Ciliary Tip Signaling Compartment Is Formed and Maintained by Intraflagellar Transport

1. Download : Download high-res image (133KB)
2. Download : Download full-size image

     

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.     

A bacterial pathogen uses dimethylsulfoniopropionate as a cue to target heat-stressed corals

Diseases are an emerging threat to ocean ecosystems. Coral reefs, in particular, are experiencing a worldwide decline because of disease and bleaching, which have been exacerbated by rising seawater temperatures. Yet, the ecological mechanisms behind most coral diseases remain unidentified. Here, we demonstrate that a coral pathogen, Vibrio coralliilyticus, uses chemotaxis and chemokinesis to target the mucus of its coral host, Pocillopora damicornis. A primary driver of this response is the host metabolite dimethylsulfoniopropionate (DMSP), a key element in the global sulfur cycle and a potent foraging cue throughout the marine food web. Coral mucus is rich in DMSP, and we found that DMSP alone elicits chemotactic responses of comparable intensity to whole mucus. Furthermore, in heat-stressed coral fragments, DMSP concentrations increased fivefold and the pathogen''s chemotactic response was correspondingly enhanced. Intriguingly, despite being a rich source of carbon and sulfur, DMSP is not metabolized by the pathogen, suggesting that it is used purely as an infochemical for host location. These results reveal a new role for DMSP in coral disease, demonstrate the importance of chemical signaling and swimming behavior in the recruitment of pathogens to corals and highlight the impact of increased seawater temperatures on disease pathways.     

Shear flow affects selective monocyte recruitment into MCP‐1‐loaded scaffolds

Novel cardiovascular replacements are being developed by using degradable synthetic scaffolds, which function as a temporary guide to induce neotissue formation directly in situ. Priming of such scaffolds with fast‐releasing monocyte chemoattractant protein‐1 (MCP‐1) was shown to improve the formation of functional neoarteries in rats. However, the underlying mechanism has not been clarified. Therefore, the goal of this study was to investigate the effect of a burst‐release of MCP‐1 from a synthetic scaffold on the local recruitment of circulating leucocytes under haemodynamic conditions. Herein, we hypothesized that MCP‐1 initiates a desired healing cascade by recruiting favourable monocyte subpopulations into the implanted scaffold. Electrospun poly(ε‐caprolactone) scaffolds were loaded with fibrin gel containing various doses of MCP‐1 and exposed to a suspension of human peripheral blood mononuclear cells in static or dynamic conditions. In standard migration assay, a dose‐dependent migration of specific CD14⁺ monocyte subsets was observed, as measured by flow cytometry. In conditions of pulsatile flow, on the other hand, a marked increase in immediate monocyte recruitment was observed, but without evident selectivity in monocyte subsets. This suggests that the selectivity was dependent on the release kinetics of the MCP‐1, as it was overruled by the effect of shear stress after the initial burst‐release. Furthermore, these findings demonstrate that local recruitment of specific MCP‐1‐responsive monocytes is not the fundamental principle behind the improved neotissue formation observed in long‐term in vivo studies, and mobilization of MCP‐1‐responsive cells from the bone marrow into the bloodstream is suggested to play a predominant role in vivo.     

Effects of Liposomal Superoxide Dismutase on Human Neutrophil Activity

Study of the effects of liposomal bovine copper superoxide dismutase on human polymorphonuclear neutrophils with respect to production of active oxygen species, chemotaxis and random migration, or bacterial killing show that no significant interference with neutrophil function is observed at levels far exceeding the clinical doses used in the treatment of various pathologies.     

Inhibition of Rac and Rac-linked functions by 8-oxo-2′-deoxyguanosine in murine macrophages

Rac is a protein involved in the various functions of macrophages (Mφ), including the production of reactive oxygen species (ROS), phagocytosis, chemotaxis and the secretion of cytokines (such as γ-INF). This study tested the effects of nucleosides containing 8-oxoguanine(8-hydroxyguanine) such as 8-oxo-2′-guanosine (8-oxoG) or 8-oxo-2′-deoxyguanosine (8-oxodG), on Rac and the above-listed Rac-associated functions of Mφ using mouse peritoneal Mφ (MpMφ). It is reported that 8-oxodG was able to effectively inhibit Rac and the Rac-associated functions of MpMφ. Compared to 8-oxodG, 8-oxoG showed negligible effects. Furthermore, normal nucleosides such as deoxyguanosine (dG), guanosine (G) and adenosine (A) did not exert any effects. These results suggest that 8-oxodG could be used as a potential tool to modulate the functions of Mφ that are intimately related to various pathological processes.     

Volatiles from soil‐borne fungi affect directional growth of roots

Volatiles play major roles in mediating ecological interactions between soil (micro)organisms and plants. It is well‐established that microbial volatiles can increase root biomass and lateral root formation. To date, however, it is unknown whether microbial volatiles can affect directional root growth. Here, we present a novel method to study belowground volatile‐mediated interactions. As proof‐of‐concept, we designed a root Y‐tube olfactometer, and tested the effects of volatiles from four different soil‐borne fungi on directional growth of Brassica rapa roots in soil. Subsequently, we compared the fungal volatile organic compounds (VOCs) previously profiled with Gas Chromatography–Mass Spectrometry (GC–MS). Using our newly designed setup, we show that directional root growth in soil is differentially affected by fungal volatiles. Roots grew more frequently toward volatiles from the root pathogen Rhizoctonia solani, whereas volatiles from the other three saprophytic fungi did not impact directional root growth. GC–MS profiling showed that six VOCs were exclusively emitted by R. solani. These findings verify that this novel method is suitable to unravel the intriguing chemical cross‐talk between roots and soil‐borne fungi and its impact on root growth.     

Ensifer alkalisoli YIC4027趋化受体基因的比较及相关蛋白序列分析

[目的] 田菁共生根瘤菌Ensifer alkalisoli YIC4027是从宿主植物田菁的根瘤中分离出来的一株新型高效的固氮菌。本研究对E.alkalisoli的趋化受体基因与其他研究透彻的物种进行比较以及相关蛋白分析。[方法] 利用NCBI的BLAST对E.alkalisoli趋化受体基因进行序列相似性搜索。以Pfam数据库为基础,用HMMR3对甲基化趋化受体蛋白（MCP）进行比较分析。[结果] E.alkalisoli有2个趋化基因簇,共有13个MCP,含有不同的信号传感结构。此外,这些MCPs的胞质结构域除了一个是由40个七肽重复序列组成,其余都是由36个七肽重复序列组成。[结论] 尽管E.alkalisoli的趋化受体与已被广泛研究的物种的趋化受体具有较高的相似性,但仍显示出其特性。通过基因的比对以及相关蛋白的分析,我们能够更好地理解E.alkalisoli是如何通过趋化系统来响应外界变化的。     

Gliadin fragments promote migration of dendritic cells

In genetically predisposed individuals, ingestion of wheat gliadin provokes a T‐cell‐mediated enteropathy, celiac disease. Gliadin fragments were previously reported to induce phenotypic maturation and Th1 cytokine production by human dendritic cells (DCs) and to boost their capacity to stimulate allogeneic T cells. Here, we monitor the effects of gliadin on migratory capacities of DCs. Using transwell assays, we show that gliadin peptic digest stimulates migration of human DCs and their chemotactic responsiveness to the lymph node‐homing chemokines CCL19 and CCL21. The gliadin‐induced migration is accompanied by extensive alterations of the cytoskeletal organization, with dissolution of adhesion structures, podosomes, as well as up‐regulation of the CC chemokine receptor (CCR) 7 on cell surface and induction of cyclooxygenase (COX)‐2 enzyme that mediates prostaglandin E2 (PGE₂) production. Blocking experiments confirmed that gliadin‐induced migration is independent of the TLR4 signalling. Moreover, we showed that the α‐gliadin‐derived 31–43 peptide is an active migration‐inducing component of the digest. The migration promoted by gliadin fragments or the 31–43 peptide required activation of p38 mitogen‐activated protein kinase (MAPK). As revealed using p38 MAPK inhibitor SB203580, this was responsible for DC cytoskeletal transition, CCR7 up‐regulation and PGE₂ production in particular. Taken together, this study provides a new insight into pathogenic features of gliadin fragments by demonstrating their ability to promote DC migration, which is a prerequisite for efficient priming of naive T cells, contributing to celiac disease pathology.     

Evaluation of cancer stem cell migration using compartmentalizing microfluidic devices and live cell imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized. Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment. We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.