The Colourful World of Fluorescent Proteins
The Colourful World of Fluorescent Proteins
Presented by: Caroline Sepiol Federici et al., 2012
3D projection of a Z-stack of confocal images of fluorescent
Arabidopsis thaliana leaf cells. A green fluorescent marker is used
to mark cells in blue and autofluorescence from chloroplasts is
shown in red.1
The Green Fluorescent Protein and Variants
life.bio.sunysb.edu; brainwindows.wordpress.com; Chalfie et al.,
1994; bcgsc.ca; Zimmer, 2009
A Small List of Variants
http://www.uniprot.org/uniprot/?query=gfP&sort=score
The Applications of Fluorescent Proteins
Arun Sampathkumar and Elliot Meyerowitz (unpublished) ; Livet et
al., 2007Fluorescent proteins enable the detection of proteins,
organelles and cells
Their applications are nearly endless. For instance they can be
used to study cancer, cell migration and connections.
They also can be used in both plants and animal cells.
A depth color-coded set of aligned confocal Z-sections of
microtubule arrays (mCherry:TUA5) in Arabidopsis leaf epidermal
pavement cells, which exhibit complex polarity.
4
The Brainbow Experiment
Litchman et al. 2008
The Brainbow Experiment
Litchman et al. 2008
Why Fluorescent Proteins Glow? Heim et al., 1994
Why Fluorescent Proteins Glow?
Webb and Miller, 2012
Photoconvertible Fluorescent Proteins
Shaner et al., 2007; Wiedenmann et al., 2006; Chudakov et al.,
2004
Rabbit Kidneys Cells; PS-CFP-hDAT tracking within filopodia of
HEK293 cells9
Photoactivable Fluorescent Proteins
Piatkevich and Verkhusha, 2010; Shaner et al., 2007; Victoria et
al., 2010
opossum kidney epithelial cells.10
Photoswitchable Fluorescent Proteins
Shaner et al., 2007; Fedor et al., 2010
opossum kidney cells. Dronpa
The movie is represented by the row of images from left to
right: red fluorescence (in red pseudocolor) image; yellow
fluorescence (in green pseudocolor) image; and the overlay between
the two. The movie contains 6 frames per one cycle in the total of
11 cycles. The data refer to the bacterial colony expressing
rsTagRFP-EYFP fusion. Note that the rsTagRFP acceptor red
fluorescence kindling is accompanied by the EYFP donor yellow
fluorescence quenching.11
*NEW* Fluorescent TimersLimited group of proteinsSlowly changes
emission from green to red or blue to red over a time period
Subach et al., 2009
created three fluorescent timers based on mCherry with slow,
medium and fast maturation rates.In this image, the clock numbers
are colonies of bacteria that have been expressing the fast
fluorescent timer protein for the number of hours indicated by the
clock numeral, yielding a real-time display of the conversion from
blue to red fluorescence12
The Ideal Candidate Checklist Expressed efficiently Bright
enough to be detected and imaged Stable fluorescence and integrity
No interaction with the protein of interest Minimal
interferenceShaner et al., 2005
EosFP and its two colours
This fluorescent proteins is photoconvertible and it is found in
stony coral Lobophyllia hemprichii .
The protein in its native state exhibits a green fluorescence
with an emission maximum at 516 nm. Following violet-blue light
exposure, the chromophore undergoes an irreversible
photoconvertible reaction and emits a red fluorescence at 581
nmWiedenmann et al., 2004; Nienhaus et al., 2005
The Ideal Candidate ChecklistFor EosFP:
Expressed efficiently Bright enough to be detected and imaged
Stable fluorescence and integrity No interaction with the protein
of interest Minimal interference
How EosFP Detects Movement and Creates ImagesLaser Scanning
Confocal Microscopy
McKinney et al., 2009
HeLa cells16
How EosFP Enables the Detection of Movement and Assists in
Creating ImagesFluorescent Recovery after Photo-bleaching
Mathur et al., 2010
17
How EosFP Enables the Detection of Movement and Assists in
Creating ImagesPhotoactivated Localization MicroscopyHow it
works:
http://zeiss-campus.magnet.fsu.edu/articles/superresolution/palm/introduction.html
How EosFP Enables the Detection of Movement and Assists in
Creating ImagesHow EosFP Detects Movement and Creates Images
Betzig et al., 2006
Acknowledgements Dr. KohalmiTravis HowesAndra Jejeran Dr.
Menassa
