Microtubule polyglutamylation is an essential regulator of cytoskeletal integrity in Trypanosoma brucei.

Tubulin polyglutamylation, catalysed by members of the tubulin tyrosine ligase-like (TTLL) protein family, is an evolutionarily highly conserved mechanism involved in the regulation of microtubule dynamics and function in eukaryotes. In the protozoan parasite Trypanosoma brucei, the microtubule cytoskeleton is essential for cell motility and maintaining cell shape. In a previous study, we showed that T. brucei TTLL6A and TTLL12B are required to regulate microtubule dynamics at the posterior cell pole. Here, using gene deletion, we show that the polyglutamylase TTLL1 is essential for the integrity of the highly organised microtubule structure at the cell pole, with a phenotype distinct from that observed in TTLL6A- and TTLL12B-depleted cells. Reduced polyglutamylation in TTLL1-deficient cells also leads to increased levels in tubulin tyrosination, providing new evidence for an interplay between the tubulin tyrosination and detyrosination cycle and polyglutamylation. We also show that TTLL1 acts differentially on specific microtubule doublets of the flagellar axoneme, although the absence of TTLL1 appears to have no measurable effect on cell motility.

Exploring generic principles of compartmentalization in a developmental in vitro model.

Self-organization of cells into higher-order structures is key for multicellular organisms, for example via repetitive replication of template-like founder cells or syncytial energids. Yet, very similar spatial arrangements of cell-like compartments ('protocells') are also seen in a minimal model system of Xenopus egg extracts in the absence of template structures and chromatin, with dynamic microtubule assemblies driving the self-organization process. Quantifying geometrical features over time, we show here that protocell patterns are highly organized with a spatial arrangement and coarsening dynamics similar to that of two-dimensional foams but without the long-range ordering expected for hexagonal patterns. These features remain invariant when enforcing smaller protocells by adding taxol, i.e. patterns are dominated by a single, microtubule-derived length scale. Comparing our data to generic models, we conclude that protocell patterns emerge by simultaneous formation of randomly assembling protocells that grow at a uniform rate towards a frustrated arrangement before fusion of adjacent protocells eventually drives coarsening. The similarity of protocell patterns to arrays of energids and cells in developing organisms, but also to epithelial monolayers, suggests generic mechanical cues to drive self-organized space compartmentalization.

Molecular Crowding: The History and Development of a Scientific Paradigm.

It is now generally accepted that macromolecules do not act in isolation but "live" in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.

Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency.

Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of four specific genomic loci. MATAC-Seq relies on preferential modification of accessible DNA by methyltransferases combined with Nanopore-Sequencing for direct readout of methylated DNA-bases. Applying MATAC-Seq to selected early-efficient and late-inefficient yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an accessible nucleosome-free region in combination with well-positioned +1 and +2 nucleosomes as a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during eukaryotic DNA replication. Consequently, our single-molecule chromatin accessibility assay will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.

Robust spatiotemporal organization of mitotic events in mechanically perturbed C. elegans embryos.

Early embryogenesis of the nematode Caenorhabditis elegans progresses in an autonomous fashion within a protective chitin eggshell. Cell-division timing and the subsequent mechanically guided positioning of cells is virtually invariant between individuals, especially before gastrulation. Here, we have challenged this stereotypical developmental program in early stages by mechanically perturbing the embryo without breaking its eggshell. Compressing embryos to about two-thirds of their unperturbed diameter only resulted in markedly slower cell divisions. In contrast, compressing embryos to half of their native diameter frequently resulted in a loss of cytokinesis, yielding a non-natural syncytium that still allowed for multiple divisions of nuclei. Although the orientation of mitotic axes was strongly altered in the syncytium, key features of division timing and spatial arrangement of nuclei remained surprisingly similar to those of unperturbed embryos in the first few division cycles. This suggests that few, very robust mechanisms provide a basic and resilient program for safeguarding the early embryogenesis of C. elegans.

Insectivorous birds and bats outperform ants in the top-down regulation of arthropods across strata of a Japanese temperate forest.

Birds, bats and ants are recognised as significant arthropod predators. However, empirical studies reveal inconsistent trends in their relative roles in top-down control across strata. Here, we describe the differences between forest strata in the separate effects of birds, bats and ants on arthropod densities and their cascading effects on plant damage. We implemented a factorial design to exclude vertebrates and ants in both the canopy and understorey. Additionally, we separately excluded birds and bats from the understorey using diurnal and nocturnal exclosures. At the end of the experiments, we collected all arthropods and assessed herbivory damage. Arthropods responded similarly to predator exclusion across forest strata, with a density increase of 81% on trees without vertebrates and 53% without both vertebrates and ants. Additionally, bird exclusion alone led to an 89% increase in arthropod density, while bat exclusion resulted in a 63% increase. Herbivory increased by 42% when vertebrates were excluded and by 35% when both vertebrates and ants were excluded. Bird exclusion alone increased herbivory damage by 28%, while the exclusion of bats showed a detectable but non-significant increase (by 22%). In contrast, ant exclusion had no significant effect on arthropod density or herbivory damage across strata. Our results reveal that the effects of birds and bats on arthropod density and herbivory damage are similar between the forest canopy and understorey in this temperate forest. In addition, ants were not found to be significant predators in our system. Furthermore, birds, bats and ants appeared to exhibit antagonistic relationships in influencing arthropod density. These findings highlight, unprecedentedly, the equal importance of birds and bats in maintaining ecological balance across different strata of a temperate forest.

Phase II trial of hypomethylating agent combined with nivolumab for acute myeloid leukaemia relapse after allogeneic haematopoietic cell transplantation-Immune signature correlates with response.

Acute myeloid leukaemia (AML) relapse after allogeneic haematopoietic cell transplantation (allo-HCT) is often driven by immune-related mechanisms and associated with poor prognosis. Immune checkpoint inhibitors combined with hypomethylating agents (HMA) may restore or enhance the graft-versus-leukaemia effect. Still, data about using this combination regimen after allo-HCT are limited. We conducted a prospective, phase II, open-label, single-arm study in which we treated patients with haematological AML relapse after allo-HCT with HMA plus the anti-PD-1 antibody nivolumab. The response was correlated with DNA-, RNA- and protein-based single-cell technology assessments to identify biomarkers associated with therapeutic efficacy. Sixteen patients received a median number of 2 (range 1-7) nivolumab applications. The overall response rate (CR/PR) at day 42 was 25%, and another 25% of the patients achieved stable disease. The median overall survival was 15.6 months. High-parametric cytometry documented a higher frequency of activated (ICOS+ , HLA-DR+ ), low senescence (KLRG1- , CD57- ) CD8+ effector T cells in responders. We confirmed these findings in a preclinical model. Single-cell transcriptomics revealed a pro-inflammatory rewiring of the expression profile of T and myeloid cells in responders. In summary, the study indicates that the post-allo-HCT HMA/nivolumab combination induces anti-AML immune responses in selected patients and could be considered as a bridging approach to a second allo-HCT. Trial-registration: EudraCT-No. 2017-002194-18.

The random walker's toolbox for analyzing single-particle tracking data.

Technological advances and a burst of new microscopy methods have boosted the use of quantitative tracking experiments, in Soft Matter and Biological Physics but also in the Life Sciences. However, in contrast to highly advanced measurement techniques and tracking tools, subsequent analyses of trajectories frequently do not exploit the data's full potential. Aiming especially at experimental laboratories and early-career scientists, we introduce, discuss, and apply in this Tutorial Review a large set of versatile measures that have proven to be useful for analyzing trajectories from single-particle tracking experiments, beyond a simple extraction of diffusion constants from mean squared displacements. To support a direct test and application of these measures, we supplement the text with a download package that comprises a low-threshold toolbox of ready-to-use routines and training data sets, hence relaxing the need to develop home-brewed solutions and/or to create suitable benchmark data.

Extracting, quantifying, and comparing dynamical and biomechanical properties of living matter through single particle tracking.

A panoply of new tools for tracking single particles and molecules has led to an explosion of experimental data, leading to novel insights into physical properties of living matter governing cellular development and function, health and disease. In this Perspective, we present tools to investigate the dynamics and mechanics of living systems from the molecular to cellular scale via single-particle techniques. In particular, we focus on methods to measure, interpret, and analyse complex data sets that are associated with forces, materials properties, transport, and emergent organisation phenomena within biological and soft-matter systems. Current approaches, challenges, and existing solutions in the associated fields are outlined in order to support the growing community of researchers at the interface of physics and the life sciences. Each section focuses not only on the general physical principles and the potential for understanding living matter, but also on details of practical data extraction and analysis, discussing limitations, interpretation, and comparison across different experimental realisations and theoretical frameworks. Particularly relevant results are introduced as examples. While this Perspective describes living matter from a physical perspective, highlighting experimental and theoretical physics techniques relevant for such systems, it is also meant to serve as a solid starting point for researchers in the life sciences interested in the implementation of biophysical methods.

Probing local chromatin dynamics by tracking telomeres.

Chromatin dynamics is key for cell viability and replication. In interphase, chromatin is decondensed, allowing the transcription machinery to access a plethora of DNA loci. Yet, decondensed chromatin occupies almost the entire nucleus, suggesting that DNA molecules can hardly move. Recent reports have even indicated that interphase chromatin behaves like a solid body on mesoscopic scales. To explore the local chromatin dynamics, we have performed single-particle tracking on telomeres under varying conditions. We find that mobile telomeres feature, under all conditions, a strongly subdiffusive, antipersistent motion that is consistent with the monomer motion of a Rouse polymer in viscoelastic media. In addition, telomere trajectories show intermittent accumulations in local niches at physiological conditions, suggesting that the surrounding chromatin reorganizes on these timescales. Reducing the temperature or exposing cells to osmotic stress resulted in a significant reduction of mobile telomeres and the number of visited niches. Altogether, our data indicate a vivid local chromatin dynamics, akin to a semidilute polymer solution, unless perturbations enforce a more rigid or entangled state of chromatin.

Microtubule polyglutamylation is important for regulating cytoskeletal architecture and motility in Trypanosoma brucei.

The shape of kinetoplastids, such as Trypanosoma brucei, is precisely defined during the stages of the life cycle and governed by a stable subpellicular microtubule cytoskeleton. During the cell cycle and transitions between life cycle stages, this stability has to transiently give way to a dynamic behaviour to enable cell division and morphological rearrangements. How these opposing requirements of the cytoskeleton are regulated is poorly understood. Two possible levels of regulation are activities of cytoskeleton-associated proteins and microtubule post-translational modifications (PTMs). Here, we investigate the functions of two putative tubulin polyglutamylases in T. brucei, TTLL6A and TTLL12B. Depletion of both proteins leads to a reduction in tubulin polyglutamylation in situ and is associated with disintegration of the posterior cell pole, loss of the microtubule plus-end-binding protein EB1 and alterations of microtubule dynamics. We also observe a reduced polyglutamylation of the flagellar axoneme. Quantitative motility analysis reveals that the PTM imbalance correlates with a transition from directional to diffusive cell movement. These data show that microtubule polyglutamylation has an important role in regulating cytoskeletal architecture and motility in the parasite T. bruceiThis article has an associated First Person interview with the first author of the paper.

A versatile cis-acting element reporter system to study the function, maturation and stability of ribosomal RNA mutants in archaea.

General molecular principles of ribosome biogenesis have been well explored in bacteria and eukaryotes. Collectively, these studies have revealed important functional differences and few similarities between these processes. Phylogenetic studies suggest that the information processing machineries from archaea and eukaryotes are evolutionary more closely related than their bacterial counterparts. These observations raise the question of how ribosome synthesis in archaea may proceed in vivo. In this study, we describe a versatile plasmid-based cis-acting reporter system allowing to analyze in vivo the consequences of ribosomal RNA mutations in the model archaeon Haloferax volcanii. Applying this system, we provide evidence that the bulge-helix-bulge motif enclosed within the ribosomal RNA processing stems is required for the formation of archaeal-specific circular-pre-rRNA intermediates and mature rRNAs. In addition, we have collected evidences suggesting functional coordination of the early steps of ribosome synthesis in H. volcanii. Together our investigation describes a versatile platform allowing to generate and functionally analyze the fate of diverse rRNA variants, thereby paving the way to better understand the cis-acting molecular determinants necessary for archaeal ribosome synthesis, maturation, stability and function.

Unscrambling exit site patterns on the endoplasmic reticulum as a quenched demixing process.

The endoplasmic reticulum (ER) is a vital organelle in mammalian cells with a complex morphology. Consisting of sheet-like cisternae in the cell center, the peripheral ER forms a vast tubular network on which a dispersed pattern of a few hundred specialized domains (ER exit sites (ERESs)) is maintained. Molecular details of cargo sorting and vesicle formation at individual ERESs, fueling the early secretory pathway, have been studied in some detail. The emergence of spatially extended ERES patterns, however, has remained poorly understood. Here, we show that these patterns are determined by the underlying ER morphology, suggesting ERESs to emerge from a demixing process that is quenched by the ER network topology. In particular, we observed fewer but larger ERESs when transforming the ER network to more sheet-like morphologies. In contrast, little to no changes with respect to native ERES patterns were observed when fragmenting the ER, indicating that hampering the diffusion-mediated coarse graining of domains is key for native ERES patterns. Model simulations support the notion of effective diffusion barriers impeding the coarse graining and maturation of ERES patterns. We speculate that tuning a simple demixing mechanism by the ER topology allows for a robust but flexible adaption of ERES patterns, ensuring a properly working early secretory pathway in a variety of conditions.

Outcomes on anti-VEGFR-2/paclitaxel treatment after progression on immune checkpoint inhibition in patients with metastatic gastroesophageal adenocarcinoma.

Through our involvement in KEYNOTE-059, we unexpectedly observed durable responses in two patients with metastatic gastroesophageal adenocarcinoma (mGEA) who received ramucirumab (anti-VEGFR-2)/paclitaxel after immune checkpoint inhibition (ICI). To assess the reproducibility of this observation, we piloted an approach to administer ramucirumab/paclitaxel after ICI in more patients, and explored changes in the immune microenvironment. Nineteen consecutive patients with mGEA received ICI followed by ramucirumab/paclitaxel. Most (95%) did not respond to ICI, yet after irRECIST-defined progression on ICI, all patients experienced tumor size reduction on ramucirumab/paclitaxel. The objective response rate (ORR) and progression-free survival (PFS) on ramucirumab/paclitaxel after ICI were higher than on the last chemotherapy before ICI in the same group of patients (ORR, 58.8% vs 11.8%; PFS 12.2 vs 3.0 months; respectively). Paired tumor biopsies examined by imaging mass cytometry showed a median 5.5-fold (range 4-121) lower frequency of immunosuppressive forkhead box P3+ regulatory T cells with relatively preserved CD8+ T cells, post-treatment versus pre-treatment (n = 5 pairs). We then compared the outcomes of these 19 patients with a separate group who received ramucirumab/paclitaxel without preceding ICI (n = 68). Median overall survival on ramucirumab/paclitaxel was longer with (vs without) immediately preceding ICI (14.8 vs 7.4 months) including after multivariate analysis, as was PFS. In our small clinical series, outcomes appeared improved on anti-VEGFR-2/paclitaxel treatment when preceded by ICI, in association with alterations in the immune microenvironment. However, further investigation is needed to determine the generalizability of these data. Prospective clinical trials to evaluate sequential treatment with ICI followed by anti-VEGF(R)/taxane are underway.

Single-Particle Tracking Reveals Anti-Persistent Subdiffusion in Cell Extracts.

Single-particle tracking (SPT) has become a powerful tool to quantify transport phenomena in complex media with unprecedented detail. Based on the reconstruction of individual trajectories, a wealth of informative measures become available for each particle, allowing for a detailed comparison with theoretical predictions. While SPT has been used frequently to explore diffusive transport in artificial fluids and inside living cells, intermediate systems, i.e., biochemically active cell extracts, have been studied only sparsely. Extracts derived from the eggs of the clawfrog Xenopus laevis, for example, are known for their ability to support and mimic vital processes of cells, emphasizing the need to explore also the transport phenomena of nano-sized particles in such extracts. Here, we have performed extensive SPT on beads with 20 nm radius in native and chemically treated Xenopus extracts. By analyzing a variety of distinct measures, we show that these beads feature an anti-persistent subdiffusion that is consistent with fractional Brownian motion. Chemical treatments did not grossly alter this finding, suggesting that the high degree of macromolecular crowding in Xenopus extracts equips the fluid with a viscoelastic modulus, hence enforcing particles to perform random walks with a significant anti-persistent memory kernel.

Carfilzomib or bortezomib in combination with lenalidomide and dexamethasone for patients with newly diagnosed multiple myeloma without intention for immediate autologous stem-cell transplantation (ENDURANCE): a multicentre, open-label, phase 3, randomised, controlled trial.

BACKGROUND: Bortezomib, lenalidomide, and dexamethasone (VRd) is a standard therapy for newly diagnosed multiple myeloma. Carfilzomib, a next-generation proteasome inhibitor, in combination with lenalidomide and dexamethasone (KRd), has shown promising efficacy in phase 2 trials and might improve outcomes compared with VRd. We aimed to assess whether the KRd regimen is superior to the VRd regimen in the treatment of newly diagnosed multiple myeloma in patients who were not being considered for immediate autologous stem-cell transplantation (ASCT). METHODS: In this multicentre, open-label, phase 3, randomised controlled trial (the ENDURANCE trial; E1A11), we recruited patients aged 18 years or older with newly diagnosed multiple myeloma who were ineligible for, or did not intend to have, immediate ASCT. Participants were recruited from 272 community oncology practices or academic medical centres in the USA. Key inclusion criteria were the absence of high-risk multiple myeloma and an Eastern Cooperative Oncology Group performance status of 0-2. Enrolled patients were randomly assigned (1:1) centrally by use of permuted blocks to receive induction therapy with either the VRd regimen or the KRd regimen for 36 weeks. Patients who completed induction therapy were then randomly assigned (1:1) a second time to either indefinite maintenance or 2 years of maintenance with lenalidomide. Randomisation was stratified by intent for ASCT at disease progression for the first randomisation and by the induction therapy received for the second randomisation. Allocation was not masked to investigators or patients. For 12 cycles of 3 weeks, patients in the VRd group received 1·3 mg/m2 of bortezomib subcutaneously or intravenously on days 1, 4, 8, and 11 of cycles 1-8, and day 1 and day 8 of cycles nine to twelve, 25 mg of oral lenalidomide on days 1-14, and 20 mg of oral dexamethasone on days 1, 2, 4, 5, 8, 9, 11, and 12. For nine cycles of 4 weeks, patients in the KRd group received 36 mg/m2 of intravenous carfilzomib on days 1, 2, 8, 9, 15, and 16, 25 mg of oral lenalidomide on days 1-21, and 40 mg of oral dexamethasone on days 1, 8, 15, and 22. The coprimary endpoints were progression-free survival in the induction phase, and overall survival in the maintenance phase. The primary analysis was done in the intention-to-treat population and safety was assessed in patients who received at least one dose of their assigned treatment. The trial is registered with ClinicalTrials.gov, NCT01863550. Study recruitment is complete, and follow-up of the maintenance phase is ongoing. FINDINGS: Between Dec 6, 2013, and Feb 6, 2019, 1087 patients were enrolled and randomly assigned to either the VRd regimen (n=542) or the KRd regimen (n=545). At a median follow-up of 9 months (IQR 5-23), at a second planned interim analysis, the median progression-free survival was 34·6 months (95% CI 28·8-37·8) in the KRd group and 34·4 months (30·1-not estimable) in the VRd group (hazard ratio [HR] 1·04, 95% CI 0·83-1·31; p=0·74). Median overall survival has not been reached in either group. The most common grade 3-4 treatment-related non-haematological adverse events included fatigue (34 [6%] of 527 patients in the VRd group vs 29 [6%] of 526 in the KRd group), hyperglycaemia (23 [4%] vs 34 [6%]), diarrhoea (23 [5%] vs 16 [3%]), peripheral neuropathy (44 [8%] vs four [<1%]), dyspnoea (nine [2%] vs 38 [7%]), and thromboembolic events (11 [2%] vs 26 [5%]). Treatment-related deaths occurred in two patients (<1%) in the VRd group (one cardiotoxicity and one secondary cancer) and 11 (2%) in the KRd group (four cardiotoxicity, two acute kidney failure, one liver toxicity, two respiratory failure, one thromboembolic event, and one sudden death). INTERPRETATION: The KRd regimen did not improve progression-free survival compared with the VRd regimen in patients with newly diagnosed multiple myeloma, and had more toxicity. The VRd triplet regimen remains the standard of care for induction therapy for patients with standard-risk and intermediate-risk newly diagnosed multiple myeloma, and is a suitable treatment backbone for the development of combinations of four drugs. FUNDING: US National Institutes of Health, National Cancer Institute, and Amgen.

Elucidating the Origin of Heterogeneous Anomalous Diffusion in the Cytoplasm of Mammalian Cells.

Diffusion of tracer particles in the cytoplasm of mammalian cells is often anomalous with a marked heterogeneity even within individual particle trajectories. Despite considerable efforts, the mechanisms behind these observations have remained largely elusive. To tackle this problem, we performed extensive single-particle tracking experiments on quantum dots in the cytoplasm of living mammalian cells at varying conditions. Analyses of the trajectories reveal a strong, microtubule-dependent subdiffusion with antipersistent increments and a substantial heterogeneity. Furthermore, particles stochastically switch between different mobility states, most likely due to transient associations with the cytoskeleton-shaken endoplasmic reticulum network. Comparison to simulations highlight that all experimental observations can be fully described by an intermittent fractional Brownian motion, alternating between two states of different mobility.

Quantifying active diffusion in an agitated fluid.

Mixing of reactants in microdroplets predominantly relies on diffusional motion due to small Reynolds numbers and the resulting absence of turbulent flows. Enhancing diffusion in microdroplets by an auxiliary noise source is therefore a topical problem. Here we report on how the diffusional motion of tracer beads is enhanced upon agitating the surrounding aqueous fluid with miniaturized magnetic stir bars that are compatible with microdroplets and microfluidic devices. Using single-particle tracking, we demonstrate via a broad palette of measures that local stirring of the fluid at different frequencies leads to an enhanced but apparently normal and homogenous diffusion process, i.e. diffusional steps follow the anticipated Gaussian distribution and no ballistic motion is observed whereas diffusion coefficients are significantly increased. The signature of stirring is, however, visible in the power-spectral density and in the velocity autocorrelation function of trajectories. Our data therefore demonstrate that diffusive mixing can be locally enhanced with miniaturized stir bars while only moderately affecting the ambient noise properties. The latter may also facilitate the controlled addition of nonequilibrium noise to complex fluids in future applications.

Statistical Analysis of Bistatic Radar Ground Clutter for Different German Rural Environments.

This article presents the statistical analysis of bistatic radar rural ground clutter for different terrain types under low grazing angles. Compared to most state-of-the-art analysis, we present country-specific clutter analysis for subgroups of rural environments rather than for the rural environment as a whole. Therefore, the rural environment analysis is divided into four dominant subgroup terrain types, namely fields with low vegetation, fields with high vegetation, plantations of small trees and forest environments representing a typical rural German environment. We will present the results for both the summer and the winter vegetation. Therefore, bistatic measurement campaigns have been carried out during the summer 2019 and the winter of 2019/20 in the aforementioned four different rural terrain types. The measurements were performed in the radar relevant X-band at a center frequency of 8.85 GHz and over a bandwidth of 100 MHz according to available transmit permission. The distinction of the rural terrain into different subgroups enables a more precise and accurate clutter analysis and modeling of the statistical properties as will be shown in the presented results. The statistical properties are derived from the calculated clutter amplitudes probability density functions and corresponding cumulative distribution functions for each of the four terrain types and the corresponding season. The data basis for the clutter analysis are the processed range-Doppler maps from the bistatic radar measurements. According to the authors' current knowledge, a similar investigation based on real bistatic radar measurement data with the division into terrain subgroups has not yet been carried out and published for a German rural environment.

Diffusion of Exit Sites on the Endoplasmic Reticulum: A Random Walk on a Shivering Backbone.

Major parts of the endoplasmic reticulum (ER) in eukaryotic cells are organized as a dynamic network of membrane tubules connected by three-way junctions. On this network, self-assembled membrane domains, called ER exit sites (ERES), provide platforms at which nascent cargo proteins are packaged into vesicular carriers for subsequent transport along the secretory pathway. Although ERES appear stationary and spatially confined on long timescales, we show here via single-particle tracking that they exhibit a microtubule-dependent and heterogeneous anomalous diffusion behavior on short and intermediate timescales. By quantifying key parameters of their random walk, we show that the subdiffusive motion of ERES is distinct from that of ER junctions, i.e., ERES are not tied to junctions but rather are mobile on ER tubules. We complement and corroborate our experimental findings with model simulations that also indicate that ERES are not actively moved by microtubules. Altogether, our study shows that ERES perform a random walk on the shivering ER backbone, indirectly powered by microtubular activity. Similar phenomena can be expected for other domains on subcellular structures, setting a caveat for the interpretation of domain-tracking data.