Karsten Kruse

Analysis of turnover dynamics of the submembranous actin cortex

Two filament subpopulations with very different turnover rates make up the actin cortex in living cells: one with fast turnover dynamics and polymerization resulting from addition of monomers to free barbed ends, and one with slow turnover dynamics with polymerization resulting from formin-mediated filament growth.

Geometry sensing by self-organized protein patterns

In the living cell, proteins are able to organize space much larger than their dimensions. In return, changes of intracellular space can influence biochemical reactions, allowing cells to sense their size and shape. Despite the possibility to reconstitute protein self-organization with only a few purified components, we still lack knowledge of how geometrical boundaries affect spatiotemporal protein patterns. Following a minimal systems approach, we used purified proteins and photolithographically patterned membranes to study the influence of spatial confinement on the self-organization of the Min system, a spatial regulator of bacterial cytokinesis, in vitro. We found that the emerging protein pattern responds even to the lateral, two-dimensional geometry of the membrane such that, as in the three-dimensional cell, Min protein waves travel along the longest axis of the membrane patch. This shows that for spatial sensing the Min system does not need to be enclosed in a three-dimensional compartment. Using a computational model we quantitatively analyzed our experimental findings and identified persistent binding of MinE to the membrane as requirement for the Min system to sense geometry. Our results give insight into the interplay between geometrical confinement and biochemical patterns emerging from a nonlinear reaction–diffusion system.

The immunological synapse controls local and global calcium signals in T lymphocytes

Summary:u2002 Cell polarization is a key feature of T‐cell function. The immunological synapse (IS) between T cells and antigen‐presenting cells is a beautiful example of how polarization of cells is used to guide cell function. Receptors, signal transducers, the cytoskeleton, and organelles are enriched at or depleted from the IS after its formation, and in many cases these re‐localizations have already been linked with certain T‐cell functions. One key step for T‐cell activation is a rise in the cytoplasmic calcium concentration. Whereas it is undisputed that the IS initiates and controls calcium signals in T cells, very little is known about the role of T‐cell polarization for calcium signals and calcium‐dependent signal transduction. We briefly summarize the basic commonly agreed principles of IS‐dependent calcium signal generation but then focus on the less well understood influence of polarization on calcium signals. The discussion of the role of polarization for calcium signals leads to a model how the IS controls local and global calcium signals and calcium‐dependent T‐cell functions. We develop a theoretical formalism based on existing spatiotemporal calcium dynamic simulations to better understand the model in the future and allow further predictions which can be tested by fast, high resolution live‐cell microscopy.

Actin kinetics shapes cortical network structure and mechanics.

Cells adjust their macroscopic mechanical properties by tuning the actin protomer concentration and activity of actin nucleators. The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs.

Still and rotating myosin clusters determine cytokinetic ring constriction

The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo.

Spiral actin-polymerization waves can generate amoeboidal cell crawling

Amoeboidal cell crawling on solid substrates is characterized by protrusions that seemingly appear randomly along the cell periphery and drive the cell forward. For many cell types, it is known that the protrusions result from polymerization of the actin cytoskeleton. However, little is known about how the formation of protrusions is triggered and whether the appearance of subsequent protrusions is coordinated. Recently, the spontaneous formation of actin-polymerization waves was observed. These waves have been proposed to orchestrate the cytoskeletal dynamics during cell crawling. Here, we study the impact of cytoskeletal polymerization waves on cell migration using a phase-field approach. In addition to directionally moving cells, we find states reminiscent of amoeboidal cell crawling. In this framework, new protrusions are seen to emerge from a nucleation process, generating spiral actin waves in the cell interior. Nucleation of new spirals does not require noise, but occurs in a state that is apparently displaying spatio-temporal chaos.

The actin cortex as an active wetting layer

Using active gel theory we study theoretically the properties of the cortical actin layer of animal cells. The cortical layer is described as a non-equilibrium wetting film on the cell membrane. The actin density is approximately constant in the layer and jumps to zero at its edge. The layer thickness is determined by the ratio of the polymerization velocity and the depolymerization rate of actin.Graphical abstract

Spontaneous Mechanical Oscillations: Implications for Developing Organisms

Major transformations of cells during embryonic development are traditionally associated with the activation or inhibition of genes and with protein modifications. The contributions of mechanical properties intrinsic to the matter an organism is made of, however, are often overlooked. The emerging field physics of living matter is addressing active material properties of the cytoskeleton and tissues like the spontaneous generation of stress, which may lead to shape changes and tissue flows, and their implications for embryonic development. Here, we discuss spontaneous mechanical oscillations to present some basic elements for understanding this physics, and we illustrate its application to developing embryos. We highlight the role of state diagrams to quantitatively probe the significance of the corresponding physical concepts for understanding development.

Load Adaptation of Lamellipodial Actin Networks

Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. Inxa0vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond toxa0mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. Inxa0axa0steady state, migrating cell filaments assume the canonical dendritic geometry, defined byxa0Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range ofxa0angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.

Self-organization in systems of treadmilling filaments

The cytoskeleton is an important substructure of living cells, playing essential roles in cell division, cell locomotion, and the internal organization of subcellular components. Physically, the cytoskeleton is an active polar gel, that is, a system of polar filamentous polymers, which is intrinsically out of thermodynamic equilibrium. Active processes are notably involved in filament growth and can lead to net filament assembly at one end and disassembly at the other, a phenomenon called treadmilling. Here, we develop a framework for describing collective effects in systems of treadmilling filaments in the presence of agents regulating filament assembly. We find that such systems can self-organize into asters and moving filament blobs. We discuss possible implications of our findings for subcellular processes.