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 Mi

n proteins dynamics Modeling:

Quantitative description How and why Min proteins regulate accurate cell division in

E. coli Open questions

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 1.5 microns in length

Length of cell cycle: ~ 1 hr From: cwx.prenhall.com

E. coli life cycle

FtsZ ring:

polymerizes on membrane 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

FtsZ ring

Accuracy of cell division in E. coli

Division accuracy: .50 +/- .02 Placement of FtsZ ring: .50 +/- .01

Big Picture Quantitative understanding of the mechanism of cell division is

important, since it is a fundamental cellular process of practical importance in development of anti-virulence

strategies

Eukaryotic (us) and prokaryotic (bacteria) cell division proceeds very differently; focus on simpler bacterial systems.

How does a single bacterial cell establish a “meterstick”?

Localization of cell division apparatus is an example of the generic problem of spatiotemporal localization of proteins inside bacterial cells to coordinate the cell’s functions.

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 MinE

Recruited by MinD:ATP onto membrane

induces ATP hydrolysis (ATPADP)

Black: MinC Red: MinD Blue: MinE

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:

Hal

e et

al.

(200

1)

MinD-GFPMinD-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.H

ale

et a

l . (2

001)

MinE-GFPMinE-GFP

Wavelength of oscillations is ~10 microns.

Ha l

e et

al .

(200

1)

MinE-GFPMinE-GFP

Filamentous cell has “zebra stripe” pattern of oscillations

MinD-GFPMinD-GFP

Raskin and

de Boer (1999)

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)

membranein ATP:MinD:MinE

membranein ATP:MinD

cytoplasmin MinE

cytoplasmin MinD

de

d

E

D

dedeEdEde

dt

d

EdEdedeEEE

dt

d 2D

ATPDdeddDDEdEd

dt

d:)]([

s

ss

ss

ED

deE

dDD

2

3

3

m 5.2

1 8.0;

m 16.0

m001.0;

m025.0

DD

ATPDdeddDD

ATPADP

ADPDATPDD

ATPD

dt

d

:

::

2:

)]([

D

ATPADP

ADPDdedeADPDD

ADPD

dt

d

:

:2: D

Result: MinD/E movie

MinE

MinDMinD

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 low.(from Huang and Wingreen)(from Huang and Wingreen)

((4 micron cell) 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 on the membrane at the middle.

MinC dynamics simply follows MinD dynamics.

MinC inhibits FtsZ ring formation.

Selection of “intrinsic” length scale by cell:

tikziii etx 0),(

Linear stability analysis around homogeneous solution:

Red curve corresponds to a normal cell

1/kmax ~ cell dimension below which there are no oscillations

Selection of “intrinsic” length scale by reaction-diffusion mechanism: Turing pattern formation

Turing instability:   diffusive, symmetry-br

eaking instability  characterized by intrins

ic wavelength (limit of large system size): "meter-stick“

Dimensional analysis L=(D/σ)^(1/2)

[D]=[L^/T], [σ]=[1/T]

Recent Developments “Recent” experiment indicate helical morphology of MinD polymers on memb

rane

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)

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

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 There are now established quantitative models of di

vision site placement in E. coli which do not rely on prescribed (and potentially unknown) topolo

gical protein markers achieved through spontaneous dynamic instability

E. coli’s oscillating Min system is NOT a universal solution; for example, B. subtillus: MinCD-DivIVA system, no oscillations, capabl

e of symmetric/asymmetric division C. crescentus: no Min system, capable of symmetric/asym

metric division

Physicists can contribute to the quantitative understanding of fundamental biological processes!

And thanks to Ned Wingreen (Princeton U.) for sharing some material for this talk with us.

Thank you!

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

Hu

Hu et al.

et al. (2002) (2002)

Min proteins in spherical cells:Neisseria gonorrhoeae

Wild typeWild typeMinDMinDNgNg

--

Sze

to

Sze

to e

t al

et a

l . (2

001)

. (2

001)

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 Marston et alet al. (1998). (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.