Research in our Lab
The Aspergillus Genetics &
Molecular Biology group is established in the
Department of Botany (1st floor, left
corridor, Microbiology lab) within the
Faculty of Biology at the Panepistimioupolis (Kaisariani/ Zografou) of
the
National and Kapodistrian
University of Athens. The
Department is relatively well equipped with major facilities for studies on
plant and microbial genetics, biochemistry, physiology and molecular
biology. Our group is primarily interested in several aspects concerning the
expression, function, cell biology and evolution of transporters. Our
model organism of choice is the non-pathogenic, filamentous fungus
Aspergillus nidulans, a classic model genetic system,
since the 1950's.
Why Transporters?
· The cellular
membranes are practically
impermeable barriers. However, all cells need to control the influx and
efflux of solutes and ions, which is
a basic biological process implicated in
nutrition,
signaling,
neurotransmission, homeostasis,
cell communication & defense.
· Transporters & channels
control both the influx and efflux of solutes and ions. Approximately 8-20%
of all genes in genomes encode transporters and channels, whereas up to 20%
of genes in eukaryotic genomes are believed to be involved in the control of
expression, cellular sorting, trafficking and turnover of transporters and
channels.
· Several human
genetic disorders caused by alterations in transport proteins (e.g
cystic fibrosis, diabetes, neurodegeneration, etc).
· Transporters also act as specific
gateways for specific drug delivery to target cells (microbes,
pathogens, cancer cells).
· Transporters are responsible for the
pleiotropic or multidrug drug
resistance (PDR/MDR), through ABC and MFS efflux proteins (antibiotic
resistance, cancer cell resistance to chemicals).
· Despite their importance, few transporter & channel molecular structures are currently known
(<450 unique PDB entries, of which 87 are ion channels/exchangers, 21 are
transporters involved in efflux of toxic metabolites or drugs, and 37 are
solute transporters, from which only 2 are eukaryotic transporter).
Thus,
for understanding how cells function and communicate there is an obvious
need for understanding transporters and channels and developing relevant
approaches to do that.
Why Aspergillus as a model system?
1953 Pontecorvo published the compendium of the genetics of the ascomycete
fungus Aspergillus nidulans1. Since 1984, after the
development of transformation protocols, A. nidulans has also become a
model microbial system for molecular biology and reverse genetics approach.
Practically, most of what can be done in
Saccharomyces cerevisiae, can be done in
A. nidulans. The beautiful and
sophisticated system of A. nidulans genetics, molecular and cell biology
led to the analysis of multiple metabolic and cellular processes. It suffices to
highlight the identification by Claudio Scazzocchio, Herb Arst and co-workers of
the crucial actors of nitrogen metabolite and carbon catabolite repression2,
the work of Ron Morris and his school3, which matches the Nobel
winning work of Paul Nurse and Lee Hartwell, the dissection of conidial
development initiated by Bill Timberlake with the introduction of the
methodology of cascade hybridisation4, and the recent work of Miguel
Angel Penalva on membrane trafficking, Golgi dynamics and endocytosis5.
Other Aspergilli are used in biotechnological processes that range from the
production of soja sauce and sake (A. soyae, A. oryzae), to the
production of citric acid (A. niger) and
the production of heterologous proteins. A. fumigatus has graduated,
thanks to immunosuppression, from rare pathogen to one of the most common causes
of fatal nosocomial infection. A. flavus, a noxious contaminant of
food-stuffs, produces aflatoxin, the most powerful carcinogen known. A.
sydowi is an insidious pathogen of gorgonian corals. A issue of Nature
carries three articles describing the genomic sequences of A. nidulans6,
A. oryzae7 and A. fumigatus8. The
differences between A. nidulans and the other two Aspergilli match the
differences found between fish and humans, which are separated by 500 million
years, suggesting a very rapid rate of divergence in this genus. The A.
oryzae genome has 12074 predicted proteins as compared with 9926 for A.
fumigatus and 9541 for A. nidulans. The excess of A. oryzae
putative genes concerns mainly secondary metabolism, and may be due to
horizontal gene transfer, including from prokaryotes. A. nidulans is
homothallic, but there are heterothallic species of Aspergillus, while no
apparent sexual cycle has been described for A. fumigatus or A. oryzae9.
What clearly distinguishes A. nidulans from
S. cerevisiae and other model yeasts
is its mode of polar life10. Cell polarity is, for example, highly
demanding in special mechanisms for rapid protein exocytosis and endocytosis
along very long cells, the apical parts of which possessing additional needs on
membrane trafficking, as compared to sub-apical parts or non-polar cells like
yeasts. In fact, genetic evidence in filamentous fungi shows that rapid
endocytosis of membrane proteins followed by their re-delivery to the plasma
membrane generates and maintains polarity11. Interestingly, the
molecular mechanisms underlying fungal polarity are analogous or/and homologous
to those found in the cells of higher animals, such as mammalian neurons12.
References
1.
Pontecorvo
G,
Roper
JA,
Hemmons
L.M,
MacDonald
KD
and
Bufton
AWJ. (1953).
The
Genetics
of
Aspergillus
nidulans.
Adv
Genet 5:
144-238.
2.
Arst
HNJr
and
Cove
DJ.
(1973).
Nitrogen
Metabolite
Repression
in
Aspergillus
nidulans.
Mol
Gen
Genet 126:111-141.
3.
Morris
NR
and
Enos
AP.
(1992).
Mitotic
gold
in
a
mold.
Trends
Genet 8:32-37.
4.
Timberlake
WE.
(1990).
Molecular
Genetics
of Aspergillus
Development.
Ann Rev Genet 24:5-36.
5.
Pantazopoulou A, Penalva MA. (2009). Organization and
dynamics of the A. nidulans
Golgi during apical extension and mitosis.
Mol Biol Cell
20: 4335-7
6.
Galagan JE.
et al. (2005). Sequencing
of Aspergillus nidulans and comparative analysis with
A. fumigatus and
A. oryzae.
Nature 438:1106-1115.
7.
Machida
M.
et
al.
(2005).
Genome
sequencing
and
analysis
of
Aspergillus
oryzae.
Nature
438:1157-11661.
8.
Nierman WC.
et al. (2005)
Genomic sequence of the pathogenic and allergenic fungus
Aspergillus fumigatus.
Nature 438:1151-1156.
9.
Geiser
DM,
Timberlake
WE
and
Arnold
ML. (1996).
Loss
of
Meiosis
in
Aspergillus.
Mol
Biol
Evol 13:809-817.
10.
Fischer
R,
Zekert
N,
Takeshita
N.
(2008).
Polarized
growth
in
fungi--interplay
between
the
cytoskeleton,
positional
markers
and
membrane domains.
Mol Microbiol
68:813-26.
11.
Taheri-Talesh N, Horio T, Araujo-Bazán L, Dou X, Espeso EA, Peñalva MA, Osmani
SA, Oakley BR. (2008). The tip growth apparatus of Aspergillus nidulans.
Mol Biol Cell
19:1439-49.
12.
Egan MJ, McClintock MA, Reck-Peterson SL. (2012).
Microtubule-based transport in filamentous fungi.
Curr
Opin Microbiol
15:637-45.
13.
Casselton
L
and
Zolan
M. (2002).
The art and
design of genetic screens: filamentous fungi.
Nat
Rev
Genet 3:683-697
Why Aspergillus particularly for studying transporters?
·
Fine knowledge of metabolism & easiness of
knock-out/reverse genetics
· Easily scored phenotypes on toxic analogues (e.g.
8-azaguanine, oxypurinol, allopurinol, 5-FC, 5-FU,5-FUd)
·
Direct genetic selection of transporter mutations or
suppressors-conditional, specificity or affinity mutants
·
Easy radiolabelled solute uptake assays with
germinating conidiospores
·
Use of GFP, RFP to distinguish functional, trafficking
& turnover mutants
· Rich repertoire of transport proteins-all basic
families found in metazoa-703 putative transporter/channels
[81.5 secondary active transporters, 11.8% primary-active transporters,
4.4% channels]
A, Growth tests of wild-type and transporter null
mutants of A. nidulans on toxic purine
analogues (5|FU, 5FUI, OX) or purines as N sources (UA, AD, HX, ALL).
B, Confocal fluorescence microscopic
view of A. nidulans expressing a
functional UapA-GFP transporter. C,
Germination of A. nidulans
conidiospores and generation of germlings (very young mycelia).
D, Radiolabelled uptake studies of purine transporter at a stage
prior germlings development.