1. Tissue Engineering & Drug Delivery BBI 4203 Lecture 3 Cell Migration

2. Cell migration videos Cells migrating across “scrape wound” https://www.youtube.com/watch?v=Jaqgov L51Cc&spfreload=10https://www.youtube.com/watch?v=Jaqgov L51Cc&spfreload=10 Neutrophil chasing a microbe http://www.biochemweb.org/neutrophil.sht mlhttp://www.biochemweb.org/neutrophil.sht ml http://en.wikipedia.org/wiki/Cell_migration

3. Cell crawling Caterpillar crawling –https://www.youtube.com/watch?v=Er6GMPd9Gx4https://www.youtube.com/watch?v=Er6GMPd9Gx4 Cell crawling –https://www.youtube.com/watch?v=t3u2_pAEB94https://www.youtube.com/watch?v=t3u2_pAEB94 Cell crawling involves great of molecular assembly and disassembly Watch “Inner Life of a Cell” to get a sense for this process –https://www.youtube.com/watch?v=yKW4F0Nu- UY&spfreload=10https://www.youtube.com/watch?v=yKW4F0Nu- UY&spfreload=10

4. Migration examples Embryogenesis (cell-cell adhesion changes, neural crest) Wound healing (fibroblast infiltration) Angiogenesis (blood vessel formation, inflammation) Immune system (microphages, lymphocytes) Cancer metastasis (no anchorage dependence, organ invasion) Bone marrow cell migration (regeneration in different organs) Cellular therapies (spinal cord, cartilage, bone) Organ transplant (microchimerism)

5. Tracking of cell migration in vivo Immunofluorescence labeling and tissue sectioning GFP labeling of cells and tissue sectioning Genetic markers (x-gal) and tissue sectioning MRI with superparamagnetic particles Two photon microscopy

6. Tracking of cell migration in vitro Time lapse microscopy Single cell assays Population assays Under-agarose assay Filter assay Collagen gel assay

7. Cell polarity Leading edge (lamellipodium filopodium) Trailing edge (uropodium) Direction of the motion (speed 0.3  m/min) Lamellopodium protrudes and retracts in a cyclic fashion (50-60 nm/s) Leading edge thinner and stiffer

8. Cell Migration Actual cell migration: https://www.youtube.com/watc h?v=TB6H8RoyPh0 Modeling cell migration: https://www.youtube.com/watch?v =Ft3ZVlDwhvU

9. Actin and Myosin Actin is structural protein that forms support scaffold of cell Actin assembly and disassembly necessary for cell extension and retraction Myosin is an ATP-dependent motor protein Myosin pulls on actin filaments to generate contraction forces of cells

10. Four stages of cell migration 1. Cell protrusion –https://www.youtube.com/watch?v=kRV7to3gst0&spfreload=10https://www.youtube.com/watch?v=kRV7to3gst0&spfreload=10 2. Adhesion of cell to substrate via integrins –https://www.youtube.com/watch?v=hcw7bwA0dTA&spfreload=10https://www.youtube.com/watch?v=hcw7bwA0dTA&spfreload=10 3. Contraction of the cytoplasm (traction) –https://www.youtube.com/watch?v=ixfOMFFaEzY&spfreload=10https://www.youtube.com/watch?v=ixfOMFFaEzY&spfreload=10 4. Retraction and cell displacement –https://www.youtube.com/watch?v=2HRscyXEVj0https://www.youtube.com/watch?v=2HRscyXEVj0

11. Phases of cell motion 1.Protrusion of lamellopodia - addition of actin monomeres - actin polymerization (by uncapping, severing or nucleation) - profilin stimulates the process - gel phase (ectoplasm, formation of actin network) - low Ca 2+ - myosin I binds actin fibers to cytoplasm 2. Adhesion to the substrate via integrines - focal adhesions - GTP-binding protein regulation

12. Phases of cell motion 2 3. Contraction of the cytoplasm (traction) - myosin II induces contraction - high Ca 2+ - gelsolin (actin network disassemble) - traction force 20 millidynes (fibroblasts) 4. Retraction and cell displacement - integrin detachment - membrane ripping (retraction fibers) - preferential displacement at the back is due to: lower stiffness (sol, endoplasm) of trailing edge fewer focal adhesions integrins that are less sticky (lower affinity of binding) - integrin recycling

13. Affect of adhesiveness Baby crawling on really slippery floor Baby crawling on really sticky floor What happens?

14. Optimal adhesiveness of substrate Adhesiveness = substrate density x ligand affinity Low adhesiveness – decreased formation of the focal adhesions High adhesiveness - decreased retraction and traction forces Medium adhesiveness - optimal Cell speed Adhesiveness

15. Person A –Heads – I take one step forward –Tails – I take one step backward Person B –Heads – I take one step to the left –Tails – I take one step to the right Example if person A gets heads and person B gets tails then I take 1 step forward and one step to the right. Where am I after 100 coin flips by both persons A and B? Have two people flipping coins at in unison.

16. Person A –Heads – I take one step forward –Tails – I take one step backward Person B –Heads – I take one step to the left –Tails – I take one step to the right Where am I after 100 coin flips by both persons A and B? Do the same thing again but both coins are now 10% more biased towards heads

17. Types of migration 1. Random migration Brownian-like motion Example: Fibroblasts migrating during cell division https://www.youtube.com/watch?v=V4tmi_Ah1uI&feature=related 2. Directed migration – taxis - chemotaxis (dissolved chemicals) - haptotaxis (bound chemicals) - contact guidance (surface topography) - galvanotaxis (electrical field) - phototaxis (light) Example: Neutrophil following chemical gradient https://www.youtube.com/watch?v=ZKNm_eMJNbs

18. Optically tracking the migration of singles cells: cell tracking Different colors are tracks of single cells. Over short times motion can appear biased (like getting a few heads in a row) but over longer periods of time behaves similar to molecular diffusion (100 coin flips)

19. Directionally biased and unbiased cell migration Cell migration consists of a series of step, probe around, step, probe around... Cell motion is biased if there is a preferred direction over time Biased cell migration shows greater “persistence” in a given direction (biased coin flip) Unbiased cell migration shows no “persistence” toward in a given direction (unbiased coin flip)

20. Cell Persistence Time Mean squared displacement of cell S is the cell speed (um/min) P is the persistence time: the time the cell remains directionally biased (min-hours) Exponential term means effect of P decays over time. For long times MSD=constant x Pt If P=0 then MSD=0

21. P roughly analogous to mass diffusion coefficient where MSD=constant x Dt Thermal agitation causes particles to move with a net displacement over time in the direction of lower concentration Thermal agitation in the absence concentration difference just keeps particles dispersed and they move with no net displacement over time

22. Except! Directed cell migration –Tends to move in direction of increasing molecular concentration –Results from active, energy consuming processes –Affected by cell- substrate interactions and factors affecting cell metabolism Particle diffusion –Tends to move in direction of decreasing molecular concentration –Results from passive kinetics caused by thermal agitation –Affected by physical factors like temperature, viscosity, solubility …

23. This week’s reading

24. Questions Who is the audience? What is the premise of the study? Why do it? What was their approach? What did they observe?

25. Main obervation