Bogomoletz Institute of PhysiologyKiev

Ukraine

Dr. Pavel Belan

Fluorescent proteins in current biology

Lecture Plan

• Development of optical microscopy

• Basics of fluorescence

• Basics of molecular biology

• Green Fluorescent Protein (GFP) in the current biology

• Gene delivery techniques

Replica of microscope by Van Leeuwenhoek

Microscopic section through one year old ash tree

Compound microscope

Current optical microscope

Olympus web site

iMIC Digital Microscopes Intravital 2P

Copyright © 2006 - 2011 TILL Photonics GmbH

State-of-the-Art microscopes

• The rapid advances made in developing live-cell imaging microscopes based upon low light level electron multiplying CCD cameras and different confocal systems, allows imaging fluorescent probes and optical highlighters over the entire visible and near-infrared spectral regions with near real-time precision and submicron space resolution.

DIC image of cultured hippocampal neurons

Fluorescent image of double labeled cultured hippocampal neurons

Biochimica et Biophysica Acta 1802(1):143-50. 2010

Basic principles of fluorescence

Marking proteins by fluorescent tags

Fluorescent tag

Protein of interest

Problems with fluorescent tags

Cell

impermeability

unspecificity

How can we specifically tag proteins by fluorescent markers?

How to do that in living cells?

Gene expression

Reporter gene expression

Gene under study

Reporter fluorescent protein

Protein under study

Himeric gene expression

FP gene

Gene under study

Himeric fluorescent protein

Gene under study

Plasmid construction

"Bioluminescent dinoflagellates (Lingulodinium polyedrum) lighting a breaking wave at midnight. The blue light is a result of a luciferase enzyme (like firefly luciferase, but the enzyme in L. polyedrum shares no similarity with that of the firefly enzyme). Under the right conditions, the dinoflagellates become so numerous that the water takes on a muddy reddish color (hence the name "Red Tide").

Image of bioluminescent tide event at a beach in Carlsbad California

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

Aequorea victoria

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

Studies of protein expression using GFP как главный репортерный белок

GFP

It is possible to report gene expression in single living cells

First study describing Green Fluorescent Protein (GFP) as a protein expression marker

Science 1994:Vol. 263. no. 5148, pp. 802 - 805DOI: 10.1126/science.8303295

Olympus website

Diversity of Fluorescent proteins

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

Examples of Labeling by Different Fluorescent Proteins

http://www.conncoll.edu/ccacad/zimmer/GFP

Pyramidal neurons of «rainbow» rats

J Biol Chem. 2011 Mar 30. [Epub ahead of print]

Mitochondrial dynamics in axons of hippocampal pyramidal neurons

llustrated is the photoconversion of a PS-CFP2 fluorescent protein fusion product with human beta-actin using a 405-nanometer diode laser for imaging and conversion, as well as the argon-ion 488-nanometer spectral line for imaging and tracking of the photoconverted protein.

Photoconversion of a PS-CFP2 fluorescent protein as a way to study protein polymers

Examples of in vivo FP expression

How can we introduce plasmids that are large circular DNA molecules into the cells?

Transfection and infection as methods to introduce gene constructs

Direct microinjection into cultured cells or nuclei

Physical Transfection Methods

Electroporation

Biolistic particle delivery

Physical Transfection Methods

Single Cell Electroporation (SCE)

Physical Transfection Methods

Various transfection technologies based on chemical reagents

Chemical Transfection Methods

Flowchart of the main procedures of Ca2+ phosphate transfection protocol

Chemical Transfection Methods

Formation of optimal DNA/Ca2+-phosphate precipitate and subsequent dissolution to stop transfection

Chemical Transfection Methods

Viral infection as a way to deliver genetic materials into the cells

Virus infection technique

Rat neocortical neurons infected with recombinant Sindbis virus-enhanced green fluorescent protein in vivo

A) Image of the injection site (arrow) and surrounding area (postnatal day [P] 11). (B) Higher magnification images (P 14). Left, layer 2/3 pyramidal neurons. Middle, layer 5 interneuron. Right, layer 5 pyramidal neurons. (C) 2-photon laser scanning microscopy image of a layer 2 pyramidal neuron in a section from a mouse (P 36).

Lern. Mem. 7, pp. 433-441, 2000.

(a1) Overlay of fluorescent and phase-contrast images illustrating a pair of transfected (green) and nontransfected neurons. (a2) Double whole-cell patch-clamp recording of the pair of neurons shown in (a1). Upper trace shows presynaptic current and lower trace the corresponding postsynaptic current. (b) A pair of neurons both transfected with EGFP.

Transfection of hippocampal neurons in low-density microisland cultures

Fast protein translocation study by means of GFP

Eur. J. Neurosci. (2010) 32:347

• Conclusions• The battery of fluorescent proteins, tools for their

genes delivery and imaging tools enables the cell biologists to readily monitor protein dynamics in living cells, continuing to provide numerous new insights into the behavior of proteins, organelles, and cells. In so doing, these remarkable probes have ushered in a new era of cell biology in which both steady-state and kinetic microscopy techniques can be used to decipher pathways and mechanisms of biological processes.

• Basics of FRET

• Combining FRET with molecular biology techniques

• Introduction to optogenetics

Lecture Plan

Can we study inter- and intramolecular interection in living

cells using GFPs?

Do we have enough spatial resolution with our conventional

microscope systems?

Optical microscopy spacial resolution

Compound optical microscopes are limited in their ability to resolve fine details by the properties of light. Optical microscope resolution depends upon the microscope objective and refraction properties of medium between the object and objective lens altogether characterized by the objective numerical aperture, AN. At the certain wavelengths of light (λ), the maximal resolution (d) is given by:

In case of λ = 550 nm (green light) and air as the medium, (the highest practical AN is 0.95), the maximal resolution is about 0.3 micrometers. At the same time due to aberrations, even the best optical microscope is limited to a resolution of around 0.5 micrometers.

http://www.olympusfluoview.com/applications/fretintro.html

http://www.olympusfluoview.com/applications/fretintro.html

The efficiency of FRET, EFRET, which is defined as probability of the occurrence of energy transfer per donor excitation event, is a steep function of the distance between the fluorophores, r, as given by the following equation:

EFRET

where Ro is the Forster distance

http://www.olympusfluoview.com/applications/fretintro.html

Experimental Setup for Ratiometric FRET Imaging

DualView

CCD camera

Inverted Microscope

The MAG Biosystems™ Dual-View utilizes a single beamsplitter to split the incident beam from the microscope into two independent beams. One beam contains all the emission reflected off of the beamsplitter; the other contains all the emission transmitted through the beamsplitter. Each of these emission channels is projected onto half of the CCD array at exactly the same point in time. Simultaneous multichannel imaging is essential to achieve quantitative emission ratiometric analysis.

http://www.olympusfluoview.com/applications/fretintro.html

Cameleons have been devised by Roger Tsien and others. They are based on fluorescence resonance energy transfer (FRET) between two fluorescent molecules that are linked by a short stretch of calmodulin, a protein that changes its shape in the presence of calcium. In the absence of calcium the two fluorescent proteins are

well separated. These calcium sensors are called Cameleons because they change color and have a long tongue (calmodulin) that retracts and extends in and out of its mouth when it binds and releases calcium.

Cameleon – FRET based Ca2+ probe

Cameleon – основанный на FRET Ca2+ зонд

Tsien’s Lab

Design and plasma membrane expression of VSFP2s

PLoS ONE, doi:10.1371/journal.pone.0000440

Protein kinase G activity measured by FRET

Tsien’s Lab

FRET summary movie

• Conclusions• Fluorescent proteins together with current biophysical

and molecular biology approaches are the powerful tool allowing to study practically any type of inter- and intramolecular interection in living functional cells and thereby they can be used to decipher pathways and mechanisms of biological molecular signaling processes.

• Can we not only study molecular signaling processes in living cells using optically active proteins but also control these processes in cell specific manner?

Optogenetics

Nature web site

Conclusions

• Optogenetics is a new experimental methodology based upon the combination of genetic and optical methods to control specific mainly electrical events in targeted cells of living tissue.

• Optogenetics allows to almost instantaneously switch on and off certain neuronal groups in order to study their functions and relationships with other cells of animal body.

• Optogenetics also allows to correct pathological changes of signaling processes being a prerequisite for treatment of many neurodegenerative disorders.