Christian Tischer

EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation

The epidermal growth factor receptor (EGFR) belongs to the receptor tyrosine kinase (RTK) superfamily and is involved in regulating cell proliferation, differentiation and motility. Growth factor binding induces receptor oligomerization at the plasma membrane, which leads to activation of the intrinsic RTK activity and trans-phosphorylation of tyrosine residues in the intracellular part of the receptor. These residues are docking sites for proteins containing Src homology domain 2 and phosphotyrosine-binding domains that relay the signal inside the cell. In response to EGF attached to beads, lateral propagation of EGFR phosphorylation occurs at the plasma membrane, representing an early amplification step in EGFR signalling. Here we have investigated an underlying reaction network that couples RTK activity to protein tyrosine phosphatase (PTP) inhibition by reactive oxygen species. Mathematical analysis of the chemical kinetic equations of the minimal reaction network detects general properties of this system that can be observed experimentally by imaging EGFR phosphorylation in cells. The existence of a bistable state in this reaction network explains a threshold response and how a high proportion of phosphorylated receptors can be maintained in plasma membrane regions that are not exposed to ligand.

Molecular recognition of a single sphingolipid species by a protein's transmembrane domain.

Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane. Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the headgroup and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein, which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.

Trapping and tracking a local probe with a photonic force microscope

An improved type of scanning probe microscope system able to measure soft interactions between an optically trapped probe and local environment is presented. Such a system that traps and tracks thermally fluctuating probes to measure local interactions is called a photonic force microscope (PFM). The instrument can be used to study two-dimensional and three-dimensional surface forces, molecular binding forces, entropic and viscoelastic forces of single molecules, and small variations in particle flow, local diffusion, and viscosities. We introduce and characterize a PFM, and demonstrate its outstanding stability and very low noise. The probe’s position can be measured within a precision of 0.2–0.5 nm in three dimensions at a 1 MHz sampling rate. The trapping system facilitates stable trapping of latex spheres with diameter D=λ0/2 at laser powers as low as 0.6 mW in the focal plane. The ratio between the trapping stiffness and laser power was able to be optimized for various trapping conditions. The measur...

Force‐ and kinesin‐8‐dependent effects in the spatial regulation of fission yeast microtubule dynamics

Microtubules (MTs) are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology. For these purposes, MT polymerisation dynamics are tightly regulated. Using automated image analysis software, we investigate the spatial dependence of MT dynamics in interphase fission yeast cells with unprecedented statistical accuracy. We find that MT catastrophe frequencies (switches from polymerisation to depolymerisation) strongly depend on intracellular position. We provide evidence that compressive forces generated by MTs growing against the cell pole locally reduce MT growth velocities and enhance catastrophe frequencies. Furthermore, we find evidence for an MT length‐dependent increase in the catastrophe frequency that is mediated by kinesin‐8 proteins (Klp5/6). Given the intrinsic susceptibility of MT dynamics to compressive forces and the widespread importance of kinesin‐8 proteins, we propose that similar spatial regulation of MT dynamics plays a role in other cell types as well. In addition, our systematic and quantitative data should provide valuable input for (mathematical) models of MT organisation in living cells.

Three-dimensional thermal noise imaging

We present a scanning probe microscope based on optical tweezers for three-dimensional imaging of the topology of transparent material in the nanometer range. A spherical nanoparticle serves as a probe. An optical trap moves it through the sample (e.g., a polymer network), while the position of the particle center is recorded by three-dimensional interferometry. Accessible volumes are reconstructed from the histogram of thermal position fluctuations of the particle. The resolution in determining the position of surfaces in three dimensions is about 20 nm.

Activation of membrane-permeant caged PtdIns(3)P induces endosomal fusion in cells

Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phospholipid residing on early endosomes, where it is proposed to be involved in endosomal fusion. We synthesized membrane-permeant derivatives of PtdIns(3)P, including a caged version that is to our knowledge the first photoactivatable phosphoinositide derivative developed so far. In living cells, photoactivation of caged PtdIns(3)P induced rapid endosomal fusion in an EEA1-dependent fashion, thus providing in vivo evidence that PtdIns(3)P is a sufficient signal for driving this process.

Lateral phosphorylation propagation: an aspect of feedback signalling?

Local stimulation with epidermal growth factor can induce lateral propagation of epidermal-growth-factor-receptor phosphorylation at the plasma membrane. We discuss mechanisms of phosphorylation propagation and its possible function. Is it the spatial transmission of a local stimulus or is it an aspect of a feedback-signalling-reaction network that generates a switch response to growth-factor stimulation?

Protein Traffic Disorders: an Effective High-Throughput Fluorescence Microscopy Pipeline for Drug Discovery

Plasma membrane proteins are essential molecules in the cell which mediate interactions with the exterior milieu, thus representing key drug targets for present pharma. Not surprisingly, protein traffic disorders include a large range of diseases sharing the common mechanism of failure in the respective protein to reach the plasma membrane. However, specific therapies for these diseases are remarkably lacking. Herein, we report a robust platform for drug discovery applied to a paradigmatic genetic disorder affecting intracellular trafficking – Cystic Fibrosis. This platform includes (i) two original respiratory epithelial cellular models incorporating an inducible double-tagged traffic reporter; (ii) a plasma membrane protein traffic assay for high-throughput microscopy screening; and (iii) open-source image analysis software to quantify plasma membrane protein traffic. By allowing direct scoring of compounds rescuing the basic traffic defect, this platform enables an effective drug development pipeline, which can be promptly adapted to any traffic disorder-associated protein and leverage therapy development efforts.

Determination and correction of position detection nonlinearity in single particle tracking and three-dimensional scanning probe microscopy.

A general method is presented for determining and correcting nonlinear position detector responses in single particle tracking as used in three-dimensional scanning probe microscopy based on optical tweezers. The method uses locally calculated mean square displacements of a Brownian particle to detect spatial changes in the sensitivity of the detector. The method is applied to an optical tweezers setup, where the position fluctuations of a microsphere within the optical trap are measured by an interferometric detection scheme with nanometer precision and microsecond temporal resolution. Detector sensitivity profiles were measured at arbitrary positions in solution with a resolution of approximately 6 nm and 20 nm in the lateral and axial directions, respectively. Local detector sensitivities are used to reconstruct the real positions of the particle from the measured position signals.

Providing Positional Information with Active Transport on Dynamic Microtubules

Microtubules (MTs) are dynamic protein polymers that change their length by switching between growing and shrinking states in a process termed dynamic instability. It has been suggested that the dynamic properties of MTs are central to the organization of the eukaryotic intracellular space, and that they are involved in the control of cell morphology, but the actual mechanisms are not well understood. Here, we present a theoretical analysis in which we explore the possibility that a system of dynamic MTs and MT end-tracking molecular motors is providing specific positional information inside cells. We compute the MT length distribution for the case of MT-length-dependent switching between growing and shrinking states, and analyze the accumulation of molecular motors at the tips of growing MTs. Using these results, we show that a transport system consisting of dynamic MTs and associated motor proteins can deliver cargo proteins preferentially to specific positions within the cell. Comparing our results with experimental data in the model organism fission yeast, we propose that the suggested mechanisms could play important roles in setting length scales during cellular morphogenesis.