Sven K. Vogel

Self-Organization of Dynein Motors Generates Meiotic Nuclear Oscillations

Meiotic nuclear oscillations in the fission yeast Schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. We report a mechanism of these oscillations on the basis of collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. By combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. The redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. Our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, owing to the intrinsic motor properties, generate regular large-scale movements in the cell.

Myosin motors fragment and compact membrane-bound actin filaments

Cell cortex remodeling during cell division is a result of myofilament-driven contractility of the cortical membrane-bound actin meshwork. Little is known about the interaction between individual myofilaments and membrane-bound actin filaments. Here we reconstituted a minimal actin cortex to directly visualize the action of individual myofilaments on membrane-bound actin filaments using TIRF microscopy. We show that synthetic myofilaments fragment and compact membrane-bound actin while processively moving along actin filaments. We propose a mechanism by which tension builds up between the ends of myofilaments, resulting in compressive stress exerted to single actin filaments, causing their buckling and breakage. Modeling of this mechanism revealed that sufficient force (∼20 pN) can be generated by single myofilaments to buckle and break actin filaments. This mechanism of filament fragmentation and compaction may contribute to actin turnover and cortex reorganization during cytokinesis. DOI: http://dx.doi.org/10.7554/eLife.00116.001

Lateral Membrane Diffusion Modulated by a Minimal Actin Cortex

Diffusion of lipids and proteins within the cell membrane is essential for numerous membrane-dependent processes including signaling and molecular interactions. It is assumed that the membrane-associated cytoskeleton modulates lateral diffusion. Here, we use a minimal actin cortex to directly study proposed effects of an actin meshwork on the diffusion in a well-defined system. The lateral diffusion of a lipid and a protein probe at varying densities of membrane-bound actin was characterized by fluorescence correlation spectroscopy (FCS). A clear correlation of actin density and reduction in mobility was observed for both the lipid and the protein probe. At high actin densities, the effect on the protein probe was ∼3.5-fold stronger compared to the lipid. Moreover, addition of myosin filaments, which contract the actin mesh, allowed switching between fast and slow diffusion in the minimal system. Spot variation FCS was in accordance with a model of fast microscopic diffusion and slower macroscopic diffusion. Complementing Monte Carlo simulations support the analysis of the experimental FCS data. Our results suggest a stronger interaction of the actin mesh with the larger protein probe compared to the lipid. This might point toward a mechanism where cortical actin controls membrane diffusion in a strong size-dependent manner.

Dynein Motion Switches from Diffusive to Directed upon Cortical Anchoring

Cytoplasmic dynein is a motor protein that exerts force on microtubules. To generate force for the movement of large organelles, dynein needs to be anchored, with the anchoring sites being typically located at the cell cortex. However, the mechanism by which dyneins target sites where they can generate large collective forces is unknown. Here, we directly observe single dyneins during meiotic nuclear oscillations in fission yeast and identify the steps of the dynein binding process: from the cytoplasm to the microtubule and from the microtubule to cortical anchors. We observed that dyneins on the microtubule move either in a diffusive or directed manner, with the switch from diffusion to directed movement occurring upon binding of dynein to cortical anchors. This dual behavior of dynein on the microtubule, together with the two steps of binding, enables dyneins to self-organize into a spatial pattern needed for them to generate large collective forces.

Minimal systems to study membrane-cytoskeleton interactions

In the context of minimal systems design, there are two areas in which the reductionist approach has been particularly successful: studies of molecular motors on cytoskeletal filaments, and of protein-lipid interactions in model membranes. However, a minimal cortex, that is, the interface between membrane and cytoskeleton, has just begun to be functionally reconstituted. A key property of living cells is their ability to change their shape in response to extracellular and intracellular stimuli. Although studied in live cells since decades, the mutual dependence between cytoskeleton and membrane dynamics in these large-scale transformations is still poorly understood. Here we report on inspiring recent in vitro work in this direction, and the promises it holds for a better understanding of key cellular processes.

Interphase Microtubules Determine the Initial Alignment of the Mitotic Spindle

In the fission yeast Schizosaccharomyces pombe, interphase microtubules (MTs) position the nucleus [1, 2], which in turn positions the cell-division plane [1, 3]. It is unclear how the spindle orients, with respect to the predetermined division plane, to ensure that the chromosomes are segregated across this plane. It has been proposed that, during prometaphase, the astral MT interaction with the cell cortex aligns the spindle with the cell axis [4] and also participates in a spindle orientation checkpoint (SOC), which delays entry into anaphase as long as the spindle is misaligned [5-7]. Here, we trace the position of the spindle throughout mitosis in a single-cell assay. We find no evidence for the SOC. We show that the spindle is remarkably well aligned with the cell longitudinal axis at the onset of mitosis, by growing along the axis of the adjacent interphase MT. Misalignment of nascent spindles can give rise to anucleate cells when spindle elongation is impaired. We propose a new role for interphase microtubules: through interaction with the spindle pole body, interphase microtubules determine the initial alignment of the spindle in the subsequent cell division.

Reconstitution of cytoskeletal protein assemblies for large-scale membrane transformation.

Membranes determine two-dimensional and three-dimensional biochemical reaction spaces in living systems. Defining size and shape of surfaces and volumes encompassed by membrane is of key importance for cellular metabolism and homeostasis, and the maintenance and controlled transformation of membrane shapes are coordinated by a large number of different protein assemblies. The orchestration of spatial elements over distances orders of magnitudes larger than protein molecules, as required for cell division, is a particularly challenging task, requiring large-scale ordered protein filaments and networks. The structure and function of these networks, particularly of cytoskeletal elements, have been characterized extensively in cells and reconstituted systems. However, their co-reconstitution with membranes from the bottom-up under defined conditions, to elucidate their mode of action in detail, is still a relatively new field of research. In this short review, we discuss recent approaches and achievements with regard to the study of cytoskeletal protein assemblies on model membranes, with specific focus on contractile elements as those based on the bacterial division FtsZ protein and eukaryotic actomyosin structures.

Intracellular nanosurgery and cell enucleation using a picosecond laser

Living cells are highly organized in space and time, which makes spatially and temporally confined manipulations an indispensable tool in cell biology. Laser‐based nanosurgery is an elegant method that allows precise ablation of intracellular structures. Here, we show cutting of fluorescently labelled microtubules and mitotic spindles in fission yeast, performed with a picosecond laser coupled to a confocal microscope. Diverse effects from photo‐bleaching to partial and complete breakage are obtained by varying the exposure time, while simultaneously imaging the structures of interest. Using this system we developed an efficient technique to generate enucleated cells without perturbing the distribution of other organelles. This enucleation method can be used to study the cytoskeleton in a nucleus‐free environment, as well as the role of the nucleus in cell growth and a variety of cellular functions.

The design of MACs (minimal actin cortices)

The actin cell cortex in eukaryotic cells is a key player in controlling and maintaining the shape of cells, and in driving major shape changes such as in cytokinesis. It is thereby constantly being remodeled. Cell shape changes require forces acting on membranes that are generated by the interplay of membrane coupled actin filaments and assemblies of myosin motors. Little is known about how their interaction regulates actin cell cortex remodeling and cell shape changes. Because of the vital importance of actin, myosin motors and the cell membrane, selective in vivo experiments and manipulations are often difficult to perform or not feasible. Thus, the intelligent design of minimal in vitro systems for actin‐myosin‐membrane interactions could pave a way for investigating actin cell cortex mechanics in a detailed and quantitative manner. Here, we present and discuss the design of several bottom‐up in vitro systems accomplishing the coupling of actin filaments to artificial membranes, where key parameters such as actin densities and membrane properties can be varied in a controlled manner. Insights gained from these in vitro systems may help to uncover fundamental principles of how exactly actin‐myosin‐membrane interactions govern actin cortex remodeling and membrane properties for cell shape changes.

Control of lipid domain organization by a biomimetic contractile actomyosin cortex

The cell membrane is a heterogeneously organized composite with lipid-protein micro-domains. The contractile actin cortex may govern the lateral organization of these domains in the cell membrane, yet the underlying mechanisms are not known. We recently reconstituted minimal actin cortices (MACs) (Vogel et al., 2013b) and here advanced our assay to investigate effects of rearranging actin filaments on the lateral membrane organization by introducing various phase-separated lipid mono- and bilayers to the MACs. The addition of actin filaments reorganized membrane domains. We found that the process reached a steady state where line tension and lateral crowding balanced. Moreover, the phase boundary allowed myosin driven actin filament rearrangements to actively move individual lipid domains, often accompanied by their shape change, fusion or splitting. Our findings illustrate how actin cortex remodeling in cells may control dynamic rearrangements of lipids and other molecules inside domains without directly binding to actin filaments. DOI: http://dx.doi.org/10.7554/eLife.24350.001