact sites: lipid transfer, ER-Mt contact, ER-mt Ca2+ regulation
Mem contact sites <30nm. Mems don’t fuse (except ER-PM). Molecular tethers involved. E.g. ER-mt contact in yeast mediated by ERMES complex (tether)
Lipid transfer: cells acquire lipids by ER synth or uptake from env (mainly endocytosis/ lipoproteins in animals- degraded in lysosomes, releasing lipids). Lipid transfer proteins form hydrophobic channels, drive selective extraction or bulk transfer between organelles- use PI4P gradient. PI4P gradient drives cholesterol transfer ER->Golgi through OSBP protein. Sac 1+ PI4K est gradient by regulating PI4P-> PI transformation.
ER-Mt contact: VAPB tether molecules show reduce motion @ ER-mt contacts, as seen in Spt-PALM (single molecule photoactivatable PALM)- VAPB mutation stops proper contact formation (disease state). ERMES in yeast proposed to transfer phosphatidylserine (PS) and phosphatidylcholine ER->mt mem. PS= precursor for phosphatidyl ethanolamine- role in mt dynamics, mem curvature, respiratory chain f(x).
ER-mt Ca2+ reg: ATP-producing enzymes+ cell survival regulators respond to [Ca2+] increase in mt matrix. [Ca2+] cytoplasm signals enter mt-> tune metabolism using targeted Ca2+ reporters. This is via ER Ca2+ channels (IP3+ ryanodine receptors)- transfer Ca2+ to mt @ contact sites. ER-MT contact disruption (dysreg/ ERMCS (=VDAC1@ mt outer mem+ GRP75+IP3r 1-3 @ER mem)) affects mt activity (Ca2+ homeostasis, autophagy, apoptosis, immune signalling+ others)
ER has other contacts w/ other organelles: ER-PM appear to juxtapose mems. Endosome contacts (up to 5% total surface) to ER for lipid transfer, trafficking, fission. Peroxisome ER contact for lipid transfer+ coordinated lipid transfer.
Degradation pathways: UQ-proteasome system and autophagy, ERAD
UQ-proteasome system (UPS)+ autophagy: UPS mostly degrades single unfolded polypeps able to enter narrow channel/proteasome. Autophagy deals w/ larger cytosolic structures (complexes, aggregates, organelles, pathogens). Both use UQ to target for degradation. UQ conjugation reactions show kinetics specific to substrate-> protein ½ lives range s to hrs. ATP-dependent UQ activation, then UQ thioester transferred to UQ-conjugating enzyme, isopeptide bond formation catalysed to UQ ligases (E3s)- substrate specific. UQ tag structures diverse, dictate outcome- can recruit accessory factors/receptors w/ UQ-binding domains, incl shuttle factors delivering substrates to proteasome.
ER-mediate degrad (ERAD): misfolded/damaged proteins recognised by E3/chaperones, retrotranslocated in cytoplasm+ UQ’d, proteasome degradation.
Autophagy: major mech in stress response (starvation, organelle damage, protein/RNA aggregates. Maintains metabolic building blocks in nutrient deprivation, eliminates unwanted cell contents. In yeast treated w/ lysosomal protease inhibitors, undegraded autophagosomal contents accum in vacuole after aa withdrawal. Microautophagy= invagination/autophagic substrates into autophagosomes or chaperone-bound denatured substrates from cytosol across lysosome mem into lysosome. Autophagosomes+ autophagolysosomes both have double mems+ incl other cell components. Autophagy integrated w/ pathways for cell growth, nutrient sensing.
mTOR
mTOR= Ser/Thr kinase, role in cell growth/size but not#. Rapamycin=antifungal found in Streptomyces strain from Easter Island- secondary activities as immunosuppressant, anti-growth activity. Genetic screens for rapamycin-resistant mutants ID’d subunit of protein complex containing protein kinase Tor. Later mammalian mTOR ID by biochem approaches. In flies: Clones of Tor homozygous cells induced in eye-> smaller cells. homozygous Tor mutants survive larval stages, but smaller. Tor -/- brains w/ mutant clones induced in head-> smaller size.
mTORC
mTORC1= autophagy regulator. When plenty of nutrients/some cancers- high mTORC1 activity inhibits autophagy, promotes cell growth+ biosynthesis. Famine- low mTORC1 activity disinhibits autophagy-> new aa/nt etc sources. mTORC1 impacts of variety/ pathways incl p300 activation, or e.g. ULK1 complex (=ATG1= apg in yeast) inhibition. ULK1= Ser/Thr kinase involved in autophagy induction- promotes formation/ pre- autophagosomal structure (PAS), Pi’s proteins+ recruits to phagophore.
mTORC1 activated by diff factors through independent, linked pathways:
1) Growth factors-> activation of PI3K/Akt or Ras/Raf/ERK pathway activation. TSC1/2 Pi’d (inhibits). Rheb released, activated by GT-DP exchange, activates mTORC1
2) Aas activate mTORC1 via TSC1/2-independent pathway involving Ras-related GTPases (Rags)+ guanine-exchange factor regulator+ lysosome assoc Rheb. When aas abundant, vacuolar H+ ATPase (V-ATPase) on lysosomes senses aa rise, activates Ragulator-> activates Rags, recruiting mTORC1 via Raptor to lysosome where mTORC1 activated.
3) AMP-activated protein kinase (AMPK) senses E status/cell. Activated when E low, -vely reg mTORC1 by Pi-ing Raptor or TSC2.
Autophagy can be constitutive od substrate specific. Proteaphagy links both.
Mitophagy, Parkinson’s disease
Mitophagy= quality ctrl mech in mt. PINK1-Parkin dependent mitophagy= molecular sensor/mt health. Defective mt recruit, activate parkin, then can be amplified by UQ-ing mt surface proteins, recognised by autophagic receptors- convert mt to autophagosomes by interacting LC3 (bound to mt outer mem proteins). PINK1-Parkin-independent mitophagy: mitophagy receptor proteins (NIX, FUNDC1, BNIP3) interact processed LC3 independent of UQ to dribe autophagosome incorporation.
2 recessive forms/ Parkinson disease caused by PINK1 (mt localised Ser/Thr kinase kinase)/Parkin (UQ ligase) mutations (LoF). Mt depolarisation blocks mt import of PINK1, stabilizing it on outer mt mem. PINK1 then Pi’s OMM proteins+ adjacent UQ molecules-> activation+ recruitment of cytosolic E3 ligase Parkin to mt surface. Parkin further activated by PINK1’s pi, continues to UQ more OMMs, escalating pathway.
Many rare diseases caused by defects in proteins for organelle f(x), dynamics, contacts. E.g Pink1 in mitophagy-> Parkinson’s neuronal degeneration. Opa1 in mt fusion-> optic atrophy/Parkinson’s vision loss+ neurodegen. Reticulon in ER org-> Hereditary spastic paraplegia (motor axon disease.
Fluorescence imaging
Fluorescence imaging: widefield (out of focus light reduced w/algorithm)+ confocal (detector pinhole reduces out of focus light). Light sheet microscopy fast w/ reduced phototoxicity- out of focus light reduced by using side-on sheet illumination. Con= Point spread function (PSF) in fluorescence microscopy: image of point source shows diffractions rings (images have res limit, can make small objects look larger due to airy halo around actual point). Physical res defined by Abbe’s equation: d=wavelength/(2n(sin(alpha))). Due to wavelength factor, res w/ blue light better than w/green. Scale/res comparisons: confocal/ light sheet res ~250nm (scale of mt). SIM ~lysosome scale. STED ~endosome. SMLM~ MT. Miniflux ~IgG, GFP.
Lattice SIM, STED, SMLS, miniflux
Lattice SIM = widefield based system using structured illumination pattern to break diffraction barrier. Diffraction grating-> overlay of non uniform illumination pattern on sample, improving visibility. Suitable for multicolour (up to 4) imaging to ~100um depth. Special config can reach 266 fps, 97nm res.
STED (simulated depletion emission microscopy)- high-power depletion laser increases res. Green excitation laser produces emission. Red depletion laser (v high power, reduces emission ‘halo’ well but also photobleaches sample faster) creates depletion donut to cancel halo around emission from actual object.
SMLS (single molecule localisation microscopy) uses widefield to produce blinking seq, Gaussian distribution fitted to image in 2D to localise point more precisely. STORM uses antibodies, Gaussian distribution fits well. PALM uses fluor. Protein (less precise than fluorophore). E.g. good for mt-MT cytoskeleton interactions.
Miniflux- nm to A scale. Single fluorophore scanning-> position correction+ probing pattern size reduction (multiple scan iterations hone in on exact position)->photon collection. E.g. can resolve nuclear pore complex.
Expansion microscopy: nm-A scale: fix sample, anchor to hydrogel+ polymerise, digest, expand hydrogel in water-> improve confocal/light res, but need to consider how expansion impacts sample. E.g. use to visualise tubulin modifications on centrioles.
Spectral imaging, vEM, cryo-electron tomography, fixing
Spectral imaging expands multicolour fluorescence capabilities: array/detectors fine tuned for specific wvlength detect emissions/partially overlapping fluorophores+ separate them post-acquisition using fluorophore calibration.
Volume EM (vEM): TEM or SEM-based. Slice+ view large volumes @ low nm res-> 3D views/ cells/tissues- e.g. visualising dynamics of airway multiciliated epithelium. Machine learning models (Automatic organelle segmentation by AI) allow automated recognition/organelles from EM images where telling by eye tricky. Can visualise, e.g. mt cristae contacts w/ cytoskeletal elements emanating from base/motile cilia (rootlets).
Cryo-electron tomography: vitrify sample, thin (100-500nm), e- beam-> computational segmentation, denoising, pattern matching+ subtomogram averaging.
Fixing: cryo fixation produces sharper res samples+ better structure preservation than chemical fixation- makes a difference when visualising, e.g. ERMES complex tethering ER+mt for lipid transfer.
