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Getting Organized
how bacterial cells move proteins and DNA
Martin Thanbichler and Lucy Shapiro
Nature Reviews 2008
ZIB presentation
January 10, 2011
Sarah Hinkelmann
Prokaryotic versus eukaryotic cells
Components of the eukaryotic cytoskeleton
Microfilaments
• G-actin monomers form F-actin filaments, show polarity
• Involved in cytokinesis, muscle contraction, cell motility
Intermediate filaments
• Can form dimer or tetramer, no polarity
• Give cellular stability, involved in cell-cell connection
Microtubules
• α- and β- monomers form dimer
• Involved in mitosis, cytokinesis,
vesicular transport
• Show polarity
Model systems in bacterial cell biology
Escherichia coli
• Long history as a bacterial model organism.
• Large collection of genetic tools, mutant strains and methods.
• Extensive body of knowledge on many aspects of its physiology.
Bacillus subtilis
• Sporulation as a model for cellular differentiation.
• Well-studied physiology.
• Large size, which facilitates the resolution of cytoskeletal structures.
Caulobacter crescentus
• Asymmetric cell division.
• One round of chromosome replication per cell division.
• Abundance of dynamically localized regulatory protein complexes.
1. Subcellular organization in bacteria
Two protein classes for subcellular organization:
1. Proteins forming large stationary complexes
2. Proteins that are part of highly dynamic interaction
networks
1. Subcellular organization in bacteria
1. Proteins forming large stationary complexes
• Based on integral membrane proteins
• Mediate establishment of cellular organelles, perform catalytic or
regulatory functions
• How do these proteins assume a defined intracellular position?
• Two localization mechanisms in bacteria:
1. Diffusion and capture
2. Targeted membrane insertion
1. Subcellular organization in bacteria
Diffusion and capture:
• Newly synthesized proteins are first inserted randomly.
• Diffusion takes place
• Proteins are captured by interaction with previously localized
membrane complexes.
• e.g. during sporulation of B.subtilis
1. Subcellular organization in bacteria
Diffusion and capture:
•
•
SpoIVFB = protein-processing enzyme
synthesized in mother cell, but
destination is septal membrane
1. Random positioning in cytoplasmic
membrane
2. Diffusion
3. Capturing by forespore protein SpoIIQ
1. Subcellular organization in bacteria
Diffusion and capture:
• Newly synthesized proteins are first inserted randomly.
• Diffusion takes place
• Proteins are captured by interaction with previously localized
membrane complexes.
• e.g. during sporulation of B.subtilis
Targeted membrane insertion:
• A protein is delivered directly to its destination by translocation to a
given cellular site.
1. Subcellular organization in bacteria
Targeted membrane insertion
•
•
•
•
SpoIIQ = protein that is
synthesized in the forspore
acts as localization factor
that recruits SpoIIIAH
(mother-cell protein) to the
forespore septal membrane
then additional factors are
synthesized and recruited
 Zipper mechanism
1. Subcellular organization in bacteria
2. Proteins that are part of highly dynamic interaction
networks
•
•
•
Adopt specific overall arangements
Provide force and directionality
Serve as tracks for the localization of other proteins
•
magnetosomes
1. Subcellular organization in bacteria
Example 1: linear arrangements of subcellular compartments
Magnetosome
• Vesicle that is filled with biomineralized magnetite or greigite
• Allows bacteria to orient themselves in the Earth‘s magnetic field
• Magnetosomes are arrayed in linear order parallel to the longitudinal
axis of the cell and centered at mid-cell
• Generates a compass needle-like structure
• Have an inherent tendeny to agglomerate to reduce their magnetostatic
energy
• The cell possesses specific mechanisms to stabilize the linear assembly
and anchor it within the cell.
1. Subcellular organization in bacteria
Example 1: linear arrangements of subcellular compartments
•
•
MamK = bacterial actin homologue
MamK polimerizes to form filamentous
structure
•
MamJ = involved in attaching
magnetosomes to the MamK filament
•
MamK functions as a track that recruits
vesicles with the help of MamJ
a) Empty and immature vesicles are randomly distributed along MamK filaments
a) Magnetostatic interactions between individual vesicles lead to their
aggregation into densily packed chains
2. Dynamic protein scaffolds and cell shape
Example 2: actin-like cytoskeleton of bacteria
Protofilament
• The basic polymeric unit of a
filamentous structure
• Consists of a linear row of
monomers
Treadmilling
• Actin protofilaments possess an
intrinsic polarity:
• Monomers are added at one
end and released from the other
end
MreB
• Actin-like proteins
• MreB cables are highly dynamic and assume several different architectures
spiral-like assembly, short coils, arcs, rings
2. Dynamic protein scaffolds and cell shape
•
MreB cables exhibit a highly dynamic localization pattern
•
Newborn cells contain spiral-like cables that extend between the two poles
•
During cell division these structures are lost and MreB condenses into a ring
at the future division site
•
Later MreB localization extends again, leading to new spirals in the two
incipient daughter cells
2. Dynamic protein scaffolds and cell shape
•
Actin-like cables consist of numerous treadmilling filaments that are
arranged side by side in a random orientation.
2. Dynamic protein scaffolds and cell shape
Example 3: regulation of cell-wall biosynthesis by actin-like proteins
•
Shape of bacterium is determined by the architecture of its cell wall
Peptidoglycan
• Rigid meshwork that is formed by linear glucan strands that are crosslinked
by short peptide bridges
Peptidoglycan biosynthesis:
• Assembly of a lipid-bound disaccharide-pentapeptide precursor
Five-amino acid peptide (interconnects neighbouring glycan
strands)
N-acetylglucosamine (GlcNAc)
N-acetylmuramic acid (MurNAc)
2. Dynamic protein scaffolds and cell shape
•
•
•
•
Precursor is transported across cytoplasmic membrane and released from lipid
carrier
Incorporation into peptidoglycan superstructure  accomplished by penicillinbinding proteins (PBP) = transglycosylase and transpeptidase
Transglycosylase = catalyzes the formation of another β-1,4-glycosidic bond
between precursor and existing glycan strand
Transpeptidase = pairwise peptide-bonds are formed
2. Dynamic protein scaffolds and cell shape
•
•
•
•
High-molecular weight PBP = contains both enzymes
Low molecular weight PBP = only transpeptidase
Growth of bacterium requires continuous remodelling of peptidoglycan
envelope
Bacteria have own autolytic enzymes to change size and shape of molecular
meshwork
3. Bacterial DNA segregation
• Replication of DNA is not a random process
• Active separation of the two copies and positioning in the daughter
cell takes place
• Partitioning relies on the activity of three different plasmid encoded
factors:
• a centromeric sequence
• a centromere-binding protein
• an ATPase that interacts with centromeric nucleoprotein
complexes
3. Bacterial DNA segregation
Example 1: plasmid segregation by actin-like proteins
•
ParM = actin-like homologue
•
ParM filaments do not perform
treadmilling, but continuously
cycle between phases of rapid
growth or complete disassembly
•
Only ATP-bound form
polymerizes efficiently
•
If integrated into a filament then nucleotide hydrolysis takes place
•
The ends continue to grow as long as there remain ATP-bound subunits
•
Then either catastrophe or stabilization takes place
3. Bacterial DNA segregation
•
ParR nucleoprotein complexes recruit
ParM filaments and promote extension
•
As a consequence plasmids are
pushed apart and moved to opposite
ends of the cell.
4. Division-site placement
The Min system
•
FtsZ = tubulin homologue
•
MinD: ATPase, interacts with MinC to form a
membrane-associated inhibitor of the FtsZ-ring
formation
•
MinE restricts MinCD complex to the polar regions
of the cell
•
Cell division occurs only close to the cell centre.
4. Division-site placement
The Min system
1. Assembly into a large polymeric patch
2. Cap-structure starts to shrink
3. Complete dissapearance
4. Concomitantly, a new MinCD assembly forms at
the opposite pole and the circle starts again
5. MinE circular structure follows the retracting edge
of MinCD
Summary I
1. Subcellular organization in bacteria
•
There are two classes of proteins: they build stationary or highly
dynamic complexes
•
Stationary complexes: diffusion and capture or targeted membrane
insertion
•
Dynamic complexes: actin-homologues function by maintaining
cellular compartments (magnetosomes)
•
 Mam-proteins
2. Dynamic protein scaffolds and cell shape
•
Actin homologues are involved in cell division or regulate cell-wall
biosynthesis
•
 MreB-proteins
Summary II
3. Bacterial DNA segregation
•
Actin-homologues have `pushing´ forces to separate the DNA
•
 Par-proteins
4. Division-site placement
•
Tubulin homologues and ATPases are involved in cell division
•
 Min- and FtsZ-proteins
Thank you for your attention !
2. Dynamic protein scaffolds and cell shape
Role of MreC in bacterial morphogenesis
• MreC can form polymeric structures
• Its inactivation results in loss of cell shape and lysis
• Interacts directly with peptidoglycan synthase pecillin-bindingprotein 2 (PBP2)
• This proteins serves as a scaffold for the formation of a multienzyme peptidoglycan biosynthetic complex, thereby organizing the
formation of new cell wall material.
• Guides peptidoglycan-synthesizing enzymes to the site of active
cell-wall growth
2. Dynamic protein scaffolds and cell shape
Crescentin:
• Protein that shares the structural characteristics of
intermediate filament proteins
• Acts as a modulator of cell shape
• Spontaneous polymerization which does not require
nucleotides (ATP)
• Tends to associate laterally into small bundles
• Responsible for establishing cell curvature
3. Bacterial DNA segregation
Example 2: Arrangement of chromosomal DNA
•
Chromosomal DNA is not distributed randomly but has a highly conserved
organization
•
Segments of chromosomal DNA are folded into supercoiled domains that
are stacked on top of each other and arranged into circular
superstructure.
•
Subcellular position of a locus correlates with its location on the circular
chromosomal map
3. Bacterial DNA segregation
Example 2: Arrangement of chromosomal DNA
•
C. crescentus : origin of replication (ORI) is found at the flagellated pole,
terminus is located at the opposite site of the cell
•
E.coli: ORI and terminus regions are placed at the cell centre and the two
arms of chromosome form separate domains that are located on opposite
sides of its transverse axis.
3. Bacterial DNA segregation
Plasmid segregation by a tubulin homologue
• TubZ = shows similarity to tubulin and is essential for
partitioning
• Assembly into highly dynamic filaments that translocate
rapidly through the cell
• Filament migration is achieved by an actin-like
treadmilling mechanism.
3. Bacterial DNA segregation
Chromosome segregation
• Cytoskeletal protein MreB is involved, it interacts with a chromosomal
region that flanks the orign of replication (in C. crescentus)
• ParB spreads into flanking chromosomal regions, forming
nucleoprotein complexes, these complexes then aggregate into a
single centromere-like superstructure in a ParA-dependent manner
• Protein assembles into a dynamic polymeric structure that seems to
pull the moving ParB-parS complex from the old pole towards the
new pole. ParA stretches throughout the cell mitotic like process
4. Division-site placement
Regulation of cell division by MipZ
•
Some organisms lack the MinCDE system
•
MipZ = ATPase, interacts with chromosome-partitioning
protein ParB
•
ParB binds to a cluster of ParS
•
Together with the origin region the complex is positioned
at the old pole in newborn cells
•
DNA replication: two copies of ParS segments are
generated, where MipZ and ParB directly bind to
•
One segment complex stays at the old pole, second
segment moves to new pole
•
MipZ = inhibitor for FtsZ polymerization
•
FtsZ is there, where the lowest concentration of MipZ is,
thus FtsZ moves to the cell centre when MipZ-ParB
complex moves to other pole
•
Then cytokinesis can be initiated