Physics and Nanotechnology to Study Bacterial Cells

Transcription

1 Physics and Nanotechnology to Study Bacterial Cells F tot k B T m ln m m m B V c m V B s Saturday Morning Physics Lecture Mar. 11, 2017 Jaan Männik

2 Impact of physics on studies of living systems 1. Application of physics based methods and techniques to experiments with living systems 2. Application of physics based theories to explain life processes.

3 Bacteria the unseen majority Majority of Earth s biomass are bacteria and archea. They are one of the main determinants how biosphere functions There are estimated 10 times more bacterial cells in human body than our own cells Recognizing bacteria and other microorganisms as disease agents have lead human life span to increase twofold over past 100 years. This gain may not be permanent.

4 Standard microbiology toolbox An agar plate Cell culture tubes The standard microbiology tools are not suitable to follow: 1. How bacteria behave in complex environments 2. Molecular process in individual cells in real time.

5 Lab-on-a-chip based tools Well controlled environment for the cells which physical and chemical characteristics can be controlled and manipulated Compatible with high resolution microscopy of cells (including super-resolution imaging, SEM)

6 Advantages of lab-on-a-chip platform Device to extract bacterial DNA In situ biochemical analysis of cells J. W. Hong et al Nat. Biotech. 21 (2003) 1179 Automation 2 m F. K. Balagadde et al Science 309 (2005) 137.

7 Applications of lab-on-a-chip in studies of cells Molecules Cells Organisms Populations Sequencing Antibiotic resistance studies Bacteria and tissue interactions Ecology, evolution Y. Mercy et al. PNAS 104 (2007) N. Q. Balaban et al. Science 305 (2004) 1622 D.Huh et al. Science 328 (2010) 1622 S. Park et al. Science 301 (2003) 188

8 Understanding how bacterial cells move in small pores using biomimetic lab-on-a-chip devices

9 Bacterial movement in channels and pores Most bacteria in different environments live in pores 10 m and smaller

10 Can t do it in patient mouth

11 Experimental setting channel 100 m chamber Fabricate on the single chip, large number of differently sized and shaped channels. Monitor bacterial movement through channels (constrictions) using fluorescent microscopy

12 Microfabrication cleanroom

13 Microchip fabrication electron beam 1 PMMA Si Use electron beam or photolithography to write pattern of channels 2 3 Develop resist Reactive ion etch Si wafer Repeat the process for different channel heights 4 Lift-off resist 5 PDMS coated glass coverslip Si Drill access holes channels Close channels

14 Channels Side view Bacterium D 1µm 1µm 1µm On single chip, channel width is made to vary between 300 nm to 5 m to using a RIE cryoetch process

15 Fluorescence Exception jellyfish Aequorea victoria UV excitation Most materials/objects do not fluoresce when excited with blue light (many materials fluoresce when exited with UV light) Excitation with blue light allows to selectively observe engineered molecules such as GFP and have very little background from other molecules.

16 Fluorescent proteins GFP (Green Fluoresccent Protein) Blue light Insert genetic code for GFP molecule into bacterial genome or in plasmid (short circular DNA that many bacteria carry). Bacteria will synthesize the protein. Shine blue light on bacteria that express GFP. They will shine green light back. There is little background at green wavelength region.

17 Gram-negative and Gram-positive bacteria Escherichia coli Gram-negative bacteria cytoplasm Bacteria lipid membrane cell wall Bacillus subtilis Gram-positive bacteria cytoplasm Mollicutes (no cell wall)

18 Two bacterial species studied Escherichia coli Gram-negative bacterium Bacillus subtilis Gram-positive bacterium E. coli RP437 D D 1µm 1µm 1µm Superfically looking the two bacterial species are similar but on molecular level they are different than humans are from roundworms.

19 Bacterial swimming E. coli RP437 1µm E. coli with fluorescently labeled flagella H. C. Berg group, Harvard University Wikipedia

20 Introduction: Bacterial motility Bacterial flagellar motor is a rotary motor superficially similar to DC electrical motor Rotation speed controlled membrane potential and ph gradient across inner membrane Bacterial sensory system (chemotaxis receptors through signaling cascade) control direction of rotations

21 Bacterial motility in channels E. coli RP437 W = 1.2 m chamber channel chamber <v> [ m/s] W [ m] E. coli and B. subtilis bacteria are motile in channels which are only 30-40% wider than their diameter In smaller channels bacteria lose their ability to swim but..

22 Growth and division is bacterial solution to penetrate narrower channels W = 0.6 m E. coli chamber channel chamber B. subtilis

23 Different layout of channels More details of cells visible in the microscope Channel ceiling soft; Bacteria can deform it

24 Bacterial movement in sub-micron channels E. coli Growth in confinement alter drastically E.coli shapes B. subtilis B. subtilis grows to filaments that buckle and finally divide

25 Re-emergence of regularly shaped bacteria 0hrs 5hrs 49 hrs 10 µm Over period 1-2 days regular rod-shaped bacteria replace initial population of aberrantly shaped bacteria

26 Modes of penetration for different channel widths E. coli but not B. subtilis bacteria are able to grow through channels which widths are smaller than their diameters

27 Mechanical properties and propagation in narrow channels E. coli B. subtilis cell wall 3 nm W channel wall channel wall D cell wall nm P osm =2-3 atm P osm =26 atm A. Boulbitch et al PRL 85 (2000) 5246 V. R. F. Matias et al Mol. Microbiol 56 (2005) 240 Cell-wall has high Young modulus but is easily bendable (think of inflated balloon) The thicker the cell wall the higher the Young modulus and the higher osmotic pressure can bacterium maintain in its interior

28 Pressure actuated valves to mechanically manipulate cells and sub-cellular structures pressure channel PDMS glass channel for bacteria Characterize the mechanical properties of bacterial cells Use it as tool to manipulate sub-cellular structures in a bacterial cell

29 Mechanical limits of bacterial deformations p max 0 p max = 2 atm p max = 3 atm Step # Bacteria can be stretched up 25% elastically Bacteria break open at isolated weak spots at cell poles

30 Short vs long time response of E. coli Elastic deformation after entry in shallow channel Slow cellular response after prolonged stay in channel

31 Summary on lab-on-a-chip and squeezing measurements Lab-on-a-chip platform allows to create biomimetic environments where cell behavior can be studied using high resolution optical microscope and bioanalytical tools. E. coli and B. subtilis are well adapted to swim in small channels despite their long flagella. Both species retain ability to swim in channels which only 30% exceed their body diameter Surprisingly, bacteria can get through even narrower channels! For that they use growth and division. Bacterial growth is robust despite drastic changes in their shapes Mechanical properties of bacteria determine how small channels they can penetrate.

32 Understanding how cells are built from molecules up using quantitative high resolution microscopy and modelling

33 Escherichia coli as living hydrogen Escherichia coli E. coli atom 1 m Jacque Monod, What is Valid for E. coli is also valid for the elephant Bacteria present the simplest systems to understand how the cellular processes unfold using basic physics principles

34 What is known Functions of about 70% genes to some degree 1 m 50% of protein structures (most based on homology) 2 nm FtsZ MatP tetramer SlmA tetramer

35 What is not known: from genes to cell DNA lipid? protein polysaccharide 1 m How nanometer-scale proteins, DNA and lipids come together and form the micro-size cell?

36 Cellular organization in bacteria Wikipedia Bag with soup of molecules Assembly of molecular machines Bacterial cells are highly organized despite their apparent simplicity

37 envelope How are chromosomes and cell division apparatus organized in a bacterial cell? How cell division proteins position relative to nucleoids?

38 Chromosome organization

39 Genetic information is tightly packed in the cell Escherichia coli circular chromosome basepairs 1 μ HupA-mCherry labelled chromosome 1.6 mm Thousand fold compaction of DNA in the cell

40 Nucleoid cytosol 1 μ HupA-mCherry labelled chromosome nucleoid DNA spreads just to a fraction of cell volume (~50%) the nucleoid (not to be confused with nucleus) There is no membrane surrounding the nucleoid

41 What compacts DNA?

42 Depletion Forces D few large molecules abundant small molecules (crowders) Volume gained (V gained ) by small molecules if two large molecules come together Molecules move so that to minimize excluded volume. This leads to appearance of an average force (depletion force) that pushes larger molecules together. Alternative view: smaller molecules exert (osmotic) pressure to big ones which pushes them together.

43 Alignment and compaction of DNA strands as result of depletion forces DNA strands are pushed together and aligned

44 Depletion forces compact DNA and lead to phase separation Cytosol phase is protein and ribosome rich Nucleoid contains DNA and occludes most proteins and ribosomes 1 μ Proteins and ribosomes are crowders. They push and compact DNA. In doing so DNA and proteins phase separate from each other (like water and ice do)

45 Dream of a bacterium is to make from one cell two cells (Jacque Monod) Bacterium living up its dream To divide bacterium needs to duplicate its DNA

46 DNA within nucleoid is dynamic Some chromosomal regions occupy well-defined locations in the cell How exactly DNA is folded in the nucleoid not yet understood.

47 Modelling replicating chromosome in a cylindrical confinement Partially replicated chromosome oric Ter oric blob 100 nm Model DNA as self-avoiding chain of 100 nm blobs that moves under thermal force (Langevin thermostat)

48 Dynamics of replication terminus region oric Ter oric starting configuration equilibrium configuration Ter Replication terminus region positions itself in the cell center as a result of entropic force

49 Cell division and organization cell division apparatus

50 Cell division in bacteria chromosome (nucleoid) Z-ring constriction Cell division proteins assemble to a ring-like structure, the Z- ring, that separates two daughters from each other.

51 Z-ring FtsZ ring Z-ring FtsZ monomer FtsZ filament H. P. Erickson et al., Microbiol. Mol. Biol. Rev. 74 (2010) 504 D. W. Adams, J. Errington, Nat. Rev. Micro. 7 (2009) 642 Assembly of FtsZ filaments is the first step in cell division FtsZ filaments act as a scaffold. This scaffold recruits about 30 other proteins to the ring (the fellowship of the ring).

52 Assembly of the Z-ring Continuous cohesive ring Patchy band model Patchy band S. Holden et al PNAS 2014 (111) 4566 Green, Red FtsZ pointing up and down How filaments are exactly organized in the Z-ring is not known

53 Positioning of cell division proteins Fellowship of the ring locates within 2% accuracy in the center of the cell

54 First view Go to the right place!!! PDB: 1FSz There is a little control freak inside every cell who knows where things need to go and it takes care of it.

55 Second view Let s get together at the middle PDB: 1FSz Self-assembly of molecules to macromolecular structures driven by diffusion, intermolecular binding, conformational entropy, excluded volume effect etc.

56 Two known molecular systems in E. coli to localize its Z-ring: The Min system Nucleoid occlusion mechanism

57 Min oscillations in E. coli cells Min operon: minc, mind, mine 2 1 y [ m] x [ m] MinD::GFP MinD, MinE and MinC proteins oscillate between two poles of rod-shaped E. coli. Their time-averaged concentration is high at the cell poles. This defines inhibitory zone for Z-ring localization.

58 Min system: Turing instability in nature Reaction-diffusion system:,, K. C. Huang et al PNAS 100 (2003) 12724

59 Nucleoid occlusion D. W. Adams, J. Errington, Nat. Rev. Micro. 7 (2009) 642 Nucleoid occlusion: presence of nucleoid produces inhibition for the Z-ring assembly

60 Nucleoid occlusion factor SlmA Origin SlmA DNA Terminus Terminus N.K. Tonhat et al EMBO J. 30 (2011) 154 SlmA is inhibitor for Z-ring formation. H. Cho et al PNAS 108 (2011) 3773 SlmA binds to specific sequences of DNA that are missing in replication terminus region. Replication terminus region positions itself in the center of the cell. No inhibition of Z-ring formation at the cell center.

61 What happens to cells that lack these positioning systems? a minicell WT minc slma slma minc Evidence for divisome localization mechanisms independent of the Min system and SlmA in Escherichia coli, M. W. Bailey, P. Bisicchia, B. T. Warren, D. J. Sherratt and J. Männik, PLoS Genet. 10 (2014) e

62 How can E. coli cells without known positioning systems still divide about its middle?

63 Localization of Z-ring in cells w/o known positioning systems slma min Intensity (arb. units) X z GFP X n phase DAPI Length along the long axes [ m] X z /L slma min X n /L counts = 66 nm slma min X z -X n [ m] Z-ring localizes the center of the nucleoid rather than to nucleoid free regions.

64 Positive regulation I [arb. units] slma min 2 m nucleoid Z-ring Length along long axes [ m] Length along long axes [ m] Length along long axes [ m] Z-ring Chromosome time [min] Apositivesignalthat guides localization of cell division proteins from the cell center to the nucleoid center.

65 Realization of the positive signal ΔslmA ΔminC ΔzapA ΔslmA ΔminC ΔzapB slma min matp Misplaced Misplaced counts = 202 nm N=145 counts = 222 nm N=218 counts = 150 nm N= X z -X n [ m] X z -X n [ m] X z -X n [ m] ZapA, ZapB and MatP proteins are required for the positive signal

66 Summary on organization of bacterial cells Min system and nucleoid occlusion are two known systems for positioning of bacterial Z-ring via a negative regulation. We have found a positive regulatory mechanism that promotes Z-ring formation at the vicinity of the Ter region of the chromosome. ZapA, ZapB and MatP proteins are needed for the positioning. Replication terminus region can spontaneously position itself in mid-cell as a result of entropic force. This spontaneous process appears ultimately to control the placement of cell division proteins.

67 Thanks Matthew Bailey, UTK George Siopsis, UTK Dan Castillo, UTK Jaana Männik, UTK Paola Bisicchia, Oxford Univ. David Sherratt, Oxford Univ. Piet de Boer, Case Western Reserve Univ. Alex Dajkovic, Univ. of Paris 5 Rodrigo Lamothe-Reyes, McGill Univ. Cees Dekker, Delft University of Tech. Conrad Woldringh, Univ. of Amsterdam Arieh Zaritsky, Ben Gurion University CNMS User Program: UTK start-up funding NSF CAREER award

68 Thank you for your attention! There is lab tour starting in 5 min.