Chemoreceptor Clustering

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Evidence from microscopy that MCPs are clustered
Maddock Shapiro

• CheA and CheW are also clustered.
• Tight clustering of MCPs requires A and W.
• Clustering of W is dependent on MCP but not A.
• 4. Clustering of A is dependent on MCP and W.

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Validation by cryo-tomography
Zhang et al., PNAS 2007
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Why put all receptors in one spot (patch/cluster)?
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Berg Turner

• Cell pole marked with Tetrazolium Red dye
• Tracked single cells as they ran and tumbled
• Monitored Dye distribution relative to the
  flagellar bundle
• Result Dye position was randomized during
  tumbles
• Conclusion Cells swam with either end in front
  i.e clusters do not serve as a nose

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If there is an advantage to localizing
chemoreceptors in patches at only one pole, that
advantage must accrue at some step after signal
detection
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A trimer of cTsrQ dimers stereo views
Kim et al., Nature 1999
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Triangular CheA-CheW vault around Tsr
keystone
Shimizu, T. S. et al. Nat. Cell Bio. 2000
Each unit made of 3 MCP dimers, 3 CheA monomers,
and 3 CheW monomers
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A trimer of cTsrQ dimers stereo views
Kim et al., Nature 1999
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Chemoreceptor Clustering
Evidence from Genetics

• Ames et al. 2002. Collaborative signaling by
  mixed chemoreceptor
• teams in Escherichia coli. Proc. Natl. Acad. Sci.
  USA 99 7060-65.

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Triangular CheA-CheW vault around Tsr
keystone
Each unit made of 3 MCP dimers, 3 CheA monomers,
and 3 CheW monomers
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• 80-aa distal tip of receptor mediates ternary
  complex formation. This region also contains
  contact sites for a t-o-d in the crystal
  structure.

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Questions

• What specific effects do mutations in the trimer
  contact sites have on Tsr signaling ability and
  at what stage in the signaling pathway do they
  act?
• Does experimental evidence support the possible
  existence of mixed chemoreceptor teams?

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Experimental Methods

• Mutations were introduced at trimer contact
  residues of Tsr
• Tsr expression levels were monitored by
  immunoblotting with anti-Tsr antibody
• Chemotaxis was assessed by observing colony size
  in semisolid agar Flagellar rotation monitored
  using Ab-tethered cells to measure time spent in
  CW rotation

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Experimental Methods (Cont.)

• Receptor clustering was observed using
  fluorescence microscopy to visualize YFP-CheZ
  clusters
• Complementation studies were undertaken to study
  genetic patterns and relationships between Tsr
  mutants and wild-type Tsr or Tar chemoreceptors
• Dithiobis crosslinking agent was used to
  crosslink receptors in order to determine whether
  direct contacts exist between heterogeneous
  receptor dimers

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Tsr Trimer Contact Sites

• 11 different residues (6 hydrophobic, 4 polar, 1
  central H-bond network) in trimer contact sites
  to be studied

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Falke, 2002
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Amino Acid Substitutions
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With one exception, P mutants showed no CW
episodes and no clustering (expected) All but
one A and W mutants showed efficient clustering,
but half of them were lt5 CW defective in
kinase activation. The others were locked Only
two (V398A and R409A) mediated serine
chemotaxis
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Summary of Clustering and Flagellar Rotation
Results

• Symbols
• Wild-type
• Alanine mutants
• Proline mutants
• Tryptophan mutants

Filled 65 of wt serine chemotaxis Open lt35
of wt serine chemotaxis
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1. No cluster, no chemotaxis 2. Cluster, but no
kinase activation 3. Kinase activation, but no
regulation (locked)
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Falke, 2002
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Testing Interaction by Complementation

• Investigated the effects of mutant Tsr on
  wild-type Tsr and Tar in trimers by using the
  soft agar assays by co-expressing the proteins in
  the same cell, varying the amount of mutant
  protein by altering inducer concentrations

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Results of Complementation

• Four different complementation patterns
• Recessive Mutations have no effect on wild-type
• Dominant Mutations impair wild-type Tsr but do
  not affect Tar
• Epistasis Mutations impair both wild-type Tsr
  and Tar
• Rescue Wild-type Tar restores serine chemotaxis
  in mutant Tsr

Epistasis An interaction between nonallelic
genes, especially an interaction in which one
gene suppresses the expression of another
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Complementation Patterns on Soft Agar Plates
A
P
W
A

• Outer ring of the rescuable mutants shows that
  serine chemotaxis has been restored

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Falke, 2002
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What can the complementation patterns reveal
about nature of defect?
Dominant mutations did not affect Tar function,
so most likely these formed Tsr/Tsr dimers,
spoiling wt subunits through mixed dimer
formation. Epistatic mutations likely spoil a
functional high-order of the dimers Rescuable
mutations also implicate such a high-order of the
dimers
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Crosslinking

• Epistatic and Rescuable complementation patterns
  predict that it should be possible to crosslink
  Tsr and Tar in higher-order complexes

Co-express Tsr and Tar-His6 is cells having no
other MCPs. Crosslink. Purify Tar-His6 and see
if Tsr comes along
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Diagnostic cross-linking sites for the
trimer-of-dimer organization.
A mixed trimer-of-dimers containing one Tsr
molecule (yellow) and two Tar molecules (light
blue). Indicated residues chosen for cysteine
replacements.
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Crosslinking Results

• The lanes which show Tsr and Tar-H bands that
  copurify indicate that these chemoreceptors are
  close enough to crosslink (Provides evidence of
  mixed complexes of Tsr and Tar dimers)

S soluble membrane fraction E eluted from Ni
resin I377P is dominant, not epistatic no
XL I1377W F373W are epistatic yes XL
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Conclusions

• Effects of mutations in trimer contact regions
  give strong circumstantial evidence that trimer
  of dimers is important in signaling
• Proline replacements prevented clustering, CheA
  activation, and chemotaxis
• Alanine and Tryptophan replacements mediated
  signaling, but not CheA regulation or chemotaxis
• Occurrence of epistatic and rescuable phenotypes
  indicate that mixed complexes of Tsr and Tar
  dimers are likely
• Probable existence of mixed complexes confirmed
  from crosslinking experiments

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Unifying Model of Chemoreceptor Teams
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Nanodiscs
Boldog, T. et. a. PNAS 2006
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If there is an advantage to localizing
chemoreceptors in patches at only one pole, that
advantage must accrue at some step after signal
detection
34
Gain
Change in rotational bias/ Change in receptor
occupancy 60
Dennis Bray, PNAS, 2002
35
Triangular CheA-CheW vault around Tsr
keystone
Shimizu, T. S. et al. Nat. Cell Bio. 2000
Each unit made of 3 MCP dimers, 3 CheA monomers,
and 3 CheW monomers
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2-D Hexagonal network
A 36-fold gain at the receptors indicates that
one receptor molecule can control the activity
of three dozen kinase molecules, implying a
functional network that links one receptor to
multiple copies of the kinase. The response to
attractant stimulation is cooperative Hill
coefficients as high as 10 have been observed
.These properties imply that receptors operate as
allosteric arrays with as many as several dozen
in the cooperative unit.
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