Single-Molecule Cellular Biophysics

Description

Recent advances in single molecule science have presented a new branch of science: single molecule cellular biophysics, combining classical cell biology with cutting-edge single molecule biophysics. This textbook explains the essential elements of this new discipline, from the state-of-the-art single molecule techniques to real-world applications in unravelling the inner workings of the cell. Every effort has been made to ensure the text can be easily understood by students from both the physical and life sciences. Mathematical derivations are kept to a minimum whilst unnecessary biological terminology is avoided and text boxes provide readers from either background with additional information. 100 end-of-chapter exercises are divided into those aimed at physical sciences students, those aimed at life science students and those that can be tackled by students from both disciplines. The use of case studies and real research examples make this textbook indispensable for undergraduate students entering this exciting field. Contents Preface Life, from the bottom up References 1 Once upon a (length and) time (scale) 1.1 Introduction 1.2 There are already many informative `multi-molecule´ methods 1.2.1 Calorimetry 1.2.2 Chromatography and dialysis 1.2.3 Circular dichroism and optical rotation 1.2.4 Electron microscopy 1.2.5 Electrophysiology 1.2.6 Fluorimetry 1.2.7 Gel eletrophoresis 1.2.8 Mass spectrometry 1.2.9 NMR and ESR spectroscopy 1.2.10 Optical interferometry 1.2.11 Optical microscopy 1.2.12 Radioactivity 1.2.13 Spectrophotometry 1.2.14 Sedimentation methods 1.2.15 X-ray, neutron and electron diffraction 1.3 American versus European coffee 1.4 Scales of length, force, energy, time and concentration 1.4.1 Length 1.4.2 Force 1.4.3 Energy 1.4.4 Time 1.4.5 Concentration 1.5 Some basic thermodynamics of life 1.6 The concept of 'functionality' 1.7 Test tube or cell? References General Advanced Questions For the life scientists For the physical scientists For those who have not made up their mind 2 The molecules of life - an idiot's guide 2.1 Introduction 2.2 The atomic components of single biological molecules 2.3 Cell structure and sub-cellular architecture 2.4 Amino acids, peptides and proteins 2.5 Sugars 2.6 Nucleic acids 2.7 Lipids 2.8 Miscellaneous 'small' molecules 2.9 The 'central dogma' of molecular biology 2.10 Molecular simulations 2.11 Importance of non-covalent forces References General Advanced Questions For the life scientists For the physical scientists For those who have not made up their mind 3 Making the invisible visible: part 1 - methods that use visible light 3.1 Introduction 3.2 Magnifying images 3.3 Generating optical contrast using scattered light or fluorescence 3.4 Organic dyes, FlAsH/ReAsH, fluorescent amino acids and quantum dots 3.5 Fluorescent proteins, SNAP/CLIP-Tags and HaloTags 3.6 Illuminating and detecting fluorescent tags 3.6.1 Widefield modes of epifluorescence and oblique epifluorescence 3.6.2 Slimfield and narrow-field epifluorescence 3.6.3 Confocal microscopy 3.6.4 Multi-photon excitation 3.6.5 Optical lock-in detection 3.6.6 Light sheet microscopy - SPIM 3.6.7 Adaptive optics 3.7 Fluorescence correlation spectrosopy (FCS) 3.8 Fluorescence lifetime imaging (FLIM) 3.9 'Super-resolution' techniques 3.9.1 Iterative fitting (FIONA-type) approaches 3.9.2 Sub-stoichiometric labelling 3.9.3 Total internal reflection fluorescence (TIRF) 3.9.4 Stochastic activation, switching and blinking of fluorescent dyes 3.9.5 Shrinking the point spread function 3.9.6 Near-field approaches 3.9.7 Structured illumination 3.9.8 Förster resonance energy transfer 3.10 'Multi-dimensional' imaging References General Advanced Questions For the life scientists For the physical scientists For those who have not made up their mind 4 Making the invisible visible: part 2 - without visible light 4.1 Introduction 4.2 Scanning probe microscopy 4.2.1 Scanning tunnelling microscopy 4.2.2 Atomic force microscopy 4.2.3 Scanning ion conductance microscopy 4.3 Electron microscopy 4.3.1 Transmission electron microscopy (TEM) 4.3.2 Scanning electron microscopy (SEM) 4.4 Ionic currents through nanopores 4.4.1 Single ion channel recording 4.4.2 Solid-state nanopores 4.4.3 Engineered protein nanopores 4.5 Raman spectroscopy 4.5.1 Surface enhanced Raman spectroscopy (SERS) 4.6 Interference-based detection References General Advanced Questions For the life scientists For the physical scientists For those who have not made up their mind 5 Measuring forces and manipulating single molecules 5.1 Introduction 5.2 Optical tweezers 5.2.1 The single-beam gradient force optical trap (i) Trapping theory (ii) The length scale of optical traps (iii) Trap stiffness (iv) Optical trapping in practice 5.2.2 Bessel beam, fibre-based and evanescent field optical traps 5.2.3 Multiple laser traps 5.2.4 Optical spanners 5.2.5 Combining optical-trapping with other single-molecule methods 5.3 Magnetic tweezers 5.4 Atomic force spectroscopy 5.4.1 AFM 'cut-and-paste' 5.4.2 Combining AFM spectroscopy with fluorescence microscopy 5.5 Using force spectroscopy to explore non-equilibrium processes 5.5.1 Detailed balance 5.5.2 Non-equilibrium processes, Kramers theory and the Jarzynski equality 5.6 Electric dipole induction in polarizable particles for torque and trapping 5.6.1 Electrorotation 5.6.2 ABEL traps References General Advanced Questions For the life scientists For the physical scientists For those who have not made up their mind 6 Single-molecule biophysics: the case studies that piece together the hidden machinery of the cell 6.1 Introduction 6.2 What makes a 'seminal' single-molecule biophysics study? 6.2.1 The (highly personal and potentially biased) `top ten´ single-molecule biophysics papers of all time 6.2.2 Analytical methods employed to objectify single-molecule experiments 6.3 Carving up the cell into a few sensible themes References General Advanced Questions For the life scientists For the physical scientists For those who have not made up their mind 7 Molecules from beyond the cell 7.1 Introduction 7.2 Receptor molecules and ligands in the cell membrane 7.2.1 Receptors relevant to normal tissue assembly (and cancers when they go wrong) 7.2.2 Sticking cells together 7.2.3 Signal transduction from activated membrane receptors 7.3 Endocytosis and exocytosis 7.3.1 Live-cell imaging of clathrin-based endocytosis 7.3.2 Single-molecule fluorescence imaging of exocytosis in functional cells 7.3.3 SNARE exocytosis proteins 7.4 Viral invasion 7.4.1 Tracking single viruses using single colour fluorophores 7.4.2 Multi-colour fluorescence tracking of viruses References General Advanced Questions For the life scientists For the physical scientists For those who have not made up their mind 8 Into the membrane 8.1 Introduction 8.2 Molecular transport via pores, pumps and carriers in membranes 8.2.1 Small sugar molecule carriers 8.2.2 Ion and water channels 8.2.3 Protein translocation 8.3 Rotary motors - the rise of the (bionano) machines 8.3.1 Counting molecular subunits in a bacterial flagellar motor 8.3.2 Measuring nanometre-scale rotary movements of the flagellar motor 8.3.3 Unravelling the mechanism of the FoF1-ATP synthase 8.4 Energizing the cell 8.4.1 Manufacturing ATP via oxidative phosphorylation 8.4.2 Using light to generate cellular energy 8.5 Architecture and shape of the cell surface 8.5.1 The cytoskeleton 8.5.2 The micro- and nano-architecture of the membrane References General Advanced Questions For the life scientists For the physical scientists For those who have not made up their mind 9 Inside cells 9.1 Introduction 9.2 Free, hindered and driven molecular diffusion in the cytoplasm 9.2.1 Using single particle tracking to study free and hindered diffusion in the cytoplasm 9.2.2 Trafficking of molecular cargo in the cytoplasm 9.3 Chromosomes and DNA: their architecture and replication 9.3.1 Probing the architecture of DNA packing using super-resolution imaging 9.3.2 Replicating DNA 9.3.3 Segregating the genetic material 9.4 Translating, transcribing and splicing the genetic code 9.4.1 Gene expression bursts and transcription factors 9.4.2 mRNA, nuclear pores and post-translational modification References General Advanced Questions For the life scientists For the physical scientists For those who have not made up their mind 10 Single-molecule biophysics beyond single cells and beyond the single molecule 10.1 Introduction 10.2 Single-molecule biophysics in complex organisms 10.2.1 Single-molecule systems biology 10.2.2 Probing even smaller length scales - sub-cellular and quantum biology 10.2.3 Single-molecule biomedicine 10.3 Bionanotechnology and 'synthetic' biology 10.3.1 DNA nanotechnology 10.3.2 Nanomedicine 10.3.3 'Biofuel' production 10.4 The outlook for single-molecule cellular biophysics References General Advanced Questions For the life scientists For the physical scientists For those who have not made up their mind Index