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Motor proteins are important for transport and force generation in a variety of cellular processes and in morphogenesis. Here, we describe a general strategy for conditional motor mutants by inserting a protease cleavage site into the 'neck' between the head domain and the stalk of the motor protein, making the protein susceptible to proteolytic cleavage at the neck by the corresponding protease. To demonstrate the feasibility of this approach, we inserted the cleavage site of the tobacco etch virus (TEV) protease into the neck of the tetrameric motor Kinesin-5. Application of TEV protease led to a specific depletion and functional loss of Kinesin-5 in Drosophila embryos. With our approach, we revealed that Kinesin-5 stabilizes the microtubule network during interphase in syncytial embryos. The 'molecular guillotine' can potentially be applied to many motor proteins because Kinesins and myosins have conserved structures with accessible neck regions.This article has an associated First Person interview with the first author of the paper.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Interfase , Proteínas Associadas aos Microtúbulos/metabolismo , Sequência de Aminoácidos , Animais , Centrossomo/metabolismo , Proteínas de Drosophila/química , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Endopeptidases , Proteínas de Fluorescência Verde/metabolismo , Proteínas Associadas aos Microtúbulos/química , Mitose , FenótipoRESUMO
The ultimate objective of a microscope of the highest resolution is to map the molecules of interest in the sample. Traditionally, linear imaging systems are characterized by their spatial frequency transfer function, which is given, in real space, by the point spread function (PSF). By extending the concept of the PSF towards the molecular contribution function (MCF), that quantifies the average contribution of a single fluorophore to the image, a straightforward concept for counting fluorophores is obtained. Using reversible saturable optical fluorescence transitions (RESOLFT), fluorophores are effectively activated only in a small, subdiffraction-sized volume before they are read out. During readout the signal exhibits an increased variance due to the stochastic nature of prior activation, which scales quadratically with the brightness of the active fluorophores while the mean of the signal scales only linearly with it. Using a two-state Markov model for the activation, showing comparable behavior to the switching kinetics of the switchable fluorescent protein rsEGFP2, we can approximate quantitatively the MCF of RESOLFT nanoscopy allowing to count the number of fluorophores within a subdiffraction-sized region of the sample. The method is validated on measurements of tubulin structures in Drosophila melagonaster larvae. Modeling and estimation of the MCF is a promising approach to quantitative microscopy.
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During the initial development of syncytial embryos, nuclei go through cycles of nuclear division and spatial rearrangement. The arising spatial pattern of nuclei is important for subsequent cellularization and morphing of the embryo. Although nuclei are contained within a common cytoplasm, cytoskeletal proteins are nonuniformly packaged into regions around every nucleus. In fact, cytoskeletal elements like microtubules and their associated motor proteins exert stochastic forces between nuclei, actively driving their rearrangement. Yet, it is unknown how the stochastic forces are balanced to maintain nuclear order in light of increased nuclear density upon every round of divisions. Here, we investigate the nuclear arrangements in Drosophila melanogaster over the course of several nuclear divisions starting from interphase 11. We develop a theoretical model in which we distinguish long-ranged passive forces due to the nuclei as inclusions in the elastic matrix, namely the cytoplasm, and active, stochastic forces arising from the cytoskeletal dynamics mediated by motor proteins. We perform computer simulations and quantify the observed degree of orientational and spatial order of nuclei. Solely doubling the nuclear density upon nuclear division, the model predicts a decrease in nuclear order. Comparing results to experimental recordings of tracked nuclei, we make contradictory observations, finding an increase in nuclear order upon nuclear divisions. Our analysis of model parameters resulting from this comparison suggests that overall motor protein density as well as relative active-force amplitude has to decrease by a factor of about two upon nuclear division to match experimental observations. We therefore expect a dilution of cytoskeletal motors during the rapid nuclear division to account for the increase in nuclear order during syncytial embryo development. Experimental measurements of kinesin-5 cluster lifetimes support this theoretical finding.
Assuntos
Núcleo Celular/metabolismo , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Fenômenos Mecânicos , Animais , Fenômenos Biomecânicos , Microtúbulos/metabolismo , Processos EstocásticosRESUMO
Canonical Wnt signaling plays a dominant role in the development of the neural crest (NC), a highly migratory cell population that generates a vast array of cell types. Canonical Wnt signaling is required for NC induction as well as differentiation, however its role in NC migration remains largely unknown. Analyzing nuclear localization of ß-catenin as readout for canonical Wnt activity, we detect nuclear ß-catenin in premigratory but not migratory Xenopus NC cells suggesting that canonical Wnt activity has to decrease to basal levels to enable NC migration. To define a possible function of canonical Wnt signaling in Xenopus NC migration, canonical Wnt signaling was modulated at different time points after NC induction. This was accomplished using either chemical modulators affecting ß-catenin stability or inducible glucocorticoid fusion constructs of Lef/Tcf transcription factors. In vivo analysis of NC migration by whole mount in situ hybridization demonstrates that ectopic activation of canonical Wnt signaling inhibits cranial NC migration. Further, NC transplantation experiments confirm that this effect is tissue-autonomous. In addition, live-cell imaging in combination with biophysical data analysis of explanted NC cells confirms the in vivo findings and demonstrates that modulation of canonical Wnt signaling affects the ability of NC cells to perform single cell migration. Thus, our data support the hypothesis that canonical Wnt signaling needs to be tightly controlled to enable migration of NC cells.
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Movimento Celular/fisiologia , Crista Neural/citologia , Fatores de Transcrição TCF/metabolismo , Fator 3 de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Hibridização In Situ , Indóis/farmacologia , Organogênese/fisiologia , Oximas/farmacologia , Crânio/embriologia , beta Catenina/metabolismoRESUMO
When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm)(2) image nanoareas can provide subdiffraction resolution (super resolution by polarization demodulation, SPoD). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, ExPAN). This shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. Our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.
Assuntos
Polarização de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Algoritmos , Animais , Células Cultivadas , Simulação por Computador , Células Epiteliais/metabolismo , Desenho de Equipamento , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/metabolismo , Microtúbulos/ultraestrutura , Modelos Teóricos , Nanosferas/química , Distribuição Normal , Fótons , Potoroidae , SoftwareRESUMO
The actin and microtubule networks form the dynamic cytoskeleton. Network dynamics is driven by molecular motors applying force onto the networks and the interactions between the networks. Here we assay the dynamics of centrosomes in the scale of seconds as a proxy for the movement of microtubule asters. With this assay we want to detect the role of specific motors and of network interaction. During interphase of syncytial embryos of Drosophila, cortical actin and the microtubule network depend on each other. Centrosomes induce cortical actin to form caps, whereas F-actin anchors microtubules to the cortex. In addition, lateral interactions between microtubule asters are assumed to be important for regular spatial organization of the syncytial embryo. The functional interaction between the microtubule asters and cortical actin has been largely analyzed in a static manner, so far. We recorded the movement of centrosomes at 1 Hz and analyzed their fluctuations for two processespair separation and individual movement. We found that F-actin is required for directional movements during initial centrosome pair separation, because separation proceeds in a diffusive manner in latrunculin-injected embryos. For assaying individual movement, we established a fluctuation parameter as the deviation from temporally and spatially slowly varying drift movements. By analysis of mutant and drug-injected embryos, we found that the fluctuations were suppressed by both cortical actin and microtubules. Surprisingly, the microtubule motor Kinesin-1 also suppressed fluctuations to a similar degree as F-actin. Kinesin-1 may mediate linkage of the microtubule (+)-ends to the actin cortex. Consistent with this model is our finding that Kinesin-1-GFP accumulates at the cortical actin caps.
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Centrossomo/metabolismo , Proteínas de Drosophila/metabolismo , Cinesinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Membrana Celular/metabolismo , Drosophila melanogaster , Modelos Biológicos , Movimento , Miosina Tipo IIRESUMO
With the advent of fluorescence superresolution microscopy, nano-sized structures can be imaged with a previously unprecedented accuracy. Therefore, it is rapidly gaining importance as an analytical tool in the life sciences and beyond. However, the images obtained so far lack an absolute scale in terms of fluorophore numbers. Here, we use, for the first time, a detailed statistical model of the temporal imaging process which relies on a hidden Markov model operating on two timescales. This allows us to extract this information from the raw data without additional calibration measurements. We show this on the basis of added data from experiments on single Alexa 647 molecules as well as GSDIM/dSTORM measurements on DNA origami structures with a known number of labeling positions.
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INTRODUCTION: Many cancer guidelines include sentinel lymph node (SLN) staging to identify microscopic metastatic disease. Current SLN analysis of melanoma patients is effective but has the substantial drawback that only a small representative portion of the node is sampled, whereas most of the tissue is discarded. This might explain the high clinical false-negative rate of current SLN diagnosis in melanoma. Furthermore, the quantitative assessment of metastatic load and microanatomical localisation might yield prognosis with higher precision. Thus, methods to analyse entire SLNs with cellular resolution apart from tedious sequential physical sectioning are required. PATIENTS AND METHODS: Eleven melanoma patients eligible to undergo SLN biopsy were included in this prospective study. SLNs were fixed, optically cleared, whole-mount stained and imaged using light sheet fluorescence microscopy (LSFM). Subsequently, compatible and unbiased gold standard histopathological assessment allowed regular patient staging. This enabled intrasample comparison of LSFM and histological findings. In addition, the development of an algorithm, RAYhance, enabled easy-to-handle display of LSFM data in a browsable histologic slide-like fashion. RESULTS: We comprehensively quantify total tumour volume while simultaneously visualising cellular and anatomical hallmarks of the associated SLN architecture. In a first-in-human study of 21 SLN of melanoma patients, LSFM not only confirmed all metastases identified by routine histopathological assessment but also additionally revealed metastases not detected by routine histology alone. This already led to additional therapeutic options for one patient. CONCLUSION: Our three-dimensional digital pathology approach can increase sensitivity and accuracy of SLN metastasis detection and potentially alleviate the need for conventional histopathological assessment in the future. GERMAN CLINICAL TRIALS REGISTER: (DRKS00015737).
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Imageamento Tridimensional/métodos , Metástase Linfática/patologia , Melanoma/patologia , Microscopia de Fluorescência/métodos , Estadiamento de Neoplasias/métodos , Linfonodo Sentinela/patologia , Humanos , Metástase Linfática/diagnósticoRESUMO
Many aspects in tissue morphogenesis are attributed to a collective behavior of the participating cells. Yet, the mechanism for emergence of dynamic tissue behavior is not well understood. Here, we report that the "yo-yo"-like nuclear movement in the Drosophila syncytial embryo displays emergent features indicative of collective behavior. Following mitosis, the array of nuclei moves away from the wave front by several nuclear diameters only to return to its starting position about 5 min later. Based on experimental manipulations and numerical simulations, we find that the ensemble of elongating and isotropically oriented spindles, rather than individual spindles, is the main driving force for anisotropic nuclear movement. ELMO-dependent F-actin restricts the time for the forward movement and ELMO- and dia-dependent F-actin is essential for the return movement. Our study provides insights into how the interactions among the cytoskeleton as individual elements lead to collective movement of the nuclear array on a macroscopic scale.
Assuntos
Núcleo Celular/fisiologia , Citoesqueleto/fisiologia , Drosophila melanogaster/fisiologia , Embrião não Mamífero/fisiologia , Mitose/fisiologia , Morfogênese , Animais , Drosophila melanogaster/embriologiaRESUMO
We study a homogeneously driven granular fluid of hard spheres at intermediate volume fractions and focus on time-delayed correlation functions in the stationary state. Inelastic collisions are modeled by incomplete normal restitution, allowing for efficient simulations with an event-driven algorithm. The incoherent scattering function F(incoh)(q,t) is seen to follow time-density superposition with a relaxation time that increases significantly as the volume fraction increases. The statistics of particle displacements is approximately Gaussian. For the coherent scattering function S(q,ω), we compare our results to the predictions of generalized fluctuating hydrodynamics, which takes into account that temperature fluctuations decay either diffusively or with a finite relaxation rate, depending on wave number and inelasticity. For sufficiently small wave number q we observe sound waves in the coherent scattering function S(q,ω) and the longitudinal current correlation function C(l)(q,ω). We determine the speed of sound and the transport coefficients and compare them to the results of kinetic theory.
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We study the velocity autocorrelation function of a driven granular fluid in the stationary state in three dimensions. As the critical volume fraction of the glass transition in the corresponding elastic system is approached, we observe pronounced cage effects in the velocity autocorrelation function as well as a strong decrease of the diffusion constant, depending on the inelasticity. At moderate densities the velocity autocorrelation function is shown to decay algebraically in time, like t(-3/2), if momentum is conserved locally, and like t(-1), if momentum is not conserved by the driving. A simple scaling argument supports the observed long-time tails.
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Wet granular materials are characterized by a defined bond energy in their particle interaction such that breaking a bond implies an irreversible loss of a fixed amount of energy. Associated with the bond energy is a nonequilibrium transition, setting in as the granular temperature falls below the bond energy. The subsequent aggregation of particles into clusters is shown to be a self-similar growth process with a cluster size distribution that obeys scaling. In the early phase of aggregation the clusters are fractals with D{f}=2, for later times we observe gelation. We use simple scaling arguments to derive the temperature decay in the early and late stages of cooling and verify our results with event-driven simulations.
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We investigate a gas of wet granular particles covered by a thin liquid film. The dynamic evolution is governed by two-particle interactions, which are mainly due to interfacial forces in contrast to dry granular gases. When two wet grains collide, a capillary bridge is formed and stays intact up to a certain distance of withdrawal when the bridge ruptures, dissipating a fixed amount of energy. A freely cooling system is shown to undergo a nonequilibrium dynamic phase transition from a state with mainly single particles and fast cooling to a state with growing aggregates such that bridge rupture becomes a rare event and cooling is slow. In the early stage of cluster growth, aggregation is a self-similar process with a fractal dimension of the aggregates approximately equal to Df approximately 2 . At later times, a percolating cluster is observed which ultimately absorbs all the particles. The final cluster is compact on large length scales, but fractal with Df approximately 2 on small length scales.
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We analyze the microscopic dynamics and transport properties of a gas of thin hard rods. Based on the collision rules for hard needles we derive a hydrodynamic equation that determines the coupled translational and rotational dynamics of a tagged thin rod in an ensemble of identical rods. Specifically, based on a pseudo-Liouville operator for binary collisions between rods, the Mori-Zwanzig projection formalism is used to derive a continued fraction representation for the correlation function of the tagged particle's density, specifying its position and orientation. Truncation of the continued fraction gives rise to a generalized Enskog equation, which can be compared to the phenomenological Perrin equation for anisotropic diffusion. Only for sufficiently large density do we observe anisotropic diffusion, as indicated by an anisotropic mean-square displacement, growing linearly with time. For lower densities, the Perrin equation is shown to be an insufficient hydrodynamic description for hard needles interacting via binary collisions. We compare our results to simulations and find excellent quantitative agreement for low densities and qualitative agreement for higher densities.