Björn Stuhrmann

Guiding neuronal growth with light

Control over neuronal growth is a fundamental objective in neuroscience, cell biology, developmental biology, biophysics, and biomedicine and is particularly important for the formation of neural circuits in vitro, as well as nerve regeneration in vivo [Zeck, G. & Fromherz, P. (2001) Proc. Natl. Acad. Sci. USA 98, 10457–10462]. We have shown experimentally that we can use weak optical forces to guide the direction taken by the leading edge, or growth cone, of a nerve cell. In actively extending growth cones, a laser spot is placed in front of a specific area of the nerves leading edge, enhancing growth into the beam focus and resulting in guided neuronal turns as well as enhanced growth. The power of our laser is chosen so that the resulting gradient forces are sufficiently powerful to bias the actin polymerization-driven lamellipodia extension, but too weak to hold and move the growth cone. We are therefore using light to control a natural biological process, in sharp contrast to the established technique of optical tweezers [Ashkin, A. (1970) Phys. Rev. Lett. 24, 156–159; Ashkin, A. & Dziedzic, J. M. (1987) Science 235, 1517–1520], which uses large optical forces to manipulate entire structures. Our results therefore open an avenue to controlling neuronal growth in vitro and in vivo with a simple, noncontact technique.

Growing Actin Networks Form Lamellipodium and Lamellum by Self-Assembly

Many different cell types are able to migrate by formation of a thin actin-based cytoskeletal extension. Recently, it became evident that this extension consists of two distinct substructures, designated lamellipodium and lamellum, which differ significantly in their kinetic and kinematic properties as well as their biochemical composition. We developed a stochastic two-dimensional computer simulation that includes chemical reaction kinetics, G-actin diffusion, and filament transport to investigate the formation of growing actin networks in migrating cells. Model parameters were chosen based on experimental data or theoretical considerations. In this work, we demonstrate the systems ability to form two distinct networks by self-organization. We found a characteristic transition in mean filament length as well as a distinct maximum in depolymerization flux, both within the first 1-2 microm. The separation into two distinct substructures was found to be extremely robust with respect to initial conditions and variation of model parameters. We quantitatively investigated the complex interplay between ADF/cofilin and tropomyosin and propose a plausible mechanism that leads to spatial separation of, respectively, ADF/cofilin- or tropomyosin-dominated compartments. Tropomyosin was found to play an important role in stabilizing the lamellar actin network. Furthermore, the influence of filament severing and annealing on the network properties is explored, and simulation data are compared to existing experimental data.

Automated tracking and laser micromanipulation of motile cells

Control over neuronal growth is a prerequisite for the creation of defined in vitro neuronal networks as assays for the elucidation of interneuronal communication. Neuronal growth has been directed by focusing a near-infrared laser beam at a nerve cell’s leading edge [A. Ehrlicher, T. Betz, B. Stuhrmann, D. Koch, V. Milner, M. G. Raizen, and J. Kas, Proc. Natl. Acad. Sci. U.S.A. 99, 16024 (2002)]. The setup reported by Ehrlicher et al. was limited to local laser irradiation and relied on a great deal of subjective interaction since the laser beam could only be steered manually. To overcome the drawbacks of the reported setup, we developed and here present a fully automated low-contrast edge detection software package, which responds to detected cell morphological changes by rapidly actuating laser steering devices, such as acousto-optical deflectors or moving mirrors, thus enabling experiments with minimum human interference. The resulting radiation patterns can be arbitrary functions of space, time, and ...

Versatile optical manipulation system for inspection, laser processing, and isolation of individual living cells

Isolation of individual cells from a heterogeneous cell population is an invaluable step in the analysis of single cell properties. The demands in molecular and cellular biology as well as molecular medicine are the selection, isolation, and monitoring of single cells and cell clusters of biopsy material. Of particular interest are methods which complement a passive optical or spectroscopic selection with a variety of active single cell processing techniques such as mechanical, biochemical, or genetic manipulation prior to isolation. Sophisticated laser-based cell processing systems are available which can perform single cell processing in a contact-free and sterile manner. Until now, however, these multipurpose turnkey systems offer only basic micromanipulation and are not easily modified or upgraded, whereas laboratory situations often demand simple but versatile and adaptable solutions. We built a flexible laser micromanipulation platform combining contact-free microdissection and catapulting capabilit...

Time-resolved microrheology of actively remodeling actomyosin networks

Living cells constitute an extraordinary state of matter since they are inherently out of thermal equilibrium due to internal metabolic processes. Indeed, measurements of particle motion in the cytoplasm of animal cells have revealed clear signatures of nonthermal fluctuations superposed on passive thermal motion. However, it has been difficult to pinpoint the exact molecular origin of this activity. Here, we employ time-resolved microrheology based on particle tracking to measure nonequilibrium fluctuations produced by myosin motor proteins in a minimal model system composed of purified actin filaments and myosin motors. We show that the motors generate spatially heterogeneous contractile fluctuations, which become less frequent with time as a consequence of motor-driven network remodeling. We analyze the particle tracking data on different length scales, combining particle image velocimetry, an ensemble analysis of the particle trajectories, and finally a kymograph analysis of individual particle trajectories to quantify the length and time scales associated with active particle displacements. All analyses show clear signatures of nonequilibrium activity: the particles exhibit random motion with an enhanced amplitude compared to passive samples, and they exhibit sporadic contractile fluctuations with ballistic motion over large (up to 30 μm) distances. This nonequilibrium activity diminishes with sample age,

Robust Organizational Principles of Protrusive Biopolymer Networks in Migrating Living Cells

Cell migration is associated with the dynamic protrusion of a thin actin-based cytoskeletal extension at the cell front, which has been shown to consist of two different substructures, the leading lamellipodium and the subsequent lamellum. While the formation of the lamellipodium is increasingly well understood, organizational principles underlying the emergence of the lamellum are just beginning to be unraveled. We report here on a 1D mathematical model which describes the reaction-diffusion processes of a polarized actin network in steady state, and reproduces essential characteristics of the lamellipodium-lamellum system. We observe a steep gradient in filament lengths at the protruding edge, a local depolymerization maximum a few microns behind the edge, as well as a differential dominance of the network destabilizer ADF/cofilin and the stabilizer tropomyosin. We identify simple and robust organizational principles giving rise to the derived network characteristics, uncoupled from the specifics of any molecular implementation, and thus plausibly valid across cell types. An analysis of network length dependence on physico-chemical system parameters implies that to limit array treadmilling to cellular dimensions, network growth has to be truncated by mechanisms other than aging-induced depolymerization, e.g., by myosin-associated network dissociation at the transition to the cell body. Our work contributes to the analytical understanding of the cytoskeletal extensions bisection into lamellipodium and lamellum and sheds light on how cells organize their molecular machinery to achieve motility.

Statistical analysis of neuronal growth: edge dynamics and the effect of a focused laser on growth cone motility

The neuronal growth cone is a small dynamic structure at the tip of neuronal extensions that guides each neurite extension to its correct partner cell. To reach the designated target, the growth cone integrates chemical signals with high accuracy and reliability. This signal detection operates close to the thermal noise limit and is, therefore of high interest not only to understand neuronal growth, but also to investigate the biological mechanisms of signalling and information processing under the influence of noise. To further investigate neuronal growth, a focused laser positioned at the leading edge of the growth cone is used to bias growth direction, however, the mechanisms of this influence are still unclear. We present a detailed measurement and analysis of the leading edge dynamics of laser treated and control growth cones. Based on the edge motility measurements, we can consistently describe neuronal growth with a stochastic model that allows a bistable potential and the noise intensity of the stochastic process to be extracted. The investigation of control growth cones that were not influenced by the laser reveals a nonlinear dependence of the noise on the overall activity of the growth cones. The presented analysis further quantifies the edge dynamics in growth cones that are manipulated by a laser. Growth cones that actively follow the laser show a tilt of the bistable potential in the direction of the laser to favour protrusions, but no significant changes in the leading edge growth velocity. This is in contrast to the potential changes observed in stationary growth cones that were influenced by the laser. Here, the laser does not tilt the potential shape, but increases the edge velocities, probably by an increase in actin polymerization velocity. These measurements provide new quantitative insight into the dynamics underlying growth cone protrusion and movement.

Optical Neuronal Guidance

We present a novel technique to noninvasively control the growth and turning behavior of an extending neurite. A highly focused infrared laser, positioned at the leading edge of a neurite, has been found to induce extension/turning toward the beams center. This technique has been used successfully to guide NG108-15 and PC12 cell lines [Ehrlicher, A., Betz, T., Stuhrmann, B., Koch, D. Milner, V. Raizen, M. G., and Kas, J. (2002). Guiding neuronal growth with light. Proc. Natl. Acad. Sci. USA 99, 16024-16028], as well as primary rat and mouse cortical neurons [Stuhrmann, B., Goegler, M., Betz, T., Ehrlicher, A., Koch, D., and Kas, J. (2005). Automated tracking and laser micromanipulation of cells. Rev. Sci. Instr. 76, 035105]. Optical guidance may eventually be used alone or with other methods for controlling neurite extension in both research and clinical applications.

Optical control of neuronal growth

Understanding and controlling neuronal growth are basic objectives in neuroscience, biology, biophysics, and biomedicine, and are vital for the formation of neural circuits in vitro, as well as for nerve regeneration in vivo. All molecular stimuli for neuronal growth eventually address the polymeric cytoskeleton, which advances a neurites leading edge also known as the growth cone. We have shown that optical forces of a highly focused infrared laser beam influence the motility of a growth cone by biasing the polymerization-driven intracellular machinery. In actively extending growth cones, a laser spot placed at specific areas of the neurites leading edge affects the growth speed, the direction taken by a growth cone, and the splitting of a growth cone. This novel optical tool manipulates a natural biological process, the cytoskeleton driven morphological changes in growth cones, with potential applications in the formation of neuronal networks and in understanding growth cone motility. The current apparatus combines optical tweezers, phase contrast and fluorescence imaging, and real-time shape detection. Automated and dynamically readjusted irradiation of the growth cone is used to examine and to influence structural and morphological changes of neuronal growth.

THE CYTOSKELETON: AN ACTIVE POLYMER-BASED SCAFFOLD

The motility of cells is a multifaceted and complicated cytoskeletal process. Significant inroads can be made into gaining a more detailed understanding, however, by focusing on the smaller, more simple subunits of the motile system in an effort to isolate the essential protein components necessary to perform a certain task. Identification of such functional modules has proven to be an effective means of working towards a comprehensive understanding of complex, interacting systems. By following a bottom-up approach in studying minimal actin-related sub-systems for keratocyte motility, we revealed several fundamentally important effects ranging from an estimation of the force generated by the polymerization of a single actin filament, to assembly dynamics and the production of force and tension of composite actin networks, to the contraction of actin networks or smaller bundled structures by the motor myosin II. While even motile keratocyte fragments represent a far more complex situation than the simple reconstituted systems presented here, clear parallels can be seen between in vivo cell motility and the idealized in vitro functional modules presented here, giving more weight to their continued focus.