• What is a cellular machine? What do they do?

- bio-molecular assemblies generating work and motion

- self assembly and emergence (including biological and non-biological examples)

- how can we visualize and examine these machines?

- ensemble vs. single molecule measurements

- introduction of the basic single-molecule experimental methods: single molecule imaging and optical tweezers

• Life at low Reynolds number

- basic concepts of mechanics: force, trajectory, velocity, friction...

- different factors are relevant at different scales

- what is important on the micrometer/nanometer-scale?

- thermal motion, diffusion

- overdamped systems

- walking in a hurricane, swimming in molasses

Further reading:

Purcell, Life at low Reynolds number, American Journal of Physics 45, 3 (1977)

• Thermal ratchets

- (macroscopic) ratchet and work

- reaction kinetics, on and off rates

- thermal ratchet

- ratcheted diffusion

- examples: protein and nucleic acid translocation through pores

Further reading:

Hepp et al, Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism. Proc Natl Acad Sci U S A. 2016;113(44):12467-12472.

Stewart, Ratcheting mRNA out of the Nucleus. Mol Cell 2007, 25:327-330

Okamoto et al, The protein import motor of mitochondria: a targeted molecular ratchet driving unfolding and translocation. EMBO J. 2002 Jul 15; 21(14): 3659–3671.

• Force generation by filament dynamics

- microtubule dynamic instability

- actin dynamics

- force generated by the growing or shrinking microtubule tip

- force generated by actin polymerization - actin comets

Further reading:

Dogterom et al, Measurement of the Force-Velocity Relation for Growing Microtubules. Science 1997, 278: 856-860

Powers et al. The Ndc80 Kinetochore Complex Forms Load-Bearing Attachments to Dynamic Microtubule Tips via Biased Diffusion. Cell (2009), 136(5), 865–875.

Theriot et al. The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature. 1992; 357(6375):257-60.

Svitkina. The Actin Cytoskeleton and Actin-Based Motility. Cold Spring Harb Perspect Biol 2018; 10:a018267

• Cytoskeletal molecular motors

- ATP hydrolysis cycle

- lever arm movement and diffusive search

- force measurements, stall force

- processivity

- kinesin-1, kinesin-5, kinesin-14, dynein examples, myosin AFM movie

Further reading:

Svoboda et al. (1994). Force and velocity measured for single kinesin molecules. Cell, 77(5), 773–784.

Schnitzer et al. (1997). Kinesin hydrolyses one ATP per 8-nm step. Nature, 388(6640), 386–390.

Rice et al. (1999). A structural change in the kinesin motor protein that drives motility. Nature, 402(6763), 778–784.

Sozański et al. (2015). Small Crowders Slow Down Kinesin-1 Stepping by Hindering Motor Domain Diffusion. Physical Review Letters, 115(21), 197–5.

Brunnbauer et al. (2012). Torque Generation of Kinesin Motors Is Governed by the Stability of the Neck Domain. Molecular Cell, 46(2), 147–158.

Kapitein et al. The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature. 2005 May 5;435(7038):114-8.

Fink et al. The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding. Nat Cell Biol. 2009 Jun;11(6):717-23.

Urnavicius et al. (2018). Cryo-EM shows how dynactin recruits two dyneins for faster movement. Nature, 554(7691), 202–206.

Kodera et al. Video imaging of walking myosin V by high-speed atomic force microscopy. Nature. 2010, 468(7320):72-6.

• Other molecular motors

- Bacterial flagellar motor, ATP synthase - rotary motors

- dynamin - twisting motor

- viral DNA packaging motors

Further reading:

Noji et al. (1997). Direct observation of the rotation of F1-ATPase. Nature, 386(6622), 299–302.

Sowa et al, Direct observation of steps in rotation of the bacterial flagellar motor

Nature, 2005, 437(7060), 916-919

Roux, et al. (2006). GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature, 441(7092), 528–531.

Smith et al. (2001). The bacteriophage straight phi29 portal motor can package DNA against a large internal force. Nature, 413(6857), 748–752.

• Entropic force generators

- harvesting thermal energy

- confined diffusion and contractile entropic forces - Ase1 example

- macromolecular crowding, depletion effects and contractile entropic forces

- cytokinetic ring constriction

Further reading:

Lansky et al. (2015). Diffusible crosslinkers generate directed forces in microtubule networks. Cell, 160(6), 1159–1168.

Hilitski et al. (2015). Measuring Cohesion between Macromolecular Filaments One Pair at a Time: Depletion-Induced Microtubule Bundling. Physical Review Letters, 114(13), 138102–6.

Braun et al. (2016). Entropic forces drive contraction of cytoskeletal networks. BioEssays, 38(5), 474–481

• Collective effects

- forces are often generated by ensembles of motors

- ensemble of motors have different properties than single motors

- friction, load-dependent off rate

- kinesin-14 examples

- cytokinetic ring closure - motors, polymer dynamics and passive crosslinkers work together

Further reading:

Braun, et al. (2017). Changes in microtubule overlap length regulate kinesin-14-driven microtubule sliding. Nature Chemical Biology, 13(12), 1245–1252.

Cheffings et al. (2016). Actomyosin Ring Formation and Tension Generation in Eukaryotic Cytokinesis. Current Biology, 26(15), R719–R737.

Furuta et al. (2013). Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors. Proceedings of the National Academy of Sciences of the United States of America, 110(2), 501–506.

• Phase separation, local concentration, microenvironment

- liquid-liquid demixing as an organising principle

- seeing each other - clients and tourists

- centrosome example

- crosslinker sorting in an actin overlap

- multivalence, avidity, local concentration and processivity of molecular motors

Further reading:

Woodruff et al. (2017). The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin. Cell, 169(6), 1066–1071

Hyman et al. (2014). Liquid-Liquid Phase Separation in Biology. Annual Review of Cell and Developmental Biology, 30(1), 39–58.

Winkelman et al. (2016). Fascin- and alpha-Actinin-Bundled Networks Contain Intrinsic Structural Features that Drive Protein Sorting. Current Biology, 26(20), 2697–2706.