How bacteria find their middle

							How E. Coli find its middle


   Journal Club talk by Xianfeng Song
       Advisor: Sima Setayeshgar
Outline

   Introduction to E. coli
   Regulation of division site placement by Min proteins
   Experiments: In vivo (qualitative) observations of
    Min proteins dynamics
   Modeling:
       Quantitative description
       How and why Min proteins regulate accurate cell division in
        E. coli
   Open questions
Big Picture

   Protein localization (in space and time) as a
    mechanism for regulating cell function in
    bacterial cells which are devoid of major
    organelles
       Examples of protein localization
About E. coli
                             E. coli is a bacterium
                              commonly found in the
                              intestinal tracts of most
                              vertebrates.
                             Studied intensively by
                              geneticists because of its
                              small genome size, normal
                              lack of pathogenicity, and
                              ease of growth in the
                              laboratory.
                             Size:
                                 0.5 microns in diameter
 From: cwx.prenhall.com          1.5 microns in length
                             Length of cell cycle: ~ 1 hr
E. coli life cycle



                                      FtsZ ring



 FtsZ ring:

    polymerizes at division site, provides framework for assembly
     of other cell division proteins
    constricts like a drawstring during cell division, splitting the cell
     in two; it disassembles after division
    accuracy of placement determines division accuracy
Accuracy of cell division in E. coli




     Division accuracy:      .50 +/- .02
     Placement of FtsZ ring: .50 +/- .01
Two systems regulate division site placement
   Nucleoid occlusion




   Min proteins
       Includes MinC, MinD, and MinE
    Function of Min Proteins
           (from experimental observations)
   MinC
     Inhibits FtsZ ring formation

     Recruited by MinD:ATP onto
      membrane
   MinD
     MinD:ATP stick onto membrane

     MinD:ADP tends to go into
      cytoplasm
     MinD:ATP recruits MinC and
      MinE to membrane               Black: MinC Red: MinD Blue: MinE
   MinE
     Recruited by MinD:ATP onto
      membrane
     induces ATP hydrolysis
      (ATPADP)
Min protein phenotypes
           (from experiments)
   Without Min proteins, get
    minicelling phenotype
    (Min-)


   If MinC is over-expressed,
    get filamentous growth,
    i.e., no division
    (Sep-)
MinD oscillations:




              MinD-GFP
MinE ring oscillation caps MinD polar region:


                                MinE ring is
                                 membrane bound.
                                Ring appears near
                                 cell center, moves to
                                 one pole, back to
                                 center, and on to next
                                 pole.



       MinE-GFP
Filamentous cell has “zebra stripe” pattern of oscillations




                                                    de Boer (1999)
                                                     Raskin and
                                  MinD-GFP

                                 Wavelength of
                                  oscillations is   ~10
                                  microns.

        MinE-GFP
Phenomenology of Min oscillations
               from in vivo observations
   MinD polar regions grow as end caps
   MinE ring caps MinD polar region
   Filamentous cell has “zebra stripe” pattern of
    oscillations
   Oscillation frequency:
       [MinE]   frequency 
       [MinD]   frequency 
   Oscillations require MinD and MinE but
    not MinC
Summary of modeling efforts
   Howard et al. (2001)
     Simple 1D model

     MinE is recruited by cytoplasmic MinD to membrane

     MinD polar region fails to reform at poles (does not agree with
      experiment)
   Meinhardt and de Boer (2001)
   Huang and Wingreen (2003)
     MinE is recruited by membrane-bound MinD:ATP

     MinD aggregation on the membrane follows a one-step process

   Kruse et al. (2005)
     Consider protein diffusion within the membrane

     MinD aggregation on the membrane follows a two-step process:
      first attachment to membrane, then self-assembly into filament
Huang and Wingreen (2003) Model
Governing equations (from Huang and Wingreen, 2003)

d D: ADP                                                    D  MinD in cytoplasm
           D D 2  D: ADP   de  de  D: ADP
  dt                                      ADP ATP           E  MinE in cytoplasm
d D: ATP                                                    d  MinD:ATP in membrane
           D D  2  D: ATP  D: ADP
  dt                           ADP ATP
                                                              de  MinE:MinD:ATP in membrane
                   [ D   dD (  d   de )] D: ATP
 d E                                                                     m 
                                                              D  0.025 
                                                                                              m 3 
                                                                              ;  dD  0.001
       D E  2  E   de  de   E  d  E                                                 s   
  dt                                                                       s                     
                                                                         m 3              1
 d de                                                        E  0.16 
                                                                         s   ;  de  0.8  
         E  d  E   de  de                                                           s
  dt
                                                                              m 2 
                                                                              s 
                                                             D D  D E  2.5        
 d d                                                                               
        E  d  E  [ D   dD (  d   de )] D: ATP
  dt
Result: MinD/E movie



             MinE




             MinD
Simulation results:
           MinD end caps and MinE ring
Mechanism for growth of MinD polar regions
               (according to Huang and Wingreen, 2003)

   MinD:ADP ejected from old end
    cap diffuses in cytoplasm.

   Slow MinD:ADP  MinD:ATP
    conversion implies uniform
    reappearance of MinD:ATP in
    the cytoplasm.

   Capture of MinD:ATP by old end
    cap leads to maximum of
    cytoplasmic MinD:ATP at
    opposite pole.
Model result I:
  Frequency of oscillations ~ [MinE]/[MinD]
                                   Relation:
                                       [MinE]   frequency ,
                                       [MinD]   frequency .


                                   Minimum oscillation
                                    period 25s.
                                       No oscillations for [MinE]
                                        too high, or for [MinD] too
    (from Huang and Wingreen)
                                        low.
      (4 micron cell)
Model result II:
    “Zebra stripe” oscillations in long cells




    Stripes form with wavelength of ~10 microns
Oscillations allow E. Coli to divide accurately

   The oscillations result in a minimum MinD
    concentration at the middle on the cell.
   MinC dynamics simply follows MinD
    dynamics.
   MinC inhibits FtsZ ring formation.
Selection of “intrinsic” length scale by cell:




               Red curve corresponds to a normal cell

Linear stability analysis around homogeneous solution:
                   
              i ( x , t )   i   i e
                                0         ikz  t


1/kmax ~ cell dimension below which there are no oscillations
Selection of “intrinsic” length scale by reaction-
diffusion mechanism: Turing pattern formation
Recent Developments
   “Recent” experiment indicate helical morphology of MinD polymers on
    membrane




               From Hu et al. (2002), Shih et al. (2003)

   Recent modeling efforts (mainly focus on cleaning some details, no
    breakthrough)
     Effect of fluctuating protein numbers (Howard, et al, 2003),
     Inclusion of membrane diffusion and more reactions (Kruse, et al, 2005)

     Min-protein oscillations in round bacteria (Huang and Wingreen, 2004)
Open questions (from the community)
   Role of helical
    polymerization of MinD
    on the membrane




   MinE ring reverses
    direction temporarily:
    stochastic effect?
Open questions from us

   Where does the precision of division come
    from? Why is it 4%, not 10% or 20%?
Conclusions
    Although bacteria such as E. coli is a very
     simple creature, it is also very complicated
     system.
    To understand this system, physicist can
     help a lot.
            Thank you!

And thanks to Ned Wingreen (Princeton U.) for
 sharing some material for this talk with us.
     Evidence from in
     vitro studies
A. Phospholipid vesicles
B. MinD:ATP binds to vesicles and
    deforms them into tubes
C.  MinD:ATP polymerizes on vesicles
D.  Diffraction pattern indicates
    well-ordered lattice of MinD:ATP
E. MinE induces hydrolysis of MinD:ATP
    and disassembly of tubes
Min proteins in spherical cells:
Neisseria gonorrhoeae
   Szeto et al. (2001)




                         Wild type   MinDNg
                                              -
Min-protein oscillations in nearly round cells
In E. coli, Min oscillations “target” MinD to
poles
Why does E. coli need an oscillator?

In B. subtilis, minicelling is prevented by MinCD
      homologs, but polar regions are static.




              Marston et al. (1998)
How E. coli find its middle
    Proteins are too small to see the caps’ curvature
    Subtilis have local proteins fixed at two ends, but
     E. coli does not have
Min protein phenotypes
           (from experiments)
   Without Min proteins, get
    minicelling phenotype
    (Min-)


   If MinC is over-expressed,
    get filamentous growth,
    i.e., no division
    (Sep-)
Predictions of model (Huang and Wingreen, 2003)


    Delay in MinD:ATP recovery is essential
     (verified by some experiments).
    Rate of hydrolysis of MinD:ATP by MinE
     sets oscillation frequency.
    Diffusion length of MinD before rebinding
     to membrane sets spatial wavelength.