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is still under development.**
One goal of cell
and tissue engineering is the targeted delivery
of therapeutic agents to cells, and toxins to
unwanted cells. Unfortunately, our understanding
of phagocytosis, or particle uptake, is situational
and empirical, hence difficult to generalize. Indeed,
the kinetics and general phenomenology of phagocytosis
are poorly characterized, largely complicated
by its speed and unpredictability of occurence. We
have circumvented these limitations by monitoring individual particles
being taken up by living cells. For the
macrophage-like cell, Dictyostelium discoideum,
laser-tracking microrheology (LTM) reveals that
forces during uptake jump >30-fold and are
fast (<10s). Complementary real-time
fluorescence measurements provide the opportunity
to resolve the signalling events and systematically
explore factors that affect particle uptake. The
implications for cell and tissue engineering
are obvious.Our studies of pathogenic bacteria
provides a powerful synergy to understanding
phagocytosis. Pathogenic bacteria have
evolved exquisite mechanisms to enhance their
uptake and subsequent vacuolar/lysosomal escape
into cytoplasm.1) What
are the kinetic events during uptake? Of
the signalling molecules involved, which ones
precede uptake?2) Which
factors enhance particle uptake?3) Can
mechanisms of pathogen escape into cytoplasm
be incorporated into artificial particles?4) What
controls the routing of particles once in the
cytoplasm?
We plan to use
optical tweezers to micromanipulate both bacteria
and particles onto cells, thus precisely initiating
particle uptake. Subsequent monitoring using
both real-time fluorescence microscopy and LTM
will quantify their uptake kinetics and forces.
By comparing wild type and mutant bacteria, we
should be able to identify the factors that control
uptake. Because most candidate proteins have
been cloned, we can attach these factors onto
particles and determine if their uptake is also
enhanced. Listeria, Shigella, and
enteropathogenic E. coli all use
different mechanisms of entry and would provide
complementary information.
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