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Overview of 130L Part 2

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

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Presentation Transcript
slide1
Week 2 (3/12-3/16)

Sub-Cloning

Week 1 (this week)

Fluorescence labeling and microscopy:

The actin and tubulin

cytoskeleton in cultured cells

Week 3 (3/19-3/23)

Transfection & vital staining:

The secretory and endocytic pathways

Overview of 130L Part 2

slide2
What you should get from today’s lecture
  • What are cultured cells and why do we use them?
  • General background on the cytoskeletonand the drug treatments used in this lab
  • How are cells fixed and stained with fluorescent reagents?
  • An introduction to microscope resolution
slide3
single cell (scanning EM)

colony of cells

dish of

cell colonies

100 mm

>0.01 mm

1 mm

How big are animal cells?

slide4
Cells in culture can undergo diverse differentiation pathways

Neuronal cell extending axons and dendrites

Mouse C2C12 muscle cells forming myofibers

slide5
embryonic stem cells

“feeder” cells

Stem cells are undifferentiated and pluripotent, meaning that they can differentiate to become multiple other cell types

slide6
Cell culture= propagation of cells outside the organism

The Good:

  • 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

The Bad:

1) Requires care and $$$ to grow & prevent contamination

2) May not always recapitulate real cellular physiology

slide8
Most cells in the body or removed from the body are MORTAL:they have a finite ability to replicate and divide

Immortal cell lines can arise by spontaneous mutation or deliberate transformation of mortal cell strains.

Embryonic stem cells are also immortal. We don’t really understand why they have this unlimited replication potential, or why most cells lack it.

slide9
Lab: Visualizing the cytoskeleton

using fluorescence microscopy

Purpose: stain cells to observe the cytoskeleton; Observe and record the effects of different drugs on cytoskeletal components and cell shape.

2 cytoskeletal components will be examined:

Actin - required for cell shape & movementsincluding translocation and cytokinesis

Tubulin - forms microtubule “tracks” that enable chromosomes, vesicles, etc.

to move in targeted ways within cells.

Your cells will be treated with 4 drugs (+ controls)

TPA/PMA

Latrunculin*

Taxol (Paclitaxel)*

Nocodazole*

*alter the equilibrium between subunits and polymersof actin or tubulin

slide10
Cells move!

all cell movement requires actin dynamics

slide13
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)

  • this leads to major changes in cell growth, cell shape and the cytoskeleton

TPA = phorbal myristate acetate = PMA

Phorbol ester

slide15
Latrunculin A

free G-actin

G-actin bound to Latrunculin A

slide16
Microtubules

DNA

Microtubules control other aspects of cell dynamics

including vesicle transport and chromosome segregation

interphase

mitosis

slide18
Microtubule dynamics are controlled by tubulin conformation, which can be modified by other proteins or drugs such as nocodazole or taxol

Eva Nogales’ lab

slide20
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).

slide21
Taxol binds to polymerized β-tubulin and inhibits depolymerization

Tubulin α/β dimer

taxol

GTP/GDP

slide22
Cell Fixation and Permeabilization

A. Chemical fixation - kills and immobilizes cells

  • Aldehydes (formaldehyde, glutaraldehyde): Cross-link amino groups in proteins.
  • Stabilizing many structures
  • Can block antibody access to targets.
  • 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)
slide23
Cell Fixation and Permeabilization
  • Permeabilization - detergents are used to solubilize cellmembranes to allow staining reagents to penetrate;makes proteins accessible to staining reagents.
  • Usually gentle, non-ionic detergents are used:

Brij-58

Tween-20

Triton X-100

  • Effect of detergents can depend upon order of steps (as in this week’s lab):
  • When added before fixative, they will often solubilize proteins (e.g. unpolymerized tubulin, as in today’s lab).
  • This can be a technical advantage - reduces background - but can also lead to experimental artifacts.
slide24
Staining of Actin

Phalloidin:a natural toxin from some mushrooms

Binds to filamentous actin (F-actin) only

For this lab, we buy phalloidin that is

covalently linked to rhodamine

Phalloidin

+

Amanita phalloides

TRITC = tetramethyl rhodamine isothiocyanate

Kidney cellstained withrhodamine-phalloidin

slide25
Variable regions

Antibodies are highly conserved molecules with variable regionsthat specify antigen recognition and affinity

Constant

region

Antibody, or Immunoglobulin

slide26
Fluorescein, or FITC

Secondary antibodies(react with primary antibodies)

Antibodies are highly conserved molecules with variable regionsthat specify antigen recognition and affinity

slide27
Fluorescein

Tubulin immobilizedby cell fixation

Secondary antibodies:Goat antibodies raised against mouse antibodies (IgGs),conjugated to a fluorophore

(fluorescein)

Primary antibodies:mouse antibodies raised against

antigen (tubulin)

Staining of Microtubules using“Indirect Immunofluorescence”

1o Ab: mouse anti-tubulin;

2o Ab: fluorescein goat anti-mouse

Note: color of the fluorescence isdetermined by the fluorophore attachedto the secondary antibody!

slide28
DAPI and Hoechst are dyes that bind directly to DNAand fluoresce brightly, with very similar spectra

DAPI (diamidino phenyl indole)

Hoechst33258/33342

DAPI bound to the minor groove

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