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Full Abstracts – CELL BIOLOGY AND CYTOSKELETON
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Talin: A Master Regulator of Cell-ECM Adhesion-Dependent Morphogenesis.
Stephanie J. Ellis, Michael J. Fairchild, Stefan Czerniecki, Mary Pines,
Guy Tanentzapf. Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada.
Morphogenesis of a complex body plan requires coordinated regulation of cell adhesion molecules and the cytoskeleton to form distinct, organized tissues.
Integrin adhesion receptors mediate ECM attachment and connect to the cytoskeleton through the adapter protein, talin. Talin interacts with many binding
partners including integrin and F-actin. A delicate balance of these multiple interactions offers a means of fine-tuning integrin function and linkage to the
cytoskeleton. Using targeted point mutations, we systematically investigate the role of different domains of talin during Drosophila embryogenesis. Our
results suggest that morphogenetic events requiring short term, transient adhesions, such as germband retraction and dorsal closure, are highly sensitive to
mutations in talin that compromise the ability to quickly disassemble adhesive contacts and linkage to the cytoskeleton. Conversely, in the embryonic and
larval musculature, where myotendinous junctions form adhesive contacts that grow and persist over several days, talin interactions that strengthen
attachment between integrins and the surrounding ECM are of greatest importance. Finally, using FRAP in the living embryo, we find that disruption of key
domains in talin alters the dynamics of talin at adhesions, suggesting talin may be a master regulator of adhesion stability and cytoskeletal dynamics.
Altogether, we demonstrate how the ability of talin to switch between multiple binding partners comprises an essential mechanism for modulating integrin
function to elicit distinct developmental outcomes.
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Septins are required for the establishment of adherens junction at the exit of mitosis of polarized epithelial cells.
Nabila Founounou, Roland Le
Borgne. CNRS UMR 6061-Institut de Génétique et Développeme, Rennes. France.
Septins are a conserved GTPases forming filament required at cytokinesis. Loss of Peanut, one of the four
Drosophila
septins, gives rise to binucleation in
various cell contexts but its precise role remains poorly understood. Here, we have investigated the function of Septins during epidermal cell divisions and
asymmetric divisions within the sensory organ (SOP) lineage. Two types of divisions are taking place within this lineage: SOP divide asymmetrically within
the plane of the epithelium giving rise to two polarized epithelial cells, while one of the SOP daughter cells divide along the apical basal axis with one
sibling that looses its epithelial characteristics. Sep2::GFP dynamics confirmed that Drosophila septins act as hetero-oligomer in vivo, and that loss of Pnut
or tissue-specific silencing of one of
septins
is sufficient to cause cytokinesis defects. Time-lapse analyses revealed that in clones of cells homozygote
mutant for pnut the early steps of SOP mitosis occur correctly but the abscission fails giving rise to a binucleated cell. Surprisingly, the binucleated cell
undergoes an asymmetric division along the apical basal axis with a complete abscission indicating that Septins are dispensable for this type of asymmetric
cell division. This is in striking contrast to the formin Diaphanous whose activity is required for the two types of asymmetric cell divisions. In both
epidermal cells and SOP cells, time-lapse analyses indicate that cytokinesis occurs asymmetrically from basal to apical pole with Septins forming a
horseshoe-shaped contractile apparatus. In a second step, an apical constriction takes place and precedes the establishment of adherens junctions as
monitored using E-Cad::GFP knock-in line. In cells mutant for pnut, while basal to apical followed by apical constrictions occur properly, we observed a
failure in the establishment of E-Cad junctions at mitosis exit. Our data reveal that septins are required in epithelial cell cytokinesis to allow adherens
junction establishment, we are currently investigating the molecular link between Septins and E-Cad.
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Actin turnover balances forces between cells during epithelial invagination.
Adam C. Martin, Frank M. Mason, Mike Tworoger. Biology, Massachusetts
Institute of Technology, Cambridge, MA.
Embryonic development requires that coordinated cell shape changes collectively deform tissues. Cell shape changes result from forces generated by actin
networks that are coupled to adhesive complexes. During Drosophila gastrulation, a ratchet-like contraction of an apical actin and myosin II network coupled
to adherens junctions drives apical constriction and generates epithelial tension, which are important for epithelial invagination. While the role of actin
filament (F-actin) turnover is well established for membrane protrusion during cell migration, it is unclear how F-actin is remodeled to allow for myosin
contraction and apical constriction during the coordinated movement of an epithelial sheet. Here, we combine live imaging, quantitative image analysis, and
drug perturbation to define the role of F-actin turnover during apical constriction. We found that in contrast to Myosin-II levels, which steadily increase, total
F-actin intensity decreases as cells constrict, suggesting actin network turnover. Pulses of Myosin-II accumulation are correlated with transient
accumulations of F-actin, consistent with these events being contractions of the actin-myosin network that are subsequently remodeled. To determine the
function of F-actin turnover, we titrated the rate of actin polymerization by injecting a wide concentration range of drugs that inhibit F-actin polymerization
(cytochalasin D) or depolymerization (phalloidin). We observed a transition from a general disruption of contractility in all cells with high drug doses, to a
mesoderm specific disruption in cell-cell connections at low doses. At low drug does neighboring actomyosin networks continually lost and reformed
connections, resulting in an unbalanced ‘tug-of-war’ between mesoderm cells. During apical constriction, transient holes continually appear in the apical F-
actin meshwork and these holes persist when F-actin turnover is inhibited. We propose that rapid F-actin turnover is required to fill holes generated during
network contraction to balance forces across adhesive contacts.