Keynote Conference - Interevent

L#5
PfCBF transcription factor, a new player for signal transduction
in melatonin-pathways in malaria parasites
Wania Rezende Lima, Miriam Moraes and Célia R. S. Garcia
Departamento de Fisiologia, Instituto de Biociências,
Universidade de São Paulo – São Paulo- Brasil
The signal transduction pathways controlling malaria parasite
development remain largely unexplored. It is now accepted that
Plasmodium senses the environment and exploits calcium and
cAMP signalling pathways to modulate cellular functions. We
want to understand how the molecular machinery for signalling
transduction is put in action in Plasmodium, how second
messengers are generated and if they play a role in the cell
cycle. We have reported that potentially important signaling
molecules from the host cell, the Red Blood Cell (RBCs) such as
ATP modulates Plasmodium falciparum progression within RBCs
through the rise of cytosolic Ca 2+ . We have used a cell-permeant
form of caged-IP3 to investigate the cytosolic IP3 levels under
physiological conditions in infected RBCs co-loaded with both
the cell-permeant caged-IP3 and Fluo4-AM. UV flash photolysis
of caged-IP3 under these conditions elicited a rapid and
transient increase in intracellular Ca 2+ in RBCs infected with P.
falciparum. Thereby providing a direct evidence that a classical
PLC-dependent intracellular Ca 2+ release pathway operates in P.
falciparum infected RBCs. We provided the first direct evidence
that the host hormone melatonin elicits a rise in intracellular IP3
levels in the malaria parasite. We have also found that the
Plasmodium kinase PfPK7 is central in the downstream
mechanism for synchronizing the parasite as a P. falciparum
clone unable to express PfPK7 does not respond to melatonin.
Taken together these data implicate that melatonin activates
Phospholipase C (PLC) to generate IP3 and open ER-localized IP3sensitive
Ca 2+ channels in P. falciparum
In our search for the molecular effectors of second messenger
signaling in P. falciparum we have search for the role of the
PfCBF. The CBF family of transcription factors are involved in
the regulation of cell cycle of many eukaryotic genes. The
molecular and functional characterization of transcription
factors in P. falciparum are yet poorly studied. In order to
determine the protein and mRNA expression levels of intraerythrocytic
PfCBF, western-blot and qRT-PCR were performed.
To localize the CBF protein distribution in the parasite, confocal
microscopy and subcellular fractionation were carried out. The
effect cAMP on PfCBF gene and protein expression during intraerythrocytic
development of the malaria parasites was followed
by qRT-PCR and western blot analysis. PfCBF is expressed
throughout the intra-erythrocytic stages but is greatly
detectable at the schizont stage at both the mRNA and protein
levels.
In conclusion, We suggest that PfCBF may have an integral role
in parasite melatonin responses and the subsequent parasite
development. Then progress in understanding the function of
PfCBF transcription factor in the context of the melatonin
response is likely to provide a better knowledge of the parasite
biology.
L#6
Signal-Adaptor Interactions that Mediate Polarized Sorting in
Neurons
Ginny G. Farías, Loreto Cuitino, Xiaoli Guo, Xuefeng Ren, Rafael
Mattera and Juan S. Bonifacino
Cell Biology and Metabolism Program, Eunice Kennedy Shriver
National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, Maryland, USA
Neurons are anatomically and functionally polarized cells that
conduct nerve impulses in a vectorial fashion. Impulses are
received by dendrites, propagated through the soma, and
eventually transmitted to other cells by axons. The plasma
membrane of each of these neuronal domains has a distinct
protein composition, but the mechanisms responsible for this
differential distribution of plasma membrane proteins remain
poorly understood. By analogy to other protein sorting
processes, we hypothesized that biosynthetic delivery of
transmembrane proteins to different neuronal domains could
be mediated by interaction of sorting signals in the cargo
proteins with adaptor proteins that are components of protein
coats. Indeed, we found that a tyrosine-based sorting signal in
the cytosolic domain of the transferrin receptor (TfR) mediates
sorting of this protein to the somatodendritic domain in both
hippocampal and cortical neurons. This signal binds to the
mu1A subunit of the clathrin-associated, heterotetrameric
adaptor protein 1 (AP-1) complex. Overexpression of a
dominant-negative mu1A mutant incapable of binding signals,
or RNAi-mediated depletion of another subunit of the AP-1
complex, gamma-adaptin, resulted in missorting of the TfR to
the axon. Various microscopic techniques revealed that sorting
occurs at AP-1-coated areas of the TGN through exclusion of the
TfR from transport carriers bound for the axon. These findings
demonstrate that interactions of sorting signals with AP-1
mediate clathrin-dependent sorting of the TfR and other cargos
to the neuronal somatodendritic domain. Together with recent
observations in other polarized cell types, our findings support
the notion that AP-1 is a global regulator of polarized sorting.
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