Bariloche protein symposium argentine society for biochemistry and molecular biology

54
BIOCELL, 27 (Suppl. I), 2003
BC-C8.
RESTRUCTURING OF FOCAL CONTACTS BY
BRADYKININ IN RAT RENAL PAPILLA
Gabriela Marquez, Diego Serrano, Laura Gagliano, Norma Sterin-
Speziale.
Cátedra Biología Celular, Facultad de Farmacia y Bioquímica,
UBA. IQUIFIB-CONICET. E-mail: [e-mail]
Focal contacts (FC) are structures of cells attachement to
extracellular matrix. We previously found that FC proteins vinculin
(V), talin (T) and paxilin (P) are associated with DRMs -detergent-
resistant membrane domains; and that bradykinin (BK) modulates
the assembly of FC. We have investigated: a) the interaction
between T and P with V in DMRs, by immunoprecipitation and
immunoblotting, and the mobilisation of V after treatment with
BK during 1, 5 and 10 minutes, and b) the “in vivo” BK effect in
collecting duct cell culture, by immunocytochemistry. Results: a)T
and P co-immunoprecipitate with V in DMRs but not in soluble
fraction and BK induces a decrease in their association after 10
minutes. b)Intense V staining was localized to FC in control cells,
with a diffuse V staining in perinuclear zone. After 1 minutes
with BK, V staining was removed from FC and concentrated in
perinuclear region, although cells remained attached, which
correlates with a dissipation of V from DRM to soluble fraction
detected by inmunoblotting. After 5 minutes, V staining dissipated
from perinuclear region to FC, and at 15 minutes the pattern
resembles that observed in control cells. Since T and P remain
associated to V, and V remain localized to FC after BK treatment,
we suggest that BK induces a restructuration of FC rather than a
dissipation.
BC-C9.
ACTH-INDUCED CAVEOLIN-1 PHOSPHORYLATION IS
RELATED TO PODOSOME ASSEMBLY IN Y1 ADRENAL
CELLS
Colonna C. and Podestá E.
Depto Bioquímica Humana, Facultad de Medicina, UBA. E-mail:
[e-mail]
Y1 adrenocortical cells respond to ACTH via protein kinase A
with an increase in steroid secretion correlated with a change in
cell morphology (rounding-up), which reflects a reorganization of
the actin cytoskeleton and focal adhesion disassembly that are
critical for transport of cholesterol to the inner mitochondrial
membrane. Caveolin is involved in cholesterol transport and cell
signaling. We previously showed that ACTH induced caveolin-1
phosphorylation on tyrosine via cAMP. In the present work we
investigated phosphocaveolin-1 subcellular distribution and its
association with the cytoskeleton. Phosphocaveolin-1 was enriched
at focal adhesions in basal conditions, which became rounded after
ACTH stimulation with a concomitant increase on the
phosphotyrosine content. These structures resembled ring-like
arrays (caveolae- rosette), which are associated with filamentous
actin. Co-localization with phalloidin showed that when cells are
flat, stress fibers are distributed along the cell cortex and
phosphocaveolin is present at the edge of actin filaments; after
rounding-up stress fibers vanishes and F-actin aggregates at the
cell periphery and are now surrounded by phosphocaveolin-1. This
effect was blocked in the presence of genistein. These observations
along with electron microscopy studies revealed these structures
as podosomes, which have a electron dense core. These results
show that ACTH induces podosome assembly in Y1 cells,
indicating that the morphological and functional responses to PKA
activation in steroidogenic cells are related to the cytoskeleton
dynamics involving phosphocaveolin-1 localization.
BC-C10.
IDENTIFICATION OF TARGETING SEQUENCES WITHIN
PROTEIN TYROSINE PHOSPHATASE 1B  (PTP1B) TO
DIFFERENT CYTOPLASMIC COMPARTMENTS
Davies Sala, Georgina, and Arregui, Carlos O.
Instituto de Investigaciones Biotecnológicas, Predio INTI, Ed. 24,
1650 San Martín, Buenos Aires, Argentina. E-mail:
[e-mail]
We recently discovered that PTP1B associates with 
β1-integrin
complexes and regulates cell-substrate adhesion. Other authors
described PTP1B associated to the endoplasmic reticulum (ER).
Here we report that PTP1B is also in mitochondria. Our aim is to
unravel the molecular determinants implicated in PTP1B targeting.
To this end we constructed different mutants of PTP1B fused to
GFP and used them to transfect fibroblasts derived from PTP1B
knockout mice. First, we examined two different substrate traps;
one in which the Asp 181 was replaced by Ala (DAPTP1B), and
another that contains the additional mutation Gln262Ala
(DAQAPTP1B). Confocal microscopy showed that both mutants
co-localize with proteins of focal adhesions such as vinculin and
paxillin. These results suggest the existence of PTP1B substrates
in adhesion complexes. A mutant PTP1B in which a SH3 binding
motif was disrupted by substituting Pro 309 and Pro 310 by Ala
(PAPTP1B) does not co-localize with vinculin and paxillin in these
sites, suggesting that this polyproline region is essential. In
addition, cells co-expressing DAPTP1B and PAPTP1B co-localize
in the ER and mitochondria; however, only DAPTP1B accumulates
in adhesion sites. Immunoprecipitation studies show that
DAPTP1B associates with p130Cas, a docking protein that
localizes in focal adhesions and contains an SH3 domain. We
presume that p130Cas may be a candidate protein that recruits
PTP1B to cell-substrate adhesion sites.
Work supported by ANPCyT, PICT 01-08039 to C.O. Arregui.
BC-C11.
INFLUENCE OF SPHINGOLIPIDS ON NICOTINIC
ACETYLCHOLINE RECEPTOR ASSEMBLY,
TRAFFICKING AND CELL-SURFACE TARGETTING
Baier, C.J. and Barrantes, F. J.
UNESCO Chair Biophys. & Molec. Neurobiol. and INIBIBB,
B8000FWB Bahía Blanca. E-mail: [e-mail]
The effect of sphingolipid (SL) modification on nicotinic
acetylcholine receptor (AChR) targeting to the cell surface was
studied in living cells using fluorescence microscopy in
combination with inhibition of SL biosynthesis at various steps. A
significant diminution of fluorescent 
α-bungarotoxin (αBTX)
labelling (60-70%) of the AChR at the plasma membrane was
observed when the mutant cell line CHO-SPB1/SPH
-
, defective
in serine-palmytoyl transferase activity, was grown at the non-
permissive temperatures (39°C). Myriocin (ISP-1), Fumonisin B-
1 and the glucosylceramide synthase inhibitor d,l-PDMP reduced
by ~30-40% Alexa
488
-
αBTX AChR staining in control, CHO-K1/
A5 cells.  Cells treated with FB-1, PDMP, or ISP-1 increased
(~50%) their intracellular AChR, which colocalized with ER
markers like calnexin and ER-tracker. Non-assembled AChR
increased ~30-40% in SL-impaired cells.  The results suggest that
assembly, trafficking and cell-surface expression of AChR is
affected by intracellular SL levels.
Work supported in part by grants from UNS, FONCYT, and FIRCA
1-RO3-TW01225-01 (NIH).

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