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SECRETORY
VESICLE ELASTICITY AND MOBILITY
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Andreas C. Chrambach, PhD, Head,
Section on Macromolecular Analysis Zsuzsanna Buzas,
PhD, Contractor Sergey Radko,
PhD, Contractor |
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While primarily focusing on a putative
proteinaceous calcium sensor in studies on biological membrane fusion, calcium-induced modulation of the mechanical
properties of the membrane and its
role in fusion are often overlooked. As a first step in studying the effects
of calcium binding to the lipid
bilayer on the mechanical properties of the membrane, the elastic moduli of
the membranes are being measured using both biological and artificial
membranous vesicles exposed to calcium. Elastic properties of the membranes of sea
urchin yolk granules Radko:
in collaboration with Zimmerberg, Blank The elastic modulus of the membrane can be derived
from measuring the osmotic swelling of the vesicles. We have investigated
osmotic swelling of isolated sea urchin yolk granules, which are fusion
competent and are abundant in sea urchin eggs (Chestkov et al., J Biol
Chem 1998;273: 2445). The osmotic gradient was produced by varying the
concentration of glycine (within the range of zero to 0.5 M) in the
medium. The size of those vesicles, measured by dynamic light scattering (DLS) was found to increase with
decreasing medium osmolarity. However, the increase was too steep to
be accounted for by swelling alone. Differential interference contrast and
phase contrast microscopy showed that, in
fact, the increase in mean size of the vesicles observed in DLS experiments resulted entirely from vesicle aggregation,
given that the decrease in osmolarity did not affect vesicle mean size
measured microscopically. The aggregation was presumably accelerated by a release of vesicle internal content of the
vesicles as a consequence to their progressive rupture with decreasing medium osmolarity. Whether the
elastic modulus may be derived for the membrane of cortical granules
by their osmotic swelling is to be tested. It is also possible that the
rupture of yolk granules masks their swelling, as assessed by averaging microscopically
measured sizes of a changing vesicle population. We will clarify the point by
microscopic sizing of the vesicles immobilized on a cover glass in a
perfusion chamber. Vesicle mobility in electrophoresis through
polymer networks as a measure of membrane elasticity Radko,
Chrambach When the mesh size of a
polymer network is close to the particle size, the particle may penetrate
into the network by either of two
mechanisms: squeezing through the mesh and occupying the available
space or disrupting polymer entanglements and locally deforming the polymer
network. The latter mechanism dominates the penetration of rigid particles
while the former mechanism may dominate
that of the elastic ones (Radko and Chrambach, 2002). Different dependencies of particle retardation on particle size, rate
of penetration, and polymer concentration are expected for those mechanisms
(Radko and Chrambach, J Chromatogr
B 1999;722:1; Radko et al., Anal Chem 2000;72:5955).
For membranous vesicles, the
switch between the mechanisms of penetration may depend on the elastic
modulus of the membrane. To test that hypothesis, we investigated the
dependencies of retardation of extruded liposomes (80 to 200 nm
in mean diameter, PC:PS: cholesterol ratio of 3:1:1), subjected to capillary
zone electrophoresis in PEG solutions, on their size, applied voltage, and
PEG concentration. We found that retardation of the liposomes increases with
vesicle size within the size range studied, thus differing from that of rigid
polystyrene latex spheres. The dependence of retardation on membrane
elasticity modulated by cholesterol content or by lipid composition
(producing lipid bilayers in either a gel or liquid state at the experimental
temperature of 25oC) is under investigation. Radko SP, Chrambach A.
Separation and characterization of submicron- and micron-sized particles by
capillary zone electrophoresis. Electrophoresis 2002;23:1957-1972. Publications Related to Other Work Buzas Z, Antal J, Gilligan
JJ, Backlund PS, Yergey AL, Chrambach A. An electroelution apparatus for sequential transfer of sodium dodecyl
sulfate-proteins into agarose and mass spectrometric identification of Li-Na-dodecyl sulfate-proteins from solubilized
agarose. Electrophoresis 2004;25:966-969. Buzas Z, Chrambach A.
Moving boundaries in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. Electrophoresis 2004;25:970-972. Cabanes-Macheteau M,
Chrambach A, Taverna M, Buzas Z, Berna P. Resolution of
8-aminonaphthalene-1,3,6-trisulfonic
acid-labeled glucose oligomers in polyacrylamide gel electrophoresis at low
gel concentration. Electrophoresis 2004;25:8-13. Chiu TC, Lin YW, Huang CC, Chrambach A, Chang
HT. A simple, rapid, and sensitive method for analysis of SYPRO Red labeled sodium dodecyl sulfate-protein complexes
by capillary electrophoresis with laser-induced
fluorescence. Electrophoresis 2003;24:1730-1736. Wheeler D, Chrambach A, Ashburn P, Jovin TM.
Discontinuous buffer systems operative at pH 2.5 - 11.0, 0 degrees C and 25 degrees C, available on the
Internet. Electrophoresis 2004;25:973-974. COLLABORATORS Paul S
Blank, PhD, Laboratory of Cellular
and Molecular Biophysics, NICHD, Joshua
Zimmerberg, MD, PhD, Laboratory of Cellular
and Molecular Biophysics, NICHD, For further information, contact acc@helix.nih.gov |
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