Cortical contractility triggers a stochastic switch to fast amoeboid cell motility.

3D amoeboid cell migration is central to many developmental and disease-related processes such as cancer metastasis. Here, we identify a unique prototypic amoeboid cell migration mode in early zebrafish embryos, termed stable-bleb migration. Stable-bleb cells display an invariant polarized balloon-like shape with exceptional migration speed and persistence. Progenitor cells can be reversibly transformed into stable-bleb cells irrespective of their primary fate and motile characteristics by increasing myosin II activity through biochemical or mechanical stimuli. Using a combination of theory and experiments, we show that, in stable-bleb cells, cortical contractility fluctuations trigger a stochastic switch into amoeboid motility, and a positive feedback between cortical flows and gradients in contractility maintains stable-bleb cell polarization. We further show that rearward cortical flows drive stable-bleb cell migration in various adhesive and non-adhesive environments, unraveling a highly versatile amoeboid migration phenotype.

Actin flows mediate a universal coupling between cell speed and cell persistence.

Cell movement has essential functions in development, immunity, and cancer. Various cell migration patterns have been reported, but no general rule has emerged so far. Here, we show on the basis of experimental data in vitro and in vivo that cell persistence, which quantifies the straightness of trajectories, is robustly coupled to cell migration speed. We suggest that this universal coupling constitutes a generic law of cell migration, which originates in the advection of polarity cues by an actin cytoskeleton undergoing flows at the cellular scale. Our analysis relies on a theoretical model that we validate by measuring the persistence of cells upon modulation of actin flow speeds and upon optogenetic manipulation of the binding of an actin regulator to actin filaments. Beyond the quantitative prediction of the coupling, the model yields a generic phase diagram of cellular trajectories, which recapitulates the full range of observed migration patterns.

Cooperative epithelial phagocytosis enables error correction in the early embryo.

Errors in early embryogenesis are a cause of sporadic cell death and developmental failure1,2. Phagocytic activity has a central role in scavenging apoptotic cells in differentiated tissues3-6. However, how apoptotic cells are cleared in the blastula embryo in the absence of specialized immune cells remains unknown. Here we show that the surface epithelium of zebrafish and mouse embryos, which is the first tissue formed during vertebrate development, performs efficient phagocytic clearance of apoptotic cells through phosphatidylserine-mediated target recognition. Quantitative four-dimensional in vivo imaging analyses reveal a collective epithelial clearance mechanism that is based on mechanical cooperation by two types of Rac1-dependent basal epithelial protrusions. The first type of protrusion, phagocytic cups, mediates apoptotic target uptake. The second, a previously undescribed type of fast and extended actin-based protrusion that we call 'epithelial arms', promotes the rapid dispersal of apoptotic targets through Arp2/3-dependent mechanical pushing. On the basis of experimental data and modelling, we show that mechanical load-sharing enables the long-range cooperative uptake of apoptotic cells by multiple epithelial cells. This optimizes the efficiency of tissue clearance by extending the limited spatial exploration range and local uptake capacity of non-motile epithelial cells. Our findings show that epithelial tissue clearance facilitates error correction that is relevant to the developmental robustness and survival of the embryo, revealing the presence of an innate immune function in the earliest stages of embryonic development.

Neuronal stretch reception - Making sense of the mechanosense.

The sensation of mechanical force underlies many of our daily activities. As the sense of touch determines the quality of life, the subconscious sense of proprioception and visceral mechanosensation is indispensible for survival. Many internal organs change shape, either as an active part of their physiology or passively due to body movements. Importantly, these shape changes need to be sensed and balanced properly to prevent organ failure and dysfunction. Consequently, a failure to properly sense volume changes of internal organs has a huge clinical relevance, manifested by a plethora of congenital and age-related diseases. Here we review novel data on mammalian stretch reception as well as classical studies from insect and nematode proprioceptors with the aim to highlight the missing link between organ-level deformation and mechanosensing on the molecular level.

Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content.

SIMPLE: Structured illumination based point localization estimator with enhanced precision.

We present a structured illumination microscopy based point localization estimator (SIMPLE) that achieves a 2-fold increase in single molecule localization precision compared to conventional centroid estimation methods. SIMPLE advances the recently introduced MINFLUX concept by using precisely phase-shifted sinusoidal wave patterns as nanometric rulers for simultaneous particle localization based on photon count variation over a 20 µm field of view. We validate SIMPLE in silico and experimentally on a TIRF-SIM setup using a digital micro-mirror device (DMD) as a spatial light modulator.

Rigidity percolation and active advection synergize in the actomyosin cortex to drive amoeboid cell motility.

Spontaneous locomotion is a common feature of most metazoan cells, generally attributed to the properties of actomyosin networks. This force-producing machinery has been studied down to the most minute molecular details, especially in lamellipodium-driven migration. Nevertheless, how actomyosin networks work inside contraction-driven amoeboid cells still lacks unifying principles. Here, using stable motile blebs from HeLa cells as a model amoeboid motile system, we imaged the dynamics of the actin cortex at the single filament level and revealed the co-existence of three distinct rheological phases. We introduce "advected percolation," a process where rigidity percolation and active advection synergize, spatially organizing the actin network's mechanical properties into a minimal and generic locomotion mechanism. Expanding from our observations on simplified systems, we speculate that this model could explain, down to the single actin filament level, how amoeboid cells, such as cancer or immune cells, can propel efficiently through complex 3D environments.

A MEC-2/stomatin condensate liquid-to-solid phase transition controls neuronal mechanotransduction during touch sensing.

A growing body of work suggests that the material properties of biomolecular condensates ensuing from liquid-liquid phase separation change with time. How this aging process is controlled and whether the condensates with distinct material properties can have different biological functions is currently unknown. Using Caenorhabditis elegans as a model, we show that MEC-2/stomatin undergoes a rigidity phase transition from fluid-like to solid-like condensates that facilitate transport and mechanotransduction, respectively. This switch is triggered by the interaction between the SH3 domain of UNC-89 (titin/obscurin) and MEC-2. We suggest that this rigidity phase transition has a physiological role in frequency-dependent force transmission in mechanosensitive neurons during body wall touch. Our data demonstrate a function for the liquid and solid phases of MEC-2/stomatin condensates in facilitating transport or mechanotransduction, and a previously unidentified role for titin homologues in neurons.

Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells.

Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In this study we addressed how cationic amphipathic peptides-in particular a CAMP with Lysine-Leucine-Lysine repeats (termed KLK)-affect the localization and dynamics of molecules in eukaryotic membranes. We found KLK to selectively inhibit the endocytosis of a subgroup of membrane proteins and lipids by electrostatically interacting with negatively charged sialic acid moieties. Ultrastructural characterization revealed the formation of membrane invaginations representing fission or fusion intermediates, in which the sialylated proteins and lipids were immobilized. Experiments on structurally different cationic amphipathic peptides (KLK, 6-MO-LF11-322 and NK14-2) indicated a cooperation of electrostatic and hydrophobic forces that selectively arrest sialylated membrane constituents.

Multiple centrosomes enhance migration and immune cell effector functions of mature dendritic cells.

Centrosomes play a crucial role during immune cell interactions and initiation of the immune response. In proliferating cells, centrosome numbers are tightly controlled and generally limited to one in G1 and two prior to mitosis. Defects in regulating centrosome numbers have been associated with cell transformation and tumorigenesis. Here, we report the emergence of extra centrosomes in leukocytes during immune activation. Upon antigen encounter, dendritic cells pass through incomplete mitosis and arrest in the subsequent G1 phase leading to tetraploid cells with accumulated centrosomes. In addition, cell stimulation increases expression of polo-like kinase 2, resulting in diploid cells with two centrosomes in G1-arrested cells. During cell migration, centrosomes tightly cluster and act as functional microtubule-organizing centers allowing for increased persistent locomotion along gradients of chemotactic cues. Moreover, dendritic cells with extra centrosomes display enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of extra centrosomes for regular cell and tissue homeostasis.

Spot variation fluorescence correlation spectroscopy allows for superresolution chronoscopy of confinement times in membranes.

Resolving the dynamical interplay of proteins and lipids in the live-cell plasma membrane represents a central goal in current cell biology. Superresolution concepts have introduced a means of capturing spatial heterogeneity at a nanoscopic length scale. Similar concepts for detecting dynamical transitions (superresolution chronoscopy) are still lacking. Here, we show that recently introduced spot-variation fluorescence correlation spectroscopy allows for sensing transient confinement times of membrane constituents at dramatically improved resolution. Using standard diffraction-limited optics, spot-variation fluorescence correlation spectroscopy captures signatures of single retardation events far below the transit time of the tracer through the focal spot. We provide an analytical description of special cases of transient binding of a tracer to pointlike traps, or association of a tracer with nanodomains. The influence of trap mobility and the underlying binding kinetics are quantified. Experimental approaches are suggested that allow for gaining quantitative mechanistic insights into the interaction processes of membrane constituents.

Comparison of additive manufactured models of the mandible in accuracy and quality using six different 3D printing systems.

The aim of this study was to analyze and compare the accuracy and quality of six 3D printing systems available on the market. Data acquisition was performed with 12 scans of human mandibles using an industrial 3D scanner and saved in STL format. These STL files were printed using six different printing systems. Previously defined distances were measured with a sliding caliper on the 72 printed mandibles. The printed models were then scanned once again. Measurements of volumes and surfaces for the STL files and the printed models were compared. Accuracy and quality were evaluated using industrial software. An analysis of the punctual aberration between the template and the printed model, based on a heat map, was also carried out. Secondary factors, such as costs, production times and expendable materials, were also examined. All printing systems performed well in terms of accuracy and quality for clinical usage. The Formiga P110 and the Form 2 showed the best results for volume, with average aberrations of 0.13 ± 0.23 cm3 and 0.12 ± 0.17 cm3, respectively. Similar results were achieved for the heat map aberration, with values of 0.008 ± 0.11 mm (Formiga P110) and 0.004 ± 0.16 mm (Form 2). Both printers showed no significant difference from the optimal neutral line (Formiga P110, p = 0.15; Form 2, p = 0.60). The cheapest models were produced by the Ultimaker 2+, with an average of 5€ per model, making such desktop printers affordable for rapid prototyping. Meanwhile, advanced printing systems with sterilizable and biocompatible printing materials, such as the Formiga P110 and the Form 2, fulfill the high expectations for maxillofacial surgery.

Mesoscale Modeling and Single-Nucleosome Tracking Reveal Remodeling of Clutch Folding and Dynamics in Stem Cell Differentiation.

Nucleosomes form heterogeneous groups in vivo, named clutches. Clutches are smaller and less dense in mouse embryonic stem cells (ESCs) compared to neural progenitor cells (NPCs). Using coarse-grained modeling of the pluripotency Pou5f1 gene, we show that the genome-wide clutch differences between ESCs and NPCs can be reproduced at a single gene locus. Larger clutch formation in NPCs is associated with changes in the compaction and internucleosome contact probability of the Pou5f1 fiber. Using single-molecule tracking (SMT), we further show that the core histone protein H2B is dynamic, and its local mobility relates to the structural features of the chromatin fiber. H2B is less stable and explores larger areas in ESCs compared to NPCs. The amount of linker histone H1 critically affects local H2B dynamics. Our results have important implications for how nucleosome organization and H2B dynamics contribute to regulate gene activity and cell identity.

A reconstituted mammalian APC-kinesin complex selectively transports defined packages of axonal mRNAs.

Through the asymmetric distribution of messenger RNAs (mRNAs), cells spatially regulate gene expression to create cytoplasmic domains with specialized functions. In neurons, mRNA localization is required for essential processes such as cell polarization, migration, and synaptic plasticity underlying long-term memory formation. The essential components driving cytoplasmic mRNA transport in neurons and mammalian cells are not known. We report the first reconstitution of a mammalian mRNA transport system revealing that the tumor suppressor adenomatous polyposis coli (APC) forms stable complexes with the axonally localized ß-actin and ß2B-tubulin mRNAs, which are linked to a kinesin-2 via the cargo adaptor KAP3. APC activates kinesin-2, and both proteins are sufficient to drive specific transport of defined mRNA packages. Guanine-rich sequences located in 3'UTRs of axonal mRNAs increase transport efficiency and balance the access of different mRNAs to the transport system. Our findings reveal a minimal set of proteins sufficient to transport mammalian mRNAs.

The nucleus measures shape changes for cellular proprioception to control dynamic cell behavior.

The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within tissues remains largely unknown. Here, using a zebrafish model, we show that the nucleus, the biggest cellular organelle, functions as an elastic deformation gauge that enables cells to measure cell shape deformations. Inner nuclear membrane unfolding upon nucleus stretching provides physical information on cellular shape changes and adaptively activates a calcium-dependent mechanotransduction pathway, controlling actomyosin contractility and migration plasticity. Our data support that the nucleus establishes a functional module for cellular proprioception that enables cells to sense shape variations for adapting cellular behavior to their microenvironment.

Cortical anchoring of the microtubule cytoskeleton is essential for neuron polarity.

The development of a polarized neuron relies on the selective transport of proteins to axons and dendrites. Although it is well known that the microtubule cytoskeleton has a central role in establishing neuronal polarity, how its specific organization is established and maintained is poorly understood. Using the in vivo model system Caenorhabditis elegans, we found that the highly conserved UNC-119 protein provides a link between the membrane-associated Ankyrin (UNC-44) and the microtubule-associated CRMP (UNC-33). Together they form a periodic membrane-associated complex that anchors axonal and dendritic microtubule bundles to the cortex. This anchoring is critical to maintain microtubule organization by opposing kinesin-1 powered microtubule sliding. Disturbing this molecular complex alters neuronal polarity and causes strong developmental defects of the nervous system leading to severely paralyzed animals.

Tracking single molecules in the live cell plasma membrane-Do's and Don't's.

In recent years, the development of fast and highly sensitive microscopy has changed the way of thinking of cell biologists: it became more and more important to study the structural origin for cellular function, and industry turned its attention to the improvement of the required instruments. Optical microscopy has now reached a milestone in sensitivity by resolving the signal of a single, fluorescence-labeled biomolecule within a living cell. First steps towards these pioneering studies were set by methods developed in the late eighties for tracking single biomolecules labeled with fluorescent latex spheres or gold-particles. Meanwhile, a time-resolution of milliseconds for imaging weakly fluorescent cellular structures like small organelles, vesicles, or even single molecules is state-of-the-art. The advances in the fields of microscopy brought new cell biological questions into reach. The investigation of a single fluorescent molecule-or simultaneously of an ensemble of individual molecules-provides principally new information, which is generally hidden in ensemble-averaged signals of molecules. In this paper we describe strategies how to make use of single molecule trajectories for deducing information about nanoscopic structures in a live cell context. In particular, we focus our discussion on elucidating the plasma membrane organization by single molecule tracking. A diffusing membrane constituent--e.g. a protein or a lipid--experiences a manifold of interactions on its path: the most rapid interactions represent the driving force for free diffusion; stronger or correlated interactions can be frequently observed as subdiffusive behavior. Correct interpretation of the data has the potential to shine light on this enigmatic organelle, where membrane rafts, protein microdomains, fences and pickets still frolic through the text-book sketches. We summarize available analytical models and point out potential pitfalls, which may result in quantitative or three even qualitative misinterpretations.

Hypolipidemic effects of herbal extracts by reduction of adipocyte differentiation, intracellular neutral lipid content, lipolysis, fatty acid exchange and lipid droplet motility.

An increase in adipose tissue is caused by the increased size and number of adipocytes. Lipids accumulate in intracellular stores, known as lipid droplets (LDs). Recent studies suggest that parameters such as LD size, shape and dynamics are closely related to the development of obesity. Berberine (BBR), a natural plant alkaloid, has been demonstrated to possess anti-obesity effects. However, it remains unknown which cellular processes are affected by this compound or how effective herbal extracts containing BBR and other alkaloids actually are. For this study, we used extracts of Coptis chinensis, Mahonia aquifolium, Berberis vulgaris and Chelidonium majus containing BBR and other alkaloids and studied various processes related to adipocyte functionality. The presence of extracts resulted in reduced adipocyte differentiation, as well as neutral lipid content and rate of lipolysis. We observed that the intracellular fatty acid exchange was reduced in different LD size fractions upon treatment with BBR and Coptis chinensis. In addition, LD motility was decreased upon incubation with BBR, Coptis chinensis and Chelidonium majus extracts. Furthermore, Chelidonium majus was identified as a potent fatty acid uptake inhibitor. This is the first study that demonstrates the selected regulatory effects of herbal extracts on adipocyte function.

Versatile analysis of single-molecule tracking data by comprehensive testing against Monte Carlo simulations.

We propose here an approach for the analysis of single-molecule trajectories which is based on a comprehensive comparison of an experimental data set with multiple Monte Carlo simulations of the diffusion process. It allows quantitative data analysis, particularly whenever analytical treatment of a model is infeasible. Simulations are performed on a discrete parameter space and compared with the experimental results by a nonparametric statistical test. The method provides a matrix of p-values that assess the probability for having observed the experimental data at each setting of the model parameters. We show the testing approach for three typical situations observed in the cellular plasma membrane: i), free Brownian motion of the tracer, ii), hop diffusion of the tracer in a periodic meshwork of squares, and iii), transient binding of the tracer to slowly diffusing structures. By plotting the p-value as a function of the model parameters, one can easily identify the most consistent parameter settings but also recover mutual dependencies and ambiguities which are difficult to determine by standard fitting routines. Finally, we used the test to reanalyze previous data obtained on the diffusion of the glycosylphosphatidylinositol-protein CD59 in the plasma membrane of the human T24 cell line.

Different types of cell-to-cell connections mediated by nanotubular structures.

Communication between cells is crucial for proper functioning of multicellular organisms. The recently discovered membranous tubes, named tunneling nanotubes, that directly bridge neighboring cells may offer a very specific and effective way of intercellular communication. Our experiments on RT4 and T24 urothelial cell lines show that nanotubes that bridge neighboring cells can be divided into two types. The nanotubes of type I are shorter and more dynamic than those of type II, and they contain actin filaments. They are formed when cells explore their surroundings to make contact with another cell. The nanotubes of type II are longer and more stable than type I, and they have cytokeratin filaments. They are formed when two already connected cells start to move apart. On the nanotubes of both types, small vesicles were found as an integral part of the nanotubes (that is, dilatations of the nanotubes). The dilatations of type II nanotubes do not move along the nanotubes, whereas the nanotubes of type I frequently have dilatations (gondolas) that move along the nanotubes in both directions. A possible model of formation and mechanical stability of nanotubes that bridge two neighboring cells is discussed.