getting organized how bacterial cells move proteins and dna martin thanbichler and lucy shapiro...

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. 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


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


• SpoIVFB = protein-processing enzyme• synthesized in mother cell, but

destination is septal membrane

1. Random positioning in cytoplasmic membrane

2. Diffusion3. Capturing by forespore protein SpoIIQ

Diffusion and capture:


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.


• 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

Targeted membrane insertion


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


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.


• 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

Example 1: linear arrangements of subcellular compartments

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


• 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


• Actin-like cables consist of numerous treadmilling filaments that are arranged side by side in a random orientation.


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

N-acetylglucosamine (GlcNAc)

N-acetylmuramic acid (MurNAc)

Five-amino acid peptide (interconnects neighbouring glycan strands)


• Precursor is transported across cytoplasmic membrane and released from lipid carrier

• Incorporation into peptidoglycan superstructure accomplished by penicillin-binding 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


• 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


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


• 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.


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 !


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-binding-

protein 2 (PBP2)• This proteins serves as a scaffold for the formation of a multi-

enzyme peptidoglycan biosynthetic complex, thereby organizing the formation of new cell wall material.

• Guides peptidoglycan-synthesizing enzymes to the site of active cell-wall growth


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


•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

Example 2: Arrangement of chromosomal DNA


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.


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.


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


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

getting organized how bacterial cells move proteins and dna martin thanbichler and lucy shapiro...

Documents

subcellular organization

synthesized proteins

bacteria diffusion

integral membrane proteins

polarity slide

subtilis slide

place proteins

localized membrane complexes