SUNDAY, DECEMBER 4- Late Abstracts 1 - Molecular Biology of the ...

SUNDAY
protein may play a critical role in maintaining neuronal homeostasis by regulating endosomal
trafficking and mitochondrial transport. Further characterization of the mechanisms of Trak1
action and its regulation will provide a better understanding of the role of Trak1 in hypertonia
and epilepsy.
1979
Post-Fusion Actin Coating of Secretory Vesicles is required for Active Content Extrusion
in Alveolar Type II Cells.
P. Miklavc 1 , E. Hecht 1 , N. Hobi 2 , O. H. Wittekindt 1 , T. Haller 2 , P. Dietl 1 , E. Felder 1 , C. Kranz 1 , M.
Frick 1 ; 1 University of Ulm, Ulm, Germany, 2 Innsbruck Medical University, Innsbruck, Austria
Exocytosis of secretory vesicles is a fundamental cellular process allowing regulated secretion
of vesicle contents in the extracellular space. Growing evidence suggests that regulation of
fusion pore dilation during the post-fusion stage of exocytosis plays an important role in release
of secretory vesicle contents. However, dependent on the nature of the cargo additional
mechanisms might be essential to facilitate effective release. We have recently described in
alveolar type II cells that surfactant-storing secretory vesicles (lamellar bodies) are coated with
actin following fusion with the plasma membrane. Surfactant, a lipoprotein-like substance, does
not readily diffuse out of fused lamellar bodies following opening and dilation of the fusion pore.
Using fluorescence microscopy, atomic force microscopy and biochemical assays we present
evidence that actin coating and subsequent compression of the actin coat is essential to
facilitate surfactant secretion. Simultaneous imaging of the vesicle membrane and the actin coat
revealed that contraction of the actin coat compresses the vesicle following fusion. This leads to
active extrusion of vesicle contents. Preventing actin coating of fused LBs with latrunculin A
inhibits surfactant secretion almost completely. Subsequent compression of the actin coat is
modulated by myosin II. In summary our data suggest that fusion pore opening and dilation is
not sufficient for “passive” release of bulky vesicle cargo in alveolar type II cells and that active
extrusion mechanisms are required.
1980
Synaptotagmin-7 - the link between fusion-activated Ca 2+ -entry and regulation of fusion
pore expansion?
N. Sharma 1 , P. Miklavc 1 , K. Thompson 1 , O. H. Wittekindt 1 , E. Felder 1 , P. Dietl 1 , M. Frick 1 ;
1 Institute of General Physiology, University of Ulm, Ulm, Germany
Ca 2+ is the key element in regulated exocytosis controlling multiple steps during the exocytic
pre- and post-fusion stages. We have recently described a “fusion-activated” Ca 2+ -entry (FACE)
via vesicular P2X4 receptors during the post-fusion stage of lamellar body exocytosis in alveolar
type II (ATII) cells. FACE regulates fusion pore expansion and vesicle content release during
the post-fusion phase of exocytosis (PNAS, 2011 Aug 30;108(35):14503-8). Yet, the question
remained how this locally restricted Ca 2+ signal is translated into mechanical force promoting
fusion pore expansion. In this study we tested whether synaptotagmins could form a molecular
link between FACE and fusion pore expansion. Members of the synaptotagmin family that
localize to secretory vesicles have been found to promote fusion pore expansion via Ca 2+
binding to C2 domains. Using RT-PCR and western-blotting we identified synaptotagmin-7 as
the Ca 2+ -binding isoform predominantly expressed in primary ATII cells. Immunofluorescence
confirmed for the first time that synaptotagmin-7 is localised on lamellar bodies. We next
performed functional studies over-expressing wt synaptotagmin-7 or mutants abolishing Ca 2+ -
binding to either the C2A, C2B or C2A and C2B domains. We measured diffusion rates of
fluorescent dyes through the fusion pore to directly assess dynamic changes in fusion pore
diameters. Our results show that overexpression of neither wt nor mutant synaptotagmin-7