Time-lapse Light Microscopy Research Articles

Symmetry Breaking in the U-Net: Hybrid Deep-Learning Multi-Class Segmentation of HeLa Cells in Reflected Light Microscopy Images

Multi-class segmentation of unlabelled living cells in time-lapse light microscopy images is challenging due to the temporal behaviour and changes in cell life cycles and the complexity of these images. The deep-learning-based methods achieved promising outcomes and remarkable success in single- and multi-class medical and microscopy image segmentation. The main objective of this study is to develop a hybrid deep-learning-based categorical segmentation and classification method for living HeLa cells in reflected light microscopy images. A symmetric simple U-Net and three asymmetric hybrid convolution neural networks—VGG19-U-Net, Inception-U-Net, and ResNet34-U-Net—were proposed and mutually compared to find the most suitable architecture for multi-class segmentation of our datasets. The inception module in the Inception-U-Net contained kernels with different sizes within the same layer to extract all feature descriptors. The series of residual blocks with the skip connections in each ResNet34-U-Net’s level alleviated the gradient vanishing problem and improved the generalisation ability. The m-IoU scores of multi-class segmentation for our datasets reached 0.7062, 0.7178, 0.7907, and 0.8067 for the simple U-Net, VGG19-U-Net, Inception-U-Net, and ResNet34-U-Net, respectively. For each class and the mean value across all classes, the most accurate multi-class semantic segmentation was achieved using the ResNet34-U-Net architecture (evaluated as the m-IoU and Dice metrics).

Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector.

Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion,which are critical for a holistic understanding of the Leishmania life cycle.

Label-Free Long-Term Methods for Live Cell Imaging of Neurons: New Opportunities.

Time-lapse light microscopy combined with in vitro neuronal cultures has provided a significant contribution to the field of Developmental Neuroscience. The establishment of the neuronal polarity, i.e., formation of axons and dendrites, key structures responsible for inter-neuronal signaling, was described in 1988 by Dotti, Sullivan and Banker in a milestone paper that continues to be cited 30 years later. In the following decades, numerous fluorescently labeled tags and dyes were developed for live cell imaging, providing tremendous advancements in terms of resolution, acquisition speed and the ability to track specific cell structures. However, long-term recordings with fluorescence-based approaches remain challenging because of light-induced phototoxicity and/or interference of tags with cell physiology (e.g., perturbed cytoskeletal dynamics) resulting in compromised cell viability leading to cell death. Therefore, a label-free approach remains the most desirable method in long-term imaging of living neurons. In this paper we will focus on label-free high-resolution methods that can be successfully used over a prolonged period. We propose novel tools such as scanning ion conductance microscopy (SICM) or digital holography microscopy (DHM) that could provide new insights into live cell dynamics during neuronal development and regeneration after injury.

CCDC88B is required for mobility and inflammatory functions of dendritic cells.

The Coiled Coil Domain Containing Protein 88B (CCDC88B) gene is associated with susceptibility to several inflammatory diseases in humans and its inactivation in mice protects against acute neuroinflammation and models of intestinal colitis. We report that mice lacking functional CCDC88B (Ccdc88bMut ) are defective in several dendritic cells (DCs)-dependent inflammatory and immune reactions in vivo. In these mice, an inflammatory stimulus (LPS) fails to induce the recruitment of DCs into the draining lymph nodes (LNs). In addition, OVA-pulsed Ccdc88bMut DCs injected in the footpad do not induce recruitment and activation of antigen-specific CD4+ and CD8+ T cells in their draining LN. Experiments in vitro indicate that this defect is independent of the ability of mutant DCs to capture and present peptide antigen to T cells. Rather, kinetic analyses in vivo of wild-type and Ccdc88bMut DCs indicate a reduced migration capacity in the absence of the CCDC88B protein expression. Moreover, using time-lapse light microscopy imaging, we show that Ccdc88bMut DCs have an intrinsic motility defect. Furthermore, in vivo studies reveal that these reduced migratory properties lead to dampened contact hypersensitivity reactions in Ccdc88b mutant mice. These findings establish a critical role of CCDC88B in regulating movement and migration of DCs. Thus, regulatory variants impacting Ccdc88b expression in myeloid cells may cause variable degrees of DC-dependent inflammatory response in situ, providing a rationale for the genetic association of CCDC88B with several inflammatory and autoimmune diseases in humans.

Estimation of individual starch granule swelling under hydro-thermal treatment

Abstract We propose an automatic algorithm to analyse the swelling kinetics of individual particles floating in a diluted suspension. Our approach is illustrated with a case study in which a diluted aqueous suspension of starch granules are submitted to various heating treatments. The evolution of modified waxy maize starch granules placed on a temperature-controlled stage was tracked using time-lapse light microscopy (one image per second). We relied on an automatic algorithm, developed for the present study, to estimate morphological and kinetic parameters for a large number of granules. Observations showed that above a given temperature, starch granules will evolve towards an equilibrium size. Due to variability of swelling kinetic, individual behaviour was different from the global one. These results suggest that modelling works should take into account the size heterogeneity and kinetic variability of starch granules.

Light-Induced Length Shrinkage of Rod Photoreceptor Outer Segments.

PurposeThis study was designed to verify light-induced outer segment (OS) length shrinkage of rod photoreceptors and to characterize its anatomic source at disc-level resolution.MethodsFrog (Rana pipiens) retinas were used for this study. Time-lapse light microscopy of freshly isolated OSs was employed to test transient rod OS changes at 10 ms temporal resolution. Histological light microscopy of dark- and light-adapted retinas was used to confirm light-induced rod OS length changes; and transmission electron microscopy (TEM) was used to quantify light-driven structural perturbation of rod OSs at disc level resolution.ResultsTime-lapse light microscopy images verified transient length shrinking responses in freshly isolated rod OSs. Histological light microscopy images confirmed reduced rod OS lengths in light-adapted retinas, compared to that of dark-adapted retinas. TEM images disclosed shortened inter-disc distances in light-adapted retinas compared to dark-adapted retinas.ConclusionsLight-induced rod OS length shrinkage was confirmed using time-lapse light microscopy of isolated rod OSs and histological light microscopy of dark- and light-adapted retinas. TEM revealed that the rod OS length shrinkage was correlated to the light-driven decrease of the space between individual discs, not the disc thickness itself.Translational RelevanceLight-induced transient rod response promises a noninvasive biomarker for early diagnosis of age-related macular degeneration and retinitis pigmentosa, in which the rod photoreceptors are known to be more vulnerable than cone photoreceptors.

Distinct shape-shifting regimes of bowl-shaped cell sheets - embryonic inversion in the multicellular green alga Pleodorina.

BackgroundThe multicellular volvocine alga Pleodorina is intermediate in organismal complexity between its unicellular relative, Chlamydomonas, and its multicellular relative, Volvox, which shows complete division of labor between different cell types. The volvocine green microalgae form a group of genera closely related to the genus Volvox within the order Volvocales (Chlorophyta). Embryos of multicellular volvocine algae consist of a cellular monolayer that, depending on the species, is either bowl-shaped or comprises a sphere. During embryogenesis, multicellular volvocine embryos turn their cellular monolayer right-side out to expose their flagella. This process is called ‘inversion’ and serves as simple model for epithelial folding in metazoa. While the development of spherical Volvox embryos has been the subject of detailed studies, the inversion process of bowl-shaped embryos is less well understood. Therefore, it has been unclear how the inversion of a sphere might have evolved from less complicated processes.ResultsIn this study we characterized the inversion of initially bowl-shaped embryos of the 64- to 128-celled volvocine species Pleodorina californica. We focused on the movement patterns of the cell sheet, cell shape changes and changes in the localization of cytoplasmic bridges (CBs) connecting the cells. The development of living embryos was recorded using time-lapse light microscopy. Moreover, fixed and sectioned embryos throughout inversion and at successive stages of development were analyzed by light and transmission electron microscopy. We generated three-dimensional models of the identified cell shapes including the localization of CBs.ConclusionsIn contrast to descriptions concerning volvocine embryos with lower cell numbers, the embryonic cells of P. californica undergo non-simultaneous and non-uniform cell shape changes. In P. californica, cell wedging in combination with a relocation of the CBs to the basal cell tips explains the curling of the cell sheet during inversion. In volvocine genera with lower organismal complexity, the cell shape changes and relocation of CBs are less pronounced in comparison to P. californica, while they are more pronounced in all members of the genus Volvox. This finding supports an increasing significance of the temporal and spatial regulation of cell shape changes and CB relocations with both increasing cell number and organismal complexity during evolution of differentiated multicellularity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0134-9) contains supplementary material, which is available to authorized users.

Exopolysaccharide microchannels direct bacterial motility and organize multicellular behavior.

The myxobacteria are a family of soil bacteria that form biofilms of complex architecture, aligned multilayered swarms or fruiting body structures that are simple or branched aggregates containing myxospores. Here, we examined the structural role of matrix exopolysaccharide (EPS) in the organization of these surface-dwelling bacterial cells. Using time-lapse light and fluorescence microscopy, as well as transmission electron microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) electron microscopy, we found that Myxococcus xanthus cell organization in biofilms is dependent on the formation of EPS microchannels. Cells are highly organized within the three-dimensional structure of EPS microchannels that are required for cell alignment and advancement on surfaces. Mutants lacking EPS showed a lack of cell orientation and poor colony migration. Purified, cell-free EPS retains a channel-like structure, and can complement EPS− mutant motility defects. In addition, EPS provides the cooperative structure for fruiting body formation in both the simple mounds of M. xanthus and the complex, tree-like structures of Chondromyces crocatus. We furthermore investigated the possibility that EPS impacts community structure as a shared resource facilitating cooperative migration among closely related isolates of M. xanthus.

Study of Osteoclast Adhesion to Cortical Bone Surfaces: A Correlative Microscopy Approach for Concomitant Imaging of Cellular Dynamics and Surface Modifications.

Bone remodeling relies on the coordinated functioning of osteoblasts, bone-forming cells, and osteoclasts, bone-resorbing cells. The effects of specific chemical and physical bone features on the osteoclast adhesive apparatus, the sealing zone ring, and their relation to resorption functionality are still not well-understood. We designed and implemented a correlative imaging method that enables monitoring of the same area of bone surface by time-lapse light microscopy, electron microscopy, and atomic force microscopy before, during, and after exposure to osteoclasts. We show that sealing zone rings preferentially develop around surface protrusions, with lateral dimensions of several micrometers, and ∼1 μm height. Direct overlay of sealing zone rings onto resorption pits on the bone surface shows that the rings adapt to pit morphology. The correlative procedure presented here is noninvasive and performed under ambient conditions, without the need for sample labeling. It can potentially be applied to study various aspects of cell-matrix interactions.

Effects of angular frequency during clinorotation on mesenchymal stem cell morphology and migration.

Aims:To determine the short-term effects of simulated microgravity on mesenchymal stem cell behaviors—as a function of clinorotation speed—using time-lapse microscopy.Background:Ground-based microgravity simulation can reproduce the apparent effects of weightlessness in spaceflight using clinostats that continuously reorient the gravity vector on a specimen, creating a time-averaged nullification of gravity. In this work, we investigated the effects of clinorotation speed on the morphology, cytoarchitecture, and migration behavior of human mesenchymal stem cells (hMSCs).Methods:We compared cell responses at clinorotation speeds of 0, 30, 60, and 75 rpm over 8 h in a recently developed lab-on-chip-based clinostat system. Time-lapse light microscopy was used to visualize changes in cell morphology during and after cessation of clinorotation. Cytoarchitecture was assessed by actin and vinculin staining, and chemotaxis was examined using time-lapse light microscopy of cells in NGF (100 ng/ml) gradients.Results:Among clinorotated groups, cell area distributions indicated a greater inhibition of cell spreading with higher angular frequency (P<0.005), though average cell area at 30 rpm after 8 h became statistically similar to control (P=0.794). Cells at 75 rpm clinorotation remained viable and were able to re-spread after clinorotation. In chemotaxis chambers, clinorotation did not alter migration patterns in elongated cells, but most clinorotated cells exhibited cell retraction, which strongly compromised motility.Conclusions:These results indicate that hMSCs respond to clinorotation by adopting more rounded, less-spread morphologies. The angular frequency-dependence suggests that a cell’s ability to sense the changing gravity vector is governed by the rate of perturbation. For migration studies, cells cultured in clinorotated chemotaxis chambers were generally less motile and exhibited retraction instead of migration.

Array tomography: characterizing FAC-sorted populations of zebrafish immune cells by their 3D ultrastructure.

For 3D reconstructions of whole immune cells from zebrafish, isolated from adult animals by FAC-sorting we employed array tomography on hundreds of serial sections deposited on silicon wafers. Image stacks were either recorded manually or automatically with the newly released ZEISS Atlas 5 Array Tomography platform on a Zeiss FEGSEM. To characterize different populations of immune cells, organelle inventories were created by segmenting individual cells. In addition, arrays were used for quantification of cell populations with respect to the various cell types they contained. The detection of immunological synapses in cocultures of cell populations from thymus or WKM with cancer cells helped to identify the cytotoxic nature of these cells. Our results demonstrate the practicality and benefit of AT for high-throughput ultrastructural imaging of substantial volumes.Lay DescriptionTo look at immune cells from zebrafish we employed array tomography, a technique where arrays of serial sections deposited on solid substrates are used for imaging. Cell populations were isolated from the different organs of zebrafish involved in haematopoiesis, the production of blood cells. They were chemically fixed and centrifuged to concentrate them in a pellet that was then dehydrated and embedded in resin. Using a custom-built handling device it was possible to place hundreds of serial sections on silicon wafers as well ordered arrays. To image a whole cell at a resolution that would allow identifying all the organelles (i.e. compartments surrounded by membranes) inside the cell, stacks of usually 50–100 images were recorded in a scanning electron microscope (SEM). This recording was either done manually or automatically using the newly released Atlas Array Tomography platform on a ZEISS SEM. For the imaging of the sections a pixel size of about 5 nm was chosen, which defines membrane boundaries very well and allows segmentation of the membrane topology. After alignment of the images, cellular components were segmented to locate the individual organelles within the 3D reconstruction of the whole cell and also to create an inventory of organelles. Based on their morphologies we could identify specific cell types in the different hematopoietic organs. We could also quantify the proportion of each cell type in the whole population isolated from a given organ.Some of these specific cells from zebrafish were grown in a culture dish together with human cancer cells. By time-lapse light microscopy we observed that the fish cells attacked the cancer cells and killed them. From this we concluded that these cells must be similar to the cytotoxic cells from humans that play an important role in defence against spontaneously arising cancer cells in our bodies. They form special structures, called immunological synapses that we could also identify on our arrays and reconstruct in 3D. This is the first time the potential of zebrafish immune cells to form immunological synapses has been demonstrated.Our study is a good example for the practicality and benefit of array tomography in high-throughput ultrastructure imaging of substantial volumes, applicable to many areas of cell and developmental biology.

Coccolithogenesis In Scyphosphaera apsteinii (Prymnesiophyceae).

Coccolithophores are the most significant producers of marine biogenic calcite, although the intracellular calcification process is poorly understood. In the case of Scyphosphaera apsteinii Lohmann 1902, flat ovoid muroliths and bulky, vase-shaped lopadoliths with a range of intermediate morphologies may be produced by a single cell. This polymorphic species is within the Zygodiscales, a group that remains understudied with respect to ultrastructure and coccolith ontogeny. We therefore undertook an analysis of cell ultrastructure, morphology, and coccolithogenesis. The cell ultrastructure showed many typical haptophyte features, with calcification following a similar pattern to that described for other heterococcolith bearing species including Emiliania huxleyi. Of particular significance was the reticular body role in governing fine-scale morphology, specifically the central pore formation of the coccolith. Our observations also highlighted the essential role of the inter- and intracrystalline organic matrix in growth and arrangement of the coccolith calcite. S. apsteinii secreted mature coccoliths that attached to the plasma membrane via fibrillar material. Time-lapse light microscopy demonstrated secretion of lopadoliths occurred base first before being actively repositioned at the cell surface. Significantly, growth irradiance influenced the coccosphere composition with fewer lopadoliths being formed relative to muroliths at higher light intensities. Overall, our observations support dynamic metabolic (i.e., in response to growth irradiance), sensory and cytoskeletal control over the morphology and secretion of polymorphic heterococcoliths. With a basic understanding of calcification established, S. apsteinii could be a valuable model to further study coccolithophore calcification and cell physiological responses to ocean acidification.

Regulated Exocytosis: Novel Insights from Intravital Microscopy

Regulated exocytosis is a fundamental process that every secretory cell uses to deliver molecules to the cell surface and the extracellular space by virtue of membranous carriers. This process has been extensively studied using various approaches such as biochemistry, electrophysiology and electron microscopy. However, recent developments in time-lapse light microscopy have made possible imaging individual exocytic events, hence, advancing our understanding of this process at a molecular level. In this review, we focus on intravital microscopy (IVM), a light microscopy-based approach that enables imaging subcellular structures in live animals, and discuss its recent application to study regulated exocytosis. IVM has revealed differences in regulation and modality of regulated exocytosis between in vitro and in vivo model systems, unraveled novel aspects of this process that can be appreciated only in in vivo settings and provided valuable and novel information on its molecular machinery. In conclusion, we make the case for IVM being a mature technique that can be used to investigate the molecular machinery of several intracellular events under physiological conditions.

Labeling Tetracysteine-Tagged Proteins with Biarsenical Dyes for Live Cell Imaging

Correlation of real-time or time-lapse light microscopy (LM) with electron microscopy (EM) of cells can be performed with biarsenical dyes. These dyes fluorescently label tetracysteine-tagged proteins so that they can be imaged with LM and, upon fluorescent photoconversion of 3,3'-diaminobenzidine tetrahydrochloride (DAB), with EM as well. In the following protocol, cells expressing tetracysteine-tagged proteins are labeled for 1 h with biarsenical dyes. The volumes indicated are for a single 30-mm culture dish containing 2 mL of labeling medium. Scale the suggested volumes up or down depending upon the size of the culture dish used in the labeling. The same procedure can be adapted for longer labeling times by lowering the amount of dye used to 50-100 nM; however, the amount of the competing dithiol EDT is maintained at 10-20 μM. Longer labeling times often produce higher signal-to-noise ratios and cause less trauma to the treated cells prior to imaging.

Correlated Live Cell Light and Electron Microscopy Using Tetracysteine Tags and Biarsenicals

Fundamental to obtaining a more complete understanding of the roles played by macromolecular complexes in cells is the ability to map their location, movement, and transient interactions at high temporal and high spatial resolution. Unfortunately, probes capable of allowing direct correlation of real-time or time-lapse light microscopy (LM) with electron microscopic observations are relatively few. Genetically encoded fluorescent reporters such as green fluorescent protein (GFP) have revolutionized live cell imaging studies but are not directly visible by electron microscopy (EM). Fluorescent nanoparticles or quantum dots are a type of label for LM that can also be visualized directly by EM, but targeting these to cytoplasmic proteins in living cells remains difficult. One method that does allow for highly correlated LM and EM with excellent preservation of cellular ultrastructure is fluorescence photoconversion, in which a fluorescent compound causes the deposition of a reaction product that can be rendered electron-dense and directly visualized by EM. We have used this method in combination with a class of genetically encoded peptide tags that can be labeled in living cells by fluorophores bearing two appropriately spaced arsenic atoms (biarsenicals). The tetracysteine motif is short, easily inserted into or attached to the termini of the host protein, and can be used in combination with other molecular tags such as GFP and its derivatives. This article presents methods to label cells with biarsenicals, conduct live cell imaging recording sessions, and generate specimens that can be evaluated by EM for a correlated LM/EM analysis.

Lithotrophic iron-oxidizing bacteria produce organic stalks to control mineral growth: implications for biosignature formation

Neutrophilic Fe-oxidizing bacteria (FeOB) are often identified by their distinctive morphologies, such as the extracellular twisted ribbon-like stalks formed by Gallionella ferruginea or Mariprofundus ferrooxydans. Similar filaments preserved in silica are often identified as FeOB fossils in rocks. Although it is assumed that twisted iron stalks are indicative of FeOB, the stalk's metabolic role has not been established. To this end, we studied the marine FeOB M. ferrooxydans by light, X-ray and electron microscopy. Using time-lapse light microscopy, we observed cells excreting stalks during growth (averaging 2.2 μm h(-1)). Scanning transmission X-ray microscopy and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy show that stalks are Fe(III)-rich, whereas cells are low in Fe. Transmission electron microscopy reveals that stalks are composed of several fibrils, which contain few-nanometer-sized iron oxyhydroxide crystals. Lepidocrocite crystals that nucleated on the fibril surface are much larger (∼100 nm), suggesting that mineral growth within fibrils is retarded, relative to sites surrounding fibrils. C and N 1s NEXAFS spectroscopy and fluorescence probing show that stalks primarily contain carboxyl-rich polysaccharides. On the basis of these results, we suggest a physiological model for Fe oxidation in which cells excrete oxidized Fe bound to organic polymers. These organic molecules retard mineral growth, preventing cell encrustation. This model describes an essential role for stalk formation in FeOB growth. We suggest that stalk-like morphologies observed in modern and ancient samples may be correlated confidently with the Fe-oxidizing metabolism as a robust biosignature.

Internalization of Nanoparticles into Spiral Ganglion Cells

The delivery of drugs or genes to the inner ear in a controlled and biocompatible manner could lead to new treatments for conditions such as Meniere’s disease, tinnitus, schwannomas of the ear, and for improving hearing. The concept of multifunctional nanoparticles, which are targetable, biodegradable, and traceable, has led to new approaches to controlled drug release and localized delivery to specific cell populations. Tissue-specific delivery can be achieved by functionally “addressed” nanostructures loaded with a therapeutic molecule. In the present study, we investigated the incorporation, distribution, and toxicology of amphiphilic block copolymer nanoparticles (NPs) in spiral ganglion (SG) cell cultures. Adult human and guinea pig SG neurons and glia/Schwann dissociated cell cultures were expanded, grown for several weeks, and then studied live using time-lapse video microscopy and high-resolution light microscopy. The cells were further characterized using immunocytochemistry for the neural marker TuJ1 and the glia cell markers S-100 and GFAP, and their morphology was studied in more detail using scanning electron microscopy (SEM). These cell cultures were exposed to fluorescently (Dil)-loaded NPs for different time periods and at different concentrations, and the uptake was studied using fluorescence microscopy. The study demonstrates that DiI-loaded NPs can be internalized into guinea pig SG neurons as well as into human and guinea pig SG glia/Schwann cells without indication of toxicity or reduced viability. After 4 hours, almost 100% of both the neurons and the glia cells had incorporated the NPs into the cytoplasm. No uptake could be detected in the nucleus and no evidence of internalization could be seen in axons or in the growth cone area of the neuron. Especially in the glia cells, the NPs were detected in small vesicles surrounding the nucleus and occasionally in the periphery of the cytoplasm. This information could lead to the development of more specialized NPs, targeting only SG neurons or Schwann cells.

Budding and direct division of adult human erythrocytes in serum-free cultures

This paper documents the budding and division process of human erythrocytes in vitro using time-lapse light microscopy of hanging-drop preparations. The erythrocytes were prepared from normal adult human peripheral blood. Red blood cells showed cytoplasmic budding, segmentation, and direct division with delayed addition of erythropoietin in serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham. These observations are thought to be useful for developing model of definitive erythropoiesis and simple expansion of human erythrocytes.

Propofol causes neurite retraction in neurones

BackgroundThe mechanism by which anaesthetic agents produce general anaesthesia is not yet fully understood. Retraction of neurites is an important function of individual neurones and neural plexuses during normal and pathological conditions, and it has been shown that such a retraction pathway exists in developing and mature neurones. We hypothesized that propofol decreases neuronal activity by causing retraction of neuronal neurites. MethodsPrimary cultures of rat cortical neurones were exposed in concentration– and time–response experiments to 0.02, 0.2, 2, and 20 μM propofol or lipid vehicle. Neurones were pretreated with the GABAA receptor (GABAAR) antagonist, bicuculline, the myosin II ATPase activity inhibitor, blebbistatin, and the F-actin stabilizing agent, phalloidin, followed by administration of propofol (20 μM). Changes in neurite retraction were evaluated using time-lapse light microscopy. ResultsPropofol caused a concentration- and time-dependent reversible retraction of cultured cortical neurone neurites. Bicuculline, blebbistatin, and phalloidin completely inhibited propofol-induced neurite retraction. Images of retracted neurites were characterized by a retraction bulb and a thin trailing membrane remnant. ConclusionsCultured cortical rat neurones retract their neurites after exposure to propofol in a concentration- and time-dependent manner. This retraction is GABAAR mediated, reversible, and dependent on actin and myosin II. Furthermore, the concentrations and times to full retraction and recovery correspond to those observed during propofol anaesthesia.

Cancer Cells Display Profound Intra- and Interline Variation following Prolonged Exposure to Antimitotic Drugs

Drugs targeting the mitotic spindle are used extensively during chemotherapy, but surprisingly, little is known about how they kill tumor cells. This is largely because many of the population-based approaches are indirect and lead to vague and confusing interpretations. Here, we use a high-throughput automated time-lapse light microscopy approach to systematically analyze over 10,000 single cells from 15 cell lines in response to three different classes of antimitotic drug. We show that the variation in cell behavior is far greater than previously recognized, with cells within any given line exhibiting multiple fates. We present data supporting a model wherein cell fate is dictated by two competing networks, one involving caspase activation, the other protecting cyclin B1 from degradation.