* * 11. GnRH. Molecular Biology of the Cell 5th Ed. Alberts et al., Garland Science, 2008

Transcription

1 I Molecular Biology of the Cell 5th Ed. Alberts et al., Garland Science, 2008 Ion Channels of Excitable Membranes 3rd Ed. Hille, B. Sinauer Associates, 2001 (ISBN ), "Neuroscience 5th Ed., Editors: Dale Purves et al., Sinauer, 2012 Companion Website (free) PDF I * * 11. GnRH *

2 ligand fast and precise slow Hydrophilic Hydrophobic (=lipophilic) Cell Animations neuro5e_0701.mov

3 Sinauer Associates, Inc.

4 G GTP serine/threonine kinases tyrosine kinases GAPs: GTPase activating proteins RGS regulators of G protein signaling GPCR desensitization Receptor inactivation Receptor sequestration Receptor down-regulation Molecular Biology of the Cell ( Garland Science 2008)

5 D2 camp IP3 camp IP3 JAK-STAT JAK SH2 STAT intracellular pool of glucose transporters in specialized recycling endosomes signal causes relocalization of glucose transporters to plasma membrane to boost glucose uptake into the cell Nuclear signaling

6 AMP G (A) (B) Animation (C) Adrenaline PDE B-adrenergic receptor camp (C)(D) (D) B-adrenergic receptor Adrenaline AMP Protein phosphatase-i Second Messenger Targets: Protein kinase C PKC

7 Second Messengers (A) Second Messenger Targets: Ca 2+ calmodulindependent (CAM) kinase X NMRCa 2+ /calmodulin (C) CaM-kinase II (B) (D) CaM-kinase II Ca 2+

8 CNG (cyclic nucleotide-gated) (A) (B) (D) (C) camp - Nuclear Signaling- UAS: upstream activator site transcriptional activator proteins =transcription factor CREB =camp response element binding protein CRE=cAMP response element (binding site on DNA) CaRE=calcium response element CRE

9 1 2 camp 3 camp RNA -Nuclear Signaling- ligand-binding domain transcriptionactivating domain coactivator proteins ligand cortisol estradiol thyroxin testosterone DNA-binding domain Vitamin D 3 inhibitory proteins receptor-binding element Transcription of target genes JAK-STAT signaling pathway Retinoic acid Nuclear receptor superfamily

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12 The constitutive and regulated secretory pathways Secretion on demand dcv dcv: dense-core vesicle Structures of putative trafficking proteins of synaptic vesicles Core complex (SNARE complex) v-snare Ca 2+ sensor NSF: N-ethyl maleimide sensitive fusion protein, ATPase SNAPs: small NSF attachment proteins (no relation to SNAP-25) SNARE: SNAP receptor (v- & t-snare) v (vesicle membrane); t (target membrane) t-snare

13 Exocytosis 2001 Gerber and Sudhof

14 Activation of SNARE complex SNARE complex (Core complex) Formation of SNARE complex Recruitment of synaptotagmin GTP Ca 2+ (closed ) (Negative regulator ) (sec1/munc18-like = SM proteins) (SNARE complex ) (syntaxin open ) (SNARE complex ) Ca Oxford University Press SNARE complexes; paring and dissociation Tetanus toxin Botulinum toxin Specific paring of t- and v-snare SNAP)

15 Endocytotic pathways and clathrin Secretory and endocytotic pathways The structure of clathrin coat Clathrin-coated pits and vesicles Clathrin triskelion

16 Clathrin coat dynamin clathrin coated vesicle pinch off (GTPase) Drosophila shibire mutant Dynamin Home Page mutant dynamin 2 Plasma membrane Model of clathrin-coated synaptic vesicle recycling at the synapse Dynamin & Dynamin-binding proteins Coated pit Clathrin coat Synaptic vesicles 2001 Sinauer Associates, Inc.

17 Exocytosis endocytosis intracellular pool of glucose transporters in specialized recycling endosomes signal causes relocalization of glucose transporters to plasma membrane to boost glucose uptake into the cell LTP 2001 Sinauer Associates, Inc. GABA GABA A GABA A GABA A Vithlani et al., 2011

18 Techniques for monitoring exocytosis 1 F/cm 2 (0.01pF/ m 2 ) 1999 Oxford University Press 1999 Oxford University Press Ic=C m dv(t)/dt V(t)=V 0 sin( t) Ic(t)= CmV 0 sin( t+90 ) de/dt=ic/c=-e/rc E=E 0 exp(-t/rc) RC= 2001 Sinauer Associates, Inc.

19 Measurements of exocytosis by monitoring membrane capacitance and the release of vesicular contents by measuring amperometric currents (serotoin) GTP S Caged Ca 2+ Mast cell V h =+700mV foot 2001 Sinauer Associates, Inc. Exocytosis observed in living cells and in EM 1999 Oxford University Press 2001 Sinauer Associates, Inc. Total Internal Reflection Microscopy (TIRM) Evanescent wave

20 Monitoring exocytosis and endocytosis in living cells kiss and run events 2001 Sinauer Associates, Inc. Imaging synaptic vesicle dynamics with the styryl dye FM Cold Spring Harbor Laboratory Press 2001 The Johns Hopkins University Press FM dyes 1) reversibly stain membranes; 2) are impermeable to membranes; 3) are more fluorescent when bound to membranes than when in solution.

21 Fertilization potential and [Ca 2+ ] i of sea urchin egg (Hemicentrotus pulcherimus) Phase II Phase I Membrane potential (mv) Voltage (mv) Time (ms) Sperm Phase III Time (s) Measurements of vesicle dynamics with activity-dependent styryl dyes RH-414 labelling of sea urchin egg at rest fertilized DIC images Differential Interference Contrast unfertilized FM1-43 fluorescence 1999 Oxford University Press