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I••••• e-mail: [email protected]://www.bs.s.u-tokyo.ac.jp/~naibunpi/lab.html

• “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 0-87893-321-2) ,

• "Neuroscience“ 5th Ed., Editors: Dale Purves et al., Sinauer, 2012

• Companion Website (free)http://sites.sinauer.com/neuroscience5e

•PDF

http://www.bs.s.u-tokyo.ac.jp/~naibunpi/Lecture_Index.html

•

•

I

1.2.3. 3-1 3-24.5.6.7.8. *9.10. *11. GnRH

*

“ligand”

fast and precise

slow

Hydrophilic Hydrophobic(=lipophilic)

2004 Sinauer Associates, Inc.

Cell Animations¥neuro5e_0701.mov

2000 Sinauer Associates, Inc. 2004 Sinauer Associates, Inc.

http://sites.sinauer.com/neuroscience5e/animations07.01.html

G

GTP

2002 Alberts et al.

Molecular Biology of the Cell (© Garland Science 2008)

• serine/threonine kinases• tyrosine kinases

• GAPs: GTPase–activating proteinsRGS regulators of G protein signaling

GPCR desensitization• Receptor inactivation• Receptor sequestration• Receptor down-regulation

IP3

IP3cAMP

D2


cAMP

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

JAK-STAT

JAK

SH2

STAT

Nuclear signaling

Adrenaline

cAMPAMP

Protein phosphatase-I

Adrenaline

2002 Alberts et al.

B-adrenergic receptor

AMPG

(A) (B)

(C)

(D)B-adrenergic receptor

PDE

Animation

(C)(D)

Second Messenger Targets: Protein kinase C


2002 Alberts et al.PKC

Second Messengers


Second Messenger Targets: Ca2+calmodulin-dependent (CAM) kinase

X NMR Ca2+/calmodulin CaM-kinase II

CaM-kinase II Ca2+

2002 Alberts et al.

(A)

(B)

(C)

(D)

CNG (cyclic nucleotide-gated)

(A) (B)

(C)(D)

http://sites.sinauer.com/neuroscience5e/animations11.02.htmlhttp://sites.sinauer.com/neuroscience5e/animations15.01.html

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

2004 Sinauer Associates, Inc. 2002 Alberts et al.

CRE

1

cAMP

2

cAMP3

RNA2004 Sinauer Associates, Inc.

cortisolestradiol

Vitamin D3

testosterone

thyroxin

Retinoic acid

Transcription of target genes

receptor-binding element

coactivator proteinsligand-binding domain

ligand

transcription-activating domain

DNA-binding domaininhibitory proteins

2002 Alberts et al.

Nuclear receptor superfamily

JAK-STAT signaling pathway

- Nuclear Signaling-

7


http://www.nobelprize.org/nobel_prizes/medicine/laureates/2013/

2002 Alberts et al.

The constitutive and regulated secretory pathways

Secretion“on demand”

2002 Alberts et al.

dcv

dcv: dense-core vesicle

Structures of putative trafficking proteins of synaptic vesicles

Core complex (SNARE complex)

t-SNARE

v-SNARE

Ca2+ sensor

NSF: N-ethyl maleimide sensitive fusion protein, ATPaseSNAPs: small NSF attachment proteins (no relation to SNAP-25)SNARE: SNAP receptor (v- & t-SNARE)v (vesicle membrane); t (target membrane)


Exocytosis


2001 Gerber and Sudhof

(sec1/munc18-like =SM proteins)

(SNARE complex)

(SNARE complex)

Ca2+

GTP

SNARE complex(Core complex)

(Negative regulator )

Activation of SNARE complex Formation of

SNARE complexRecruitment of synaptotagmin

(closed )

(syntaxin open )

Ca2+

1999 Oxford University Press

SNARE complexes; paring and dissociation

Tetanus toxinBotulinum toxin

SNAP)Specific paring of t- and v-SNARE

2002 Alberts et al.

2002 Alberts et al. 2004 Sinauer Associates, Inc.

Endocytotic pathways and clathrin

Clathrin-coated pits and vesicles

The structure of clathrin coat

Secretory and endocytotic pathways

Clathrin triskelion2002 Alberts et al.

Clathrin coat

dynamin

clathrin coated

vesiclepinch off

Drosophila shibire mutant

mutant dynamin

(GTPase)

2002 Alberts et al.

Dynamin Home Page

2

Clathrin coat

Coated pit

Dynamin & Dynamin-binding proteins

Synaptic vesicles

Plasma membrane

Model of clathrin-coated synaptic vesicle recycling at the synapse


LTP

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

Exocytosis endocytosis

2002 Alberts et al.


GABA

GABAA

GABAA

GABAA

Vithlani et al., 2011

Techniques for monitoring exocytosis

1 F/cm2 (0.01pF/ m2)


dE/dt=Ic/C=-E/RC

E=E0 exp(-t/RC) RC=

Ic=Cm dV(t)/dtV(t)=V0sin( t)Ic(t)= CmV0sin( t+90 )



Measurements of exocytosis by monitoring membrane capacitance and the release of vesicular contents by measuring amperometric currents

(serotoin)

Vh=+700mV

foot”

Mast cell

Caged Ca2+GTP S


Exocytosis observed in living cells and in EM

Total Internal Reflection Microscopy (TIRM)“Evanescent wave”



Monitoring exocytosis and endocytosis in living cells

“kiss and run” events


Imaging synaptic vesicle dynamics with the styryl dye FM1-43

FM dyes 1) reversibly stain membranes; 2) are impermeable to membranes; 3) are more fluorescent when bound to membranes than when in solution.

2000 Cold Spring Harbor Laboratory Press

2001 The Johns Hopkins University Press

Time (s)

Mem

bran

e po

tent

ial (

mV

)

Sperm

200 400 600

-10

-20

-30

-40

-50

-60

-70

-80

0

Fertilization potential and [Ca2+]i of sea urchin egg (Hemicentrotus pulcherimus)

Phase I

Phase II

Phase III

600005000040000Time (ms)

Volta

ge(m

V)

-80

-70

-60

-50

-40

-30

-20

-10

1 2 3

4 5 6

7 8 9

12 3 4

5678

9

Measurements of vesicle dynamics with activity-dependent styryl dyes

RH-414 labelling of sea urchin egg at rest

fertilized

unfertilized

DIC imagesDifferential

Interference Contrast

FM1-43 fluorescence


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