L5Exptal design and cell migration

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L5-Exptal design andcell migration
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Exptal design

• We think A -gt B
• A causes B.
• i.e. A is necessary for B to
  occur!
• How can we test this?
• we have an assay for B
• we can somehow interfere with or remove A.
• Conclusions
• If B still occurs, A wasnt all that necessary!
• If B changes/doesnt occur, A must be involved.

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Exptal design

• How do we interfere with A?
• So many ways!
• inhibitory drugs
• downregulate protein expression (promoters, etc.)
• deletion mutants
• siRNA (morpholinos)knockdown
• knockout mice
• etc.

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Exptal design

• Controls
• C (assay should work, no changes in B)
• - cells with functional A (sometimes called
  wt, wild type)
• mock knockdown (no siRNA)
• knockdown with nonsense/unrelated siRNA
• C-
• - is there something else known to regulate A?

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Exptal design

• How do we get things into living cells?
• microinjection
• transfection (lipofectamine, electroporation
  vectors include plasmids, adenovirus, etc.)
• cell permeant dyes and drugs
• etc.

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Cell migration

• cell migration
• development
• immune system
• wound healing
• but
• - metastasis

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Cancers

• Cancers
• disregulated dividing and crawling
• hard to find cancer-specific markers and
  behaviors
• Basics of life divide, crawl!
• - Development
• - Maintenance of body
• - Healing
• What gives you life, kills you. And heals you.
  (Dr. Giorgi, thesis)

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Cell migration- resources

• http//www.cellmigration2008.org/ Frontiers in
  cell migration meeting, Sept. 2008
• http//www.cellmigration.org/ Cell Migration
  Consortium website
• http//www.cellmigration.org/science/index.shtml
  migration 101
• http//cellix.imba.oeaw.ac.at/ cell migration
  primer

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Cell migration

• Stereotypical migration (fibroblast in 2-D
  culture)

view from side
leading edge
view from top
direction of migration
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Cell migration

• Some key players in cell migration
• Actin (and actin regulators, including cofilin)
• Cell matrix junctions focal adhesions,
  including vinculin, integrins, etc.
• Microtubules (for cell polarity)
• ECM

cell-cell junctions
cell-matrix junctions
ECM
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Paul Martin lab- wound healing

• Background
• - Embryos heal without scarring
• Purse string mechanism actin fibers pull the
  wound closed
• http//www.embryowound.info/
• Movie on embryonic wound healing.

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Paul Martin lab- wound healing

• Inflammation and scarring
• macrophages go to wound, remodel ecm., may cause
  fibrosis/scarring
• Wound healing gel
• slow release of osteopontin (glycoprotein, also
  upregulated in many tumors).
• ECM is much looser.
• Less scarring
• Quicker healing

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Paul Martin lab- wound healing

• DIC movie of macrophage extravasation!
• - Zebrafish (transparent embryos)
• - macrophages exit blood vessel, head right
  for wound
• http//www.embryowound.info/
• Directionality of migration by choice of
  pseudopods
• ? Wasp1 protein with morpholino and slower, less
  direct, same amount of pseudopods

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Paul Martin lab- wound healing

• http//www.embryowound.info/
• http//en.wikipedia.org/wiki/Osteopontin
• http//news.bbc.co.uk/2/hi/health/7199897.stm
  wound healing gel

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John Condeelis-breast cancer
Intravital imaging breast cancer tumors in
live mice real tumors, not xenografts Excellent
explaination of benefits of multiphoton http//ww
w.aecom.yu.edu/aif/intravital_imaging/introduction
.htm 2p 100 cells deep, less tissue damage,
no pinhole (more light), better z
resolution Confocal few cells deep, more toxic
byproducts, emission pinhole captures small of
light
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John Condeelis-breast cancer

• See individual cells exiting the tumor and
  heading for blood vessels
• crawl along ECM fibers, inchworm style
• 100x faster than see in vitro
• seem to be able, tragically, to get blood
  vessels to turn towards the tumor
• puts a catheter in with ECM like a blood vessel
  and sees tumor cells there in 90 min

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John Condeelis-breast cancer
confocal excitation
2 photon excites a spot
http//www.aecom.yu.edu/aif/intravital_imaging/cuv
ette.htm
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John Condeelis-breast cancer

http//www.aecom.yu.edu/aif/intravital_imaging/rat
.htm
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John Condeelis-breast cancer

• See individual cells exiting the tumor and
  heading for blood vessels
• crawl along ECM fibers, inchworm style
• 100x faster than see in vitro
• seem to be able, tragically, to get blood
  vessels to turn towards the tumor
• puts a catheter in with ECM like a blood vessel
  and sees tumor cells there in 90 min
• Movie http//www.aecom.yu.edu/aif/intravital_imag
  ing/introduction.htm
• Sequence 2 tumor cells run towards a blood
  vessel

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John Condeelis-breast cancer

• Macrophages may help the invasion
• paracrine signaling
• each tumor cell surrounded by a perivascular
  macrophage on either side entry point into blood
  vessel for tumor cell
• new anatomical landmark, signals trouble
• overexpression of certain proteins (mena)

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John Condeelis-breast cancer

• Cofilin low in benign tumors, high in metastatic
  tumors
• Cofilin senses subtle pH and PIP2 gradients (hard
  to find in most assays), turns them into
  migration on/off switch.
• Note most tumors have high pH.
• Autofluorescence of ECM fibers is increased in
  tumors.
• Could be diagnostic marker.

http//www.aecom.yu.edu/aif/intravital_imaging/int
roduction.htm
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John Condeelis-breast cancer

• Daniel Larson developing photoactivable promoter!
  Light activated gene expression, using ecdysone
  promoter.
• shine light --gt turn on a selected gene!
• activation persists for 5 days

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Denise Montell-embryos
Conventional model for cell migration during
development is EMT epithelial to
mesenchyme transition
cell-cell adhesion ----gt cell matrix
adhesion Works well for some cases, but need to
expand models, more diversity. Ovaries of adult
drosophila produce egg chambers. In each one, a
small group of border cells predictably undergo a
200 um migration towards the oocyte, in response
to hormones and cell signaling.
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Denise Montell-embryos

• 6 of 150 cells acquires ability to migrate in
  response to steroid hormones at a certain time in
  development.
• Questions what causes this? How do they move?
• Assay cells migrate.
• Unusual
• Migrate as a coherent cluster
• 2 non migratory cells in the center retain
  polarity
• 4-6 migratory cells surround them (and carry
  them along)
• Interesting cell-adhesion challenges inside the
  cluster!

oocyte
cluster
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Denise Montell-embryos
Movie - GFP labeled border cells migrate towards
oocyte http//www.hopkinsmedicine.org/dmontell/
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Some opportunities

• Diane Barber pH affects cell migration.
• Diane Cox, Albert Einstein College of Medicine
• Surp summer intern program, Albert Einstein
  College of Medicine
• Claire Brown, job posting