Natalie Busta
BIO410
Dr. Cooper
30 Apr 2012
The Effects of an Unknown Drug on the Cytoskeleton of Dictyostelium discoideum using
Actin Staining with Rhodamine-Conjugated Phalloidin.
A
B
C
D
Figure 1: Actin of Dictyostelium discoideum stained and viewed with a fluorescence
microscope. Dictyostelium discoideum cells were mixed with 800ul PBM in an eppendorf tube
and distributed onto four round coverslips. Two coverslips were designated to receive the drug
solution and two were designated as the control group of cells. After 20 minutes, the liquid was
removed from the “drug” coverslips and replaced with 200ul drug solution. The liquid was
removed after 10 minutes and replaced with 200ul fix solution and the cells were left to fix for 5
minutes. The fix solution was removed and replaced with 200ul PBS. The PBS was removed and
replaced with 150ul rhodamine-phalloidin and left to sit in the dark for 30-40 minutes. After
incubation, the rhodamine-phalloidin solution was removed from all coverslips and replaced with
200ul distilled water. Four drops of Prolong Gold Antifade mounting solution with DAPI were
put on a dry slide and the treated coverslips were placed on the drops (one coverslip per drop). A
week later, the coverslips were sealed with clear fingernail polish and observed under a
fluorescence microscope and pictures of the cells were taken under phase contrast and
fluorescence settings.
Figure 1A is a picture taken of the Dictyostelium discoideum cells under fluorescence
microscopy after treatment with the drug and staining with rhodamine. The illuminated (white)
areas show actin filaments present in the cells after treatment with the drug. Figure 1B shows the
fluoresced nuclei of the Dictyostelium discoideum cells treated with the drug and viewed with
fluorescence microscopy after staining with DAPI. Figure 1C is a picture of the actin filaments
of Dictyostelium discoideum control cells also stained with rhodamine and viewed under
fluorescence. Figure 1D shows the DAPI stained nuclei of Dictyostelium discoideum control
cells viewed with a fluorescence microscope.
Questions
1) What is Phalloidin?
Phalloidin is a toxin produced by fungi that binds to and stabilizes actin filaments without
binding to actin monomers by preventing actin from depolymerizing. It can be labeled with a
fluorescent analog in addition to staining actin filaments. This allows for the actin filaments to be
more easily stained for and observed under fluorescence microscopy (Alberts et al., 2010).
2) What are drugs that affect actin filaments and what would they do to the cytoskeleton?
* Phalloidins bind to F-actin filaments (1 molecule for 1-2 actin promoters) and lock actin
subunits together. Phalloidins favor actin filament formation and reduce the concentration of Gactin monomers that are needed to maintain filament elongation (Glossary Terms: Actin Binding
Drugs. MBinfo.).
* Latrunculin A sequesters actin monomers thus preventing polymerization and favoring
filament disassembly of actin filaments (Glossary Terms: Actin Binding Drugs. MBinfo.).
* Cytochalasin D binds to actin filaments at the barbed ends of the filaments which blocks actin
monomers from adding on therefore inhibits elongation of F-actin (Glossary Terms: Actin
Binding Drugs. MBinfo.).
*Jasplakinolide has many effects. It can induce spontaneous nucleation of actin monomers and
actin polymerizations, stabilize actin filaments by inhibiting filament disassembly, and deplete
the actin monomer supply (Glossary Terms: Actin Binding Drugs. MBinfo.).
3) What is rhodamine?
Rhodamines are a group of red fluorescent dyes that label proteins in many immunofluorescence
techniques. It binds to F-actin, making it possible for a cell’s cytoskeleton to be observed under
fluorescence microscopy (Rhodamine Phalloidin Conjugate – Molecular Probes® Superior
Performance. Life Technologies). Rhodamine was used as the fluorescent analog in combination
with phalloidin to stain the actin filaments in the cells to view under fluorescence.
4) What is DAPI?
DAPI is a counterstain of rhodamine that is used on fixed cells and binds to DNA. DAPI allows
for the visualization of the cell nuclei by illuminating the DNA within the nucleus when viewed
with fluorescence microscopy (DAPI Nuclear Counterstain. Thermo Scientific: Pierce Protein
Research Products).
5) How does fluorescence microscopy work?
In fluorescence microscopy, samples are labeled with fluorescent substances and then
illuminated by a higher energy source. Fluorophores (ex. rhodamine and DAPI) are attached to
the sample via staining techniques and absorb the illumination light. The illumination light
excites the fluorophores and causes them to emit light of lower energy wavelength. Filters
specific for a certain wavelength allow for the viewer to see only the light waves desired. This
filter only lets through radiation that is specific for the fluorescing material applied to the sample
(Rice, 2012). So, under one filter we were able to see the rhodamine stained actin filaments and
under the other filter we were able to view the DAPI stained DNA in the nuclei of the
Dictyostelium discoideum cells.
Discussion: What drug was used?
Latrunculin-A drug may be responsible for the differences between the experimental and
control groups of Dictyostelium discoideum cells. Fluorescence microscopy shows decreased
levels of rhodamine stained actin filaments in Fig1A (experimental group) than Fig1C (control
group). Since Latrunculin-A sequesters G-actin monomers thus preventing polymerization and
promoting F-actin disassembly (MBinfo) it makes sense that the amount of rhodamine fluoresced
F-actin filaments would be decreased after treatment with the drug.
Works Cited
Rhodamine Phalloidin Conjugate – Molecular Probes® Superior Performance. Life
Technologies. Retrieved from <http://www.invitrogen.com/site/us/en/home/Productsand-Services/Applications/Cell-Analysis/Cell-Structure/Cytoskeleton/Phalloidin-andPhalloidin-Conjugates-For-Staining-Actin/Rhodamine-Phalloidin-Conjugate.html>.
Glossary Terms: Actin Binding Drugs. MBinfo. Retrieved from
<http://www.mechanobio.info/Home/glossary-of-terms/mechano-glossary--a/actinbinding-drugs>.
Rice, G. (2012). Fluorescent Microscopy: What is Fluorescent Microscopy? Microbial Life
Educational Resources. Accessed 25 Apr 2012 from
<http://serc.carleton.edu/microbelife/research_methods/microscopy/fluromic.html>.
DAPI Nuclear Counterstain. Thermo Scientific: Pierce Protein Research Products. Retrieved
from <http://www.piercenet.com/browse.cfm?fldID=01041204>.
Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P.
(2010). Essential Cell Biology. New York: Garland Science.