Manfred Lindau
Professor
School of Applied and Engineering Physics
217 Clark Hall
Tel (607)
255-5264
email ml95@cornell.edu
This is the Lindau
Research Group
Qinghua Fang, Khajak
Berberian, Raymond Molloy, Paromita Majumder*, Kassandra Kisler, Sunitha
Bandla, Anita Ngatchou, Dominik Ho, Rong Dong, Manfred Lindau, Joan Lenz**
*visiting from University
of Sao Paulo, Brazil
**took the picture
Research Publications
Much insight into biological function has come from the
development of new biophysical techniques and instrumentation development.
Essential to the development of techniques with significant impact has been
that the driving force is an important specific biological questions.
After a formal training in physics I
directed my own interest towards biophysics and chose to study the function of
the visual pigment rhodopsin. After light absorption intramolecular charge
movements give rise to a major conformational change that allows activation of
an enzymatic cascade inside the cell, eventually leading to a change in the rate of
neurotransmitter release. I developed a new method to spread the native
membranes containing rhodopsin on a filter (1) such that direct electrical
measurements could be made and the charge movement could be characterised
(3-5).
Fascinated
by exploring cellular and molecular mechanisms in biology using biophysical
methods, I had the privilege to work as a post-doc with Erwin Neher in 1984/85.
In his lab I learned patch clamping and worked on secretory cells (6-10). To
determine if ion channels play a role in stimulation of histamine release
release from mast cells I developed a novel patch-clamp configuration which we
named "slow whole cell" where electrical access to the cell was not
by patch disruption but by creating small pores in the patch under the pipette
such that the biochemistry inside was minimally disturbed (6). This was the
basis of what is now called "permeabilized patch" recordings.
Since then, my activities were
mainly driven by an interest in the mechanisms of exocytosis and transmitter
release, which represent currently one of the most exciting topics in cell
biology, neurobiology and biophysics. The process of regulated exocytosis is
responsible for release of neurotransmitters and neuropeptides by nerve
terminals and endocrine cells, release of enzymes or cytotoxic proteins by
granulo‚cytes, or release of histamine and other mediators by mast cells.
During exocytosis the membrane of secretory granules fuses with the plasma
membrane of the cell thereby releasing their contents through the fusion pore.
Although biochemical studies revealed a set of proteins that are essential for
exocytosis, the specific molecular mechanisms of fusion are still obscure. As
for ion channels, functional studies of the fusion processes are essential to
elucidate the molecular mechanisms of fusion.
We
investigate single exocytotic fusion events by measurements of membrane
capacitance using the patch clamp technique exploiting the fact that exocytosis
and endocytosis are associated with changes in plasma membrane area leading to
proportional changes of electrical membrane capacitance (11,20,49,51). In
addition, release of oxidizable substances from single vesicles is monitored by
amperometric techniques. We developed an improved method that allows to
investigate single vesicle exocytosis by patch capacitance measurements
(36,49). To investigate directly the relation of fusion pore dynamics and
transmitter release in neuronal cell types we developed the method of patch
amperometry, which combines high resolution patch capacitance measurements with
amperometric detection of transmitter release inside the patch pipette (41).
My work in this field is
internationally widely recognized. Besides of essential technical developments
in this area we characterized a variety of aspects of membrane fusion and its
regulation applying these methods. We recently published the first
characterization of exocytotic events in neurosecretion and the regulation of
the mode of exocytosis by calcium (41,46). Our methods of patch capacitance measurements
allow for the investigation of the opening of single exocytotic fusion pores in
neurosecretory vesicles using cell-attached and excised patches (41,46,55) with
a resolution similar to that obtained in conventional single channel
recordings. We are presently investigating the effect of certain protein
mutations on the properties of the fusion pore. We anticipate that these
studies will reveal the molecular machinery of fusion analogous to the way
single channel recordings were instrumental to understand ion channel function.
Using this method we recently discovered a novel mechanism of vesicular
membrane dynamics by which secretory vesicles change their volume depending on
their transmitter loading state (59).
Other important steps presumably of essential
importance are the tethering and docking events which vesicles undergo
preceding the actual fusion event. We have very recently succeeded in measuring
the forces tethering vesicles together using horse eosinophil granules. These
granules perform homotypic fusion inside the cell (61,62). We have isolated
such granules and manipulated them using optical tweezers. We are in the
process of characterising these forces and their modulation. In the future we
will move on to study docking forces in vesicle-plasma membrane interactions
using atomic force microscopy.
Another approach to investigate the
role of molecular interactions in fusion pore formation and expansion employs
Fluorescence Resonance Energy Transfer (FRET). FRET provides direct evidence of
changes in molecular interactions. To correlate such changes directly with
fusion pore formation and expansion in single vesicles we have developed a
microfabricated electrochemical detector array on a glass coverslip. This
allows for observation of the cell surface under a fluorescence microscope as
well as electrochemical imaging of fusion pore openings (54). For optimal
resolution of fluorescence changes we are combining this with Total Internal
Reflection Fluorescence (TIRF) microscopy. During my recent sabbatical I
developed an approach using fluorescent nanoparticles (quantum dots) to
investigate intracellular trafficking of synaptic proteins in neurons (A87). I
developed a method to efficiently inject quantum dots functionalized with
specific antibodies into living cells and to follow the intracellular movement
of single molecules.
My
future work will be focused on the study of exocytosis, endocytosis, and
intracellular trafficking in combination of biophysical and biochemical methods
including patch-clamp capacitance measurements, amperometry, video imaging,
optical tweezers, nanotechnology and molecular biology. One major aim of these
experiments is a mechanistic molecular understanding of fusion. Eventually,
isolated granules will be fused with plasma membrane patches and tethering and fusion among granules will
be studied in vitro. The molecular reconstitution of fusion between vesicles and plasma mem‚brane
patches with properties resembling those of natural exocytotic events will
provide a clear picture of the molecular machinery analogous to the previous
demonstration that isolated protein complexes form functional ion channels when
reconstituted into lipid membranes. In addition to these activities we are
developing novel nanobioelectronic devices for biosensors to be used in cell-
and neurobiological research, medical diagnostics, environmental testing and
possibly microscopic implants and neural prosthetics. Such devices will include
on-chip electrochemical detector arrays with integrated electronics,
incorporation of electrochemical detectors into planar patch clamp devices and
electrochemical detector devices to be used in the investigation and treatment
of Parkinson's disease. We will also further develop the application of
fluorescent nanoparticles in the study of intracellular trafficking.
For references see Publication
List
Teaching
Fall only:
A&EP 470
also BIONB 470, VETMM 470, BMEP 570: Biophysical Methods
Spring only:
A&EP 571:
Biophysical Methods Advanced Laboratory Course
Taught
daily during the first 3 weeks of January.
My other web pages at Cornell University:
School
of Applied and Engineering Physics
Biomedical
Engineering
Graduate
Field of Pharmacology