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Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. In vitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. In vivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44+/+ and Cd44−/− mice but not Cd14−/− mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.

Nature chemical biology 16, 1-19 (2020). doi:10.1038/s41589-020-0600-3, The p53 homolog TAp63$α$ is the transcriptional key regulator of genome integrity in oocytes. After DNA damage, TAp63$α$ is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer that initiates apoptosis. By measuring activation kinetics in ovaries and single-site phosphorylation kinetics in vitro with peptides and full-length protein, we show that TAp63$α$ phosphorylation follows a biphasic behavior. Although the first two CK1 phosphorylation events are fast, the third one, which constitutes the decisive step to form the active conformation, is slow. Structure determination of CK1 in complex with differently phosphorylated peptides reveals the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63$α$ substrate interaction in which the product of one phosphorylation step acts as an inhibitor for the following one., Published by Nature Publishing Group, Basingstoke

Phosphatidic acid (PA) lipids have a dual role as building blocks for membrane biogenesis and as active signaling molecules. This study establishes the molecular details of selective PA recognition by the transcriptional regulator Opi1 from baker’s yeast., A key event in cellular physiology is the decision between membrane biogenesis and fat storage. Phosphatidic acid (PA) is an important intermediate at the branch point of these pathways and is continuously monitored by the transcriptional repressor Opi1 to orchestrate lipid metabolism. In this study, we report on the mechanism of membrane recognition by Opi1 and identify an amphipathic helix (AH) for selective binding of PA over phosphatidylserine (PS). The insertion of the AH into the membrane core renders Opi1 sensitive to the lipid acyl chain composition and provides a means to adjust membrane biogenesis. By rational design of the AH, we tune the membrane-binding properties of Opi1 and control its responsiveness in vivo. Using extensive molecular dynamics simulations, we identify two PA-selective three-finger grips that tightly bind the PA phosphate headgroup while interacting less intimately with PS. This work establishes lipid headgroup selectivity as a new feature in the family of AH-containing membrane property sensors.

The adherens junction associated protein 1 (AJAP1, aka shrew-1) is presumably a type-I transmembrane protein localizing and interacting with the E-cadherin-catenin complex. In various tumors, AJAP1 expression is reduced or lost, including hepatocellular and esophageal squamous cell carcinoma, and glial-derived tumors. The aberrant expression of AJAP1 is associated with alterations in cell migration, invasion, increased tumor growth, and tumor vascularization, suggesting AJAP1 as a putative tumor suppressor. We show that AJAP1 attenuates sprouting angiogenesis by reducing endothelial migration and invasion capacities. Further, we show for the first time that endogenous AJAP1 is associated with the microtubule cytoskeleton. This linkage is independent from cell confluency and stable during angiogenic sprouting in vitro. Our work suggests that AJAP1 is a putative negative regulator of angiogenesis, reducing cell migration and invasion by interfering with the microtubule network. Based on our results and those of other authors, we suggest AJAP1 as a novel tumor suppressor and diagnostic marker., Summary: Depletion of AJAP1 in endothelial cells can increase angiogenic-like sprouting and cell migration. In addition, AJAP1 associates with microtubules in these cells.

The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of the Tc-foxQ2 forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types of Tc-foxQ2 positive neural progenitor cells based on differential co-expression with Tc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tc-ey, Tc-rx and Tc-fez1. An enhancer trap line built by genome editing marked Tc-foxQ2 positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, in Tc-foxQ2 RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishes foxQ2 as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.

During the mammalian preimplantation phase, cells undergo two subsequent cell fate decisions. During the first cell fate decision, cells become either part of an outer trophectoderm or part of the inner cell mass. Subsequently, the inner cell mass (ICM) segregates into an embryonic and an extraembryonic lineage, giving rise to the epiblast and the primitive endoderm, respectively. Inner cell mass organoids represent an experimental model system for preimplantation development, mimicking the second cell fate decision taking place inin vivomouse embryos. In a previous study, the spatial pattern of the different cell lineage types was investigated. The study revealed that cells of the same fate tend to cluster stronger than expected for purely random cell fate decisions. Three major processes are hypothesised to contribute to the final cell fate arrangements at the mid and late blastocysts or 24 h old and 48 h old ICM organoids, respectively: 1) intra- and intercellular chemical signalling; 2) a cell sorting process; 3) cell proliferation. In order to quantify the influence of cell proliferation on the emergence of the observed cell lineage type clustering behaviour, we developed an agent-based model. Hereby, cells are mechanically interacting with direct neighbours, and exert adhesion and repulsion forces. The model was applied to compare several current assumptions of how inner cell mass neighbourhood structures are generated. We tested how different assumptions regarding cell fate switches affect the observed neighbourhood relationships. The model supports the hypothesis that initial cell fate acquisition is a stochastically driven process, taking place in the early development of inner cell mass organoids. The model further shows that the observed neighbourhood structures can emerge due to cell fate heredity during cell division and allows the inference of a time point for the cell fate decision.STATEMENT OF SIGNIFICANCECell fate decisions in early embryogenesis have been considered random events, causing a random cell fate distribution. Using an agent-based mathematical model, fitted to ICM organoid data, we show that the assumed random distribution of cell fates occurs only for a short time interval, as cell fate heredity and cell division quickly lead to spatial cell fate clustering. Our results show that neighbourhood clustering can emerge without specific neighbourhood interactions affecting the cell fate decision. The approach indicates four consecutive phases of early development: 1) co-expression of cell fate markers, 2) cell fate decision, 3) division and local cell fate clustering, and 4) phase separation, whereby only the phases 1-3 occur in ICM organoids during the first 24h of growth.

Cell fate decisions such as apoptosis require cells to translate signaling input into a binary yes/no response. A tight control of the process is required to avoid loss of cells by accidental activation of cell death pathways. One particularly critical situation exists in primary oocytes because their finite number determines the reproductive capacity of females. On the one hand a stringent genetic quality control is necessary to maintain the genetic integrity of the entire species; on the other hand an overly stringent mechanism that kills oocytes with even minor DNA damage can deplete the whole primary oocyte pool leading to infertility. The p53 homolog TAp63α is the key regulator of genome integrity in oocytes. After DNA damage TAp63α is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer. By measuring activation kinetics in ovaries and single site phosphorylation kineticsin vitrowith peptides and full length protein we show that TAp63α phosphorylation follows a biphasic behavior. While the first two CK1 phosphorylation events are fast, the third one that constitutes the decisive step to form the active conformation is slow. We reveal the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63α substrate interaction and demonstrate by quantitative simulation that the slow phosphorylation phase determines the threshold of DNA damage required for induction of apoptosis.

The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of theTc-foxQ2forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types ofTc-foxQ2positive neural progenitor cells based on differential co-expression withTc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tc-ey, Tc-rxandTc-fez1. An enhancer trap line built by genome editing markedTc-foxQ2positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, inTc-foxQ2RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishesfoxQ2as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.Summary statementAn ancestral neuroendocrine center contributes to the evolution of the central complex.foxQ2is a gene required for the development of midline structures of the insect brain, which distinguish protocerebrum from segmental ganglia.

In vitro cell culture models used to study epithelia and epithelial diseases would benefit from the recognition that organs and tissues function in a three-dimensional (3D) environment. This context is necessary for the development of cultures that more realistically resemble in vivo tissues/organs. Our aim was to establish and characterize biologically meaningful 3D models of epithelium. We engineered 3D epithelia cultures using a kidney epithelia cell line (MDCK) and spherical polymer scaffolds. These kidney epithelia were characterized by live microscopy, immunohistochemistry and transmission electron microscopy. Strikingly, the epithelial cells displayed increased physiological relevance; they were extensively polarized and developed a more differentiated phenotype. Using such a growth system allows for direct transmission and fluorescence imaging with few restrictions using wide-field, confocal and Light Sheet Fluorescence Microscopy. We also assessed the wider relevance of this 3D culturing technique with several epithelial cell lines. Finally, we established that these 3D micro-tissues can be used for infection as well as biochemical assays and to study important cellular processes such as epithelial mesenchymal transmission. This new biomimetic model could provide a broadly applicable 3D culture system to study epithelia and epithelia related disorders.

During mammalian preimplantation, cells of the inner cell mass (ICM) adopt either an embryonic or an extraembryonic fate. This process is tightly regulated in space and time and has been studied previously in mouse embryos and embryonic stem cell models. Current research suggests that cell fates are arranged in a salt-and-pepper pattern of random cell positioning or a spatially alternating pattern. However, the details of the three-dimensional patterns of cell fate specification have not been investigated in the embryo nor in in vitro systems. We developed ICM organoids as a, to our knowledge, novel three-dimensional in vitro stem cell system to model mechanisms of fate decisions that occur in the ICM. ICM organoids show similarities to the in vivo system that arise regardless of the differences in geometry and total cell number. Inspecting ICM organoids and mouse embryos, we describe a so far unknown local clustering of cells with identical fates in both systems. These findings are based on the three-dimensional quantitative analysis of spatiotemporal patterns of NANOG and GATA6 expression in combination with computational rule-based modeling. The pattern identified by our analysis is distinct from the current view of a salt-and-pepper pattern. Our investigation of the spatial distributions both in vivo and in vitro dissects the contributions of the different parts of the embryo to cell fate specifications. In perspective, our combination of quantitative in vivo and in vitro analyses can be extended to other mammalian organisms and thus creates a powerful approach to study embryogenesis.

The survival rate of cancer patients is steadily increasing, owing to more efficient therapies. Understanding the molecular mechanisms of chemotherapy-induced premature ovarian insufficiency (POI) could identify targets for prevention of POI. Loss of the primordial follicle reserve is the most important cause of POI, with the p53 family member p63 being responsible for DNA-damage-induced apoptosis of resting oocytes. Here, we provide the first detailed mechanistic insight into the activation of p63, a process that requires phosphorylation by both the priming kinase CHK2 and the executioner kinase CK1 in mouse primordial follicles. We further describe the structural changes induced by phosphorylation that enable p63 to adopt its active tetrameric conformation and demonstrate that previously discussed phosphorylation by c-Abl is not involved in this process. Inhibition of CK1 rescues primary oocytes from doxorubicin and cisplatin-induced apoptosis, thus uncovering a new target for the development of fertoprotective therapies.

During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterised by the transcription factors NANOG and GATA6, respectively. Here, we present quantitative three-dimensional single cell-based neighbourhood analyses to investigate the spatial distribution of NANOG and GATA6 expression in the ICM of the mouse blastocyst. The cell neighbourhood is characterised by the expression levels of the fate markers in the surrounding cells, together with the number of surrounding cells and cell position. We find that cell neighbourhoods are established in early blastocysts and different for cells expressing different levels of NANOG and GATA6. Highest NANOG expressing cells occupy specific positions within the ICM and are surrounded by 9 neighbours, while GATA6 expressing cells cluster according to their GATA6 levels. The analysis of mutants reveals that NANOG local neighbourhood is regulated by GATA6.Summary statementThree-dimensional cell neighbourhood, which includes fate marker levels, number of neighbouring cells and cell position, determines cell fate decision in early mouse embryos.

Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.

Insect development has contributed significantly to our understanding of metazoan development. However, most information has been obtained by analyzing a single species, the fruit fly Drosophila melanogaster. Embryonic development of the red flour beetle Tribolium castaneum differs fundamentally from that of Drosophila in aspects such as short-germ development, embryonic leg development, extensive extra-embryonic membrane formation and non-involuted head development. Although Tribolium has become the second most important insect model organism, previous live imaging attempts have addressed only specific questions and no long-term live imaging data of Tribolium embryogenesis have been available. By combining light sheet-based fluorescence microscopy with a novel mounting method, we achieved complete, continuous and non-invasive fluorescence live imaging of Tribolium embryogenesis at high spatiotemporal resolution. The embryos survived the 2-day or longer imaging process, developed into adults and produced fertile progeny. Our data document all morphogenetic processes from the rearrangement of the uniform blastoderm to the onset of regular muscular movement in the same embryo and in four orientations, contributing significantly to the understanding of Tribolium development. Furthermore, we created a comprehensive chronological table of Tribolium embryogenesis, integrating most previous work and providing a reference for future studies. Based on our observations, we provide evidence that serosa window closure and serosa opening, although deferred by more than 1 day, are linked. All our long-term imaging datasets are available as a resource for the community. Tribolium is only the second insect species, after Drosophila, for which non-invasive long-term fluorescence live imaging has been achieved.

The filamentous ascomycete Podospora anserina is a well-established aging model in which a variety of different pathways, including those involved in the control of respiration, ROS generation and scavenging, DNA maintenance, proteostasis, mitochondrial dynamics, and programmed cell death have previously been demonstrated to affect aging and life span. Here we address a potential role of autophagy. We provide data demonstrating high basal autophagy levels even in strains cultivated under noninduced conditions. By monitoring an N-terminal fusion of EGFP to the fungal LC3 homolog PaATG8 over the lifetime of the fungus on medium with and without nitrogen supplementation, respectively, we identified a significant increase of GFP puncta in older and in nitrogen-starved cultures suggesting an induction of autophagy during aging. This conclusion is supported by the demonstration of an age-related and autophagy-dependent degradation of a PaSOD1-GFP reporter protein. The deletion of Paatg1, which leads to the lack of the PaATG1 serine/threonine kinase active in early stages of autophagy induction, impairs ascospore germination and development and shortens life span. Under nitrogen-depleted conditions, life span of the wild type is increased almost 4-fold. In contrast, this effect is annihilated in the Paatg1 deletion strain, suggesting that the ability to induce autophagy is beneficial for this fungus. Collectively, our data identify autophagy as a longevity-assurance mechanism in P. anserina and as another surveillance pathway in the complex network of pathways affecting aging and development. These findings provide perspectives for the elucidation of the mechanisms involved in the regulation of individual pathways and their interactions.

Fluorescence long-term imaging of cellular processes in three-dimensional cultures requires the control of media supply, temperature, and pH, as well as minimal photodamage. We describe a system based on a light sheet fluorescence microscope (LSFM), which is optimized for long-term, multi-position imaging of three-dimensional in-gel cell cultures. The system integrates a stable culture condition control system in the optical path of the light-sheet microscope. A further essential element is a biocompatible agarose container suitable for the LSFM, in which any cell type can be cultured in different gel matrices. The TC-LSFM allows studying any in vitro cultured cell type reacting to, dividing in, or migrating through a three-dimensional extracellular matrix (ECM) gel. For this reason we called it "tissue culture-LSFM" (TC-LSFM). The TC-LSFM system allows fast imaging at multiple locations within a millimeter-sized ECM gel. This increases the number of analyzed events and allows testing population effects. As an example, we show the maturation of a cyst of MDCK (canine kidney epithelial) cells over a period of three days. Moreover, we imaged, tracked, and analyzed MDCK cells during the first five days of cell aggregate formation and discovered a remarkable heterogeneity in cell cycle lengths and an interesting cell death pattern. Thus, TC-LSFM allows performing new long-term assays assessing cellular behavior in three-dimensional ECM-gel cultures. For example migration, invasion or differentiation in epithelial cell systems, stem cells, as well as cancer cells can be investigated.

*† SUMMARY Most plant growth occurs post-embryonically and is characterized by the constant and iterative formation of new organs. Non-invasive time-resolved imaging of intact, fully functional organisms allows studies of the dynamics involved in shaping complex organisms. Conventional and confocal fluorescence microscopy suffer from limitations when whole living organisms are imaged at single-cell resolution. We applied light sheet-based fluorescence microscopy to overcome these limitations and study the dynamics of plant growth. We designed a special imaging chamber in which the plant is maintained vertically under controlled illumination with its leaves in the air and its root in the medium. We show that minimally invasive, multi-color, three-dimensional imaging of live Arabidopsis thaliana samples can be achieved at organ, cellular and subcellular scales over periods of time ranging from seconds to days with minimal damage to the sample. We illustrate the capabilities of the method by recording the growth of primary root tips and lateral root primordia over several hours. This allowed us to quantify the contribution of cell elongation to the early morphogenesis of lateral root primordia and uncover the diurnal growth rhythm of lateral roots. We demonstrate the applicability of our approach at varying spatial and temporal scales by following the division of plant cells as well as the movement of single endosomes in live growing root samples. This multi-dimensional approach will have an important impact on plant developmental and cell biology and paves the way to a truly quantitative description of growth processes at several scales.

For genomic integrity to be maintained, the cell cycle and DNA damage responses must be linked. Cdt1, a G1-specific cell-cycle factor, is targeted for proteolysis by the Cul4-Ddb1Cdt2 ubiquitin ligase following DNA damage. Using a laser nanosurgery microscope to generate spatially restricted DNA damage within the living cell nucleus, we show that Cdt1 is recruited onto damaged sites in G1 phase cells, within seconds of DNA damage induction. PCNA, Cdt2, Cul4, DDB1 and p21Cip1 also accumulate rapidly to damaged sites. Cdt1 recruitment is PCNA-dependent, whereas PCNA and Cdt2 recruitment are independent of Cdt1. Fitting of fluorescence recovery after photobleaching profiles to an analytic reaction-diffusion model shows that Cdt1 and p21Cip1 exhibit highly dynamic binding at the site of damage, whereas PCNA appears immobile. Cdt2 exhibits both a rapidly exchanging and an apparently immobile subpopulation. Our data suggest that PCNA provides an immobile binding interface for dynamic Cdt1 interactions at the site of damage, which leads to rapid Cdt1 recruitment to damaged DNA, preceding Cdt1 degradation.

During development, different cell types must undergo distinct morphogenetic programs so that tissues develop the right dimensions in the appropriate place. In early eye morphogenesis, retinal progenitor cells (RPCs) move first towards the midline, before turning around to migrate out into the evaginating optic vesicles. Neighbouring forebrain cells, however, converge rapidly and then remain at the midline. These differential behaviours are regulated by the transcription factor Rx3. Here, we identify a downstream target of Rx3, the Ig-domain protein Nlcam, and characterise its role in regulating cell migration during the initial phase of optic vesicle morphogenesis. Through sophisticated live imaging and comprehensive cell tracking experiments in zebrafish, we show that ectopic expression of Nlcam in RPCs, as is observed in Rx3 mutants, causes enhanced convergence of these cells. Expression levels of Nlcam therefore regulate the migratory properties of RPCs. Our results provide evidence that the two phases of optic vesicle morphogenesis: slowed convergence and outward-directed migration, are under different genetic control. We propose that Nlcam forms part of the guidance machinery directing rapid midline migration of forebrain precursors, where it is normally expressed, and that its ectopic expression upon loss of Rx3 imparts these migratory characteristics upon RPCs.

A long-standing goal of biology is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 hours of development. Multiview in vivo imaging at 1.5 billion voxels per minute provides “digital embryos,” that is, comprehensive databases of cell positions, divisions, and migratory tracks. Our analysis of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo's cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.

Precise control over organelle shapes is essential for cellular organization and morphogenesis. During yeast meiosis, prospore membranes (PSMs) constitute bell-shaped organelles that enwrap the postmeiotic nuclei leading to the cellularization of the mother cell's cytoplasm and to spore formation. Here, we analysed how the PSMs acquire their curved bell-shaped structure. We discovered that two antagonizing forces ensure PSM shaping and proper closure during cytokinesis. The Ssp1p-containing coat at the leading edge of the PSM generates a pushing force, which is counteracted by a novel pathway, the spore membrane-bending pathway (SpoMBe). Using genetics, we found that Sma2p and Spo1p, a phospholipase, as well as several GPI-anchored proteins belong to the SpoMBe pathway. They exert a force all along the membrane, responsible for membrane bending during PSM biogenesis and for PSM closure during cytokinesis. We showed that the SpoMBe pathway involves asymmetric distribution of Sma2p and does not involve a GPI-protein-containing matrix. Rather, repulsive forces generated by asymmetrically distributed and dynamically moving GPI-proteins are suggested as the membrane-bending principle.

Moving from cell monolayers to three-dimensional (3D) cultures is motivated by the need to work with cellular models that mimic the functions of living tissues. Essential cellular functions that are present in tissues are missed by 'petri dish'-based cell cultures. This limits their potential to predict the cellular responses of real organisms. However, establishing 3D cultures as a mainstream approach requires the development of standard protocols, new cell lines and quantitative analysis methods, which include well-suited three-dimensional imaging techniques. We believe that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.

Filopodia are thin, spike-like cell surface protrusions containing bundles of parallel actin filaments. So far, filopodial dynamics has mainly been studied in the context of cell motility on coverslip-adherent filopodia by using fluorescence and differential interference contrast (DIC) microscopy. In this study, we used an optical trap and interferometric particle tracking with nanometer precision to measure the three-dimensional dynamics of macrophage filopodia, which were not attached to flat surfaces. We found that filopodia act as cellular tentacles: a few seconds after binding to a particle, filopodia retract and pull the bound particle toward the cell. We observed F-actin-dependent stepwise retraction of filopodia with a mean step size of 36 nm, suggesting molecular motor activity during filopodial pulling. Remarkably, this intracellular stepping motion, which was measured at counteracting forces of up to 19 pN, was transmitted to the extracellular tracked particle via the filopodial F-actin bundle and the cell membrane. The pulling velocity depended strongly on the counteracting force and ranged between 600 nm/s at forces 15 pN. This result provides an explanation of the significant differences in filopodial retraction velocities previously reported in the literature. The measured filopodial retraction force–velocity relationship is in agreement with a model for force-dependent multiple motor kinetics.

Background The complex cellular networks within tumors, the cytokine milieu, and tumor immune escape mechanisms affecting infiltration and anti-tumor activity of immune cells are of great interest to understand tumor formation and to decipher novel access points for cancer therapy. However, cellular in vitro assays, which rely on monolayer cultures of mammalian cell lines, neglect the three-dimensional architecture of a tumor, thus limiting their validity for the in vivo situation. Methods Three-dimensional in vivo-like tumor spheroid were established from human cervical carcinoma cell lines as proof of concept to investigate infiltration and cytotoxicity of NK cells in a 96-well plate format, which is applicable for high-throughput screening. Tumor spheroids were monitored for NK cell infiltration and cytotoxicity by flow cytometry. Infiltrated NK cells, could be recovered by magnetic cell separation. Results The tumor spheroids were stable over several days with minor alterations in phenotypic appearance. The tumor spheroids expressed high levels of cellular ligands for the natural killer (NK) group 2D receptor (NKG2D), mediating spheroid destruction by primary human NK cells. Interestingly, destruction of a three-dimensional tumor spheroid took much longer when compared to the parental monolayer cultures. Moreover, destruction of tumor spheroids was accompanied by infiltration of a fraction of NK cells, which could be recovered at high purity. Conclusion Tumor spheroids represent a versatile in vivo-like model system to study cytotoxicity and infiltration of immune cells in high-throughput screening. This system might proof useful for the investigation of the modulatory potential of soluble factors and cells of the tumor microenvironment on immune cell activity as well as profiling of patient-/donor-derived immune cells to personalize cellular immunotherapy. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1321-y) contains supplementary material, which is available to authorized users.

The formation of functional neuronal circuits relies on accurate migration and proper axonal outgrowth of neuronal precursors. On the route to their targets migrating cells and growing axons depend on both, directional information from neurotropic cues and adhesive interactions mediated via extracellular matrix molecules or neighbouring cells. The inactivation of guidance cues or the interference with cell adhesion can cause severe defects in neuronal migration and axon guidance. In this study we have analyzed the function of the MAM domain containing glycosylphosphatidylinositol anchor 2A (MDGA2A) protein in zebrafish cranial motoneuron development. MDGA2A is prominently expressed in distinct clusters of cranial motoneurons, especially in the ones of the trigeminal and facial nerves. Analyses of MDGA2A knockdown embryos by light sheet and confocal microscopy revealed impaired migration and aberrant axonal outgrowth of these neurons; suggesting that adhesive interactions mediated by MDGA2A are required for the proper arrangement and outgrowth of cranial motoneuron subtypes., Biology Open, 4 (2), ISSN:2046-6390

During sporulation in Saccharomyces cerevisiae, the four daughter cells (spores) are formed inside the boundaries of the mother cell. Here, we investigated the dynamics of spore assembly and the actin cytoskeleton during this process, as well as the requirements for filamentous actin during the different steps of spore formation. We found no evidence for a polarized actin cytoskeleton during sporulation. Instead, a highly dynamic network of non-polarized actin cables is present underneath the plasma membrane of the mother cell. We found that a fraction of prospore membrane (PSM) precursors are transported along the actin cables. The velocity of PSM precursors is diminished if Myo2p or Tpm1/2p function is impaired. Filamentous actin is not essential for meiotic progression, for shaping of the PSMs or for post-meiotic cytokinesis. However, actin is essential for spore wall formation. This requires the function of the Arp2/3p complex and involves large carbohydrate-rich compartments, which may be chitosome analogous structures.

Spindle elongation segregates chromosomes and occurs in anaphase, an essential step in mitosis. Dynein-mediated pulling forces position the spindle, but their role in anaphase is a matter of debate. Here, we demonstrate that dynein is responsible for rapid spindle elongation in the model fungus Ustilago maydis. We show that initial slow elongation is supported by kinesin-5, which is located in the spindle mid-zone. When the spindle reaches approximately 2 microm in length, the elongation rate increases four-fold. This coincides with the appearance of long and less-dynamic microtubules (MTs) at each pole that accumulate dynein at their tips. Laser-mediated nanosurgery revealed that these MTs exert pulling forces in control cells, but not in dynein mutants. In addition, dynein mutants undergo initial slow anaphase, but fail to establish less-dynamic MTs and do not perform rapid spindle elongation, suggesting that dynein drives anaphase B. This is most likely mediated by cortical sliding of astral MTs along stationary dynein, which is off-loaded from the MT plus-end to the cortex.

Nud1p, a protein homologous to the mammalian centrosome and midbody component Centriolin, is a component of the budding yeast spindle pole body (SPB), with roles in anchorage of microtubules and regulation of the mitotic exit network during vegetative growth. Here we analyze the function of Nud1p during yeast meiosis. We find that a nud1-2 temperature-sensitive mutant has two meiosis-related defects that reflect genetically distinct functions of Nud1p. First, the mutation affects spore formation due to its late function during spore maturation. Second, and most important, the mutant loses its ability to distinguish between the ages of the four spindle pole bodies, which normally determine which SPB would be preferentially included in the mature spores. This affects the regulation of genome inheritance in starved meiotic cells and leads to the formation of random dyads instead of non-sister dyads under these conditions. Both functions of Nud1p are connected to the ability of Spc72p to bind to the outer plaque and half-bridge (via Kar1p) of the SPB.

Spindle pole bodies (SPBs) provide a structural basis for genome inheritance and spore formation during meiosis in yeast. Upon carbon source limitation during sporulation, the number of haploid spores formed per cell is reduced. We show that precise spore number control (SNC) fulfills two functions. SNC maximizes the production of spores (1–4) that are formed by a single cell. This is regulated by the concentration of three structural meiotic SPB components, which is dependent on available amounts of carbon source. Using experiments and computer simulation, we show that the molecular mechanism relies on a self-organizing system, which is able to generate particular patterns (different numbers of spores) in dependency on one single stimulus (gradually increasing amounts of SPB constituents). We also show that SNC enhances intratetrad mating, whereby maximal amounts of germinated spores are able to return to a diploid lifestyle without intermediary mitotic division. This is beneficial for the immediate fitness of the population of postmeiotic cells.

We report on the manipulation of intracellular filaments using a nanosurgery system based on a subnanosecond pulsed UV laser optimized for the localized severing of biological polymers. By inducing artificial catastrophe of selected microtubules (MTs), we perform shrinkage-rate measurements in interphase Ptk-2 cells throughout the entire cell. We quantify the impact of two labeling methods and three fluorescent markers, showing a 25% faster depolymerization with Alexa-488 tubulin compared with Rhodamine and yellow fluorescent protein (YFP) tubulins and a 20% higher variability induced by microinjection compared with stable transfection. Using EB3-GFP as a tip marker, we establish a new protocol to measure shrinkage rate, growth rate and rescue frequency simultaneously with high temporal and spatial specificity in live cells. As our analysis shows, laser-induced MT dynamics are physiologically relevant. The high statistical efficiency that the method offers in terms of numbers of measured events and therefore reduced standard deviations represents an important quantitative improvement in the measurement of dynamic instability parameters in vivo. We extend the application of the method by demonstrating induced dynamic behavior of actin-stress fibers after severing. This new method enables the quantitative investigation of cytoskeleton dynamics in a local confinement.

During unequal cell divisions a mitotic spindle is eccentrically positioned before cell cleavage. To determine the basis of the net force imbalance that causes spindle displacement in one-cell Caenorhabditis elegans embryos, we fragmented centrosomes with an ultraviolet laser. Analysis of the mean and variance of fragment speeds suggests that the force imbalance is due to a larger number of force generators pulling on astral microtubules of the posterior aster relative to the anterior aster. Moreover, activation of heterotrimeric guanine nucleotide– binding protein (Gprotein) subunits is required to generate these astral forces. Cell divisions creating daughter cells of unequal sizes are widespread in developing organisms, where they contribute to the generation of cell fate diversity (1). During such divisions, the mitotic spindle is off-center at the end of anaphase. The cleavage furrow then bisects the spindle in the middle, generating two daughter cells of unequal size (2). During the first division of a C. elegans embryo, the polarity of the cell is established

The dynamic organization of eukaryotic genomes in cell nuclei recently came into the focus of research interest. The kinetics of genome dynamics can be addressed only by approaches involving live cell microscopy. Different methods are available to visualize chromatin, specific chromatin fractions, or individual chromosome territories within nuclei of living mammalian cells. Appropriate labeling procedures as well as cell chamber systems and important controls for live cell microscopy are described.

The actin system forms a supramolecular, membrane-associated network that serves multiple functions in Dictyostelium cells, including cell motility controlled by chemoattractant, phagocytosis, macropinocytosis, and cytokinesis. In executing these functions the monomeric G-actin polymerizes reversibly, and the actin filaments are assembled into membrane-anchored networks together with other proteins involved in shaping the networks and controlling their dynamics. Most impressive is the speed at which actin-based structures are built, reorganized, or disassembled. We used GFP-tagged coronin and Arp3, an intrinsic constituent of the Arp2/3 complex, as examples of proteins that are recruited to highly dynamic actin-filament networks. By fluorescence recovery after photobleaching (FRAP), average exchange rates of cell-cortex bound coronin were estimated. A nominal value of 5 s for half-maximal incorporation of coronin into the cortex, and a value of 7 s for half-maximal dissociation from cortical binding sites has been obtained. Actin dynamics implies also flow of F-actin from sites of polymerization to sites of depolymerization, i.e. to the tail of a migrating cell, the base of a phagocytic cup, and the cleavage furrow in a mitotic cell. To monitor this flow, we expressed in Dictyostelium cells a GFP-tagged actin-binding fragment of talin. This fragment (GFP-TalC63) translocates from the front to the tail during cell migration and from the polar regions to the cleavage furrow during mitotic cell division. The intrinsic dynamics of the actin system can be manipulated in vivo by drugs or other probes that act either as inhibitors of actin polymerization or as stabilizers of filamentous actin. In order to investigate structure–function relationships in the actin system, a technique of reliably arresting transient network structures is in demand. We discuss the potential of electron tomography of vitrified cells to visualize actin networks in their native association with membranes.

Background Small Rab GTPases are important regulators of vesicular trafficking in plants. AtRabA1d, a member of the RabA1 subfamily of small GTPases, was previously found in the vesicle-rich apical dome of growing root hairs suggesting a role during tip growth; however, its specific intracellular localization and role in plants has not been well described. Results The transient expression of 35S::GFP:RabA1d construct in Allium porrum and Nicotiana benthamiana revealed vesicular structures, which were further corroborated in stable transformed Arabidopsis thaliana plants. GFP-RabA1d colocalized with the trans-Golgi network marker mCherry-VTI12 and with early FM4-64-labeled endosomal compartments. Late endosomes and endoplasmic reticulum labeled with FYVE-DsRed and ER-DsRed, respectively, were devoid of GFP-RabA1d. The accumulation of GFP-RabA1d in the core of brefeldin A (BFA)-induced-compartments and the quantitative upregulation of RabA1d protein levels after BFA treatment confirmed the association of RabA1d with early endosomes/TGN and its role in vesicle trafficking. Light-sheet microscopy revealed involvement of RabA1d in root development. In root cells, GFP-RabA1d followed cell plate expansion consistently with cytokinesis-related vesicular trafficking and membrane recycling. GFP-RabA1d accumulated in disc-like structures of nascent cell plates, which progressively evolved to marginal ring-like structures of the growing cell plates. During root hair growth and development, GFP-RabA1d was enriched at root hair bulges and at the apical dome of vigorously elongating root hairs. Importantly, GFP-RabA1d signal intensity exhibited an oscillatory behavior in-phase with tip growth. Progressively, this tip localization dissapeared in mature root hairs suggesting a link between tip localization of RabA1d and root hair elongation. Our results support a RabA1d role in events that require vigorous membrane trafficking. Conclusions RabA1d is located in early endosomes/TGN and is involved in vesicle trafficking. RabA1d participates in both cell plate formation and root hair oscillatory tip growth. The specific GFP-RabA1d subcellular localization confirms a correlation between its specific spatio-temporal accumulation and local vesicle trafficking requirements during cell plate and root hair formation. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0252-0) contains supplementary material, which is available to authorized users.

This protocol describes how to observe gastrulation in living mouse embryos by using light-sheet microscopy and computational tools to analyze the resulting image data at the single-cell level. We describe a series of techniques needed to image the embryos under physiological conditions, including how to hold mouse embryos without agarose embedding, how to transfer embryos without air exposure and how to construct environmental chambers for live imaging by digital scanned light-sheet microscopy (DSLM). Computational tools include manual and semiautomatic tracking programs that are developed for analyzing the large 4D data sets acquired with this system. Note that this protocol does not include details of how to build the light-sheet microscope itself. Time-lapse imaging ends within 12 h, with subsequent tracking analysis requiring 3-6 d. Other than some mouse-handling skills, this protocol requires no advanced skills or knowledge. Light-sheet microscopes are becoming more widely available, and thus the techniques outlined in this paper should be helpful for investigating mouse embryogenesis.

Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 (Hsp70) and 90 (Hsp90) assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins with respect to the cytosolic chaperone-dependent regulation. Some preproteins such as pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins such as pSSU is more strongly dependent on Hsp70. The E3 ligase, C-terminus of Hsp70-interacting protein (Chip), appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable with the cytosolic unfolded protein response.

We present a 3D assay for the quantification of the autophagic flux in live cell spheroids by using the fluorescent reporter mRFP-GFP-LC3. The protocol describes the formation of the spheroids from the astrocytoma cell line U343, live long-term 3D fluorescence imaging of drug-treated spheroids, and the image processing workflow required to extract quantitative data on the autophagic flux.

We investigated the chromatin organization of living cells with a combination of recently developed approaches for histone and DNA labeling. Nucleosomal DNA was labeled with a histone H2B-GFP (green fluorescent protein) fusion protein and the chromatin organization of living HeLa cells was analyzed by high resolution confocal microscopy. Within the perinuclear and perinucleolar regions chromatin was organized into large-scale fibers of 2 to 8 microm in length and 300 to 500 nm in diameter. Within the nuclear interior we observed similar large-scale fibers, but in addition focal as well as diffuse forms of organization. Comparison with standard labeling and detection procedures revealed major differences in the chromatin organization observed. Chromatin organization revealed by the distribution of histone H2B-GFP was directly compared with the functional organization of chromatin by Cy3-dUTP labeling of DNA replicating at a specific time. DNA regions replicating at a specific time display characteristic physical and functional properties. Analysis of Cy3-labeled foci revealed that they are associated with all three forms of chromatin organization (fibrillar, focal and diffuse). In particular, Cy3-labeled foci appeared as discontinuous regions of large-scale fibers. These results demonstrate that large-scale chromatin fibers have discontinuous functional characteristics.

Cell biologists have used photobleaching to investigate the lateral mobility of fluorophores on the cell surface since the 1970s. Fusions of green fluorescent protein (GFP) to specific proteins extend photobleaching techniques to the investigation of protein dynamics within the cell, leading to renewed interest in photobleaching experiments. This article revisits general photobleaching concepts, reviews what can be learned from them and discusses applications illustrating the potential of photobleaching GFP fusion proteins inside living cells.

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export. Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

During gastrulation in the mouse embryo, dynamic cell movements including epiblast invagination and mesodermal layer expansion lead to the establishment of the three-layered body plan. The precise details of these movements, however, are sometimes elusive, because of the limitations in live imaging. To overcome this problem, we developed techniques to enable observation of living mouse embryos with digital scanned light sheet microscope (DSLM). The achieved deep and high time-resolution images of GFP-expressing nuclei and following 3D tracking analysis revealed the following findings: (i) Interkinetic nuclear migration (INM) occurs in the epiblast at embryonic day (E)6 and 6.5. (ii) INM-like migration occurs in the E5.5 embryo, when the epiblast is a monolayer and not yet pseudostratified. (iii) Primary driving force for INM at E6.5 is not pressure from neighboring nuclei. (iv) Mesodermal cells migrate not as a sheet but as individual cells without coordination.

In Arabidopsis , lateral root primordia (LRPs) originate from pericycle cells located deep within the parental root and have to emerge through endodermal, cortical, and epidermal tissues. These overlaying tissues place biomechanical constraints on the LRPs that are likely to impact their morphogenesis. This study probes the interplay between the patterns of cell division, organ shape, and overlaying tissues on LRP morphogenesis by exploiting recent advances in live plant cell imaging and image analysis. Our 3D/4D image analysis revealed that early stage LRPs exhibit tangential divisions that create a ring of cells corralling a population of rapidly dividing cells at its center. The patterns of division in the latter population of cells during LRP morphogenesis are not stereotypical. In contrast, statistical analysis demonstrated that the shape of new LRPs is highly conserved. We tested the relative importance of cell division pattern versus overlaying tissues on LRP morphogenesis using mutant and transgenic approaches. The double mutant aurora1 ( aur1 ) aur2 disrupts the pattern of LRP cell divisions and impacts its growth dynamics, yet the new organ’s dome shape remains normal. In contrast, manipulating the properties of overlaying tissues disrupted LRP morphogenesis. We conclude that the interaction with overlaying tissues, rather than the precise pattern of divisions, is most important for LRP morphogenesis and optimizes the process of lateral root emergence.

Cell-based assays are essential in both basic research and drug discovery. Three-dimensional cellular spheroids are more realistic models of tumors and healthy tissues compared to standard two-dimensional cultures. Employing spheroids improves the reliability and the physiological significance of cell-based assays. We present a detailed drug assay protocol performed with live cellular spheroids. We employ automated epifluorescence live microscopy to investigate the effects of drugs on the spheroids over several days. We describe the spheroid preparation, manipulation, live fluorescence imaging, and data processing. We quantify the autophagy-triggering effects of the drugs (-)-Gossypol and Rapamycin in glioma cell spheroids. The formation of the autophagosomes and the fusion of the autophagosomes with lysosomes in the treated spheroids are monitored over time and space with a mRFP:GFP:LC3 fusion protein.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun. Electronic supplementary material The online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

Conventional two-dimensional cell monolayers do not provide the geometrical, biochemical and mechanical cues found in real tissues. Cells in real tissues interact through chemical and mechanical stimuli with adjacent cells and via the extracellular matrix. Such a highly interconnected communication network extends along all three dimensions. This architecture is lost in two-dimensional cultures. Therefore, at least in many cases, two-dimensional cell monolayers do not represent a suitable in vitro tool to characterize accurately the biology of real tissues. Many studies performed over the last few years have demonstrated that the differences between three-dimensional and two-dimensional cultured cells are striking at the morphological and molecular levels and that three-dimensional cell cultures can be employed in order to shrink the gap between real tissues and in vitro cell models. End-point and long-term imaging of cellular and sub-cellular processes with fluorescence microscopy provides direct insight into the physiological behavior of three-dimensional cell cultures and their response to chemical or mechanical stimulation. Fluorescence imaging of three-dimensional cell cultures sets new challenges and imposes specific requirements concerning the choice of a suitable microscopy technique. Deep penetration into the specimen, high imaging speed and ultra-low intensity of the excitation light are key requirements. Light-sheet-based fluorescence microscopy (LSFM) offers a favorable combination of these requirements and is therefore currently established as the technique of choice for the study of three-dimensional cell cultures. This review illustrates the benefits of cellular spheroids in the life sciences and suggests that LSFM is essential for investigations of cellular and sub-cellular dynamic processes in three-dimensions over time and space.

The growing interest in the investigation of endocytosis, vesicular transport routes, and corresponding regulatory mechanisms resulted in the exploitation of cell biological, genetic, biochemical, and proteomic approaches. Methods and techniques such as site-directed and T-DNA insertional mutagenesis, RNAi, classical inhibitor treatments, and recombinant GFP technology combined with confocal laser scanning microscopy (CLSM) and electron and immune-electron microscopy were routinely employed for investigation of endocytosis in plant cells. However, new approaches such as high-throughput confocal microscopy screens on mutants and proteomic analyses on isolated vesicular compartments and root cells treated with vesicular trafficking inhibitors (both focused on the identification of new endosomal proteins), together with chemical genomics and advanced microscopy approaches such as Forster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), light sheet-based fluorescence microscopy, and super-resolution microscopy provided a significant amount of new data and these new methods appear as extremely promising tools in this field.

The Golgi complex has a central role in the secretory pathway of all higher organisms. To explain the synthesis of its unique stacked structure in mammalian cells, two major models have been proposed. One suggests that it is synthesized de novo from the endoplasmic reticulum. The second model postulates a pre-existing Golgi template that serves as a scaffold for its biogenesis. To test these hypotheses directly, we have developed an approach in which we deplete the Golgi complex from living cells by laser nanosurgery, and subsequently analyze the ‘Golgi-depleted’ karyoplast using time-lapse and electron microscopy. We show that biosynthetic transport is blocked after Golgi depletion, but is restored 12 hours later. This recovery of secretory transport coincides with an ordered assembly of stacked Golgi structures, and we also observe the appearance of matrix proteins before that of Golgi enzymes. Functional experiments using RNA interference-mediated knockdown of GM130 further demonstrate the importance of the matrix during Golgi biogenesis. By contrast, the centrosome, which can also be removed by laser nanosurgery and is not reformed within the considered time frame, is not required for this process. Altogether, our data provide evidence that de novo Golgi biogenesis can occur in mammalian cells.

In eukaryotic cells, the onset of mitosis involves cyclin molecules which interact with proteins of the cdc2 family to produce active kinases. In vertebrate cells, cyclin A dependent kinases become active in S- and pro-phases, whereas a cyclin B-dependent kinase is mostly active in metaphase. It has recently been shown that, when added to Xenopus egg extracts, bacterially produced A- and B-type cyclins associate predominantly with the same kinase catalytic subunit, namely p34cdc2, and induce its histone H1 kinase activity with different kinetics. Here, we show that in the same cell free system, both the addition of cyclin A and cyclin B changes microtubule behavior. However, the cyclin A-dependent kinase does not induce a dramatic shortening of centrosome-nucleated microtubules whereas the cyclin B-dependent kinase does, as previously reported. Analysis of the parameters of microtubule dynamics by fluorescence video microscopy shows that the dramatic shortening induced by the cyclin B-dependent kinase is correlated with a several fold increase in catastrophe frequency, an effect not observed with the cyclin A-dependent kinase. Using a simple mathematical model, we show how the length distributions of centrosome-nucleated microtubules relate to the four parameters that describe microtubule dynamics. These four parameters define a threshold between unlimited microtubule growth and the establishment of steady-state dynamics, which implies that well defined steady-state length distributions can be produced by regulating precisely the respective values of the dynamical parameters. Moreover, the dynamical model predicts that increasing catastrophe frequency is more efficient than decreasing the rescue frequency to reduce the average steady state length of microtubules. These theoretical results are quantitatively confirmed by the experimental data.

The analysis of all cell movements and all cell interactions in a vertebrate during the entire period of embryonic development is a fundamental goal in biology. Using DSLM, we recorded the development of entire zebrafish embryos in vivo and with sub-cellular resolution. By imaging at a speed of 1.5 billion volume elements per minute, image data in the order of several terabytes were acquired for each embryo over the time course of an entire day, i.e. up to a stage, in which the embryo comprises 20,000 cells and major organs are in a functional state. By using automated image processing algorithms the image data of each embryo were converted into a digital representation of the embryo (the "digital embryo"), i.e. a database with comprehensive information about migratory tracks and divisions of the embryo's cells. The digital embryos permit to follow single cells as a function of time such that the "fate" as well as the origin of the cells can be reconstructed. By means of these analyses, developmental blueprints of tissues and organs can be determined in a whole-embryo context. Defects in embryonic development or disease models can now be analyzed and understood on a quantitative level.

The mechanics of the actin cytoskeleton have a central role in the regulation of cells and tissues, but the details of how molecular sensors recognize deformations and forces are elusive. By performing cytoskeleton laser nanosurgery in cultured epithelial cells and fibroblasts, we show that the retraction of stress fibers (SFs) is restricted to the proximity of the cut and that new adhesions form at the retracting end. This suggests that SFs are attached to the substrate. A new computational model for SFs confirms this hypothesis and predicts the distribution and propagation of contractile forces along the SF. We then analyzed the dynamics of zyxin, a focal adhesion protein present in SFs. Fluorescent redistribution after laser nanosurgery and drug treatment shows a high correlation between the experimentally measured localization of zyxin and the computed localization of forces along SFs. Correlative electron microscopy reveals that zyxin is recruited very fast to intermediate substrate anchor points that are highly tensed upon SF release. A similar acute localization response is found if SFs are mechanically perturbed with the cantilever of an atomic force microscope. If actin bundles are cut by nanosurgery in living Drosophila egg chambers, we also find that zyxin redistribution dynamics correlate to force propagation and that zyxin relocates at tensed SF anchor points, demonstrating that these processes also occur in living organisms. In summary, our quantitative analysis shows that force and protein localization are closely correlated in stress fibers, suggesting a very direct force-sensing mechanism along actin bundles.