Our
laboratory is interested in the fundamental microdomain of membranes,
what factors determine the lateral mobility of
membrane proteins and lipids, and how such mobility is related to the
functions that membranes carry out. To investigate these issues we
use a combination
of microscope imaging tools and cell and molecular biology techniques.
By tracking the individual movements of single membrane lipids and tagged
with 40 nm gold particles or quantum dots, we have found that the two-dimensional
Brownian motion of components in the plane of the membrane can be directly
observed. This technology reveals subtle features of the dynamic lateral
organization of the membrane on the molecular scale. For example, a current
idea is that a class of microdomains, termed lipid rafts, exist in the
plane of the membrane; this notion is both attractive and controversial.
Attractive because such domains could have important functional significance
such as being hot spots for signal transduction. Controversial because
the concept is derived from biochemical extraction data and the in vivo
correlate of these procedures is not known. We have found that raft-like
domains can be reconstituted in various lipid bilayer model membranes
that remarkably recapitulate the properties hypothesized for lipid
rafts but
such concrete evidence is missing for membranes of the living cell. Current
work involves characterizing the dynamics of micro- and nanodomains in
the living cell membrane that either are induced to form or are known
to exist from other techniques. We are currently investigating microdomains
composed of C-type lectins
that have been demonstrated to possess quite unexpected characteristics.
DC-SIGN, a Ca2+-dependent C-type transmembrane lectin, is found assembled
in microdomains on the plasma membranes of dendritic cells (DC). These
microdomains bind a large variety of pathogens and facilitate their uptake
for subsequent antigen presentation. Fluorescence imaging has indicated
that DC-SIGN microdomains may contain other C-type lectins. Fluorescence
recovery after photobleaching (FR/-\P), line-scan fluorescence correlation
spectroscopy and defined valency quantum dot single particle tracking
measurements showed that full-length and cytoplasmically truncated
DC-SiGN is essentially
immobilized in microdomains. By contrast, FRAP indicated that inner leaflet
lipids are able to move through DC-SIGN microdomains suggesting that
the domain is composed of elemental subdomains on the nanoscale. lndeed,
super-resolution
Blink Microscopy has indicated that component DC-SIGN nanodomains are
very small (~65 nm in diameter) and are arranged randomly on the cell
surface.
Calibrated
TIRFM single molecule counting studies using either GFP or antibody
as a tag suggest that the small subdomains are occupied
by 10 or fewer tetramers and that the tetramers are not close packed.
DC-SIGN, when ectopically expressed in a variety of cells including
murine fibroblasts, Raji cells and HeLa cells, forms microdomains.
Studies on ectopically expressed DC-SlGN and its mutants, indicate
that the cytoplasmic domain is not required for domain formation; however,
the tandem repeats in the ectodomain appear to be necessary for domain
formation and immobilization. The surprising stability of DC-SIGN microdomains
may reflect structural features that enhance pathogen uptake by providing
high-avidity platforms. Moreover, the domain must remain intact when
transporting pathogenic cargo from the leading edge of the DC to the
ultimate sites of endocytosis where subsequent antigen processing is
initiated. Future work will involve super-resolution studies of the
lateral organization of these C-type lectin domains, proteomic and
lipidomic studies to get at the origin of the remarkable stability
of these microdomains, and studies to investigate the mechanism of
the very rapid retrograde transport of these cargo-laden domains for
the purpose of antigen presentation.
Papers describing or reviewing this new technology and research have
been published in Science, Biochemistry, Biophys. J., Current Opinion
in Cell Biology, J. Cell Biol., Nature Cell Biology, Trends in Cell
Biol. and Proc. Natl. Acad. Sci.
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different
modes of lateral transport:
