Taylor Allen Gafni/Steel Lab June â Aug 2011 - umich.edu and www ...

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Taylor Allen Gafni/Steel Lab June – Aug 2011

•  Form of Demen=a -‐ Problems with memory, behavior, thinking -‐ Most common (50%-‐80%) -‐ 6th leading cause of death -‐ Debilita=ng… eventually impairs motor func=ons -‐ De-‐condi=oning… death Alzheimer’s Associa=on: alz.org

•  Small hydrophobic pep=de •  Interacts w/ hydrophobic tails of lipid membranes •  Has been linked to neuronal cell death Asp -‐ Ala -‐ Glu -‐ Phe -‐ Arg -‐ His -‐ Asp -‐ Ser -‐ Gly -‐ Tyr -‐ Glu -‐ Val -‐ His -‐ His -‐ Gln -‐ Lys -‐ Leu -‐ Val -‐ Phe -‐ Phe -‐ Ala -‐ Glu -‐ Asp -‐ Val -‐ Gly -‐ Ser -‐ Asn -‐ Lys -‐ Gly -‐ Ala -‐ Ile -‐ Ile -‐ Gly -‐ Leu -‐ Met -‐ Val -‐ Gly -‐ Gly -‐ Val -‐ Val -‐ Ile -‐ Ala

Shaked, G.M, Kummer, M.P. (2007) NIH(Public access)

•  Amyloid precursor protein (APP) •  Cleaved by secretase •  Releases Aß into space -‐ Oligomeriza=on & aggrega=on -‐ Cell perfora=on, Ion homeostasis disrup=on, cell death -‐ Fibril forma=on & plaques

Shaked, G.M, Kummer, M.P. (2007) NIH(Public access)

•  Types associated w/ AD -‐ 40 length pep=de -‐ 42 length pep=de •  Aß40 -‐ Most common length pep=de •  Aß42 -‐ Less common -‐ More toxic (faster aggrega=on) Shaked, G.M, Kummer, M.P. (2007) NIH(Public access)

•  Aß perfora=ons disrupt ion homeostasis (Ca2+) •  Fluo calcium indicators allow for imaging and characterizing of Ca2+ signaling and leakage •  Loading: Uncharged acetoxymethyl (AM) esters allow dye to permeate, which are cleaved by esterases to load the cell •  Objec=ve: Induce ﬂuorescence and Ca2+ signaling, characterize signaling & perfora=on Molecular ProbesTM: Product Informa=on, Fluo Calcium Indicators

•  Aliquots: 1) 50mL HBSS (washing) 2) 10mL HBSS (Fluo4)  Water bath at 37oC for 15 min •  Bring up Fluo4 (3μM), Aß42 (1μM), Ionomycin (60μM – 1:10), Buﬀer (500μL) •  1) Wash cells with ~ 2mL of HBSS 2)Incubate with ~ 2mL of Fluo4 for 15 min 3) Wash twice more with ~ 2mL of HBSS Molecular ProbesTM: Product Informa=on, Fluo Calcium Indicators

•  Prep sample holders (vacuum grease) •  Transfer slides to holders and apply 950 μL HBSS •  Imaging: 488 nm…bf exposure 0.1 & gain 1… ﬂuorescent exposure 0.5 & gain 100 •  Adding Reagents: Aß42/Buﬀer ~ frame 95 … Ionomycin ~ frame 400 (50 μL both) •  Quan=fying: Normalized by [Ca2+]free= [F-‐ BG]/[$Fi-‐$BGi] Molecular ProbesTM: Product Informa=on, Fluo Calcium Indicators

• Added Aß42 ~ frame 95 •  No iono... evidently

Mean Fluorescence w/ AB42 10 9 8 Fluorescence

7 6 5 4 3 2 1 0 0

50

100

150

200

250

300

350

400

450

Frame

Aß42 ~ 95

Ionomycin ~ 400

•  Buﬀer ~ frame 95 •  Ionomycin ~ frame 400

Mean Fluobuﬀer_1_488 9.6 8.6 Fluorescence

7.6 6.6 5.6 4.6 3.6 2.6 1.6 0.6

0

100

200

300

400

500

Frame

Buﬀer ~ 95

Ionomycin ~ 400

600

Mean Fluobuﬀer_1_488

Mean Fluorescence w/ AB42 10

9.6

9

8.6

8

7.6 Fluorescence

Fluorescence

7 6 5 4

6.6 5.6 4.6

3

3.6

2

2.6

1

1.6

0 0

50

100

150

200

250

300

350

400

450

0.6

0

100

200

400

Frame

Frame

Aß42 ~ 95

300

Ionomycin ~ 400

Buﬀer ~ 95

Ionomycin ~ 400

500

600

•  Aß42 ~ frame 95 •  Iono ~ frame 400

Mean Fluorescence AB42_2 3

Fluorescence

2.5 2 1.5 1 0.5 0 0

50

100

150

200

250

300

350

400

450

500

Frame

Aß42 ~ 95

Ionomycin ~ 400

•  Buﬀer ~ 95 •  Ionomycin ~ 400

Mean Fluorescence Everything

Mean Fluorescence “Lesser Fluorescing Cells”

7 7

5

6

4

5

Fluorescence

Fluorescence

6

3 2

4 3 2

1

1

0

0 0

50

100

150

Aß42 ~ 95

200

250

300

350

400

450

500

0

100

Frame

200

300

400

500

Time (Sec)

Ionomycin ~ 400

Aß42 ~ 95

Ionomycin ~ 400

Mean Fluorescence Everything

7

7

6

6

5

5 Fluorescence

Fluorescence

Mean Fluorescence AB42_2

4 3

4 3 2

2

1

1

0

0 0

50

100

150

200

250

300

350

400

450

500

Time (sec)

Aß42 ~ 95

0

50

100

150

250

300

350

400

450

500

Frame

Buﬀer ~ 95 Ionomycin ~ 400

200

Ionomycin ~ 400

•  Dynasore: Small molecule inhibitor of dynamin-‐guanosine triphosphate (GTP); blocking endocytosis (non-‐compe=ng) •  Block lipid-‐binding, endocytosis to characterize whether it increases ability Aß to perforate membrane •  If doesn’t take up, maybe con=nuing aggrega=on on membrane will perforate. Biovision.com Macia, E, Ehrlich M, Massol R, Brunner C, Kirchhausen T. (2006) Developmental Cell

•  Aliquots: 1) 40mL HBSS (washing) 2) 10mL HBSS (Fluo4) 3) 15mL HBSS (Dynasore)  Water bath at 37oC for 15 min •  Bring up Fluo4 (3μM), Aß42 (1μM), Buﬀer (500μL), Dynasore(80 μM, 0.2% DMSO), Meliun (4μM) •  1) Incubate with ~ 2mL Dynasore for 30 min 2) Incubate with ~ 2mL Fluo4 for 15 min 3) Incubate again with ~ 2mL Dynasore 15 min

Mean Fluorescence All 4

A

Fluorescence

3.5 3

B

2.5

C

2

D

1.5

E

1

F

0.5

G

0 0

100

200

300

400

500

Meliun ~ 400

Frame

Aß42 ~ 95 Mean Fluorescence A,B,C

H I

4 Fluorescence

3.5 3 2.5 2

A

1.5

B

1

C

0.5 0 0

100

Aß42 ~ 95

200

300 Frame

400

500

Meliun ~400

Mean Fluorescence Buﬀer 3 2.5

Fluorescence

2 1.5 1 0.5 0 0

100

-‐0.5

Buﬀer ~ 95

200

300

400

500

Frame

Meliun ~ 400

600

Mean Fluorescence AB42

Mean Fluorescence Buﬀer

4

3

3.5 2.5 2

2.5 Fluorescence

Fluorescence

3

2 1.5 1

1.5 1 0.5

0.5 0

0 0

100

200

300

400

500

100

200

-‐0.5

Frame

Aß42 ~ 95

0

Meliun ~ 400

Buﬀer ~ 95

300

400

500

Frame

Meliun ~ 400

600

•  MßCD: Hydrophobic ring structure allows complex forma=on with other hydrophobic structures, i.e. cholesterols -‐ Cholesterol extrac=on(1mM) •  Looking to increase extracellular ion exchange by weakening membrane •  Interfering with endocytosis and lipid ravs Depry, C, Allen, M.D, Zhang, J. (2010) RSC Publishing

Mean Fluorescence AB42_2 3 Fluorescence

2.5 2 1.5 1 0.5 0 0

10

20

30

40

50

Frame

Aß42 ~ 10

Meliun ~ 40

60

Mean Fluorescence Buﬀer 3

Fluorescence

2.5 2 1.5 1 0.5 0 0

10

20

30

40

Frame

Buﬀer ~ 10

Meliun ~ 40

50

60

Mean Fluorescence Buﬀer

3

3

2.5

2.5

2

2

Fluorescence

Fluorescence

Mean Fluorescence AB42_2

1.5 1

1.5 1 0.5

0.5

0

0 0

10

20

30

40

50

60

0

10

Meliun ~ 40

30

40

Frame

Frame

Aß42 ~ 10

20

Buﬀer ~ 10

Meliun ~ 40

50

60

•  Italian Muta=on Aß42: Lysine subs=tuted for glutamic acid at posi=on 22. Aggregates more rapidly and is more toxic than wild-‐type Aß42. •  Hoping to see increased ﬂuorescence, more cells responding, and calcium leakage. H -‐ Asp -‐ Ala -‐ Glu -‐ Phe -‐ Arg -‐ His -‐ Asp -‐ Ser -‐ Gly -‐ Tyr -‐ Glu -‐ Val -‐ His -‐ His -‐ Gln -‐ Lys -‐ Leu -‐ Val -‐ Phe -‐ Phe -‐ Ala -‐ Lys -‐ Asp -‐ Val -‐ Gly -‐ Ser -‐ Asn -‐ Lys -‐ Gly -‐ Ala -‐ Ile -‐ Ile -‐ Gly -‐ Leu -‐ Met -‐ Val -‐ Gly -‐ Gly -‐ Val -‐ Val -‐ OH AnaSpec: Product Data Sheet [Lys22]-‐ß-‐Amyloid (1-‐42), Italian Muta=on

Mean Fluorescence Italian AB42_1 16 14

Fluorescence

12 10 8 6 4 2 0 -‐2 0

20

Aß42 ~ 10

40

60

80

100

Time (sec)

Meliun ~ 90

120

Mean Fluorescence Buﬀer_2 3.5 Fluorescence

3 2.5 2 1.5 1 0.5 0 0

20

Buﬀer~ 10

40

60

80

100

120

Frame

Meliun ~ 90

Mean Fluorescence Buﬀer_2

16

16

14

14

12

12

Fluorescence

Fluorescence

Mean Fluorescence Italian AB42_1

10 8 6

10 8 6

4

4

2

2

0

0

-‐2 0

20

40

60

80

100

Time (sec)

Aß42 ~ 10

Meliun ~ 90

120

0

20

Buﬀer ~ 10

40

60 Frame

80

100

Meliun ~ 90

120

•  Outdated cell media led to unhappy cells. Thus causing odd perfora=ons and leaking (Italian) •  Ways to bexer the technique in the future... -‐ Regular Aß42 Ca2+ indicator: Trying diﬀerent laser and camera seungs, like recording =mes… maybe try shorter videos or longer ones? -‐ Ca2+ indicator+MßCD: 1mM might have been too weak. So try higher concentra=on. Also maybe try diﬀerently =med video segments and seungs. (Wash oﬀ to reduce binding to Aß?)

-‐  Ca2+ indicator+Dynasore: Trouble going into solu=on… might be dissolved bexer by lowering concentra=on slightly. Also, try with iItalian also?. Faster aggrega=on might show interes=ng ﬂuorescence. -‐  Ca2+ indicator Italian: Trying diﬀerent laser and camera seungs, like recording =mes… maybe try shorter videos or longer ones.

•  SHSY5Y – Human Derived Nueroblastoma •  Cell Media: DMEM:F-‐12 Phenol Red Free (stored 4oC) : 10% Fetal Bovine Serum (stored -‐20oC) : 200 units/mL Pen/Strep (10k units/mL, 1mL/50mL media) (stored -‐20oC) •  Unlabeled ß-‐Amyloid (1-‐42): (3&8 μg) •  Italian ß-‐ Amyloid(1-‐42): (5 μg)(Muta=on) •  Fluo-‐4 AM (Acetoxymethyl): Brought up in DMSO •  Ionomycin: (Ionophore) Pore forming •  Meliun: Pore forming pep=de. •  HBSS Buﬀer: •  Sodium Phosphate Buﬀer: pH 7.4 (NO salt) •  Dynasore: Dynamin endocytosis blocking (0.2% DMSO) •  Methyl-‐ß-‐Cyclodextrin: Cholesterol Extrac=on

•  Alzheimer’s Associa=on: alz.org •  AnaSpec: Product Data Sheet [Lys22]-‐ß-‐Amyloid (1-‐42), Italian Muta=on •  Depry, C, Allen, M.D, Zhang, J. (2010) RSC Publishing •  Macia, E, Ehrlich M, Massol R, Brunner C, Kirchhausen T. (2006) Developmental Cell •  Molecular ProbesTM: Product Informa=on, Fluo Calcium Indicators •  Shaked, G.M, Kummer, M.P. (2007) NIH(Public access)

•  NSF REU

•  University of Michigan Biophysics Department •  Gafni/Steel Laboratories •  Robin Johnson, Kathleen Wisser, Joe Schauerte, Andrew Chang, Pavi Aravamudhan