Apology from Zhenbiao Yang

					       Apology from

      Zhenbiao Yang
University of California-Riverside
  Center for Plant Cell Biology
                &
 China Agricultural University
      Zhenbiao Yang’s Lectures
          August 9, 2005

•Lecture 1: Methods in Plant Cell Biology

• Lecture 2: The Cytoskeleton in Cell Polarity
and Morphogenesis

•Lecture 3: G Proteins/Rho and ROP GTPases

•Lecture 4: ROP Signaling Networks in Polar
growth
References:
•   Szymanski DB.2005. Breaking the WAVE complex:
    the point of Arabidopsis trichomes. Curr Opin Plant
    Biol. 2005 Feb;8(1):103-12.
•   Gu Y, Wang Z, Yang Z. 2004. ROP/RAC GTPase: an
    old new master regulator for plant signaling. Curr
    Opin Plant Biol. 7:527-36
•   Yang, Z. 2002. Small GTPases: Versatile Signaling
    Switches in Plants. Plant Cell 14: S375-388.
•   Fu Y, Gu Y, Zheng Z, Wasteneys G, Yang Z. 2005.
    Arabidopsis interdigitating cell growth requires two
    antagonistic pathways with opposing action on cell
    morphogenesis. Cell. 2005 Mar 11;120(5):687-700.
•   Gu Y, Fu Y, Dowd P, Li S, Vernoud V, Gilroy S, Yang
    Z. 2005. A Rho family GTPase controls actin
    dynamics and tip growth via two counteracting
    downstream pathways in pollen tubes. J Cell Biol.
    2005 Apr 11;169(1):127-38.
Genes, RNAs, proteins, metabolites, and ions
 (Genetics, molecular biology, biochemistry,
              ion physiology)




             Cellular/subcellular
           processes (Cell Biology)




     Growth    Development    Responses to
                              Environment
                    Cell Biology:
 An interface between studies at molecular levels and
physiological, morphogenetic and developmental levels
    Internal Organization of the Cell
    --Organellar and sub-organellar structures: functions and
    dynamics
    --Mechanisms for the biogenesis of subcellular structures
    --Mechanisms for the dynamics of subcellular structures
    --The cytoskeleton: functions and regulation
    Cell growth, division, and development
    --Cell cycle control
    --Cytokinesis/cell division
    --Cell polarity/polar growth/morphogenesis

   Cells in Their Social Context
   --Cell walls (extracellular matrix)
   --Cell-cell communication
   --Signaling networks
 Why Plant Cell Biology?
Plant-specific cellular processes,
      Cell wall biogenesis and function in cell-cell
             communication
      Chloroplast biogenesis
      Organization of microtubules without
      centrosomes
      Pollen development
      Self-incompatibility

Fundamental cellular processes
     Protein degradation regulation (signalosome)
     Apical cell growth (tip growth)
     Intercalary cell growth
     Pollen tube growth guidance
     Leaf pavement cells




•Intercalary cell growth is also present in animals
                                Fine F-
                                 Actin
                                          Lobe
          RIC4
        ROP
                                                         Neck
        GTP RIC1
ROP
GDP                                                     RIC1
           Spatial Cue                           RIC1


      Localized ROP2 activation           MT

        RIC4             RIC1             ?

      Localized    Transverse MT
      Actin MFs     arrangement


      Lobed pavement cells           Fu et al. 2005. Cell 120:687-800
Cell Preview
Intercalating Arabidopsis Leaf Cells: A Jigsaw Puzzle
of Lobes, Necks, ROPs, and RICs

Jeffrey Settleman, MGH Cancer Center and Harvard
Medical School, Charlestown, Massachusetts 02129
Available online 10 March 2005.

Intercalation of cells is an evolutionarily conserved
strategy used for a variety of developmental processes in
animals. In this issue of Cell, Fu et al. have uncovered an
elaborate Rho GTPase-mediated mechanism by which
cytoskeletal-dependent intercalation of Arabidopsis leaf
cells is achieved, suggesting that conserved Rho GTPase
signaling pathways may similarly regulate tissue
morphogenesis in animals and plants.
                    Cell Biology:
 An interface between studies at molecular levels and
physiological, morphogenetic and developmental levels
    Internal Organization of the Cell
    --Organellar and sub-organellar structures: functions and
    dynamics
    --Mechanisms for the biogenesis of subcellular structures
    --Mechanisms for the dynamics of subcellular structures
    --The cytoskeleton: functions and regulation
    Cell growth, division, and development
    --Cell cycle control
    --Cytokinesis/cell division
    --Cell polarity/polar growth/morphogenesis

   Cells in Their Social Context
   --Cell walls (extracellular matrix)
   --Cell-cell communication
   --Signaling networks
         Lecture 1
Methods in Plant Cell Biology
           How are cells studied?
Cellular structures are small, complex and dynamics.
How to visualize the structures and their dynamics and
how to study their functions?
  --Microscopy
  --Imaging
      •Marker development and introduction
      •Digital imaging and analysis
  --Biochemical analysis
      •Separation and isolation of structures and proteins
      •Co-immunoprecipitation/complex purification
  --Genetic studies
      •Alteration of specific genes or proteins
  --Genomics and proteomics approaches
      •High throughput studies of many genes or the whole
      genome
Topics to be covered

Essentials in microscopy
Confocal laser scanning microscopy
Real-time confocal microscopy
Live marker technologies
Fluorescence recovery after photobleaching (FRAP)
Fluorescence resonance energy transfer (FRET)
      Applications of FRET
              Microscopy
              Light microscopes (resolution-0.2 µm)
              •       Bright field--white light
              •       Epifluorescent--fluorescent light
              •       Confocal--laser
              Technologies to improve light microscopy
              resolution
              •       Fluorescence tagging/marker
chloroplast
mitochodria   •       Total internal fluorescence reflection
                              microscopy

              Electron microscopes (theoretically 1A)
                 •Transmission (TEM)-internal
                 structure/2-D view
                 •Scannng (SEM)-surface/3-D view
Milestones in LM
  Plant cell
   Nucleus
   (orchids)




 Imaging of proteins


 Confocal microscopy
Basic Light Microscope
Bright-field                Epifluorescent




               A fluorescent compound absorbs light with a specific
               wavelength (e.g, blue) and emits fluorescent light
               with a longer wavelength (e.g. green)
Limitations with conventional epifluorescent microscopy
   •Image blurring due to deflection of fluorescent light
   from out-of-focus images.
   •Inability to detect images from thick tissues
   •Inability to obtain a high resolution 3-D image
Confocal Laser-Scanning Microscopy (CLSM)
                   Epifluorescent   Confocal




CLSM
 High resolution
CLSM allows reconstruction of 3-D images from laser-scanning sections




  Laser scanning sections (2 µm thick)




                                              Reconstructed 3-D image
Limitations of CLSM
•   Line scanning is slow
•   3-D images come from reconstruction of
          many laser sections--one 3-D image
          can take up to several minutes
•   Unable to obtain real-time 3-D images
Spinning disc-based real-time
    confocal microscopy




             QuickTime?and a
         TIFF (LZW) decompressor
      are needed to see this picture.
                    Cellular Imaging
Why?
--Structures are too small to be seen with bright-field microscope
--Localization of a specific molecule in the cell
--Quantification of a specific molecule in the cell
--Spatiotemporal dynamics of a molecule or structure

Most structures and molecules in the cell do not fluoresce or
do not have a unique optic property

Imaging depends on the use of markers
-Some structures can be stained with a specific dye
-Use of a fluorescent indicator (e.g., Fura-2)
-Immunocytochemical methods
-Proteins tagged with a fluorescence protein
Imaging of calcium using the fluorescent Fura-2 dye
 Free Fura-2: excitation-362 nm, emission-518 nm
 Calcium-bound Fura-2: excitation-335, emission-510 nm
 Ratio of 510 nm/518 nm intensities indicates calcium concentration




Ca2+ in neurons            Ca2+ oscillates during pollen tube growth
                           (spatiotemporal changes)
                      Cellular Imaging
 Why?
 --Structures are too small to be seen with bright-field microscope
 --Localization of a specific molecule in the cell
 --Quantitation of a specific molecule in the cell
 --Spatiotemporal dynamics of a molecule or structure

Most structures and molecules in the cell do not fluoresce
or do not have a unique optic property

 Imaging depends on the use of markers
 -Some structures can be stained with a specific dye
 -Use of a fluorescent indicator (e.g., Fura-2)
 -Immunocytochemical methods
 -Proteins tagged with a fluorescence protein
Imaging a protein using immunocytochemical methods




Cells are fixed       Primary antibodies are        Labeled second antibodies are
with a chemical     reacted with a target protein     reacted with the primary
                                                              antibody

     The second antibodies can be labeled (marked)with either
     •Gold particles (for TEM)
     •or a fluorescent dye
Two commonly used fluorescent dyes

   Fluorescein
      Is excited with blue light
      Emits green light



  Rhodamine
     Is excited with green-yellow light
     Emits red light


  These two dyes can be simultaneously
  used as markers for co-localization of
  two different molecules
A)                             B)                               C)
1st antibodies:                1st antibodies:                  A+B
     --Anti-Rop from rabbits        --Anti-annexin from rats
2nd ant-bodies:                2nd ant-bodies:
    --Anti-rabbit Igg               --Anti-rat Igg
    --2nd antibodies are            --2nd antibodies are
      labeled with fluorescein       labeled with rhodamine




   Rop GTPase                  Vacuolar Annexin                Co-localization
                      Cellular Imaging
 Why?
 --Structures are too small to be seen with bright-field microscope
 --Localization of a specific molecule in the cell
 --Quantitation of a specific molecule in the cell
 --Spatiotemporal dynamics of a molecule or structure

Most structures and molecules in the cell do not fluoresce
or do not have a unique optic property

 Imaging depends on the use of markers
 -Some structures can be stained with a specific dye
 -Use of a fluorescent indicator (e.g., Fura-2)
 -Immunocytochemical methods
 -Proteins tagged with a fluorescence protein
Live Imaging Using GFP (green fluorescence protein)

 What is GFP?
   From the Pacific jellyfish (Aequoria victoria)
   Spontaneously fluorescent
   27 kD (238 amino acids)
   Excited at 470 nm and emits at 528 nm.
   Active in all organisms

  GFP variants allow simultaneous imaging of several proteins
     Enhanced GFP (EGFP)
     Cyan GFP (CFP)
     Blue GFP (BFP)
     Yellow GFP (YFP)
     Red fluorescence protein (RFP-mDsRed, cherry RFP)
  Live Imaging Using GFP (green fluorescence protein)
Method: GFP gene is fused with a gene encoding a protein under
investigation, and the fusion gene is introduced into a cell or an organism

     GFP                 target protein

                  transformation



                                                      QuickTime?and a
                                                         decompressor
                                                are needed to see this picture.
                       imaging
Advantages over conventional imaging methods
  -Live imaging
   -Non-invasive
   -Allows the study of the structural and molecular dynamics
   -No need for antibodies
Live Imaging Using GFP (green fluorescence protein)

 What is GFP?
   From the Pacific jellyfish (Aequoria victoria)
   Spontaneously fluorescent
   27 kD (238 amino acids)
   Excited at 395-500 nm and emits at 508 nm-550.
   Active in all organisms

  GFP variants allow simultaneous imaging of several proteins
     Enhanced GFP (EGFP)
     Cyan GFP (CFP)
     Blue GFP (BFP)
     Yellow GFP (YFP)
     Red fluorescence protein (RFP, dsRed)
Triple labeling (DsRed-Mito, YFP-Tub, and CFP-Nuc)
Overlapping
excitation and
emission
spectra
among
different GFP
variants make
it technically
challenging to
conduct
colocalization
Key elements for GFP-based
colocalization studies

  •Selection of GFP variants/DsRed
  •Microscopes
  •Skills/experience
    •Expression levels for fusion proteins
    •Laser power
    •Emission gain control
    •Tricks for colocalization of GFP
    fusions with overlapping exicitation
    and emission spectra
Confocal microscopy has
high detection specificity
and sensitivity

          Excite       Emit
Conv.
           band      band path
Fluo

CLSM      Single λ    Spectral

BFP   370 nm          380-450
CFP   434/442        450-500
GFP 458              500-550
YFP   488            525-575
DsRed 554            575-625
Key elements for GFP-based
colocalization studies

  •Selection of GFP variants/DsRed
  •Microscopes
  •Skills/experience
    •Expression levels for fusion proteins
    •Laser power
    •Emission gain control
    •Tricks for colocalization of GFP
    fusions with overlapping exicitation
    and emission spectra
Experimental tricks for GFP/YFP colocalization
                     RIC1-YFP
αtubulin-GFP         (transiently
(Stably expressed)   expressed)       Merged




     RIC1 is colocalized with cortical MTs
Fluorescence resonance energy
transfer (FRET) analysis

Emission light (higher energy) from one
molecule can excite another molecule if the
second molecule can be excited by the
emission light and is within a distance of 50 A.
Thus FRET is used to detect physical
interaction between two molecules

Pairs of GFP proteins suitable for FRET
•CFP and YFP fusions
•GFP and DsRed fusions
CLSM   Single λ    Spectral

BFP   370 nm       380-450
CFP   434/442     450-500
GFP 458           500-550
YFP   488         525-575
DsRed 554         575-625
               Fluorescence resonance energy transfer (FRET)
                  analysis of in vivo ROP1-RIC4 interaction


                        Settings
                      CFP     YFP              ROP1     RIC4
                                         442
      ROP1            Ex: 442
442
                      Em:450 Em:570            CFP          YFP
      CFP                -525  -600


            450-
            525                Ex:514                             570-600
                                                  450-525
                      Em:570   Em:570
                        -600      -600
       RIC4
514
       YFP

            570-600                               FRET Settings
                                                  Ex:442
                                                  Em: 570-600
FRET analysis of in vivo interaction


            CFP-ROP1/YFP-RIC4




           CFP-DN-rop1/YFP-RIC4




             CFP-ROP1/YFP-RIC3




           CFP-DN-rop1/YFP-RIC3
Common applications of FRET analysis

•In vivo protein-protein interaction
•Protein activity in vivo
•Ion levels in vivo
Measurements of Cellular Protein Activity or Ion Levels
       Based on protein-protein interactions
Why my FRET experiment did
not work?
•No interaction in vivo
•Transient interaction in vivo
•Problems with fusion proteins
   • Inactive
   • The distance between the two
           fluorescence proteins is
           greater than 50 A
   Fluorescence recovery after
      photobleaching (FRAP)

•Continuously exciting a fluorescent molecule
can bleach the fluorescence
•Recovery of GFP fluorescence in a locally
photo-bleached area suggests movement of the
GFP fusion protein from unbleached area to the
bleached area
FRAP applications
    Detect movement of proteins
         between cells or subcellular
         compartments
    Measure rates and kinetics of protein
         movement
  A word of caution for GFP-based
              imaging
•Making a right construct
   •N-terminal, C-terminal, or internal fusion
•Functional test--complementation of mutants
•Expression levels
•Cell type- and time-specific localization

				
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