细胞生物学

Dozens of Mycoplasma species belonging to the class Mollicutes bind to solid surfaces through the organelle formed at a cell pole and glide in its direction by a unique mechanism. In Mycoplasma mobile, the fastest gliding species in Mycoplasma, the force for gliding is generated by ATP hydrolysis on an internal structure. However, the spatial and temporal behaviors of the internal structures in living cells were unclear. High-speed atomic force microscopy (HS-AFM) is a powerful method to monitor the dynamic behaviors of biomolecules and cells that can be captured while maintaining their active state in aqueous solution. In this protocol, we describe a method to detect their movements using HS-AFM. This protocol should be useful for the studies of many kinds of microorganisms.

Graphic abstract:

Scannnig Mycoplasma cell

Cell migration is a vital process in the development of multicellular organisms. When deregulated, it is involved in many diseases such as inflammation and cancer metastisation. Some cancer cells could be stimulated using chemoattractant molecules, such as growth factor Heregulin β1. They respond to the attractant or repellent gradients through a process known as chemotaxis. Indeed, chemotactic cell motility is crucial in tumour cell dissemination and invasion of distant organs. Due to the complexity of this phenomenon, the majority of available in vitro methods to study the chemotactic motility process have limitations and are mainly based on endpoint assays, such as the Boyden chamber assay. Nevertheless, in vitro time-lapse microscopy represents an interesting opportunity to study cell motility in a chemoattracting gradient, since it generates large volume image-based information, allowing the analysis of cancer cell behaviours. Here, we describe a detailed time-lapse imaging protocol, designed for tracking T47D human breast cancer cell line motility, toward a gradient of Heregulin β1 in a Dunn chemotaxis chamber assay. The protocol described here is readily adapted to study the motility of any adherent cell line, under various conditions of chemoattractant gradients and of pharmacological drug treatments. Moreover, this protocol could be suitable to study changes in cell morphology, and in cell polarity.

Swarming – swift movement across a surface via flagella propulsion – is a unique property of many bacteria. The role of swarming, particularly among bacterial populations of the human gut microbiome, is not yet fully understood; although, it is becoming an area of increased scientific and clinical inquiry. To further characterize bacterial swarming in human health, an effective assay for swarming that utilizes complex material, such as fecal matter, is necessary. Until now, the vast majority of swarming assays have only been able to accommodate bacteria grown in culture, most often Pseudomonas. These assays tend to use a standard lysogenic broth (LB) agar medium; however, the reagents involved have not been tailored to the inoculation of complex material. In this paper, we offer a specialized protocol for eliciting the swarming of bacteria from frozen human fecal samples. We describe the simple, yet reproducible steps required to perform the assay, identifying an ideal volume of 7.5 μl for inoculation of material, as well as an ideal agar concentration of 0.4%. This protocol typically allows researchers to identify swarming within 24 h after incubation in a standard incubator.

Surface-associate motility on biotic and abiotic environments is a key mechanism used by the model bacterium Bacillus subtilis and its closest relatives (i.e., B. amyloliquefaciens, B. thuringiensis, B. cereus, B. pumilus) for surface colonization and spreading across surfaces. The study of this mechanism in a research, industrial or clinic laboratory is essential; however, precautions should be taken for the reproducibility of the results, for example, the procedure to inoculate the bacteria on the testing plate, the humidity of the plate and the agar concentration. In this protocol, we describe, using Bacillus subtilis, how to perform these assays and, in addition, we show how by varying the agar concentration in the plate, you can make a first approximation of what type of motility has other bacterial species.

Vigorous sperm flagellar motility is essential for fertilization, and so the quantitative measurement of motility is a useful tool to assess the intrinsic fertility potential of sperm cells and explore how various factors can alter sperm’s ability to reach the egg and penetrate its protective layers. Human sperm beat their flagella many times each second, and so recording and accurately quantifying this movement requires a high-speed camera. The aim of this protocol is to provide a detailed description of the tools required for quantitative beat frequency measurement of tethered human sperm at the single-cell level and to describe methods for investigating the effects of intracellular or extracellular factors on flagellar motion. This assay complements bulk measurements of sperm parameters using commercially-available systems for computer-assisted sperm analysis (CASA).

A method was developed to allow the quantification and mapping of relative bacterial twitching motility in dense samples, where tracking of individual bacteria was not feasible. In this approach, movies of bacterial films were acquired using differential interference contrast microscopy (DIC), and bacterial motility was then indirectly quantified by the degree to which the bacteria modulated the intensity of light in the field-of-view over time. This allowed the mapping of areas of relatively high and low motility within a single field-of-view, and comparison of the total distribution of motility between samples.

Lipopeptides is an important class of biosurfactants having antimicrobial and anti-adhesive activity against pathogenic bacteria. These include surfactin, fengycin, iturin, bacillomycin, mycosubtilin, lichenysin, and pumilacidin (Arima et al., 1968; Naruse et al., 1990; Yakimov et al., 1995; Steller and Vater, 2000; Roongsawang et al., 2002; Vater et al., 2002). To date, none of these lipopeptides have been reported to possess any anti-motility activity. We isolated, purified and characterized two novel cyclic lipopeptides (CLPs) from Bacillus sp. 176 using high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. CLPs dramatically suppress the motility of pathogenic bacterium Vibrio alginolyticus 178, and promote cellular aggregation without inducing cell death. Cell aggregation assay was performed with the modification according to methods described by Dalili for anti-biofilm assay (Dalili et al., 2015). In future, this assay can be adapted to test both the cell aggregation and anti-biofilm activity of lipopeptide-like active substances derived from bacteria.

Motile male gametes (spermatozoids) of land plants are coiled and contain a modified and precisely organized complement of organelles that includes a locomotory apparatus with two to thousands of flagella. Each flagellum is generated from a basal body that originates de novo as a centriole in spermatogenous cell lineages. Much of what is known about the diversity of plant male gametes was derived from detailed transmission electron microscopic studies. Because the process of spermatogenesis results in complete transformation of the shape and organization of these cells, TEM studies have yielded a wealth of information on cellular differentiation. Because green algal progenitor groups contain centrioles and a variety of motile cells, land plant spermatozoids also provide a plethora of opportunities to examine the evolution and eventual loss of centrioles and locomotory apparatus during land colonization.

Here we provide a brief overview of the studies and methodologies we have conducted over the past 20 years that have elucidated not only the structural diversity of these cells but also the development of microtubule organizing centers, the de novo origin of centrioles and the ontogeny of structurally complex motile cells.

Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro.

The Actin-Related Protein 2/3 (ARP2/3) complex is an actin nucleator that generates a branched actin network in mammalian cells. In addition to binding nucleation promoting factors, LeClaire et al. demonstrated that its phosphorylation state is essential key for its activity (LeClaire et al., 2008). In cells, the ARP2/3 complex is phosphorylated on threonine and tyrosine residues of the ARP2, ARP3, and ARPC1 subunits (Vadlamudi et al., 2004; LeClaire et al., 2008; Narayanan et al., 2011; LeClaire et al., 2015). In particular, phosphorylation of threonine 237 and 238 of the ARP2 subunit is necessary to allow a change in the ARP2/3 complex structure to its active conformation (Narayanan et al., 2011; LeClaire et al., 2015). While important for many functions in eukaryotic cells, ARP2/3 complex activity also benefits several cellular pathogens (Haglund and Welch, 2011; Welch and Way, 2013). Recently, we demonstrated that the bacterial pathogen, Legionella pneumophila, manipulates ARP2/3 complex phosphorylation state using a bacterial protein kinase injected in host cell cytoplasm (Michard et al., 2015). Here, we describe how to test the ability of a bacterial protein kinase or another protein kinase to phosphorylate the ARP2/3 complex in an in vitro context. First, the ARP2/3 complex and the bacterial protein kinase are produced and purified. Then, the purified proteins are incubated in the presence of ATP, and the ARP2/3 phosphorylation level is analyzed by Western blot.