ACTIN STAINING OF FIBROBLASTS

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ACTIN STAINING OF FIBROBLASTS
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Housekeeping

• Be vigilant about spills
• Wipe up as soon as possible without compromising
  sterility or activities
• Watch for splatters on back of hood

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Lab Objectives

• Understand the cellular biology ( size, structure
  and functions) of
• Microtubulues, Intermediate Filaments
• G-Actin and F-actin
• Stress Fibers, Focal Contact/Adhesion, Adhesion
  Belt, Contractile Ring
• Become familiar with fluorescent microscopy
  concepts and reagents used this week
• Specific reagents such as formaldehyde, glycine,
  Triton-X 100, PBS and Rhodamine phalloidin
• Fluorescent microscope itself and components, be
  able to diagram all parts and the light path
  (show diagram)
• Alternatives to organic dyes

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Cytoskeleton

• Gives the cell mechanical strength, controls its
  shape, drives and guides its movement
• Four classes
• Muscle thick filament
• Microfilaments
• Intermediate filaments
• Microtubules

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Appearance of cytoskeletal components in cultured
fibroblast cell
Transmission Electron Microscopy TEM
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Comparison of cytoskeletal components
Rope-like bars Long, hollow cylinders
Helical polymers of actin about 10 nm made
from tubulin flexible, concentrated in
about 25nm the cortex of the
cell about 7nm
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Microtubules

• Most rigid of cytoskeletal elements
• May occur singly or in groups
• Responsible for movement of materials within cell
• Gives cells shape
• Form cilia and flagella
• Helps cells divide

Negative stain
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Placement of tubulin in non-metaphase and
metaphase cell

• Microtubules help with intracellular traffic in
  non-dividing cell
• Aid in splitting apart sister chromatids during
  cell division

Fluorescently labeled antibody against tubulin
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Intermediate Filaments

• Strengthen cell against mechanical stress
• Keratin is found in epithelial cells
• Vimentin is found in connective tissue, muscle
  and support cells of the nervous system
• Neurofilaments are found in nerve cells

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Microfilaments

• Are the thinnest at 7nm in diameter
• Also called actin
• Ubiquitous, multifunctional
• Responsible for cell crawling
• Needed to produce contractile forces
• Interacts with myosin for muscle contraction
• Actin filaments are thin and flexible
• Usually found in bundles rather than individually
• Usually associated with the plasma membrane

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Placement of actin in cells
Contractile ring In dividing cells
Microvilli cores And terminal web
Leading edge of Motile cell
Stress fibers And cell cortex
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Functions of Actin in Non-Muscle Cells

• Contractile ring
• important in cell division

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• Adhesion belts
• Intercellular anchoring junctions found in
  epithelia

• Stress fibers
• Important for a cells structure and movement
• Are involved in forming adhesion plaques (points
  of cell adhesion to the ECM)

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• Actin bundles
• Bundles of actin, parallel in microvilli on many
  columnar epithelial cells
• Important for the structure and movement of the
  microvilli, to help keep them clean

• Cell cortex
• Actin fibers connected to the plasma membrane
• Play a role in providing mechanical strength to
  the surface, allowing for movement.

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Lamellipodia, filipodia and blebs
Pinocytotic cup

• These membrane protrusions are under the control
  of actin
• Can view these as cells move across a surface

lamellipodia
Bridge still connecting recently divided cells
filipodia
bleb
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Actin (microfilaments)

• The most abundant protein in eukaryotic cells
• 2 forms
• G-actin
• globular, a protein monomer associated with ATP
• ATP needed to bind units together to form a
  filament.
• F-actin
• filamentous, a polymerized chain of actin
  monomers
• Polymerization occurs on the positive end of the
  filament

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Actin Assembly/Polymerization
Direction of Growth
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Solutions and Equipment

• PBS
• Used to wash the cells
• 4 Formaldehyde
• Cross-links and fixes proteins in cells, prevents
  degradation
• Glycine
• Blocks background (noise) by binding ketones
• 0.4 Triton X-100
• A detergent that permeabilizes cell membrane for
  dye penetration

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• Mounting Media
• Specially formulated to not cause interference in
  the fluorescent image
• Rhodamine Phalloidin
• Phalloidin
• From the poisonous mushroom Amanita phalloides
  (Death Cap)
• Binds specifically to f-actin
• Rhodamine
• Fluorescent marker conjugated to phalloidin
• Excited by green light l 520-560 nm
• Emits red light l 650-720nm

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Fluorescence Microscopy

• Green light photons promote rhodamine electron
  to a higher energy level. When they return to
  original level, they emit a lower energy photon.

• What you can expect to see
• Black background, with red stress fibers visible
• Keep sample protected from light after rhodamine
  phalloidin is added.
• If a fluorescent sample is left exposed too long
  to light, the fluorescence will fade. This is
  called quenching or photobleaching

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Fluorescence Microscope Diagram
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A formula for those who like them
The fluorescence intensity in a stained object
can be related to a series of factors to which it
is directly proportional as elucidated in the
following formula  
  Equation 1 F fluorescence intensity f(q) is
a geometric factor g(l) is the quantum
efficiency of the detector and I0 denotes the
intensity of the excitation light. FF denotes the
quantum efficiency of the fluorescence dye, e is
the extinction coefficient of the dye, b the
optical path length (thickness with dye) and c
the concentration of the dye. All the factors in
the M bracket are to do with the imaging and
illumination side, the C bracket contains those
factors concerned with dye concentration sample
thickness, etc..
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Other Fluorescent Dyes and Techniques

• Other chemicals with specificity for particular
  parts of the cell
• DAPI, Hoechst, or ethidium bromide for DNA
• Fluorescently tagged antibodies
• Used anywhere you have a protein you have an
  antibody for.
• Anti-tubulin antibody conjugated to fluorescein
• (excited by blue and glows green)
• GFP
• Must be expressed by the cells.
• Can localize a particular protein or entire cell

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Quantum dots

• Tiny light emitting particles on the nanometer
  scale
• Inorganic semiconductors
• Cadmium selenide
• 2-8nm (200-1000atoms)
• Size determines the color
• Synthesized to emit l between 400-2000nm by
  changing composition and size

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QD advantages over organic dyes

• Same size regime as biological macromolecules
• Resistant to photobleaching
• Improved brightness
• Multicolor light emission from single light
  source
• Ultrasensitive chemical analysis and cellular
  imaging
• Can image within an organism

QD
Texas red
QD
Fluorescein -FITC
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Multi colored fluorescence images require
changing excitation sources and layering the
images

• Blue DAPI (for DNA)
• Red Rhodamine phalloidin (actin)
• Green HPA lectin conjugated to Alexa Fluor 488
  for golgi
• Pink Alexa Fluor 680 conjugated to primary
  antibody for mitochondrial protein

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Disadvantages of QD

• Expensive
• Not refined yet
• Can be toxic
• Resistance to change on the part of scientists

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Fluorescence tutorials from Molecular Probes, Inc.

• TAs will show these in class
• http//probes.invitrogen.com/resources/education/t
  utorials/1Introduction/player.html
• http//probes.invitrogen.com/resources/education/t
  utorials/3Light_Sources_Filters/player.html

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Application to Tissue Engineering

• Cytoskeletal changes are seen in
• Cell migration (chemotaxis)
• Physiological functions
• Organogenesis and embryonic development
• Wound healing and angiogenesis
• Understanding tissue development and cellular
  changes helps the tissue engineer create new
  tissues.

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Cellular response to topography

• SEM images A 13nm islands, B 35nm, C 95nm
  islands
• Cells more spread out on 13nm islands compared to
  flat polystyrene, No difference on 35nm islands,
  less spread on 95mm islands
• Distinct interactions between cell filipodia and
  islands. Interactions increased with island size
• Larger islands produced more amoeboid-like
  pseudopodia

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Cytoskeletal response to topography

• Actin red (rhodamine phalloidin), tubulingreen
  (Fluorescein anti tubulin), nucleusblue (DAPI)
• 13nm islands (a) supported fibroblasts with well
  defined actin and tubulin skeletons
• 35nm islands (b) less distinct cytoskeleton
• 95nm islands (c) had poorly organized
  cytoskeleton
• Time studies show that cells adhere and spread
  better initially on the large islands but by 24
  hours of culture the cells take on the amoeboid
  morphology and proliferate very slowly.

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Cell Morphology on Scaffolds
Lamellopodia Filopodia
Images courtesy of Aylin Sendemir
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On Chitosan On Chitosan/BCP
Images courtesy of Aylin Sendemir
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By The Way...

• If no times are given for washes, just after
  solution is put on, take it off.
• Incubation temp is RT
• Keep cells moist at all times during prep.
• Step 10 Put coverslip and parafilm in moist,
  dark box, then tell TA. TA will distribute
  phalloidin due to toxicity.
• When adding warmed mounting media, you may want
  to cut end of yellow pipette tip to have a larger
  opening for easier dispensing.

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Other exercises this week

• Groups 4, 5, 6 complete exercises B and C before
  starting actin staining exercise
• B-Add induction media to all three wells of 6
  well plate
• Remove mediaGENTLY
• No rinsing!
• Add induction media from TA--GENTLY
• C-Create at least one stock plate of 3T3-L1
• calculate cells for 10cm dish