SCIENTIFIC REPORT 2004 - Sylvester Comprehensive Cancer Center

T U M O R C E L L B I O L O G Y P R O G R A M
PEDRO J. I. SALAS, M.D., PH.D.
Associate Professor of Cell Biology
and Anatomy
DESCRIPTION OF RESEARCH
Centrosomes are an essential piece of the mitotic
machinery. In polarized epithelial cells,
centrosomes and other non-centrosomal microtubule
organizing centers (MTOC) are distributed
in a subapical localization. During mitosis, centrosomes
migrate to the lateral domain, from
where they organize the spindle. This orientation
of the spindle is crucial for the maintenance of
epithelial polarity since it determines that the cytokinesis
will proceed in a plane perpendicular to
the plane of the epithelial layer. Likewise, the polarization
of MTOCs during interphase is essential
to the polarization because it ensures that the
minus ends of microtubules will be aligned under
the apical domain.
Dr. Salas’ research has demonstrated that
centrosomes and non-centrosomal MTOCs colocalize
with the apical intermediate filament (IF)
cytoskeleton by using high-resolution confocal
microscopy, near-neighbor deconvolution, and
3D image reconstruction. At the electron microscopy
level, co-localization indicated that
pericentriolar material containing g-tubulin and
the cytokeratin (CK) 19 intermediate filaments
approach up to 10 nm. Using sonication, homogenization,
and immunoprecipitation coupled
with immunoblot, his laboratory also demonstrated
that CKs 18 and 19 co-immunoprecipitate
with g-tubulin in fragments that cannot
sustain physical trapping. The down-regulation of
CK19 IF using anti-sense oligonucelotides resulted
in changes in localization of the centrosomes.
The analysis of the sonication
fragments indicated that only a few proteins
other than CKs and g-tubulin are present, so that
two potential candidates identified by yeast twohybrid
and MS-MS microsequencing to fulfill
the role of the “glue” attaching centrosomes, are
now under consideration. Interestingly, one of
those proteins is phosphorylated by p34cdc2. Because
the IF do not depolymerize during mitosis
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in epithelial cells, the attachment of centrosomes
to IF must be necessarily broken at the onset of
mitosis. Current laboratory projects include the
isolation and identification of the protein(s) involved
in the apical attachment of centrosomes to
IF and their function during mitosis. Theoretically,
a manipulation of this mechanism may halt
the cell cycle in actively dividing epithelial cells.
In addition, the relevance of this mechanism during
ischemia or ATP depletion also is under
investigation.
SELECTED PUBLICATIONS
2002
Yang, X, Salas, PJ , Pham, TV, Wasserlauf, BJ,
Smets, MJ, Myerburg, RJ, Gelband, H,
Hoffman, BF, and Bassett, AL. Cytoskeletal actin
microfilaments and the transient outward potassium
current in hypertrophied rat ventriculocytes.
Journal of Physiology 541:411-21, 2002.
Figueroa, Y, Wald, FA, and Salas, PJ . p34cdc2-
mediated phosphorylation mobilizes microtubule-organizing
centers from the apical
intermediate filament scaffold in CACO-2 epithelial
cells. Journal of Biological Chemistry
277:37848-54, 2002.
2003
Ramsauer, VP, Carothers Carraway, CA, Salas,
PJ, and Carraway, KL. MUC4/Sialomucin complex,
the intramembrane ErbB2 ligand, translocates
ErbB2 to the apical surface in polarized
epithelial cells. Journal of Biological Chemistry
278:30142-47, 2003.
Ameen, NA, Marino, C, and Salas, PJ . cAMPdependent
exocytosis and vesicle traffic regulate
CFTR and fluid transport in rat jejunum in vivo.
American Journal of Physiology Cell Physiology
284:C429-38, 2003.
Wald, FA, Figueroa, Y, Oriolo, AS, and Salas, PJ .
Membrane repolarization is delayed in proximal
tubules after ischemia-reperfusion: possible role
of microtubule-organizing centers. American
Journal of Physiology Renal Physiology
285:F230-40, 2003.
UM/Sylvester Comprehensive Cancer Center Scientific Report 2004