Chemoreceptor Clustering

1
Chemoreceptor Clustering

• BIO 339M
• March 3rd, 2009 Lecture 10
• V. Nieto

2
Polarization in Caulobacter cresentus

• Inspiration for polar localization studies in E.
  coli came from Caulobacter crescentus, as well as
  eukaryotic cells.
• E. coli chemoreceptors were localized to the
  correct polar location when expressed in C.
  crescentus.

3
Sensing Efficiency

• Berg and Parcell theorized a even distribution to
  be the most efficient for sensing attractant and
  repellent molecules.
• For optimized sensitivity to half of the
  molecules that pass an E. coli cell, you would
  need 3000 receptors evenly spaced with a radius
  of 1nm.

4
Are MCPs clustered or evenly distributed?
5
How are you going to resolve this uncertainty?

• An electron microscope is a type of microscope
  that uses a particle beam of electrons to
  illuminate a specimen and create a
  highly-magnified image.
• Together with gold labeled polyclonal antibodies,
  an observer will be able to perform
  immunoelectron microscopy.

http//en.wikipedia.org/wiki/Electron_microscope
6
(No Transcript)
7
Negative Control.
8
Why did they indentify the location of MBP?
Positive Control.
9
In strains devoid of MCPs, where do CheA CheW
localize?
Diffuse in the cytoplasm.
10
81 of gold particles are localized to the
membrane, 80 of those membrane gold particles
are at the pole, and 81 of particles at the pole
are in clusters.
11
There is 9 times the amount of polar localized
particles when compared to nonpolar particles.
12

• Septating cells had a higher occurrence of
  clusters, but nonseptating cells had a higher
  occurrence of polar clustering.

13

• Polar clusters contained a larger amount of gold
  particles than nonpolar clusters.

14
More accurate visualization of CheA and CheW
requirement of MCPs for proper polar localization.
15
Will CheA and CheW Localize With Presence of MCP?
Tar
CheA
CheA
CheW
CheW
pTar
MCPs
MCPs pTar
After 30, and 90 minutes, CheA and CheW localize
to the poles and laterally.
16
Without the transmembrane domains, CheA and CheW
localize within inclusion bodies. Interaction is
independent of transmembrane domains.
17
Conclusions

• The majority MCPs, and MCP-CheA-CheW ternarary
  complexes are localized in clusters to the cell
  poles.
• Mutants void of MCPs are void of polar clusters,
  and decrease the amount of CheA and CheW at the
  poles.
• Mutants void of or with hindered expression of
  CheA or CheW effect MCP polar clustering as well.

18
Two Models

• Lateral Movement

• Polar Anchoring

• MCPs, along with CheW and CheA interact at the
  inner-membrane, and through lateral diffusion,
  reach the poles.
• Upon reaching the pole, the ternary complex
  associates and stabilizes its localization with
  other polar components.

• MCPs localize to cell pole, and randomly
  associate with diffuse CheW and CheA.
• Lateral localization and clusters occur from
  inappropriate dislodging and redistribution of
  the ternary complexes.

19
Does E. coli have a nose?

• Does one pole of the cell serve as the sensor,
  and the other serve as the propeller?
• Berg and Turner decide to tackle issue with use
  of polar marking using the granule forming dye,
  tetrazolium red.
• If the cell does have a predisposed nose, a
  single pole should be limited to one side.

20
Tetrazolium Red
21
(No Transcript)
22
Run intervals, whether in H or T orientation,
last for similar durations.
23
(No Transcript)
24
Conclusions

• Cells do not have a designated nose or
  orientation bias necessary for proper chemo-taxis
  or motility.
• Tumbles occur with equal probability regardless
  of the orientation of the cell body, meaning that
  which controls the direction of flagellar
  rotation is not sensitive to cell orientation.
• Runs of both orientations last for a similar
  amount of time.