Work Smart Not Hard: Instant Visualization of Organelles

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Work Smart Not Hard Instant Visualization of
Organelles

• Scott Hartsel, David Lewis, Lori Scardino
• UW-Eau Claire

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How did we get here?Studies on Amphotericin B

• Mechanism of action
• Mechanism of toxicity/efficacy effect of
  different drug delivery systems on induced ion
  currents,inflammatory response and uptake

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We think route of uptake might have a profound
effect on AmB toxicity/efficacy and PK

• Serum effects
• Clathrin uptake (LDL-assoc)
• Caveolar (direct interaction with raft domains)
• Transcytosis (HSA)
• Phagocytosis
• Other?

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Experimental Approach

• We wanted to colocalize different labeled
  formulae with different organelles, membrane
  domains and compartments
• Design of AmB fluorescent analogs-tricky!
• Early problems with bleaching of commercial
  probes, e.g. Lysotracker Red
• Enter naphthalimide probes and the extensive
  collection of David Lewis

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Overview of Organelles
Endoplasmic Reticulum
Nucleus
Nucleolus
Mitochondria
Golgi
Plasma Membrane
Cytosol
Lysosome
Lipid Rafts
Image from Molecular Probes Invitrogen website
was modified in Adobe Photoshop
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InstantLyso LLT1

• Max Excitation 421, Max Emission 533
• Weak base should accumulate in acidic organelles
• Resistant to photobleaching
• Non-cytotoxic
• High quantum yield
• Exhibits very little self quenching
• Loads rapidly into live cells
• Fixable with paraformaldehyde

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InstantLyso LLTImpressive Stokes Shift
Lysotracker green
InstantLyso
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THP-1 Lysosomes Labeled with InstantLyso

• Lysosomes are labelled,verified through
  colocalization with LysoTracker Red
• Should increase fluorescence 10x for every 1 ? pH
  (even 0.5 pH unit would give 3 x increase)
• We noted that the MTOC was visiblecould the
  Golgi or other compartments also be labeled?

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InstantLysoWhere does it go?

• Many compartments have relatively acidic pH,
  including Golgi

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InstantLyso Labeling of Golgi
BODIPY TR C5-ceramide
InstantLyso1

• Trans-Golgi (more acidic) is plainly labeled as
  well as lysosomes in fibroblasts.
• InstantLyso may also get to the Golgi by caveolar
  transport

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InstantLyso Colocalization
BODIPY TR C5-ceramide
InstantLyso1
But notice there are places where there is no
overlap
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Immunofluorescence Using Anti-Golgin 97 to
Label the Golgi
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Immunofluorescence Using Anti-Golgin to Label
the Golgi works well-Why bother with a new label?
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Comparing InstantLyso location with other probes

• REALimage

FFT filtered
Endoplasmic Reticulum in human foreskin
fibroblasts. Probe used ER Tracker Blue-White
DPX from Molecular Probes/ Invitrogen.ImageJ FFT
filter
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Comparison
ER

• REAL

BODIPY ceramide

• ER blue/white

Instant Lyso
InstantLipo
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What specificity is due to just the
naphthalimide?InstantLipo

• Max Excitation 432, Max Emission 533
• Resistant to photobleaching
• Non-cytotoxic
• High quantum yield
• Exhibits very little self quenching
• Loads rapidly into live cells

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THP-1 Labeled with InstantLipo

• A very different pattern than InstantLyso. The
  MTOC is much brighter but also many other areas
  are stained
• Could this probe have an affinity for raft-type
  domains?
• Sometimes looks like an alien!

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Lipid Rafts Localize with InstantLipo
Merge
InstantLipo
CTB
CTB
InstantLipo
Merge
CTB
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1
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Cells were treated with InstantLipo Sep-1 and
Molecular Probes Vybrant Alexa Fluor 594 Lipid
Raft Labeling Kit based on the cholera toxin
subunit B. 1) InstantLipo 2) Merged image 3) CTB
kit fluorescence
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Deranged Cholesterol Metabolism
Without U18666A
With U18666A

• U18666A is an aminosterol that inhibits
  cholesterol trafficking
• InstantLipo labels cholesterol inclusions
  potential for FACS/ high throughput screening of
  sterol metabolism

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Golgi Labeling with InstantLipo

• Golgi can be seen in fibroblasts stained with
  InstantLipo Sep-1, but not because they are
  acidic
• Golgi is rich in lipid raft components such as
  cholesterol and sphingolipids
• The naphthalimide chromophore must have an
  intrinsic affinity for raft-like domains

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Why would InstantLipo go there anyway?
Cholesterol
Very cozy!
InstantLipo
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Other probes InstantMito Dyes
Human Fibroblasts with Filamentous Mitochondria
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Other probes InstantMito Dyes

• Smallest delocalized cationic system we know of
  thus highly soluble
• Compare with Mitotracker Green
• Should show 10X fluorescence increase per -60 mV
  ??
• Even -20 mV should have 2X increase

InstantMito2

• Length of conjugated, delocalized cation system
  is only 4.2Å, less than 1/2 of mitotracker

Mitotracker Green
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Confirming that we are localizing in mitochondria
MitoTracker Red Commercially available
mitochondrion dye
Merge Yellow areas show where both dyes occupy
the same place in the cell
InstantMito1 in THP-1 monocytes
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Possible Uses/Applications of Probes

• InstantMito
• Apoptosis assays by FACS

• InstantLyso
• Real time tracking of acidic organelles in live
  cells
• Colocalization for drug uptake studies
• InstantLipo
• Diagnostic for lipid storage diseases
• Cholesterol accumulation trafficking probe, e.g.
  for cholesterol lowering drug development
• Histological or in vivo visualization of
  cholesterol deposits
• Studies of lipid microdomains

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Acknowledgements

• Dr. Lloyd Turtinen, Dr. David Lewis, Kristy
  McNitt, Grant Sormunen, Jessica Walters, and
  Robyn Laskowski for synthesis of probes
• Lori Scardino, Damon Campbell, Vinay Rao, and
  Kavita Sachdeva
• NSF, UW-Eau Claire Center of Excellence for
  Faculty and Undergraduate Student Research
  Collaboration, WiSys, UW-System Applied Research
  Grant.

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What is Amphotericin B?

• The Swiss Army Knife of drugs
• Antifungal (1st line for serious fungal
  infections)
• Antiparasitic (Leishmania)
• Antiviral (HIV)
• Antimicrobial (indirect?)
• Antiprion (e.g. scrapie andmad cow disease !!)
• Anticancer?
• BUT, It is extremely toxic.

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• cholesterol

• ergosterol

• AmB

• Lipid

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A model based on aggregation state and reduction
of chemical potential in serum and hence ion
channel formation in cholesterol vs ergosterol
containing membranes
AmB delivery from liposomes reduces side effects
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• Possible models of immune cell modulation.

Recent evidence Shows that TLR-2 and CD-14
are necessary for AmBs Inflammatory response
We have shown that HUVECS (with no TLR-2) do not
respond to AmB, but THP-1s do. TLR-4 does not
seem to matter.
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Some Puzzles.

• Lipid-based delivery systems do produce less
  direct membrane ionophoric activity, but toxicity
  is not always correlated with ion fluxes.
• AmB is highly toxic at the organismal level. This
  is assumed to be due to ion leakage, BUT 20X
  fatal dose (and 50X ion current producing dose)
  can be easily tolerated by kidney cell cultures
  and some other cultured cells.
• Immune overstimulation is a major untoward
  side effect (fever, chills, nausea). AmB may also
  reactivate HIV infections.
• What else could be happening besides ion leakage?

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AmB aggravates the inflammatory response,but how?

• Different lipid associated drug delivery systems
  induce different responses-Studies with cytokine
  antibody arrays of monocytes.