Overview of 130L Part 2 Week 3 (3/19-3/23) Week 3 (3/19-3/23) Transfection & vital staining: Transfection & vital staining: The secretory and endocytic pathways The secretory and endocytic pathways Week 2 (3/12-3/16) Sub-Cloning Sub-Cloning Week 1 (this week) Week 1 (this week) Fluorescence labeling and microscopy: Fluorescence labeling and microscopy: The actin and tubulin The actin and tubulin cytoskeleton in cultured cells cytoskeleton in cultured cells
The secretory and endocytic pathwaysThe secretory and endocytic pathways
Week 2 (3/12-3/16)Sub-CloningSub-Cloning
Week 2 (3/12-3/16)Sub-CloningSub-Cloning
Week 1 (this week)Week 1 (this week)Fluorescence labeling and microscopy: Fluorescence labeling and microscopy:
The actin and tubulin The actin and tubulin cytoskeleton in cultured cellscytoskeleton in cultured cells
Week 1 (this week)Week 1 (this week)Fluorescence labeling and microscopy: Fluorescence labeling and microscopy:
The actin and tubulin The actin and tubulin cytoskeleton in cultured cellscytoskeleton in cultured cells
What you should get from today’s lectureWhat you should get from today’s lecture
1. What are cultured cells and why do we use them?1. What are cultured cells and why do we use them?
3. How are cells fixed and stained with fluorescent reagents?3. How are cells fixed and stained with fluorescent reagents?
4. An introduction to microscope resolution4. An introduction to microscope resolution
2. General background on the cytoskeletonand the drug treatments used in this lab2. General background on the cytoskeletonand the drug treatments used in this lab
dish of cell colonies
100 mm
single cell (scanning EM)
>0.01 mm
colony of cells
1 mm
How big are animal cells?
Cells in culture can undergo diverse differentiation pathways
Neuronal cell extending axons and dendrites
Mouse C2C12 muscle cells forming myofibers
Stem cells are undifferentiated and pluripotent, meaning that they can
differentiate to become multiple other cell types
embryonic stem cells
“feeder” cells
The Bad:The Bad:1) Requires care and $$$ to grow & prevent contamination 1) Requires care and $$$ to grow & prevent contamination 2) May not always recapitulate real cellular physiology2) May not always recapitulate real cellular physiology
Cell culture = propagation of cells outside the organismCell culture = propagation of cells outside the organism
The Good:The Good:1)Cellular environment can be easily observed and manipulated
a. Injectionb. Transfection -introduction of genesc. 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 - biochemistry4) Diverse cellular functions can be investigated5) Noninvasive way to study mammalian cells
1)Cellular environment can be easily observed and manipulated
a. Injectionb. Transfection -introduction of genesc. 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 - biochemistry4) Diverse cellular functions can be investigated5) Noninvasive way to study mammalian cells
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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.
2 cytoskeletal components will be examined:2 cytoskeletal components will be examined:
Actin - required for cell shape & movementsincluding translocation and cytokinesis
Actin - required for cell shape & movementsincluding translocation and cytokinesis
TubulinTubulin - forms microtubule “tracks” - forms microtubule “tracks” that enable chromosomes, vesicles, etc.that enable chromosomes, vesicles, etc.to move in targeted ways within cells.to move in targeted ways within cells.
TubulinTubulin - forms microtubule “tracks” - forms microtubule “tracks” that enable chromosomes, vesicles, etc.that enable chromosomes, vesicles, etc.to move in targeted ways within cells.to move in targeted ways within cells.
• DAG plus Ca++ activate protein kinase C (PKC)• 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
• this leads to major changes in cell growth, cell shape and the cytoskeleton
Phorbol esterPhorbol ester
A schematic of the normal activation of PKCA schematic of the normal activation of PKC
MARCKS
Latrunculin A free G-actinG-actin bound to Latrunculin A
interphase mitosis
Microtubules
DNA
Microtubules control other aspects of cell dynamics
including vesicle transport and chromosome segregation
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Microtubules are Comprised of Many Tubulin Heterodimers
Microtubule dynamics are controlled by tubulin conformation, which can be modified by other
proteins or drugs such as nocodazole or taxol
Eva Nogales’ lab
Microtubule dynamics during interphase
QuickTime™ and aSorenson Video decompressorare needed to see this picture.
Microtubule dynamics at the cell edge
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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).
Taxol binds to polymerized β-tubulin and inhibits depolymerization
taxol GTP/GDPTubulin α/β
dimer
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 PermeabilizationCell Fixation and Permeabilization
A. Chemical fixation - kills and immobilizes cellsA. Chemical fixation - kills and immobilizes cells
2.Effect of detergents can depend upon order of steps (as in this week’s lab):
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).
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)This can be a technical advantage - reduces background - but can also lead to experimental artifacts.
B.Permeabilization - detergents are used to solubilize cellmembranes to allow staining reagents to penetrate;makes proteins accessible to staining reagents.
B.Permeabilization - detergents are used to solubilize cellmembranes to allow staining reagents to penetrate;makes proteins accessible to staining reagents.
1.Usually gentle, non-ionic detergents are used:1.Usually gentle, non-ionic detergents are used:Triton X-100Triton X-100 Tween-20Tween-20 Brij-58Brij-58
Cell Fixation and PermeabilizationCell Fixation and Permeabilization
For this lab, we buy phalloidin that iscovalently linked to rhodamine
Staining of ActinStaining of Actin
Amanita phalloidesAmanita phalloides
Phalloidin
Phalloidin:a natural toxin from some mushroomsBinds to filamentous actin (F-actin) only
+
TRITC = tetramethyl rhodamine isothiocyanate
Kidney cellstained withrhodamine-phalloidin
Variable regions
Antibodies are highly conserved molecules with variable regionsthat specify antigen recognition and affinity
Antibody, or Immunoglobulin
Constant region
Antibodies are highly conserved molecules with variable regionsthat specify antigen recognition and affinity
Fluorescein, or FITC
Secondary antibodies(react with primary antibodies)
Note: color of the fluorescence isdetermined by the fluorophore attached
to the secondary antibody!
Fluorescein
Staining of Microtubules using“Indirect Immunofluorescence”
1o Ab: mouse anti-tubulin;
Tubulin immobilizedby cell fixation
Primary antibodies:mouse antibodies raised against antigen (tubulin)
2o Ab: fluorescein goat anti-mouse
Secondary antibodies:Goat antibodies raised against
mouse antibodies (IgGs),conjugated to a fluorophore
(fluorescein)
DAPI and Hoechst are dyes that bind directly to DNAand fluoresce brightly, with very similar spectra
DAPI and Hoechst are dyes that bind directly to DNAand fluoresce brightly, with very similar spectra
DAPI bound to the minor grooveDAPI bound to the minor groove
