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Super-resolution fluorescence microscopy has become a powerful tool in cell biology to observe sub-cellular organization and molecular details below the diffraction limit of light. Super-resolution methods are generally classified into three main concepts: stimulated emission depletion (STED), single molecule localization microscopy (SMLM) and structured illumination microscopy (SIM). Here, we highlight the novel concept of modulation-enhanced localization microscopy (meLM) which we designate as the 4th super-resolution method. Recently, a series of modulation-enhanced localization microscopy methods have emerged, namely MINFLUX, SIMPLE, SIMFLUX, ModLoc and ROSE. Although meLM combines key ideas from STED, SIM and SMLM, the main concept of meLM relies on a different idea: isolated emitters are localized by measuring their modulated fluorescence intensities in a precisely shifted structured illumination pattern. To position meLM alongside state-of-the-art super-resolution methods we first highlight the basic principles of existing techniques and show which parts of these principles are utilized by the meLM method. We then present the overall novel super-resolution principle of meLM that can theoretically reach unlimited localization precision whenever illumination patterns are translated by an arbitrarily small distance. L R acknowledges support of a fellowship from 'la Caixa' Foundation (ID 100010434, LCF/BQ/IN18/11660032) and funding from the European Union´s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 713673. V R acknowledges support from the Spanish Ministry of Economy and Competitiveness through the Program 'Centro de Excelencia Severo Ochoa 2013-2017', the CERCA Programme/Generalitat de Catalunya, MINECO's Plan Nacional (BFU2017-86296-P) and support from the CRG Advanced Light Microscopy Facility. S W acknowledges support from the Spanish Ministry of Economy and Competitiveness through the 'Severo Ochoa' program for Centres of Excellence in R&D (SEV-2015-0522), from Fundació Privada Cellex, and from Generalitat de Catalunya through the CERCA program.

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. Funding: V.V. acknowledges support from the ICFOstepstone PhD Programme funded by the European Union’s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement 665884. F.P. and Q.T.-R. acknowledge grants funded by Ministerio de Ciencia, Innovación y Universidades and Fondo Social Europeo (FSE) (BES2017-080523-SO, PRE2018-084393). M.A.V. acknowledges support from the Spanish Ministry of Science, Education and Universities through grant RTI2018-099718-B-100 and an institutional “Maria de Maeztu Programme” for Units of Excellence in R&D (CEX2018-000792-M) and FEDER funds.S.W. and M.K. acknowledge support from the Spanish Ministry of Economy and Competitiveness through the Severo Ochoa program for Centres of Excellence in R&D (CEX2019-000910-S), from Fundació Privada Cellex, Fundación Mig-Puig, and from Generalitat de Catalunya through the CERCA program and LaserLab (654148). M.K. acknowledges support through Spanish Ministry of Economy and Competitiveness (RYC-2015-17935, EQC2018-005048-P, AEI-010500-2018-228, and PGC2018-097882-A-I00),Generalitat de Catalunya (2017 SGR 1012), the ERC (715243), and the HFSPO (CDA00023/2018). S.W. acknowledges support through the Spanish Ministry of Economy and Competitiveness via MINECO’s Plan Nacional (PGC2018-098532-A-I00). V.R. acknowledges support from the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programme/Generalitat de Catalunya, and the MINECO’s Plan Nacional (BFU2017-86296-P)

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 their local environment remains largely unknown. Here we show that the nucleus, the biggest cellular organelle, functions as a non-dissipative cellular shape deformation gauge that enables cells to continuously measure shape variations on the time scale of seconds. Inner nuclear membrane unfolding together with the relative spatial intracellular positioning of the nucleus provides physical information on the amplitude and type of cellular shape deformation. This adaptively activates a calcium-dependent mechano-transduction pathway, controlling the level of 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 behaviour to their microenvironment.One Sentence SummaryThe nucleus functions as an active deformation sensor that enables cells to adapt their behavior to the tissue microenvironment.

SUMMARYNeurons are among the most highly polarized cell types. They possess structurally and functionally different processes, axon and dendrites, to mediate information flow through the nervous system. 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 little 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 cell 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.

In vitro reconstitutions reveal the essential components of a mammalian, cytoplasmic mRNA transport system and their functions., 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 β-actin and β2B-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.

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., Research highlights ► Cationic antimicrobial peptide KLK affects eukaryotic host cells ► KLK induces accumulation of plasma mebrane proteins/lipids ► Dependent on sialic acid residues ► Application of KLK leads to a recycling inhibition of sialyted molecules

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.

International audience; In the past decade carbon nanotubes (CNTs) have been widely studied as a potential drug-delivery system, especially with functionality for cellular targeting. Yet, little is known about the actual process of docking to cell receptors and transport dynamics after internalization. Here we performed single-particle studies of folic acid (FA) mediated CNT binding to human carcinoma cells and their transport inside the cytosol. In particular, we employed molecular recognition force spectroscopy, an atomic force microscopy based method, to visualize and quantify docking of FA functionalized CNTs to FA binding receptors in terms of binding probability and binding force. We then traced individual fluorescently labeled, FA functionalized CNTs after specific uptake, and created a dynamic 'roadmap' that clearly showed trajectories of directed diffusion and areas of nanotube confinement in the cytosol. Our results demonstrate the potential of a single-molecule approach for investigation of drug-delivery vehicles and their targeting capacity.

International audience; 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.

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.

In recent years increasing evidence has been reported for the inherent heterogeneity of cell populations. Cell-to-cell variability was particularly found when analyzing protein expression patterns or the responses of cells to different stimuli. Yet, structural features – in particular below the resolution limit of light microscopy – have so far eluded in-depth statistical analysis. We report here for the first time a detailed investigation of the variations in protein mobility between nominally identical cells. Our reasoning was that mobility probes nanometer-sized properties of the moving protein and its local environment, which may be subject to cell-to-cell variability. Single-molecule tracking was employed to characterize the diffusion constant of the glycosylphosphatidylinositol- (GPI-) anchored protein CD59 and the transmembrane protein CD147 in the plasma membrane of T24 cells. Automated and tailored data analysis routines allowed for the analysis of the required large data sets: ∼200000 trajectories obtained on ∼350 cells were analyzed in total. We found up to five-fold higher variability of the diffusion constant between cells compared to the uncertainty for the determination of the diffusion constant on a single cell.

There has been emerging interest whether plasma membrane constituents are moving according to free Brownian motion or hop diffusion. In the latter model, lipids, lipid-anchored proteins, and transmembrane proteins would be transiently confined to periodic corrals in the cell membrane, which are structured by the underlying membrane skeleton. Because this model is based exclusively on results provided by one experimental strategy—high-resolution single particle tracking—we attempted in this study to confirm or amend it using a complementary technique. We developed a novel strategy that employs single molecule fluorescence microscopy to detect confinements to free diffusion of CD59—a GPI-anchored protein—in the plasma membrane of living T24 (ECV) cells. With this method, minimum invasive labeling via fluorescent Fab fragments was sufficient to measure the lateral motion of individual protein molecules on a millisecond timescale, yielding a positional accuracy down to 22nm. Although no hop diffusion was directly observable, based on a full analytical description our results provide upper boundaries for confinement size and strength.