细胞生物学

Several filamentous cyanobacteria like Nostoc differentiate specialized cells in response to changes in environmental factors, such as low light or nutrient starvation. These specialized cells are termed heterocysts and akinetes. Under conditions of nitrogen limitation, nitrogen-fixing heterocysts form in a semi-regular pattern and provide the filament with organic nitrogen compounds. Akinetes are spore-like dormant cells, which allow survival during adverse unfavorable conditions. Both cell types possess multilayered thick envelopes mainly composed of an outermost polysaccharide layer and inner layers of glycolipids, that are important for stress adaptation. To study these envelope glycolipids, a method for the isolation, separation and analysis of lipids from heterocysts and akinetes is essential. The present protocol describes a method involving the extraction of lipids from cyanobacteria using solvents and their separation and visualization on silica plates, to render analysis simple and easy. This protocol is relevant for studying mutants that are defective in glycolipid layer formation and for the comparison of glycolipid composition of heterocysts and akinetes under different environmental stresses.

Cell surface protrusions include F-actin rich, wave-like ruffles that are erected transiently in response to stimuli and during cell migration. Macrophages are innate immune cells that ruffle constitutively and more dramatically in cells activated by pathogens. Dorsal ruffles and their resulting macropinosomes are key sites for environmental sampling, pathogen detection and immune signaling. Quantitative assessment of ruffling is important for assessing pathogen responses in macrophages and for analysis of growth factor responses in other cell types but automated and quantitative methods are lacking, and rely on manual and qualitative assessments. Here we present an automated ImageJ macro for quantifying dorsal cell surface protrusions from 3D microscope images. The assay presented here is suitable for high-throughput screening applications to detect drug, pathogen, or growth factor induced changes in cell ruffling by measuring ruffle area and intensity and providing normalized values in an easy to read combined spreadsheet.

All bacteria, fungi and plant cells are surrounded by a cell wall. This complex network of polysaccharides and glycoproteins provides mechanical support, defines cell shape, controls cell growth and influences the exchange of substances between the cell and its surroundings. Despite its name, the cell wall is a flexible, dynamic structure. However, due to the lack of non-invasive methods to probe the structure, relatively little is known about the synthesis and dynamic remodeling of cell walls. Here, we describe a non-invasive method that quantifies a key physiological parameter of cell walls, the porosity, i.e., the size of spaces between cell wall components. This method measures the porosity-dependent decrease of the plasma membrane-localized fluorescent dye FM4-64 in the presence of the extracellular quencher Trypan blue. This method is applied to bacteria, fungi and plant cell walls to detect dynamic changes of porosity in response to environmental cues.

In polarised epithelial cells the midbody forms at the apical cell surface during cytokinesis. Once severed, the midbody is inherited by one of the daughter cells remaining tethered to the apical plasma membrane where it participates in non-cytokinetic processes, such as primary ciliogenesis. Here, we describe a novel method to physically remove the midbody remnant from cells and assess the possible effects caused by its loss (Bernabé-Rubio et al., 2016).

Angiogenesis, the growth of new blood vessels from pre-existing vessels, is a critical process that occurs during normal development and tumor formation. Targeting tumor angiogenesis by blocking the activity of vascular endothelial growth factor (VEGF) has demonstrated some clinical benefit; nevertheless there is a great need to target additional angiogenic pathways. We have found that the human umbilical vein endothelial cell (HUVEC) fibrin bead sprouting assay (FBA) is a robust and predictive in vitro assay to evaluate the activity of angiogenesis inhibitors. Here, we describe an optimized FBA protocol for the assessment of biological inhibitors of angiogenesis and the automated quantification of key endpoints.

α2β1-integrin clustering experiment can be used to trigger internalization of α2β1-integrin. When clustering is performed with sequential administration of primary and fluorescent secondary antibodies, the entry kinetics of integrin can be followed into the cell. The idea is first to allow binding of primary antibodies (recognizing the extracellular epitope) to the α2β1-integrins and then to cluster the α2β1-integrin-bound primary antibodies together by the means of the secondary antibody. Binding is done on ice so that the α2β1-integrins will not internalize before both sets of antibodies are bound. Clustering is known to trigger α2β1-integrin internalization efficiently from the cell surface to the cytoplasm. In this protocol we used antibody-induced clustering of α2β1-integrin in order to quantitate the amount of internalized α2β1-integrins in comparison to cell surface-associated α2β1-integrin.

Examination of interactions between a transmembrane protein and a soluble protein by pull-down or immunoprecipitation assays can be tricky and complicated due to the detergent extraction of membrane proteins during the lysate preparation step. The choice and concentration of detergents must be determined empirically and the procedure can be burdensome. Here, we describe a simplified binding assay by expressing the membrane protein of interest in COS-7 cells and applying detergent-free solutions containing an extracellular protein to be tested. The binding is then examined by immunocytochemistry.

Stomata embedded in the epidermis of terrestrial plants are important for CO₂ absorption and water transpiration, and are possible points of entry for pathogens. Thus, the regulation of stomatal apertures is extremely important for the survival of plants. Furthermore, stomata can respond via accurate change of stomatal apertures to a series of extracellular stimuli such as phytohormones, pathogens, ozone, drought, humidity, darkness, CO₂, visible light and UV-B radiation, so stomatal bioassay is widely used to dissect signal transduction mechanisms of plant cells in responses to multiple stimuli. This protocol describes how to measure stomatal apertures in leaves of model plant Arabidopsis thaliana under multiple treatments.

A great way to specifically isolate and quantify proteins in the cell surface membrane is to take advantage of the biotinylation technique. It consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pull-down. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a membrane protein (as control).