Distinct Morphological States Research Articles

LRRK2 and WAVE2 regulate microglial-transition through distinct morphological phenotypes to induce neurotoxicity in a novel two-hit in vitro model of neurodegeneration.

We report a novel in vitro classification system that tracks microglial activation state and their potential neurotoxicity. Mixed live-cell imaging was used to characterize transition through distinct morphological phenotypes, production of reactive oxygen species (ROS), formation of reactive microglial aggregates, and subsequent cytokine production. Transwell cultures were used to determine microglial migration (control and lipopolysaccharide (LPS) treated) to glutamate pre-stressed or healthy neurons. This two-hit paradigm was developed to model the vast evidence that neurodegenerative conditions, like Parkinson's disease (PD), may stem from the collective impact of multiple environmental stressors. We found that healthy neurons were resistant to microglial-mediated inflammation, whereas glutamate pre-stressed neurons were highly susceptible and in fact, appeared to recruit microglia. The LPS treated microglia progressed through distinct morphological states and expressed high levels of ROSand formed large cellular aggregates. Recent evidence implicates leucine-rich repeat kinase 2 (LRRK2) as an important player in the microglial inflammatory state, as well as in the genesis of PD. We found that inhibition of the LRRK2 signaling pathway using the kinase inhibitor cis-2,6-dimethyl-4-(6-(5-(1-methylcyclopropoxy)-1H-indazol-3-yl)pyrimidin-4-yl)morpholine (MLi2) or inhibition of the actin regulatory protein, Wiskott-Aldrich syndrome family Verprolin-homologous Protein-2 (WAVE2), stunted microglial activation and prevented neurotoxicity. Furthermore, inhibition of LRRK2 kinase activity reduced pro-inflammatory chemokines including MIP-2, CRG-2, and RANTES. These data together support the notion that LRRK2 and WAVE2 are important mediators of cytokine production and cytoskeletal rearrangement necessary for microglial-induced neurotoxicity. Furthermore, our model demonstrated unique microglial phenotypic changes that might be mechanistically important for better understanding neuron-microglial crosstalk.

Aggregate morphology of active Brownian particles on porous, circular walls.

We study the motility-induced aggregation of active Brownian particles (ABPs) on a porous, circular wall. We observe that the morphology of aggregated dense-phase on a static wall depends on the wall porosity, particle motility, and the radius of the circular wall. Our analysis reveals two morphologically distinct, dense aggregates; a connected dense cluster that spreads uniformly on the circular wall and a localized cluster that breaks the rotational symmetry of the system. These distinct morphological states are similar to the macroscopic structures observed in aggregates on planar, porous walls. We systematically analyze the parameter regimes where the different morphological states are observed. We further extend our analysis to motile circular rings. We show that the motile ring propels almost ballistically due to the force applied by the active particles when they form a localized cluster, whereas it moves diffusively when the active particles form a continuous cluster. This property demonstrates the possibility of extracting useful work from a system of ABPs, even without artificially breaking the rotational symmetry.

Abstract PHB01: The zebrafish as a mechanical filter: Using zebrafish xenografts to model Ewing sarcoma metastasis in vivo

Abstract Ewing Sarcoma (ES), a pediatric cancer driven by the oncogenic fusion protein EWS-FLI1, recurs or metastasizes in 1 in 3 patients with no clear correlation to accumulation of secondary mutations, raising the possibility that environmental cues may drive metastasis. Previous work in the field has observed distinct cellular morphologies of ES cells in cell culture as a function of EWS-FLI1 level and posited the idea that this may correlate with distinct functional cellular states. In this work, we use zebrafish xenografts to investigate the effect of variable microenvironments on cellular morphologies of ES cells. We use high resolution light-sheet microscopy to focus on specific recurring seeding sites within the 3-day old zebrafish larvae. We then implement a unique cell-shape classification method to distinguish discrete 3D cellular morphologies in these datasets. Using this workflow, we observe a variety of cellular morphologies within the fish, including elongated cellular protrusions, blebbing, and rounded cells. We demonstrate that ES cells take on distinct morphological states as a function of the region in which they reside. We find cell proliferation, but not cell death, vary greatly in the different regions, suggesting differences in the functional state of the ES cells. Interestingly, knockdown of EWS-FLI1 in xenografted cells leads to discrete switches between cellular morphologies, but only as a function of seeding site within the fish, suggesting a previously undescribed environmental cue that modulates cellular effects of EWS-FLI1. Overall, the xenograft model for ES in zebrafish embryos can be viewed as a “mechanical filter”, which provides unique, complex 3-dimensional environments to study cell behavior within an organism, while leveraging the advantages of the zebrafish system, namely higher throughput and high resolution in vivo imaging capabilities. This work establishes a pipeline for classification and distinction of morphological states within a living organism, and, more specifically, helps our understanding of cellular plasticity of ES cells in response to microenvironment. Citation Format: Dagan Segal, Bo-Jui Chang, Hanieh Mazloom-Farsibaf, Reto Fiolka, James Amatruda, Gaudenz Danuser. The zebrafish as a mechanical filter: Using zebrafish xenografts to model Ewing sarcoma metastasis in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PHB01.

Morphological State Transition Dynamics in EGF-Induced Epithelial to Mesenchymal Transition.

Epithelial to Mesenchymal Transition (EMT) is a multi-state process. Here, we investigated phenotypic state transition dynamics of Epidermal Growth Factor (EGF)-induced EMT in a breast cancer cell line MDA-MB-468. We have defined phenotypic states of these cells in terms of their morphologies and have shown that these cells have three distinct morphological states—cobble, spindle, and circular. The spindle and circular states are the migratory phenotypes. Using quantitative image analysis and mathematical modeling, we have deciphered state transition trajectories in different experimental conditions. This analysis shows that the phenotypic state transition during EGF-induced EMT in these cells is reversible, and depends upon the dose of EGF and level of phosphorylation of the EGF receptor (EGFR). The dominant reversible state transition trajectory in this system was cobble to circular to spindle to cobble. We have observed that there exists an ultrasensitive on/off switch involving phospho-EGFR that decides the transition of cells in and out of the circular state. In general, our observations can be explained by the conventional quasi-potential landscape model for phenotypic state transition. As an alternative to this model, we have proposed a simpler discretized energy-level model to explain the observed state transition dynamics.

Linking Cellular Morphogenesis with Antifungal Treatment and Susceptibility in Candida Pathogens.

Fungal infections are a growing public health concern, and an increasingly important cause of human mortality, with Candida species being amongst the most frequently encountered of these opportunistic fungal pathogens. Several Candida species are polymorphic, and able to transition between distinct morphological states, including yeast, hyphal, and pseudohyphal forms. While not all Candida pathogens are polymorphic, the ability to undergo morphogenesis is linked with the virulence of many of these pathogens. There are also many connections between Candida morphogenesis and antifungal drug treatment and susceptibility. Here, we review how Candida morphogenesis—a key virulence trait—is linked with antifungal drugs and antifungal drug resistance. We highlight how antifungal therapeutics are able to modulate morphogenesis in both sensitive and drug-resistant Candida strains, the shared signaling pathways that mediate both morphogenesis and the cellular response to antifungal drugs and drug resistance, and the connection between Candida morphology, drug resistance, and biofilm growth. We further review the development of anti-virulence drugs, and targeting Candida morphogenesis as a novel therapeutic strategy to target fungal pathogens. Together, this review highlights important connections between fungal morphogenesis, virulence, and susceptibility to antifungals.

Exploring the Stability Limits of Actin and its Suprastructures

Actin is the main component of the microfilament system in eukaryotic cells and can be found in distinct morphological states. G-actin is able to assemble into highly organized, supramolecular cellular structures, filamentous (F) and bundled (B) actin. To evaluate the structure and establish the stability of G-, F- and B- actin over a wide range of temperatures and pressures, FTIR spectroscopy in combination with differential scanning and pressure perturbation calorimetry, small-angle X-ray scattering, laser confocal scanning and transmission electron microscopy was used. On the one hand, our analysis was designed to provide new insights into the stabilizing forces of actin self-assembly and to reveal the stability of the actin polymorphs, including conditions encountered in extreme environments. On the other hand, our data help understand the limited pressure stability of actin self-assembly observed in vivo. G-actin is not only the least temperature-stable, but also the least pressure-stable actin species. At abyssal conditions, where temperatures as low as 1-4°C and pressures up to 1 kbar are reached, G-actin is hardly stable anymore. The supramolecular assemblies of actin are stable enough to withstand the extreme conditions usually encountered on Earth, however. Beyond ∼3-4 kbar, filamentous structures disassemble, and beyond ∼4 kbar, complete dissociation of F-actin structures is observed. Between about 1 and 2 kbar, some disordering of actin assemblies commences already, in agreement with in vivo observations. The limited pressure stability of the monomeric building block seems to be responsible for the suppression of actin assembly in the kbar pressure range.

Exploring the Stability Limits of Actin and Its Suprastructures

Actin is the main component of the microfilament system in eukaryotic cells and can be found in distinct morphological states. Global (G)-actin is able to assemble into highly organized, supramolecular cellular structures known as filamentous (F)-actin and bundled (B)-actin. To evaluate the structure and stability of G-, F-, and B-actin over a wide range of temperatures and pressures, we used Fourier transform infrared spectroscopy in combination with differential scanning and pressure perturbation calorimetry, small-angle x-ray scattering, laser confocal scanning microscopy, and transmission electron microscopy. Our analysis was designed to provide new (to our knowledge) insights into the stabilizing forces of actin self-assembly and to reveal the stability of the actin polymorphs, including in conditions encountered in extreme environments. In addition, we sought to explain the limited pressure stability of actin self-assembly observed in vivo. G-actin is not only the least temperature-stable but also the least pressure-stable actin species. Under abyssal conditions, where temperatures as low as 1–4°C and pressures up to 1 kbar are reached, G-actin is hardly stable. However, the supramolecular assemblies of actin are stable enough to withstand the extreme conditions usually encountered on Earth. Beyond ∼3–4 kbar, filamentous structures disassemble, and beyond ∼4 kbar, complete dissociation of F-actin structures is observed. Between ∼1 and 2 kbar, some disordering of actin assemblies commences, in agreement with in vivo observations. The limited pressure stability of the monomeric building block seems to be responsible for the suppression of actin assembly in the kbar pressure range.

Transcriptome analysis of amoeboid and ramified microglia isolated from the corpus callosum of rat brain

BackgroundMicroglia, the resident immune cells of the central nervous system (CNS), have two distinct phenotypes in the developing brain: amoeboid form, known to be amoeboid microglial cells (AMC) and ramified form, known to be ramified microglial cells (RMC). The AMC are characterized by being proliferative, phagocytic and migratory whereas the RMC are quiescent and exhibit a slow turnover rate. The AMC transform into RMC with advancing age, and this transformation is indicative of the gradual shift in the microglial functions. Both AMC and RMC respond to CNS inflammation, and they become hypertrophic when activated by trauma, infection or neurodegenerative stimuli. The molecular mechanisms and functional significance of morphological transformation of microglia during normal development and in disease conditions is not clear. It is hypothesized that AMC and RMC are functionally regulated by a specific set of genes encoding various signaling molecules and transcription factors.ResultsTo address this, we carried out cDNA microarray analysis using lectin-labeled AMC and RMC isolated from frozen tissue sections of the corpus callosum of 5-day and 4-week old rat brain respectively, by laser capture microdissection. The global gene expression profiles of both microglial phenotypes were compared and the differentially expressed genes in AMC and RMC were clustered based on their functional annotations. This genome wide comparative analysis identified genes that are specific to AMC and RMC.ConclusionsThe novel and specific molecules identified from the trancriptome explains the quiescent state functioning of microglia in its two distinct morphological states.

Sponge budding is a spatiotemporal morphological patterning process: Insights from synchrotron radiation-based x-ray microtomography into the asexual reproduction of Tethya wilhelma

BackgroundPrimary agametic-asexual reproduction mechanisms such as budding and fission are present in all non-bilaterian and many bilaterian animal taxa and are likely to be metazoan ground pattern characters. Cnidarians display highly organized and regulated budding processes. In contrast, budding in poriferans was thought to be less specific and related to the general ability of this group to reorganize their tissues. Here we test the hypothesis of morphological pattern formation during sponge budding.ResultsWe investigated the budding process in Tethya wilhelma (Demospongiae) by applying 3D morphometrics to high resolution synchrotron radiation-based x-ray microtomography (SR-μCT) image data. We followed the morphogenesis of characteristic body structures and identified distinct morphological states which indeed reveal characteristic spatiotemporal morphological patterns in sponge bud development. We discovered the distribution of skeletal elements, canal system and sponge tissue to be based on a sequential series of distinct morphological states. Based on morphometric data we defined four typical bud stages. Once they have reached the final stage buds are released as fully functional juvenile sponges which are morphologically and functionally equivalent to adult specimens.ConclusionOur results demonstrate that budding in demosponges is considerably more highly organized and regulated than previously assumed. Morphological pattern formation in asexual reproduction with underlying genetic regulation seems to have evolved early in metazoans and was likely part of the developmental program of the last common ancestor of all Metazoa (LCAM).

Helicobacter pylori produces unique filaments upon host contact in vitro

Helicobacter pylori exists in 2 distinct morphological states, helicoid and coccoid. Both have been observed in in vitro culture and in gastric biopsies. We visualized H. pylori during AGS cell infections using immunofluorescence microscopy. Anti-H. pylori mouse serum as well as human serum from H. pylori-positive patients recognized long, thin bacterial filaments, which formed on helicoids and more frequently on coccoids. These filaments reached lengths of 59 microm and often connected bacteria. Periodate oxidation abolished antibody recognition, suggesting that carbohydrates compose a major antigenic component of the filaments. Similar to results obtained using immunofluorescence microscopy, scanning electron microscopy imaging revealed thin filamentous structures, which were absent on uninfected cells. Both coccoid conversion and filament development increased over the time course of infection with peak filament formation at 4 h. The number of visible filaments then decreased as bacteria clustered on the apical surface of AGS cells. Since the observed filaments were clearly distinct from previously described surface structures, including flagella and the cag type IV secretion system, our results demonstrate that these filaments represent a unique, previously unrecognized, organelle.

Cdc42p GTPase regulates the budded-to-hyphal-form transition and expression of hypha-specific transcripts in Candida albicans.

The yeast Candida albicans is a major opportunistic pathogen of immunocompromised individuals. It can grow in several distinct morphological states, including budded and hyphal forms, and the ability to make the dynamic transition between these forms is strongly correlated with virulence. Recent studies implicating the Cdc42p GTPase in hypha formation relied on cdc42 mutations that affected the mitotic functions of the protein, thereby precluding any substantive conclusions about the specific role of Cdc42p in the budded-to-hypha-form transition and virulence. Therefore, we took advantage of several Saccharomyces cerevisiae cdc42 mutants that separated Cdc42p's mitotic functions away from its role in filamentous growth. The homologous cdc42-S26I, cdc42-E100G, and cdc42-S158T mutations in C. albicans Cdc42p caused a dramatic defect in the budded-to-hypha-form transition in response to various hypha-inducing signals without affecting normal budded growth, strongly supporting the conclusion that Cdc42p has an integral function in orchestrating the morphological transition in C. albicans. In addition, the cdc42-S26I and cdc42-E100G mutants demonstrated a reduced ability to damage endothelial cells, a process that is strongly correlated to virulence. The three mutants also had reduced expression of several hypha-specific genes, including those under the regulation of the Efg1p transcription factor. These data indicate that Cdc42p-dependent signaling pathways regulate the budded-to-hypha-form transition and the expression of hypha-specific genes.

Fracture toughness and morphology study of ternary blends of epoxy, poly(ether sulfone) and acrylonitrile-butadiene rubber

An epoxy resin based on diglycidyl ether of bisphenol A has been modified with poly(ether sulfone) (PES) and poly(acrylonitrile- co-butadiene) rubber (NBR) and cured with 4,4′-diaminodiphenylsulfone. The fracture behaviour of the ternary blend was examined using a fracture mechanics approach. Depending on the combination of PES and NBR, two distinct morphological states were shown, and the fracture behaviour varied from brittle to ductile with changing morphology. From fracture surface observations by scanning electron microscopy, the ternary blends that showed ductile fracture behaviour have a phase-inverted morphology in which the NBR-rich phase formed a continuous matrix, the epoxy-rich phase formed particles and the PES-rich phase also formed particles around the epoxy particles. Dynamic mechanical thermal analysis revealed that the ternary blends exhibited high T g, as high as the T g of the unmodified epoxy resin.

Lysosomes and the modulation of cell morphology in the isthmus region of the outer mantle epithelium in the estuarine clam, Rangia cuneata

In bivalves, the isthmus region of the outer mantle epithelium lies adjacent to the hinge ligament. In Rangia cuneata, the columnar cells of the epithelium manifest two distinct morphological states which are a function of the animal's environment. In state 1 the cells are uniformly filled with glycogen and in state 2 they contain large spherical whorls of rough endoplasmic reticulum (RER) and are depleted in glycogen. In both states the cells contain large dense inclusions which are residual bodies based on their morphology and small but definite acid phosphatase activity. Based on the stucture of the RER and the cytoplasmic dispositions of residual bodies and other organelles, it is suggested that (1) the residual bodies are derived from autophagic activity originating at the center of RER whorls and (2) that autophagy is responsible for the destruction of the whorls when the cells pass from state 2 to state 1.