The electron microscope offers a look at complex membrane systems. This image shows theinfrastructure of cells from an adrenal gland tumor in a rat. The double membrane forms acrescent-shaped arch around the truncated cell nucleus. Extending from the upper left to thelower right corner, the membranes separate two cells from each other.

Biological membranes are flexible barriers that permit only specific substances to pass. Theinside of their double lipid layer resembles an oily film. That is why water-soluble moleculescan only pass through when specific pore proteins open up to let them go through. On theone hand, membranes split the cell into separate compartments designed for differentmetabolic processes. On the other hand, the cell makes contact with its surroundingsthrough its outer membrane. In this function, the cellmembrane can transmit as well asreceive outside signals.

Schematic drawing of a biological membrane,consisting of lipids (blue, red and white) andproteins (green).

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departments and research groups 51www.gwgd.de/˜theimbu

Professor Thomas Heimburg

Thomas Heimburg received his Ph.D.from the University of Göttingen

in 1989. Subsequently, he worked as apostdoctoral fellow at the University ofVirginia in Charlottesville for two years.

In 1995, he received his ›Habilitation‹from the University of Göttingen.

In 1997, he was granted a Heisenbergfellowship that allowed him to

establish the Membrane Biophysics and Thermodynamics Group

at the Max Planck Institute for Biophysical Chemistry.

Since 2002, Thomas Heimburg has been professor at the

University of Göttingen.

theimbu@gwdg.de

Our group is dedicated to studying the biophysics of bio-

logical membranes. To this end, we apply a large variety

of thermodynamical methods, both experimental and

theoretical.

Biomembranes are flexible and dynamic barriers that define the in-

side and the outside of cells as well as cell organelles. They control

the transport of molecules in and out of the cell and serve to main-

tain ion gradients. Membranes consist of hundreds of different

lipids (small molecules that form the membrane structure) and

proteins (which are embedded in the lipid membranes). The lipids

in particular exhibit a large diversity in different membranes, even

among membranes of organelles located in the same cell. The

reason for this phenomenon is as yet largely unknown. In recent

years, however, it has become increasingly clear that biomem-

branes are micro-structured into domains of different compositions

and physical states, which may also fluctuate over time. The inter-

est in domain formation is especially nurtured by the hypothesis

that they may play an important role in influencing communication

between different parts of the membrane or between different pro-

teins. In this respect, the fact that the lipids of membranes have the

ability to melt is of particular interest. This means that they are

able to exist in a rigid state at low temperatures and in a flexible

state at high temperatures. In many intact biomembranes, melting

temperatures are interestingly very close to human body tempera-

ture. One of our aims is to understand the role of this melting

process in biological processes.

We also study domains and various interesting physical aspects

that are closely linked to their formation, such as elastic constants,

relaxation properties and diffusion behavior. Among the methods

we use and develop are calorimetry, densitometry, and especially

single molecule fluorescence experiments. Most of our experiments

are accompanied by theoretical modeling, in particular by com-

Left: Confocal fluorescencemicroscopy of a lipid vesicle with adiameter of 30µm showing domainformation (in red and green). Right: Computer simulation ofdomain formation in membranes(red and green regions).

puter simulations. The key motivation of our

studies is to understand the biophysics of

cooperative interactions of many molecules,

and to investigate how the function of bio-

logical membranes is controlled by physical

parameters such as temperature and pH.

This also includes interactions of mem-

branes with other molecules, such as soluble

proteins or neurotransmitters.

Membrane Biophysics and Thermodynamics

T. Heimburg: Coupling of chain melting andbilayer structure: domains, rafts, elasticity andfusion. In: Planar lipid bilayers and their appli-cations. (Eds.) H.T.Tien and A.Ottova-Leitmannova. Elsevier, Amsterdam, 2003,269–293 .

Professor Dieter Gallwitz

Dieter Gallwitz obtained an M.D.degree from the University ofFrankfurt/Main in 1964. Subsequently,he conducted research in biochemistryat the University of Marburg (1965–1967and 1969–1971) and at the University ofWisconsin in Madison (1967–1969).After that, he taught as Professor ofPhysiological Chemistry at MarburgUniversity (1972–1986) and as GuestProfessor at the University of Californiain San Francisco (1977). In 1986, he wasappointed director at the Max PlanckInstitute for Biophysical Chemistry and head of the Department ofMolecular Genetics. Dieter Gallwitz is also an HonoraryProfessor at the University of Göttingenand a member of the AmericanAcademy of Microbiology. Among other awards, he received the Aronson Prize for Microbiology and Immunology in 1986.

Internal research groups:Dr. Manfred KonradDr. Hans-Dieter Schmitt

Dieter.Gallwitz@mpi-bpc.mpg.de

Proteins can be viewed as the working horses of living

cells. Proteins, for example, facilitate chemical reactions,

form channels in biological membranes, allow different

cellular compartments to communicate with each other, or they de-

termine cell shape and function. Many newly synthesized proteins

required to act in the cell’s different membrane-enclosed compart-

ments, or destined to reach the outside walls of the cell, are first

imported into the lumen or inserted into the membrane of the

endoplasmic reticulum (ER). From there, small vesicles mediate

transportation to their final destinations (Fig.1). Transport vesicles

travel different routes, and in order to deliver their cargo to the cor-

rect cellular location, they must be specifically marked and guided.

Vesicular protein transport is a vital cellular activity and requires a

multitude of proteins to secure cargo selection in donor compart-

ments, formation of cargo containers, and their fusion with target

membranes. Proteins regulating vesicular traffic and membrane fu-

sion, such as membrane receptors (SNAREs), SNARE-binding pro-

teins (also termed SM proteins), and GTPases (Ypt/Rab proteins),

are remarkably well conserved from yeast to man – to an extent

that a human protein can functionally replace its yeast counterpart.

In the past, we have discovered a number of such proteins in yeast,

a single-celled organism amenable to genetic and biochemical in-

vestigations. In particular, specific and structurally related GTP-

Molecular Genetics

Fig.1. Electron microscopy of part of a yeast cell showing the nucleus harboring thegenetic material, ribosomes, which are involved in protein biosynthesis, and 80-100 nmtransport vesicles.

Nucleus

Ribosomes

Transport vesicles

binding proteins, forming a family of regulators, have

been found to assemble distinct but related protein

complexes at any given transport step that permit the

fusion of vesicles with different target membranes

(Fig.2). Present studies are aimed at understanding

how GTPases act and how their activity is regulated

by other components. Specifically, we would like to

know how a given GTPase identifies the target mem-

brane where it fulfils its function, and how and when

during vesicle traffic the nucleotide bound to the

GTPase is hydrolyzed. Research efforts are also

focused on elucidating how SNARE-binding proteins

affect the specificity of SNARE/SNARE interactions as

a prerequisite for transport vesicle fusion with the

correct cellular membranes. Such studies in yeast are

also of value to trace transport defects in human cells

that might lead to fatal diseases.

Another direction of research concerns nucleotide

kinases, a species of enzymes involved in the genera-

tion of nucleotides, the building blocks of nucleic

acids. One goal of these investigations is to create

enzymes that can be used in chemotherapy of human

cancer.

www.mpibpc.mpg.de/abteilungen/150/

Fig.2. A »road map« of vesicular protein transport in yeast and the GTPases (Ypt proteins) that regulate differenttransport steps in the secretory and the endocytic pathway. ER, endoplasmic reticulum; e.E., l.E., early and lateendosomes.

Albert, S., E. Will and D. Gallwitz: Identification of the catalytic domains andtheir functionally critical arginine residues of two yeast GTPase-activatingproteins specific for Ypt/Rab transport GTPases. EMBO J. 18, 5216–5225(1999).

Rak, A., R. Fedorov, K. Alexandrov, S. Albert, R.S. Goody, D. Gallwitz and A.J.Scheidig: Crystal structure of the GAP domain of Gyp1p: first insights intointeraction with Ypt/Rab proteins. EMBO J. 19, 5105–5113 (2000).

Votsmeier, C. and D. Gallwitz: An acidic sequence of a putative yeast Golgimembrane protein binds COPII and facilitates ER export. EMBO J. 20,6742–6750 (2001).

De Antoni, A., J. Schmitzová, H.-H. Trepte, D. Gallwitz and S. Albert:Significance of GTP hydrolysis in Ypt1p-regulated endoplasmic reticulum toGolgi transport revealed by the analysis of two novel Ypt1-GAPs. J. Biol.Chem. 277, 41023–41031 (2002).

Peng, R. and D. Gallwitz, D.: Sly1 protein bound to Golgi syntaxin Sed5pallows assembly and contributes to specificity of SNARE fusion complexes. J.Cell Biol. 157, 645–655 (2002).

Sabini, E., S.Ort, C.Monnerjahn, M.Konrad and A.Lavie: Structure of humandCK suggests strategies to improve anticancer and antiviral therapy. NatureStruct. Biol. 10, 513–519 (2003).

departments and research groups 53

54 departments and research groups

Professor Erwin Neher

Erwin Neher studied physics in Munichand at the University of Wisconsin beforehe redirected his interests to biophysics.He completed his doctoral studies at theMax Planck Institute for Psychiatry (nowMPI for Neurobiology) in Munich andjoined the Max Planck Institute forBiophysical Chemistry in 1972. After aresearch stay at Yale University during the1975/76 Academic Year, he became adirector at the Max Planck Institute forBiophysical Chemistry and head of theDepartment of Membrane Biophysics in1983. Erwin Neher is an HonoraryProfessor at Göttingen University andChairman of the Board of the EuropeanNeuroscience Institute – a joint programoperated by the University of Göttingen’sMedical School and the local Max PlanckInstitutes. He also is a member of theEuropean Research Advisory Board inBrussels. In 1991, he was awarded theNobel Prize in Physiology or Medicine for his discovery of ion channel currents in biological membranes(together with Bert Sakmann).

Internal research groups:Dr. Jakob SørensenDr. Takeshi SakabaDr. Holger Taschenberger

eneher@gwdg.de

Cellular membranes composed of lipids and proteins serve

a broad variety of functions in living cells. They consti-

tute a diffusion barrier and provide electrical insulation

between the cell interior and the outside world. Also, they sub-

divide the cell into specific sub-compartments, and form small

vesicles as storage containers for a variety of substances. The cargo

that such vesicles carry may be very diverse, e.g. digestive en-

zymes in the pancreas, the stress hormone adrenalin in the adrenal

gland, and neurotransmitters in synaptic nerve endings. The mech-

anisms by which such vesicles are transported and how the con-

tents are released on demand – processes that occur in a highly

regulated fashion – are quite similar in the different cell types con-

cerned. The molecules involved in these processes are also well

conserved between different cell types and different membrane

compartments. Research on membranes and the regulatory mecha-

nisms that shape them and govern their interactions requires an

intricate combination of knowledge and tools from the fields of

biophysics, physiology, and molecular biology.

Techniques for studying exocytosisIn order to release their contents, storage vesicles need to fuse with

the cell membrane in the process of exocytosis (Fig.1). This can

happen within a fraction of a millisecond at nerve terminals, i.e.,

when a nerve impulse arrives. In order to monitor this process and

the regulatory signals that control exocytosis, rapid measurement

techniques are required. The Max Planck Institute for Biophysical

Chemistry has a long tradition in kinetic measurements. One novel

approach that adds to this tradition allows the monitoring of exo-

cytosis at the single cell level, in some cases even at the single

vesicle level. This approach takes advantage of the fact that the

total cell surface area increases when vesicles fuse with the outer

membrane. This can be monitored as an increase in electrical

capacitance due to the fact that membranes behave like capacitors

in an electrical measurement. In another approach, the released

substance is detected electrochemically by a micrometer-sized

carbon fiber (Fig.2).

Exocytosis in nerve terminals and neurosecretory cells, i.e. the

fusion of a vesicle with the outer membrane, is triggered by an

increase in the cellular concentration of Calcium ions ([Ca++]).

Calcium usually enters the cell through special ion channels,

which open whenever a nerve impulse arrives. However, for the

study of exocytosis, this trigger is not precise enough. We do not

yet know the exact relative position of channels and vesicles and,

therefore, cannot predict how high [Ca++] will rise at the site of the

Membrane Biophysics

www.mpibpc.mpg.de/abteilungen/140/

Xu, T., B. Rammner, M. Margittai, A.R. Artalejo,E. Neher, and R. Jahn: Inhibition of SNAREcomplex assembly differentially affects kineticcomponents of exocytosis. Cell 99, 713–722(1999).

Voets, T., T. Moser, P.-E. Lund, R.H. Chow, M.Geppert, T.C. Suedhof, and E. Neher:Intracellular calcium dependence of largedense-core vesicle exocytosis in the absence ofsynaptotagmin I. Proc. Natl. Acad. Sci. USA 98,11680–11680 (2001).

Sakaba, T. and E. Neher: Calmodulin mediatesrapid recruitment of fast-releasing synapticvesicles at a calyx-type synapse. Neuron 32,1119–1131 (2001).

Nagy, G., U. Matti, R.B. Nehring, T. Binz, J.Rettig, E. Neher and J.B. Soerensen: Proteinkinase C-dependent phosphorylation of synap-tosome-associated protein of 25 kDa at Ser187potentiates vesicle recruitment. J. Neurosci. 22,9278–9286 (2002).

Rettig, J. and E. Neher: Emerging roles of presy-naptic proteins in Ca++-triggered exocytosis.Science 298, 781–785 (2002).

departments and research groups 55

vesicle when channels open. For a better-defined

stimulus, we therefore load cells with so-called caged-

Ca++, a compound that releases Ca++ when exposed

to UV light. A flash of UV light then can elicit a rapid,

incremental increase in [Ca++], which causes the re-

lease of neurotransmitter and a concomitant increase

in surface area. We are also interested in the study of

the kinetics of this release and the fact that it proceeds

in several well-defined phases – a rapid ›exocytotic

burst‹ followed by a slow, continuous further increase

in membrane surface area.

Molecules involved in vesicle priming andtriggering

The exocytotic burst, which is observed upon a rapid

increase in [Ca++], is interpreted as the Ca++-

dependent fusion of those vesicles that have docked

at the membrane and have advanced to a mature (so-

called ›primed‹) state at the time of the [Ca++] step.

The speed of this step depends on [Ca++] and on the

molecules involved in Ca++-dependent triggering. The

sustained phase of release following the exocytotic

burst, on the other hand, represents priming (and

immediate exocytosis) of new vesicles after the

primed vesicles have been released. We therefore can

distinguish the effects of molecular manipulations on

priming from those on Ca++ triggering. We employ

this distinction to study the consequences of muta-

tions in secretory proteins and the defects in the

secretory mechanisms in cells from transgenic ani-

mals, which lack one or the other of the molecules be-

lieved to play a role in priming or triggering. By using

this approach, we have been able to assign a distinct

role in priming to the protein munc13, and to deter-

mine the Ca++-affinity of several variations of the

putative Ca++-sensor synaptotagmin.

The biophysical analysis of these molecular processes

not only advances our understanding of neuronal sig-

naling and hormone release. In fact, isoforms of the

relevant molecules are found in numerous cell types

in our bodies as well as in animals and plants. They

fulfill their specialized tasks wherever vesicles bud

from biological membranes and fuse with each other

or with other membranes in order to deliver their

cargo.

Fig.2. Two cells from theadrenal medulla, specialized

on the release of the stresshormone adrenaline. One of

these is contacted by twotypes of electrodes: A micro-carbon fiber (left) measuresthe adrenaline upon release

into its surroundings.Simultaneously, a ›patch

pipette‹ (right) establishes aconnection to the cell interiorand measures the cell surface

area, which increases uponfusion of vesicles with the

outer membrane.

Fig.1. A secretory vesicle fuses with thecell membrane and releases its contents