Getting Organized how bacterial cells move proteins and DNA

1. Getting Organized how bacterial cells move proteins and DNA Martin Thanbichlerand Lucy Shapiro Nature Reviews 2008 ZIB presentationJanuary 10, 2011 Sarah Hinkelmann

2. Prokaryotic versus eukaryotic cells

3. Components of the eukaryotic cytoskeleton • Microfilaments • G-actinmonomers form F-actinfilaments, showpolarity • Involved in cytokinesis, musclecontraction, cellmotility • Intermediate filaments • Can form dimer ortetramer, nopolarity • Givecellularstability, involved in cell-cellconnection • Microtubules • α- andβ- monomers form dimer • Involved in mitosis, cytokinesis, vesiculartransport • Show polarity

4. 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.

5. 1. Subcellular organization in bacteria Twoproteinclassesforsubcellularorganization: • Proteins forming large stationarycomplexes • Proteins thatarepartofhighlydynamicinteractionnetworks

6. 1. Subcellular organization in bacteria • Proteins forming large stationarycomplexes • Based on integral membraneproteins • Mediate establishmentofcellularorganelles, performcatalyticorregulatoryfunctions • How do theseproteinsassume a definedintracellularposition? • Twolocalizationmechanisms in bacteria: • Diffusion andcapture • Targetedmembraneinsertion

7. 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

8. 1. Subcellular organization in bacteria Diffusion and capture: • SpoIVFB = protein-processingenzyme • synthesized in mothercell, but destinationisseptalmembrane • Random positioning in cytoplasmicmembrane • Diffusion • CapturingbyforesporeproteinSpoIIQ

9. 1. Subcellular organization in bacteria Diffusion andcapture: • Newlysynthesizedproteinsarefirstinsertedrandomly. • Diffusion takesplace • Proteins arecapturedbyinteractionwithpreviouslylocalizedmembranecomplexes. • e.g. duringsporulationofB.subtilis Targetedmembraneinsertion: • A proteinisdelivereddirectlytoitsdestinationbytranslocationto a givencellularsite.

10. 1. Subcellular organization in bacteria Targeted membrane insertion • SpoIIQ = proteinthatissynthesized in theforspore • actsaslocalizationfactorthatrecruitsSpoIIIAH(mother-cellprotein) totheforesporeseptalmembrane • then additional factorsaresynthesizedandrecruited •  Zipper mechanism

11. 1. Subcellular organization in bacteria • Proteins thatarepartofhighlydynamicinteractionnetworks • Adoptspecificoverallarangements • Provideforceanddirectionality • Serveastracksforthelocalizationofotherproteins • magnetosomes

12. 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.

13. 1. Subcellular organization in bacteria Example 1: linear arrangements of subcellular compartments • MamK = bacterialactinhomologue • MamKpolimerizesto form filamentousstructure • MamJ = involved in attachingmagnetosomestotheMamKfilament • MamKfunctionsas a trackthatrecruitsvesicleswiththehelpofMamJ Empty and immature vesicles are randomly distributed along MamK filaments Magnetostatic interactions between individual vesicles lead to their aggregation into densily packed chains

14. 2. Dynamic protein scaffolds and cell shape Example 2: actin-like cytoskeleton of bacteria • Protofilament • The basicpolymericunitof a filamentousstructure • Consistsof a linear rowofmonomers • Treadmilling • Actinprotofilamentspossess an intrinsicpolarity: • Monomers areaddedatone end andreleasedfromtheother end • MreB • Actin-likeproteins • MreBcablesarehighlydynamicandassumeseveral different architecturesspiral-likeassembly, shortcoils, arcs, rings

15. 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

16. 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.

17. 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)

18. 2. Dynamic protein scaffolds and cell shape • 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

19. 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

20. 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

21. 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

22. 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.

23. 4. Division-site placement • The Min system • FtsZ= tubulinhomologue • MinD:ATPase, interactswithMinCto form a membrane-associatedinhibitoroftheFtsZ-ring formation • MinErestrictsMinCDcomplextothe polar regionsofthecell • Celldivisionoccursonlyclosetothecellcentre.

24. 4. Division-site placement The Min system Assemblyinto a large polymeric patch Cap-structurestartstoshrink Completedissapearance Concomitantly, a newMinCDassemblyformsattheopposite pole andthecirclestartsagain MinEcircularstructurefollowstheretractingedgeofMinCD

25. Summary I • Subcellularorganization in bacteria • Therearetwoclassesofproteins: theybuildstationaryorhighlydynamiccomplexes • Stationarycomplexes: diffusionandcaptureortargetedmembraneinsertion • Dynamic complexes: actin-homologuesfunctionbymaintainingcellularcompartments (magnetosomes) •  Mam-proteins • Dynamic proteinscaffoldsandcellshape • Actinhomologuesareinvolved in celldivisionorregulatecell-wall biosynthesis •  MreB-proteins

26. Summary II • Bacterial DNA segregation • Actin-homologues have `pushing´ forces to separate the DNA •  Par-proteins • Division-site placement • Tubulin homologues and ATPases are involved in cell division •  Min- and FtsZ-proteins

27. Thank you for your attention !

28. 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-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

29. 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

30. 3. Bacterial DNA segregation Example 2: Arrangement of chromosomal DNA • Chromosomal DNA is not distributedrandomly but has a highlyconservedorganization • Segments ofchromosomal DNA arefoldedintosupercoileddomainsthatarestacked on top ofeachotherandarrangedintocircularsuperstructure. • Subcellularpositionof a locuscorrelateswithitslocation on thecircularchromosomalmap

31. 3. Bacterial DNA segregation Example 2: Arrangement of chromosomal DNA • C. crescentus: originofreplication (ORI) isfoundattheflagellated pole, terminusislocatedattheoppositesiteofthecell • E.coli: ORI andterminusregionsareplacedatthecellcentreandthetwoarmsofchromosome form separate domainsthatarelocated on oppositesidesofitstransverseaxis.

32. 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.

33. 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

34. 4. Division-site placement • Regulation ofcelldivisionbyMipZ • Someorganisms lack theMinCDEsystem • MipZ = ATPase, interactswithchromosome-partitioningproteinParB • ParBbindsto a clusterofParS • Togetherwiththeoriginregionthecomplexispositionedattheold pole in newborncells • DNA replication: twocopiesofParSsegmentsaregenerated, whereMipZandParBdirectly bind to • Onesegmentcomplexstaysattheold pole, secondsegmentmovestonew pole • MipZ = inhibitorforFtsZpolymerization • FtsZisthere, wherethelowestconcentrationofMipZis, thusFtsZmovestothecellcentrewhenMipZ-ParBcomplexmovestoother pole • Thencytokinesiscanbeinitiated