VISUALIZATION OF

ACTIN FILAMENTS
Group 2 - Cruz, Tan Palanca, Rayos del Sol
Group 4 - Carreon, Enriquez, Lingao
Group 5 - Benig, Brillantes, Yu

ICE BREAKER

ACTIN FILAMENTS (AF)

The cytoskeleton is made up of cytoskeletal

filaments; one of those is the AF

Functions include mechanical support and

whole cell locomotion

Thinnest cytoskeletal filament (~7 nm)

ACTIN FILAMENTS (AF)

2 strands of protein twisted like a rope

F-actin = linear polymer of G-actin

(globular) subunits

Present throughout the cell but mostly

concentrated in the plasma membrane

VISUALIZATION OF ACTIN
FILAMENTS

Actin antibodies

Fluorescent
phalloidin

Electron microscopy

BACKGROUND

In 1937, Feodor Lynen and Ulrich Wieland

crystallized phalloidin from the toxic Amanita
phalloides or death cap mushroom

In 1979, Wulf et al. used a fluorescent derivative

of phalloidin to visualize cellular filamentous
actin; in 1981, Wehland and Weber; actin
rearrangement in living cells

BACKGROUND

Cooper first
reported in 1981
that fluorescent
phalloidin is a
useful tool in
investigating F-actin
distribution in cells
(basing from two
previous studies)

FLUORESCENCE
MICROSCOPY
Fluorescence

absorption and re-radiation of light by

certain molecules

Irradiate with desired specific wavelengths

(excitation filter) -> block unwanted excitation
wavelengths (barrier filter)

FLUORESCENCE
MICROSCOPY
Multiple fluorescence
labeling allows for
simultaneous
identification of several
target molecules

FLUORESCENT PHALLOIDIN

Fluorescent derivative of
phalloidin, a highly toxic
heptapeptide phallotoxin
from A. philloides

Phallotoxins stabilize the

F-actin

FLUORESCENT PHALLOIDIN

Binds to native
quaternary structure of
F-actin (more tightly than
to G-actin)

Produces low
background staining

STAINS USED

Alexa Fluor 488 Phalloidin

Phalloidin conjugated to a bright, greenfluorescent Alexa Fluor 488 dye

Excitation: 495 nm

Emission: 519 nm

STAINS USED

DAPI (4,6-diamidino-2-phenylindole)

Nuclear and chromosome counterstain; binds

to AT-rich regions

Excitation: 350 nm

Emission: 470 nm

METHODOLOGY

GENERAL STEPS
1. Treatment
2. Fixation
3. Permeabilization
4. Blocking

5. Staining
6. Mounting
7. Fluorescence
Microscopy

permeabilization

fixation

blocking

RESULTS

Primary culture of mouse liver cells viewed at 10X magnification using

Olympus IX51 inverted microscope with (L) phalloidin-AlexaFluor488
(InvitrogenTM) conjugated stain through FITC filter in fluorescence; (M) 4,
6-diamidino-2-phenylindole (DAPI) counterstain through the DAPI filter in
fluorescence; (R) both stains superimposed.

RESULTS

Primary culture of mouse liver cells viewed at 20X magnification using

Olympus IX51 inverted microscope with (L) phalloidin-AlexaFluor488
(InvitrogenTM) conjugated stain through FITC filter in fluorescence; (M) 4,
6-diamidino-2-phenylindole (DAPI) counterstain through the DAPI filter in
fluorescence; (R) both stains superimposed.

TROUBLESHOOTING

COMMON PROBLEMS:

Overstaining

Autofluorescence

Photobleaching

Optimal microscope criteria for excitation

TROUBLESHOOTING

Overstaining (non specific fluorescence)

decrease concentration of dye to prevent

overstaining

Autofluorescence

wash cells thoroughly to remove excess fluorochrome

TROUBLESHOOTING

Photobleaching

Use of a different dye during staining

neutral-density filters

reducing the exposure of the specimen to the

excitation light

TROUBLESHOOTING

Optimal microscope criteria for excitation

High energy light sources

Proper barrier filters

APPLICATIONS

Monitoring cancer cell invasion and metastasis

Lorente G., Syriani E., Morales, M. (2014). Actin Filaments at the Leading
Edge of Cancer Cells Are Characterized by a High Mobile Fraction and
Turnover Regulation by Profilin I. PLoS ONE 9(1): e85817. doi:10.1371/
journal.pone.0085817

Creating models or paradigms of protein-cytoskeleton

interactions and cytoskeleton-dependent protein expression

Mullins, R. D. (2000). How WASP-family proteins and the Arp2/3 complex

convert intracellular signals into cytoskeletal structures. Current Opinion in
Cell Biology 12(1):91-96.
http://dx.doi.org/10.1016/
S0955-0674(99)00061-7

APPLICATIONS

Tracing cellular transportation

Toomre, D., Keller, P., White, J., Olivo, J. C., & Simons, S. (1999).
Dual-color visualization of trans-Golgi network to plasma
membrane traffic along microtubules in living cells. Journal of Cell
Science 112: 21-33. Retrieved from http://jcs.biologists.org/content/
112/1/21.long.

Tracing cytoskeletal regulation of signalling pathway

Fletcher, G., Elbediwy, A., Khanal, I., Ribeiro, P., Tapon, N., &
Thomson, B. (2015).The Spectrin cytoskeleton regulates the Hippo
signalling pathway. The EMBO Journal: e201489642. doi:10.15252/
embj.201489642

REFERENCES

Alberts, B., Johnson, A., Lewis,thJ., Morgan, D., Raff, M., Roberts, K., & Walter, P. (2015).
Molecular biology of the cell (6 ed.). New York, NY: Garland Science.

Abramowitz, M. & Davidson, M.W. (2012). Optimization and Troubleshooting. Olympus

Microscopy Resource Centre. Retrieved 12 September 2016 from http://
www.olympusmicro.com/primer/techniques/fluorescence/troubleshoot.html

Buchwalow, I. B., & Bcker, W. (2010). Immunohistochemistry: Basics and methods.

Heidelberg: Springer.

Thermo Fisher Scientific. (2016). Photobleaching in Fluorescence Imaging and Ways to

Reduce It. Retrieved 12 September 2016 from https://www.thermofisher.com/ph/en/
home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-offluorescence/protocols-troubleshooting/troubleshooting/photobleaching.html

Wulf, E., Deboben, A., Bautz, F. A., Faulstich, H., & Wieland, T. (1979). Fluorescent
phallotoxin, a tool for the visualization of cellular actin. Proceedings of the National
Academy of Sciences, 76(9), 4498-4502. doi:10.1073/pnas.76.9.4498

REFERENCES

Alberts, B., Johnson,thA., Lewis, J., Morgan, D., Raff, M., Roberts, K., & Walter, P. (2015). Molecular
biology of the cell (6 ed.). New York, NY: Garland Science.

Abramowitz, M. & Davidson, M.W. (2012). Optimization and Troubleshooting. Olympus Microscopy
Resource Centre. Retrieved 12 September 2016 from http://www.olympusmicro.com/primer/
techniques/fluorescence/troubleshoot.html

Buchwalow, I. B., & Bcker, W. (2010). Immunohistochemistry: Basics and methods. Heidelberg:
Springer.

Thermo Fisher Scientific. (2016). Photobleaching in Fluorescence Imaging and Ways to Reduce It.
Retrieved 12 September 2016 from https://www.thermofisher.com/ph/en/home/life-science/cellanalysis/cell-analysis-learning-center/molecular-probes-school-of-fluorescence/protocolstroubleshooting/troubleshooting/photobleaching.html

Wulf, E., Deboben, A., Bautz, F. A., Faulstich, H., & Wieland, T. (1979). Fluorescent phallotoxin, a
tool for the visualization of cellular actin. Proceedings of the National Academy of Sciences, 76(9),
4498-4502. doi:10.1073/pnas.76.9.4498

http://www.olympusmicro.com/primer/photomicrography/fluorescenceerrors.html

https://www.microscopyu.com/techniques/fluorescence/introduction-to-fluorescence-microscopy