Misc 331

• Synaptic vesicles


  • Metabolite transporters, Rabs, SNAREs
• V Type ATPase


  Present in other organelles (ie secretory vesicles, late endosome, lysosome)
• Phalloidin

  F-actin marker. Conjugate to rhodamine, visualize by flu microscopy
• γ tubulin

  Marker for centrosome. Use anti-γ-tubulin, secondary conjugated to fluorophore, and flu microscopy
• Fungi and diatoms lack centrosomes but use γ tubulin to nucleate still
• Tubulin

  GFP-tubulin, Flu
• DNA

  DAPI, Flu, stains blue
• Lamellipodia


  Ab for ARP2/3 and cofilin, Flu, also mark F actin
• Apoptotic cells


  Annexin V conjugated to flu, Annexin V binds to PS on outer membrane. Colocalize with cytC and mito
• Tight junction


  Tracer molecule→ heavy metal, View by EM
• Glycocalyx


  Lectin conjugated to flu or enzyme
• Mitochondria

  • Translocation complexes: TOM, TIMs, OXA, SAM, Ab or flu
• Peroxisomes


  Pex5→ nuclear import receptor
• Lysosomes

  Acid hydrolase, Incubate with the substrate of an acid hydrolase in the presence of heavy metal salt; electron dense heavy metal salt will deposit if enzyme is present thereby labeling the lysosomes of different sizes
• Cis golgi

  Osmium, Reduced by cis golgi
• Trans golgi


  Nucleoside diphosphatase
• TGN

  Acid phosphatase, Ab to enzyme. Add substrate, add metal, see deposition in TEM
• CGN

  GlcNAc phosphotransferase
• Early and recycling endosome

  LDLR, TFR+TF, LDLR does not need ligand bound
• Early and late endosome

  EGFR, opioid receptors
• Mitotracker red


  Taken up in mito due to membrane potential across IMM, Visualize by flu microscopy
• Rhodamine 123

  Flu microscopy, mitochondria
• TUNEL technique


  Deoxynucleotidyl transferase transfers flu labelled dUTP to 3'OH ends of DNA fragments, Inc intensity→ inc death/apoptotic cells bc more fragmented DNA
• TMRM, JC-1

  Loss of mitochondrial potential can be visualized, Loss of flu when dead cell
• Lysotracker Red

  pH dependent, aggregates in low pH area of live cell
• Ruthenium red


  EM stain
• Peroxisomes, No marker/stain needed, Urate oxidase and catalase are electron dense, can visualize by EM. Primates don't have urate oxidase
• M6P patch

  Lysosome, Acid phosphatase removes signal when in endosome via removing P
• Nuclear Localization Signal (NLS)


  Cluster of basic AAs: Lys and Arg (internal), If adaptor protein has it, cargo is imported, if one subunit has it, complex is imported
• KDEL

  ER residents, Soluble proteins, Membrane bound→KKXX (best characterized is C terminal)

  
• Mitochondrial signal sequences


  N terminal→ matrix, Internal→ membrane protein
• Peroxisomal signal sequence


  SKL on C term, Some have N term, Proteins do not need to be unfolded

  
• ER signal sequence


  Hydrophobic stretch, N term→ Lumenal, Internal→ transmembrane
• GPI anchor addition in ER

  Hydrophobic C term and a few adjacent aas on the lumenal side, Phospholipases may remove from the anchor upon signalling
• Ran (Ras related protein)

  Cytosol, Nuclear import, GEF is attached to chromatin, Gap is in cytosol

  
• Coat Recruitment GTPases

  Cytosol, Control assembly of clathrin coat on endosome and COPII on ER and COPI on Golgi, ARF- COPI and CCVs at golgi, Sar1-COPII at ER→ no GAP
• Rabs (Ras related protein)

  Cytosol, Specificity in vesicle fusion→ direct vesicles to specific sites on target membranes, Located on vesicle membranes and target membranes (membrane patch formation), activated by GEFs on the membranes→ rab-GTP membrane bound via lipid anchor, GDP form is bound to GDI to remain soluble, Effectors facilitate vesicle transport, tethering, fusion, motor proteins

  
• v SNARE-synaptobrevin

  Transport vesicle, Vesicle docking
• t SNARE-syntaxin+3xSNAP25

  Target organelle, Vesicle docking
• Complexin

  Synaptic vesicle, Freeze SNAREs in metastable state to partially pair- priming to release neurotransmitters