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HORIZON DISCOVERY
Application Note
Immunofluorescent microscopy: Applications for secondary profiling of compounds that
target the cytoskeleton
David Sorrell, Ceri Wiggins and Rebecca Foster
Introduction
Fluorescent microscopy is the main tool used for examining the
cytoskeleton. Horizon Discovery offers custom immunofluorescence
imaged-based assays to support the secondary cellular profiling of
compounds originating from cytoskeletal-targeting drug discovery
programs (e.g. Aurora and Polo-like kinases, microtubule-binding
agents, or kinesins) or where a compound series is suspected of having
off-targets against the cytoskeleton.
Here the use of fluorescence microscopy to characterize the on-target
effects of an Aurora kinase inhibitor and two tubulin-binding agents,
and the off-target effects of a broad-spectrum kinase inhibitor, on the
cytoskeleton is described.
Methods
Cells were grown on glass coverslips. After treatment they were fixed,
stained with tubulin antibodies or Alexa Fluor 488-phalloidin to visualize
F-actin, mounted in anti-fade mounting medium containing DAPI and
imaged using a Zeiss LSM500 Meta confocal microscope.
Control
Interphase
VX680
*
*
*
Paclitaxel
Unintended disruption of the cytoskeleton is also a commonly
encountered off-target effect of small molecules, which can result in
undesirable cytotoxity3. This is most likely due to the promiscuous
nature of tubulin in binding a structurally diverse range of molecules
and because the cytoskeleton is regulated by a complex network of
upstream regulators.
Mitosis
Nocodazole
The cytoskeleton is a target for several classes of important
pharmacological agents. Anti-mitotics target microtubules, either by
binding directly to tubulin, or indirectly by inhibiting upstream
regulators such as mitotic kinases or kinesin motor proteins1. This
results in disruption of the bipolar spindle and mitotic arrest. Similarly,
actin filaments can be modulated by direct binding of inhibitors or by
indirect inhibition of upstream regulators, which can result in effects on
cell motility and shape2.
Figure 1. Differential phenotypes resulting from anti-mitotic
treatment. HeLa cells were treated with VX680 (0.5 µM), paclitaxel
(0.1 µM) or nocodazole (1 µM) for 24h. Mini-spindles are indicated
with asterisks. Cells were fixed and stained to reveal microtubules
(α-tubulin; red), centrosomes (ϒ-tubulin; green) and DNA (blue).
Results & Discussion
The on-target effects of three anti-mitotic agents, paclitaxel (microtubule stabilizing agent), nocodazole (tubulin polymerization inhibitor) and VX680
(MK-0457; pan-Aurora kinase inhibitor) on mitotic spindle microtubules were investigated in HeLa cells after 24h of treatment. VX680 induced a
number of spindle defects consistent with previous reports4. Most striking was a novel phenotype in which bi- or tripolar mini-spindles, that were
typically separated from the main chromosomal mass, were formed (Figure 1). Increased numbers of multinucleated interphase cells were also
apparent, which is consistent with the known ability of Aurora B inhibition to induce polyploidy4 (Figure 1). Nocodazole induced the formation of
abnormal multipolar spindles consisting of short thick microtubules, whereas paclitaxel resulted in the formation of well-developed ‘bushy’
monopolar and multipolar spindles (Figure 1). These agents also distorted the microtubules of interphase cells compared to control and VX680treated cells, which exhibited long and relatively straight microtubules.
Control
Staurosporine
α-tubulin
α-tubulin
These observations are consistent with published literature5,6. To
investigate the use of immunofluorescence to identify off-target
phenotypes, we examined the non-specific effects of the broadspectrum kinase inhibitor staurosporine, on microtubules and actin
filaments in MCF10A cells after a 2h treatment (Figure 2).
Staurosporine-treated cells adopted a striking star-like morphology due
to the presence of multiple cytoplasmic extensions. Staurosporine
treatment also resulted in the disappearance of stress fibers that were
present in control cells and the accumulation of F-actin at the periphery
of the extensions. These phenotypes are consistent with the
uncoordinated lamellipodial activity reported for other cell lines in
response to staurosporine7.
Conclusion
Fluorescent confocal microscopy is an important tool for studying the
cytoskeleton since it can extract detailed information at the single cell
level, which cannot be achieved with Western blot, flow cytometry or
plate-reader-based approaches commonly favored for drug discovery.
Two important applications of these methods are to confirm the ontarget mechanism-of-action of anti-mitotic agents and for identifying
unintended ‘off-target’ effects on the cytoskeleton.
F-actin
F-actin
References
1.
2.
3.
4.
5.
6.
7.
Merge + DAPI
Merge + DAPI
Figure 2. Staurosporine induces an altered morphology
and F-actin redistribution. MCF10A cells were treated
for 2 h with 2 µM staurosporine, fixed and stained to
reveal microtubules (α-tubulin; red), F-actin (Alexa Fluor
488-phalloidin; green) and DNA (blue).
Jackson et al., Nature Rev Cancer 2007 (7) p107
Fenteany and Zhu, Curr Topics in Med Chem 2003 (3) p593
Ross-Macdonald et al., Mol Cancer Ther 2008 (7) p3490
Tyler et al., Cell Cycle 2007 (6) p2846
Ywon et al., Mol Biol Cell 1999 (10) p947
Jordan et al., J Cell Sci 1992 (102) p401
Mannherz et al., Eur J Cell Biol 2006 (85) p785
Horizon Support
Horizon supplies genetically-defined cell lines, custom cell line
generation, in vivo models, reporter gene assay kits, molecular
reference standards and assay development and screening services to
organizations engaged in academic research; drug discovery and
development; clinical diagnostics; and biopharmaceutical process
optimization. Please contact us to learn more about how Horizon can
support your work.
Cell lines similar to those listed in this Application Note include:
Name
Description
Catalogue No.
p53 (R175HInducible)
DLD-1 Over-expression of GFP-tagged mutant (R175H) p53 induced in the absence
of tetracycline
HD R00-010
p53 (wtInducible)
DLD-1 Over-expression of GFP-tagged wild type p53 induced in the absence of
tetracycline
HD R00-012
PUMA (wtInducible)
DLD-1 Over-expression of GFP-tagged PUMA induced in the absence of tetracycline HD R00-014
TGFβRII (wtInducible)
DLD-1 Over-expression of HA and GFP dual tagged TGFβRII induced in the absence
of tetracycline
Over-expression lines
HD R00-016
HaloTag® reporter kits
K-Ras (+Halotag/+)
SW48 Heterozygous HaloTag fusion to wild type K-Ras
K-Ras (G12CHaloTag®/+)
SW48 Heterozygous HaloTag reporter fusion to K-Ras activating mutation (G12C)
K-Ras (G12DHaloTag®/+)
SW48 Heterozygous HaloTag reporter fusion to K-Ras activating mutation (G12D)
K-Ras (G12VHaloTag®/+)
SW48 Heterozygous HaloTag reporter fusion to K-Ras activating mutation (G12V)
K-Ras (G13DHaloTag®/+)
SW48 Heterozygous HaloTag reporter fusion to K-Ras activating mutation (G13D)
Contact Horizon or go to
www.horizondiscovery.com
for more information.
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