1. A Good Death: Programmed Cell Death of Algae
and its Influence on the Microbial Loop
Jennifer L. Duncan-Taylor Port Angeles High School, Port Angeles WA
What do mouse toes, tadpole tails and diatoms in Antarctica have in common? Apoptosis. Programmed Cell Death.
Cells designed to die for the benefit of others.
Programmed Cell Death (“Apoptosis”) is a system-organizing process
used by multicellular organisms to shape body structures. Much like
erecting scaffolding to facilitate the construction of a building and then
disassembling the scaffolding when the building is finished, the
“scaffolding” cells will dramatically die off when the intended structure is
complete and they are no longer needed.
INTRODUCTION
Cells undergoing apoptosis glow orange due to a fluorescent dye that adheres to their
membranes. Photo credits: Amitabha Bandyopadhyay, PLoS Genet.: 2006
If Programmed Cell Death (PCD) is a normal process in healthy biological
systems, is it possible for PCD to play a beneficial role in building,
organizing and maintaining other systems as well – perhaps even
ecological systems? Further, does PCD occur in single celled organisms?
Recently, it has been shown that PCD of the algae Dunalliela salina in
hypersaline environments (The Great Salt Lake) benefits the overall
ecosystem and maintains the microbial loop by recycling vital nutrients
and minerals (Orellana et al., 2013). Biochemical pathways of different
organisms (Halobacterium salinarum and other microbes) become
metabolically interconnected and these unrelated individuals
end up impacting each other’s fitness directly through their
metabolic activities.
D. salina: Annexin V-FITC and Sytox® Blue fluorochromes are used to show the process of PCD. The cell
membrane emits green as evidence of apoptosis and blue detects nucleic acids. (Orellana et al. 2013)
MICROBIAL
LOOP
O2
CO2
Phytoplankton
POM (particulate
organic matter)
DOM (dissolved
organic matter)
Bacteria &
Archaea
Zooplankton
C, N, P, Si, Fe
Fish
Fecal matter
Protozoans
INVESTIGATIVE QUESTION
Fragilariopsis is a dominant diatom species in the Ross
Sea that has well documented rapid population growth such
that these enormous “blooms” can be seen from satellites.
Equally remarkable, is the sudden drop in these populations.
Is this caused by PCD on a massive scale? And like the
microbial loop in the closed ecosystem of the Great Salt Lake, might PCD
of Fragilariopsis cylindra play a role in nutrient cycling of polar
ecosystems, specifically that of the Ross Sea in Antarctica?
Photo credit:
J. Duncan-Taylor
and A. Lee
RESULTS and DISCUSSION
References:
Orellana, Mónica V., Wyming L. Pang, Pierre M. Durand, Kenia Whitehead, and Nitin S. Baliga. "A Role for Programmed Cell Death in the Microbial
Loop." PLOS One 8.5 (2012): n. pag. Web.
Azam F. & Malfatti, F. Microbial structuring of marine ecosystems Nat Rev. Microbiol.5:782-791
Givan, Alice Longobardi. Flow Cytometry: First Principles. New York: Wiley-Liss, 1992. Print.
"Antibodies & AssaysCancer." Cancer Antibodies. Merck KGaA, 2013. Web. 02 Aug. 2013. <http://www.millipore.com/antibodies/flx4/cancer_antibodies>.
Christie, William W. "Phosphatidylserine and Related Lipids Structure, Biochemistry and Analysis." Lipid Library. AOCS, 4 Apr. 2013. Web.
<http:lipidlibrary.aocs.org/lipids/ps/index.htm>.
"SYTOX Blue Dead Cell Stain." Molecular Probes. Invitrogen, 20 June 2005. Web. <http://www.mobitec.com/probes/docs/sections/0801.pdf>.
Flow Cytometry and Deconvolution Fluorescence Microscopy are both utilized to gather
qualitative and quantitative data of Fragilariopsis undergoing PCD. Two probes,
Annexin V-FITC (Fluorescein Isothiocyanate) and Sytox® Blue are used to stain cells
filtered from Antarctic seawater. Fluorescent-labeled cells are quantified using an
InFlux Flow Cytometer (BD Biosciences).
METHODS
Excited at 488nm, the chlorophyll of
living cells emit red light at 715nm.
Phosphotidylserine (PS) is found on the external
surface of the plasma membrane of cells that have
undergone PCD. Annexin V-FITC is a protein that
binds to PS and emits green light at 530nm as
evidence of PCD.
Sytox ®Blue is a secondary indicating dye of PCD
and detects the cell’s nucleic acids when the plasma
membrane has been disrupted by externalization of
PS. It emits blue light at 520nm.
Fragilariopsis cylindra
Photo credit:
J. Duncan-Taylor and A. Lee.
Photo credit:
J. Duncan-Taylor and A. Lee.
Photo credit:
J. Duncan-Taylor and A. Lee.
Photo credit:
J. Duncan-Taylor and A. Lee.
Acknowledgements:
PS “flips” to the outside
of the membrane of cells
undergoing PCD
Outside of cell membrane
Phosphotidylserine (PS) is localized in the inner
layer of the bilipid membrane of intact, living cells.
Annexin V
FITC
Laser 488nm Emission 530nm
Diatom Bloom in the Ross Sea via satellite
© Norman Kuring, Ocean Color Team, NASA
Acquired January 22, 2011.
Inside of cell membrane
Figure A Figure B Figure C
Unstained Cells Annexin V-FITC + Sytox Blue Annexin V-FITC + Sytox Blue
Samples from the Ross Sea were collected at depth intervals from the
surface to 500 meters. After fluorescent probes are added, the algae
cells are analyzed by flow cytometry.
A) Unstained: Sample shows living cells fluorescing at 715nm.
B) At a depth of 20 meters, cells stained with Annexin V-FITC
fluoresce at 530nm as evidence of PCD.
C) At a depth of 500 meters, cells are virtually non-existent.
To date, preliminary data indicates Programmed Cell Death may be
occurring in this algae bloom. Continuing the analysis of the remaining
300+ samples will reveal the extent to which PCD is occurring in algae
populations in the Ross Sea.
Beads
Depth: 20 Meters Depth: 20 Meters Depth: 500 Meters
Mónica Orellana, PhD.
Senior Research Scientist
Institute for Systems Biology.
Thank you for this amazing
opportunity to participate in
truly remarkable research!
Allison Lee
Research Associate 2
Institute for Systems Biology.
Thank you for welcoming me to your bench
and for generously, patiently teaching me so
many new skills! Thank you for encouraging
me every day.
Cora Chadick
Core Facility Coordinator
Institute for Systems Biology.
Thanks for your brilliant expertise and
gracious willingness to drop whatever
you’re doing and keep our equipment
running. Thanks for making me laugh!
SPECIAL THANKS TO:
•NSF 11-532- Antarctic Astrophysics and
Geospace Sciences NSF Tracing the fate of Algal
Carbon Export in the Ross Sea (TRACERS)
•MJ Murdock Charitable Trust
•Institute for Systems Biology
•Nitin Baliga PhD. Professor & Director
•Claudia Ludwig, M.Ed. Program Manager
FUTURE WORK
Azam and Malfatti, 2007