organelle structure
DESCRIPTION
Organelle structure. Nucleus Sarco(endo)plasmic reticulum Mitochondria Peroxisome Autophagosome. Electron microscope. Diffraction limit Wave nature of light passing through lens Optical: l ~0.6 um, resolution ~0.2 um Confocal, FT, nanohole arrays De Broglie wavelength l = h/mv - PowerPoint PPT PresentationTRANSCRIPT
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Organelle structure
• Nucleus• Sarco(endo)plasmic reticulum• Mitochondria• Peroxisome• Autophagosome
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Electron microscope
• Diffraction limit– Wave nature of light passing through lens– Optical: ~0.6 um, resolution ~0.2 um– Confocal, FT, nanohole arrays
• De Broglie wavelength– = h/mv– Electron: = 1.2(V- ½) nm (V: accelerating voltage)
B~/(2 NA)
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EM imaging
• Essentially a CRT– Vacuum– Focus e-beam on
specimen– Expand onto screen
• Sample prep– Dehydrate & fix– Heavy metal stain
(uranium)– 90 nm slices
(Graham Colm, wikipedia)
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Structure
Myofibril Nucleus
Mitochondria
Artifact
Out of plane
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Eisenberg et al., 1974
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Common features
• Densely packed myofibrils• Subsarcolemmal space
– Nuclei– Mitochondria
• Intermyofibrillar I-band space– Mitochondria– SR/T-tubule triads
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Fractional composition
• Stereology– Statistical model of 3-D structure– 2-D images & discrete sampling
Compartment Guinnea pig Toadfish swimbladder
Rattlesnake tail shaker
Myofibrils 85-90% 70-80% 32%
SR 3.5-4.5% 26%
Mitochondrial 2-5% (18% SS, 3-4% core)
1-4% (female-male)
26%
T-system 0.15-0.25%
Nuclei 0.9%
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Rattlesnake tail muscle: prominent extensive glycogen & mitochondria; relatively few myofibrilsClark & Schultz, 1980
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Nucleus
• Multiple– MND 3e4-5e4 um3/nucleus– Fiber volume 5e5-5e6 um3 (10-1000 nuclei)
• Cylindrical, ~30 um3
• Nucleolus• Cajal bodies• DNA• RNA processing
Terada et al., 2010
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RNA processing
• Nucleolus: rRNA synthesis & ribosome assy• Cajal bodies: Spliceosome assembly• Speckles: RNA splicing
Pollard & Earnshaw, 2008
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Ribosomal biogenesis
• Multiple (300+) copies of rRNA strand– Transcribed– Cleaved to 28S, 18S, 5.8S strands– Ribose methylation– Uridine isomerization to pseudouridine
• Ribosomal proteins– Imported to nucleus– Anneal to rRNA in nucleolus
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Nuclear Envelope
• Dual membrane• Control nuclear import/export• Nuclear pore complex
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Nuclear membrane proteins
• Chromatin anchors– Lamin, lamin B receptor, emerin
• Transport proteins– Importin, Exportin, Ran
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Mitochondria
• Dual membrane• Oxidative ATP synthesis
– Citric acid cycle– Electron transport
chain• Separate genome
– Most Mt proteins come from nuclearDNA
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Mitochondrial syncytium
• Network, not bacterium-like
• Regulated growth– Division– Fusion
• Apoptosis
Manella, 2000
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Muscle mitochondria
• Extensive subsarcolemmal syncitia• Elongated tubules, perpendicular to fiber
– Frequently doubled around Z-disks
Ogata & Yamasaki, 1997
Subsarcolemmalmitochondria
Intermyofibrillarmitochondria
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Inner membrane
• Cristae– “Shelf-like”– Tubular network
Manella, 2008
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Oxidative ATP Synthesis
• Electron transport chain– NADH– FADH2– Ubiquinone– Cytochrome C
• Reactive oxygen
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Peroxisome
• Single membrane• Controlled H2O2 chemistry
– Oxidases generate H2O2– Peroxidases degrade H2O2– Fatty acid metabolism– Cholesterol & bile synthesis
• Peroxins (PEX) control protein composition and biogenesis
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Sarcoplasmic Reticulum
• Single membrane– Lamellar, fenestrated– Terminal cisternae
• Synthesis of transmembrane and extracellular proteins
• Calcium storage
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SR T-tubule communication
• Terminal cisternae wrapT-tubule
• Communication of membrane events to SR
Ogata & Yamasaki, 1997
SEM of I-band region of a myofiber with myofibrils extracted, leaving only membrane-bounded skeletons of T-tubule (T), SR (blue), terminal cisternae (arrowheads), and mitochondria. Fiber axis is vertical.
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Triad
• Physical association between T-Tubule and SR
• “Feet”– Dihydropyridine receptor
(DHPR)– Ryanodine Receptor (RyR)
Franzini-Armstrong, 1970
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“white” vs “red” muscle
• Red: larger, more extensive mitochondria• White: (slightly) more extensive SR
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Autophagosome
• Double membrane– Small (1 um), spherical
• Self-engulfment• Recruit lysosome & degrade
Mizushima & al., 2002
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Autolysis
• Degradation of large structures• Starvation defense• Infection defense• Targeting
– Apg5/Apg12 targeting complex– LC3 microtubule associated light chain– PI3k, FOXO3
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Summary
• Specialized chemistry is constrained to isolated cellular domains
• Nucleus• Mitochondria• ER/SR• Autophagosome• Peroxisome