Roland Wedlich-Söldner

Lifeact: a versatile marker to visualize F-actin.

Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.

Rapid leukocyte migration by integrin-independent flowing and squeezing

All metazoan cells carry transmembrane receptors of the integrin family, which couple the contractile force of the actomyosin cytoskeleton to the extracellular environment. In agreement with this principle, rapidly migrating leukocytes use integrin-mediated adhesion when moving over two-dimensional surfaces. As migration on two-dimensional substrates naturally overemphasizes the role of adhesion, the contribution of integrins during three-dimensional movement of leukocytes within tissues has remained controversial. We studied the interplay between adhesive, contractile and protrusive forces during interstitial leukocyte chemotaxis in vivo and in vitro. We ablated all integrin heterodimers from murine leukocytes, and show here that functional integrins do not contribute to migration in three-dimensional environments. Instead, these cells migrate by the sole force of actin-network expansion, which promotes protrusive flowing of the leading edge. Myosin II-dependent contraction is only required on passage through narrow gaps, where a squeezing contraction of the trailing edge propels the rigid nucleus.

Processive Movement of MreB-Associated Cell Wall Biosynthetic Complexes in Bacteria

Bacteria elongation involves moving synthetic complexes around the cell wall. The peptidoglycan cell wall and the actin-like MreB cytoskeleton are major determinants of cell shape in rod-shaped bacteria. The prevailing model postulates that helical, membrane-associated MreB filaments organize elongation-specific peptidoglycan-synthesizing complexes along sidewalls. We used total internal reflection fluorescence microscopy to visualize the dynamic relation between MreB isoforms and cell wall synthesis in live Bacillus subtilis cells. During exponential growth, MreB proteins did not form helical structures. Instead, together with other morphogenetic factors, they assembled into discrete patches that moved processively along peripheral tracks perpendicular to the cell axis. Patch motility was largely powered by cell wall synthesis, and MreB polymers restricted diffusion of patch components in the membrane and oriented patch motion.

Endocytosis optimizes the dynamic localization of membrane proteins that regulate cortical polarity

Diverse cell types require the ability to maintain dynamically polarized membrane-protein distributions through balancing transport and diffusion. However, design principles underlying dynamically maintained cortical polarity are not well understood. Here we constructed a mathematical model for characterizing the morphology of dynamically polarized protein distributions. We developed analytical approaches for measuring all model parameters from single-cell experiments. We applied our methods to a well-characterized system for studying polarized membrane proteins: budding yeast cells expressing activated Cdc42. We found that a balance of diffusion, directed transport, and endocytosis was sufficient for accurately describing polarization morphologies. Surprisingly, the model predicts that polarized regions are defined with a precision that is nearly optimal for measured endocytosis rates and that polarity can be dynamically stabilized through positive feedback with directed transport. Our approach provides a step toward understanding how biological systems shape spatially precise, unambiguous cortical polarity domains using dynamic processes.

Robust cell polarity is a dynamic state established by coupling transport and GTPase signaling

Yeast cells can initiate bud formation at the G1/S transition in a cue-independent manner. Here, we investigate the dynamic nature of the polar cap and the regulation of the GTPase Cdc42 in the establishment of cell polarity. Using analysis of fluorescence recovery after photobleaching, we found that Cdc42 exchanged rapidly between the polar caps and cytosol and that this rapid exchange required its GTPase cycle. A previously proposed positive feedback loop involving actomyosin-based transport of the Cdc42 GTPase is required for the generation of robust cell polarity during bud formation in yeast. Inhibition of actin-based transport resulted in unstable Cdc42 polar caps. Unstable polarity was also observed in mutants lacking Bem1, a protein previously implicated in a feedback loop for Cdc42 activation through a signaling pathway. When Bem1 and actin were both inhibited, polarization completely failed. These results suggest that cell polarity is established through coupling of transport and signaling pathways and maintained actively by balance of flux.

Lifeact mice for studying F-actin dynamics.

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A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis.

We identified a temperature‐sensitive mutant of the plant pathogenic fungus Ustilago maydis that is defective in the polar distribution of cell wall components and shows abnormal morphology. The affected gene, yup1, was cloned by complementation. It encodes a putative target soluble N‐ethylmaleimide‐sensitive fusion protein attachment protein receptor (t‐SNARE), suggesting a function in membrane fusion. A Yup1–GFP fusion protein localized to vesicles that showed rapid saltatory motion along microtubules. These vesicles are part of the endocytic pathway and accumulate at sites of active growth, thereby supporting the expansion of the hyphal tip. In yup1ts cells, endocytosis is impaired and accumulation of Yup1‐carrying endosomes at cell poles is abolished, resulting in apolar distribution of wall components and morphological alterations. This suggests that a membrane recycling process via early endosomes supports polar growth of U.maydis.

A balance of KIF1A-like kinesin and dynein organizes early endosomes in the fungus Ustilago maydis

In Ustilago maydis, bidirectional transport of early endosomes is microtubule dependent and supports growth and cell separation. During early budding, endosomes accumulate at putative microtubule organizers within the bud, whereas in medium‐budded cells, endosome clusters appear at the growing ends of microtubules at the distal cell pole. This suggests that motors of opposing transport direction organize endosomes in budding cells. Here we set out to identify these motors and elucidate the molecular mechanism of endosome reorganization. By PCR we isolated kin3, which encodes an UNC‐104/KIF1‐like kinesin from U.maydis. Recombinant Kin3 binds microtubules and has ATPase activity. Kin3–green fluorescent protein moves along microtubules in vivo, accumulates at sites of growth and localizes to endosomes. Deletion of kin3 reduces endosome motility to ∼33%, and abolishes endosome clustering at the distal cell pole and at septa. This results in a transition from bipolar to monopolar budding and cell separation defects. Double mutant analysis indicates that the remaining motility in Δkin3‐mutants depends on dynein, and that dynein and Kin3 counteract on the endosomes to arrange them at opposing cell poles.

Spontaneous cell polarization: undermining determinism

It is widely observed that eukaryotic cells can polarize spontaneously in the absence of pre-established asymmetric cues. This phenomenon indicates that the principle of self-organization may be central to the establishment of cell polarity. Modelling work, as well as recent experimental data from several organisms, suggests that a combination of local positive feedback loops and global inhibitors could result in robust cell symmetry breaking through amplification of minute, stochastic variations.

ADF/Cofilin-Mediated Actin Retrograde Flow Directs Neurite Formation in the Developing Brain

Neurites are the characteristic structural element of neurons that will initiate brain connectivity and elaborate information. Early in development, neurons are spherical cells but this symmetry is broken through the initial formation of neurites. This fundamental step is thought to rely on actin and microtubule dynamics. However, it is unclear which aspects of the complex actin behavior control neuritogenesis and which molecular mechanisms are involved. Here, we demonstrate that augmented actin retrograde flow and protrusion dynamics facilitate neurite formation. Our data indicate that a single family of actin regulatory proteins, ADF/Cofilin, provides the required control of actin retrograde flow and dynamics to form neurites. In particular, the F-actin severing activity of ADF/Cofilin organizes space for the protrusion and bundling of microtubules, the backbone of neurites. Our data reveal how ADF/Cofilin organizes the cytoskeleton to drive actin retrograde flow and thus break the spherical shape of neurons.