1. Overview of 130L Part 2 Week 3 (3/19-3/23) Transfection & vital staining: The secretory and endocytic pathways Week 3 (3/19-3/23) Transfection & vital staining: The secretory and endocytic pathways Week 2 (3/12-3/16)Sub-Cloning Sub-Cloning Week 1 (this week) Fluorescence labeling and microscopy: The actin and tubulin cytoskeleton in cultured cells Week 1 (this week) Fluorescence labeling and microscopy: The actin and tubulin cytoskeleton in cultured cells

2. What you should get from today’s lecture 1. What are cultured cells and why do we use them? 3. How are cells fixed and stained with fluorescent reagents? 4. An introduction to microscope resolution 2. General background on the cytoskeleton and the drug treatments used in this lab

3. dish of cell colonies 100 mm single cell ( scanning EM) >0.01 mm colony of cells 1 mm How big are animal cells?

4. Cells in culture can undergo diverse differentiation pathways Neuronal cell extending axons and dendrites Mouse C2C12 muscle cells forming myofibers

5. Stem cells are undifferentiated and pluripotent, meaning that they can differentiate to become multiple other cell types embryonic stem cells“feeder” cells

6. The Bad: 1) Requires care and $$$ to grow & prevent contamination 2) May not always recapitulate real cellular physiology Cell culture = propagation of cells outside the organism The Good: 1)Cellular environment can be easily observed and manipulated a. Injection b. Transfection -introduction of genes c. Pharmaceutical or genetic manipulation (RNAi) d. Fluorescent tracers (live and fixed cells) 2) Cell type can be well defined (How?) 3) Large quantities of cells can be obtained - biochemistry 4) Diverse cellular functions can be investigated 5) Noninvasive way to study mammalian cells 1)Cellular environment can be easily observed and manipulated a. Injection b. Transfection -introduction of genes c. Pharmaceutical or genetic manipulation (RNAi) d. Fluorescent tracers (live and fixed cells) 2) Cell type can be well defined (How?) 3) Large quantities of cells can be obtained - biochemistry 4) Diverse cellular functions can be investigated 5) Noninvasive way to study mammalian cells

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8. Immortal cell lines can arise by spontaneous mutation or deliberate transformation of mortal cell strains. Most cells in the body or removed from the body are MORTAL: they have a finite ability to replicate and divide Embryonic stem cells are also immortal. We don’t really understand why they have this unlimited replication potential, or why most cells lack it.

9. 2 cytoskeletal components will be examined: Actin - required for cell shape & movements including translocation and cytokinesis Tubulin - forms microtubule “tracks” that enable chromosomes, vesicles, etc. to move in targeted ways within cells. Tubulin - forms microtubule “tracks” that enable chromosomes, vesicles, etc. to move in targeted ways within cells. TPA/PMA Latrunculin* TPA/PMA Taxol (Paclitaxel)* Nocodazole* Nocodazole* *alter the equilibrium between subunits and polymers of actin or tubulin Your cells will be treated with 4 drugs (+ controls) Purpose: stain cells to observe the cytoskeleton; Observe and record the effects of different drugs on cytoskeletal components and cell shape. Lab: Visualizing the cytoskeleton using fluorescence microscopy Lab: Visualizing the cytoskeleton using fluorescence microscopy

10. Cells move! all cell movement requires actin dynamics

11. Actin dynamics in a motile cell

12. ARP 2/3 complex actin monomer actin filaments form three-dimensional branching networks

13. TPA = phorbal myristate acetate = PMA mimics 1,2 diacylglycerol (DAG) DAG plus Ca ++ activate protein kinase C (PKC) PKC activation results in phosphorylation of MANY PKC targets (i.e. MARCKS myristolated alanine rich C kinase substrate) PKC activation results in phosphorylation of MANY PKC targets (i.e. MARCKS myristolated alanine rich C kinase substrate) this leads to major changes in cell growth, cell shape and the cytoskeleton Phorbol ester

14. A schematic of the normal activation of PKC MARCKS

15. Latrunculin A free G-actin G-actin bound to Latrunculin A

16. interphasemitosis Microtubules DNA Microtubules control other aspects of cell dynamics including vesicle transport and chromosome segregation

17. 17 Microtubules are Comprised of Many Tubulin Heterodimers

18. Microtubule dynamics are controlled by tubulin conformation, which can be modified by other proteins or drugs such as nocodazole or taxol Eva Nogales’ lab

19. Microtubule dynamics during interphase

20. Microtubule dynamics at the cell edge Individual microtubules are constantly growing and shrinking. Their rate of growth and the frequency of switching between growth and shrinkage are controlled by a large number of factors, including MAPs (microtubule associated proteins).

21. Taxol binds to polymerized β-tubulin and inhibits depolymerization taxol GTP/GDP Tubulin α/β dimer

22. 1.Aldehydes (formaldehyde, glutaraldehyde): Cross-link amino groups in proteins. Stabilizing many structures Can block antibody access to targets. 1.Aldehydes (formaldehyde, glutaraldehyde): Cross-link amino groups in proteins. Stabilizing many structures Can block antibody access to targets. 2.Alcohols (methanol or ethanol, with or without acetic acid): Denatures and precipitates proteins in place. Alcohol fixation does not retain soluble proteins Cell/protein morphology not preserved Fast and easy Good for some labile structures (microtubules) 2.Alcohols (methanol or ethanol, with or without acetic acid): Denatures and precipitates proteins in place. Alcohol fixation does not retain soluble proteins Cell/protein morphology not preserved Fast and easy Good for some labile structures (microtubules) Cell Fixation and Permeabilization A. Chemical fixation - kills and immobilizes cells

23. 2.Effect of detergents can depend upon order of steps (as in this week’s lab): a)When added before fixative, they will often solubilize proteins (e.g. unpolymerized tubulin, as in today’s lab). b)This can be a technical advantage - reduces background - but can also lead to experimental artifacts. B.Permeabilization - detergents are used to solubilize cell membranes to allow staining reagents to penetrate; makes proteins accessible to staining reagents. 1.Usually gentle, non-ionic detergents are used: Triton X-100 Tween-20 Brij-58 Cell Fixation and Permeabilization

24. For this lab, we buy phalloidin that is covalently linked to rhodamine Staining of Actin Amanita phalloides Phalloidin Phalloidin:a natural toxin from some mushrooms Binds to filamentous actin (F-actin) only + TRITC = tetramethyl rhodamine isothiocyanate Kidney cell stained with rhodamine- phalloidin

25. Variable regions Antibodies are highly conserved molecules with variable regions that specify antigen recognition and affinity Antibody, or Immunoglobulin Constant region

26. Antibodies are highly conserved molecules with variable regions that specify antigen recognition and affinity Fluorescein, or FITC Secondary antibodies (react with primary antibodies)

27. Note: color of the fluorescence is determined by the fluorophore attached to the secondary antibody! Fluorescein Staining of Microtubules using “Indirect Immunofluorescence” 1 o Ab: mouse anti-tubulin; Tubulin immobilized by cell fixation Primary antibodies: mouse antibodies raised against antigen (tubulin) 2 o Ab: fluorescein goat anti-mouse Secondary antibodies: Goat antibodies raised against mouse antibodies (IgGs), conjugated to a fluorophore (fluorescein)

28. DAPI and Hoechst are dyes that bind directly to DNA and fluoresce brightly, with very similar spectra DAPI bound to the minor groove Hoechst 33258/33342 DAPI (diamidino phenyl indole)