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Pb 2+. Pb 2+. Lead Uptake and Accumulation in Rat Cerebellar Granules by Two-Photon Microscopy. Title. A.Esposito*', F.Pellistri*, A.Cupello ***, C.Marchetti**, M.Robello*. * INFM - National Institute for the Physics of the Matter (Genoa)

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Title

Pb2+

Pb2+

Lead Uptake and Accumulation

in Rat Cerebellar Granules

by Two-Photon Microscopy

Title

A.Esposito*', F.Pellistri*, A.Cupello ***, C.Marchetti**, M.Robello*

* INFM - National Institute for the Physics of the Matter (Genoa)

** IFB-CNR - National Research Council - BioPhysics Institute (Genoa)

*** Istituto di Bioimmagini e Fisiologia Molecolare CNR(Genoa)

' Current address: [email protected] Cell Biophysics Group - European Neuroscience Institute (Göttingen)


Lead introduction

Lead introduction

Ca2+

Calcium, a second messenger

Pb2+

Lead, interferes with calcium signalling

Ca2+

Pb2+

Uptake machinery

Lead action

Pb2+

Pb2+

Ca2+

?

Lead Methods Results Discussion

Lead

H.A.Godwin, Curr.Opinion Chem.Bio. 5, 223-227 (2001)

M.Mazzolini,S.Traverso,C.Marchetti, J.Neurochem.2001 Oct;79(2):407-16

2


Probe model and microscopy choice

Pb2+

Pb2+

Pb2+

Lead Methods Results Discussion

Methods

Probe, model and microscopy choice

Ratcerebellar granule cells

Membrane permeant high affinity fluorescent dye: Indo-1

Two-photon laser scanning microscopy (TPLSM)

45´ Indo-1 AM - 1M

30´ Standard solution

@37°C

3

TPEN (100M)


Indo spectra and binding

Pb2+

Lead Methods Results Discussion

Methods

Indo spectra and binding

Autofluorescence <<1%

Residual Indo1 fluorescence: <5%

M.E.Legare, R.Barhoumi, E.Hebert, G.R.Bratton, R.C.Burghardt and E.Tiffany-Castiglioni, “Analysis of Pb2+ Entry into Cultured Astroglia”, Toxicological Sciences 46, 90-100 (1998)

4

Figura ‑5 a) rappresentazione dell’eccitazione a doppio fotone di una molecola di Indo-1. b) spettro dell’Indo-1 a varie concentrazioni di calcio, si nota una lunghezza d’onda non sensibile alla variazione di tale ione; c) sezioni d’urto d’azione dell’Indo-1. In azzurro la sonda libera, in violetto quella legata; d) Spettri relativi della sonda libera (in azzurro), legata al calcio (in violetto) e legata al piombo (in nero). Si evidenziano i picchi a 405nm (punto a) e 485nm (punto b) corrispondenti rispettivamente alla sonda legata al calcio e libera. Inoltre a 460nm (punto c) il punto isosbestico, insensibile alla concentrazione del calcio, ma dipendente da quella del piombo (d).


Tplsm

TPLSM

Lead Methods Results Discussion

Methods

Indo-1 UV excitation

Reduced photoxicity and photodamages

High resolution and confocality

TPM

Resolution

Axial: 700nm Lateral:350nm Temporal: 3s/pxl

Wavelength: 700nm (680-1050nm)

Mean power on the sample: 5-7mW Pulsed laser: 80MHz ; 100-200fs

A.Diaspro, “Confocal and Two-Photon Microscopy”, Wiley-Liss (New York, 2002)

F.M. Wahl, “Digital Image Signal Processing”, Artech House (1987)

5


Microscope

Microscope

Microscope

TE300Eclipse

(Nikon)

PMT1

PMT2

Scanning Head

(PCM2000)

Tsunami

(Spectra Physics)

Millennia

Lead Methods Results Discussion

Methods

A.Diaspro et al. (1999b), “Adapting a compact confocal […]”, Microsc. Res. Tech. 47, 196-205

A.Diaspro […] M.Robello and F.Olivini (2001), “Two-photon microscopy […]”, J.Biomed.Opt.

6


Protocol

KCl 75mM

La3+ (25M)

2‘

Fluorescence intensity

Pb2+ (20M)

TPEN (100M)

time

Lead Methods Results Discussion

Methods

Protocol

7


Photobleaching

photobleaching

quenching

S1

T1

photochemistry

S0

FLUORESCENCE

time

PHOSPHO_

RECENCE

(3.40.5)10-4 % s-1 mW-2 frm-1

Bleaching rate ~3-8°/00frm-1

Fluorescence variations

2 control frames

Photobleaching curve

Bleaching estimation

2

4

6

0

8

-0%

Time (min)

-10%

-20%

Lead Methods Results Discussion

Results

Photobleaching

8


Indo1 concentration calibration

Indo1 concentration calibration

0

255

Intensity (a.u.)

5mm

Lead Methods Results Discussion

Results

Autofluorescence <<1%

Mean Indo-1: ~90mM

50/100mM

Brust-Mascher and Webb, “Calcium Induced by Large Voltage Pulses in Fish Keratocytes”,Biophy.J. Vol75 1998

9


Lead uptake

Var: 54±6%

Raw: 67±9%

Sat: 63±7%

75

Pb2+ (20µM)

Pb2+ (20µM)

65

55

45

35

Estimated concentration (µM)

25

~

15

bound

5

0

2

4

Time (min)

Lead Methods Results Discussion

Results

Lead uptake

Time (min)

0

2

4

6

8

0%

-15%

-30%

Fluorescence variations (%)

-45%

Pb2+(20µM)

-60%

Fluorescence variations

Photobleaching

-75%

10


Lanthanum inhibition

75

Pb2+ (20µM)

Pb2+ (20µM)

65

M)

55

Pb2+

45

35

Concentration (

La3+

25

15

+La3+ (25µM)

5

0

2

4

Time (min)

Lead Methods Results Discussion

Results

Lanthanum inhibition

11


Free cytosolic lead

Pb2+ (20µM)

Pb2+ (20µM)

Lead Methods Results Discussion

Results

Free cytosolic lead

12


Conclusions

Conclusions

Rapid lead uptake in the micromolar range (bound)

Free cytosolic lead in the picomolar range

Probable saturation of transport

Partial lanthanum inhibition (~70%)

Pb2+

Pb2+

Pb2+

Pb2+

Pb2+

La3+

C2 (PKC 2pM, sinaptogamin)

EF-hand helic (calmodulin, calcineurin @ 100pM)

Cys3(zinc proteins in pM range)

Pb2+

Pb2+

Pb2+

Pb2+

Pb2+

Lead Methods Results Discussion

Discussion

R.H.S.Westerink et al., “Ca2+-indipendent vesicular catecholamine release in PC12 cells by nanomolar concentrations of Pb2+”, Journal of Neurochemistry, 2002 Vol.80 861-873

13


Aknoledgment

Aknoledgment

Acknowledgment

Acknowledgment

Mauro Robello

Carla Marchetti

Francesca Pellistri

Aroldo Cupello

Alberto Diaspro

Paola Ramoino

Camilla Luccardini

Silvia Casagrande

Marzia Pisciotta

Marco Raimondo

Federico Federici

Mauro Robello

Carla Marchetti

Fred Wouters

Cell Biophysics Group

European Neuroscience Institute

Göttingen - Germany

www.eni.gwdg.de

Contact

Contact

Alessandro Esposito - PhD stud. @ ENI-G (CGB)

FLIM (FD/TD) development, Molecular Biology

www.gwdg.de/~aesposi [email protected]


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