Biomedical Engineering Reference
In-Depth Information
•
Annexin V is a recombinant protein that interacts specifi cally and
strongly with phosphatidylserine residues. Fluorescently labeled
Annexin V is used to detect the externalization of phosphatidylserine
on the outer leafl et of the plasma membrane during apoptosis.
Membranes of necrotic cells are also labeled by Annexin V. To
differentiate between necrotic and apoptotic cells, the Annexin
V-positive cells are co-stained with membrane-impermeable nucleic
acid dyes such as propidium iodide (PI), which are excluded from
cells with intact membranes and therefore stain only necrotic cells.
Fungal cells cannot be directly stained with Annexin V because of
their cell wall and must therefore be fi rst treated with cell-wall-
degrading enzymes to release protoblasts (Baek et al. 2004, Li et al.
2006).
•
Changes in caspase activity can be detected in a number of ways.
Modifi ed caspase substrates are used, which, when cleaved by
caspases, release a fl uorescent product that can be detected and
quantifi ed.
Fungal Apoptosis
Fungal apoptosis was disregarded for many years due to lack of genetic
evidence of homologs of known apoptotic genes and the assumption that
PCD cannot exist in unicellular organisms. In 1997 Madeo and colleagues
published 'A Yeast Mutant Showing Diagnostic Markers of Early and
Late Apoptosis' which was one of the fi rst reports of PCD in yeast. The
researchers reported that a
Saccharomyces cerevisiae
mutant (a mutated cell
division cycle gene
CDC48)
showed typical markers of apoptosis including
chromatin condensation and nuclear fragmentation. Positive staining with
Annexin V and TUNEL was reported. The coordinate occurrence of these
events at different locations in the cell, which had no obvious connection
except their relation to apoptosis, implied that the molecular machinery
required for PCD was present in yeast.
This discovery forced the rethinking of PCD in budding yeast and other
systems, and signalled a new phase in apoptosis research in fungi as well as
in other lower eukaryotes. Apoptotic-like PCD has since been demonstrated
in a variety of organisms, including plants, fungi and protists (Maercker et
al. 1999, Vardi et al. 1999, Al-Olayan et al. 2002, Jin and Reed 2002).
Fungal apoptotic cells exhibit many of the morphological and
biochemical changes seen in mammalian apoptosis, and therefore, the assays
used to monitor fungal apoptosis are similar to those used in mammals.
However, not all apoptotic markers are readily detected in fungi, and most
methods require some modifi cations due to structural differences, one of
the main differences being the presence of the fungal cell wall. Differences
