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Full Abstracts – CELL BIOLOGY AND CYTOSKELETON
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Mechanisms of Epithelial Wound Repair.
Jeffrey M. Verboon, Maria-Teresa Abreu-Blanco, James J. Watts, Raymond Liu, Susan M. Parkhurst. Division
of Basic Sciences. Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Upon injury, an epithelial tissue must have a rapid and robust repair mechanism to prevent invasion by microorganisms as well as to preserve tissue
integrity. In other organisms, this repair response has been characterized to use either dynamic protrusions to crawl the wound edge forward or an
actomyosin purse-string to cinch the wound closed. These mechanisms have been loosely correlated to large and smaller wounds respectively. We find that
wound repair is achieved utilizing a combination of these mechanisms: an actomyosin cable circumferentially contracting and actin protrusions that knit the
wound closed regardless of the wound size. Using 4D, high-resolution microscopy we set out to examine the relative contribution of the actin cable versus
the protrusions using fluorescent expression constructs and genetic perturbations to components of the cytoskeleton in wounded fly embryos. Wounded
embryos mutant for the core cytoskeletal proteins myosin and cadherin, as well as a transmembrane protein, Echinoid, have reduced actin cable in varying
degrees. These mutants close using increased amounts of cellular protrusions, however, repair is delayed. In contrast, embryos mutant for Cdc42 decrease
the quantity of protrusions during wound repair without affecting the cable. These wounded embryos are delayed and often unable to completely reseal the
epithelial such that a small hole remains. Thus, protrusions play an indispensible role in completely fusing the final wound edges together. We are currently
investigating additional components, which affect the actin cable and protrusions and their role in wound repair.
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A Novel Mechanism for Actin Filament Disassembly Mediated by the Semaphorin/Plexin Axon Guidance Signaling Protein Mical.
Ruei-Jiun Hung
1
,
Chi Pak
2
, Jonathan Terman
1
. 1) Neuroscience; 2) Biochemistry, UT Southwestern Medical Center, Dallas, TX.
Semaphorins are one of the largest families of axon guidance cues and were characterized for their ability to rapidly disassemble actin filaments (F-actin)
and “collapse” elongating neuronal growth cones. Mical, a multidomain cytosolic protein associated with cell-surface Semaphorin receptors (Plexins), is
critical for Sema/Plexin-mediated neural connectivity and actin cytoskeletal rearrangements. Recently, we have found that Mical directly binds F-actin and
provides a conduit between Semaphorin/Plexin and the actin cytoskeleton. Interestingly, Mical belongs to a class of flavoprotein
monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide
phosphate (NADPH) in oxidation-reduction (Redox) reactions. Although MICALs have no known substrate/s, they employ their Redox region to bind F-
actin and disassemble filaments in an NADPH-dependent manner. These observations suggest the possibility that MICALs are direct actin regulatory
enzymes. To further address this hypothesis, we utilized in vitro biochemical assays and found that only very low, substochiometric levels of Mical were
required for F-actin disassembly, lending additional support for a catalytic/post-translational mechanism underlying Mical-mediated F-actin disassembly.
Moreover, this Mical-treated actin failed to re-polymerize even after removal of Mical/NADPH, indicating that Mical stably modifies actin to alter
polymerization. Indeed, we found that actin was a specific substrate of Mical. Specifically, mass spectrometric analyses identified that F-actin subunits were
directly modified by Mical on their conserved pointed-end that is critical for filament assembly. Mical post-translationally oxidized a conserved amino acid
within the D-loop of actin, simultaneously severing filaments and decreasing polymerization. These results together with in vivo analyses using the
Drosophila model bristle process present a specific oxidation-dependent mechanism that selectively regulates actin dynamics and cellular behavior.