Name: quantification of acanthamoeba spp. motility
Uploaded: 2024-09-20T12:40:00.000+00:00
Duration: 7 min 33 s
Description: Acanthamoeba keratitis is a severe ocular infection that poses treatment challenges and can lead to blindness. Despite its ubiquity and potential ...

<meta charset="utf8"></head><body>棘 阿米巴 物种运动的长期跟踪和定量

<ol>
	<li>Randag, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rising+incidence+of+Acanthamoeba+keratitis:+A+7-year+nationwide+survey+and+clinical+assessment+of+risk+factors+and+functional+outcomes.">The rising incidence of Acanthamoeba keratitis: A 7-year nationwide survey and clinical assessment of risk factors and functional outcomes.</a> PLoS One. 14 (9), e0222092 (2019).</li><li>Szentmary, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba+keratitis+-+Clinical+signs,+differential+diagnosis+and+treatment.">Acanthamoeba keratitis - Clinical signs, differential diagnosis and treatment.</a> J Curr Ophthalmol. 31 (1), 16-23 (2019).</li><li>Carnt, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba+keratitis:+confirmation+of+the+UK+outbreak+and+a+prospective+case-control+study+identifying+contributing+risk+factors.">Acanthamoeba keratitis: confirmation of the UK outbreak and a prospective case-control study identifying contributing risk factors.</a> Br J Ophthalmol. 102 (12), 1621-1628 (2018).</li><li>Verani, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+outbreak+of+Acanthamoeba+keratitis+associated+with+use+of+a+contact+lens+solution,+United+States.">National outbreak of Acanthamoeba keratitis associated with use of a contact lens solution, United States.</a> Emerg Infect Dis. 15 (8), 1236-1242 (2009).</li><li>Tu, E. Y., Joslin, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+outbreaks+of+atypical+contact+lens-related+keratitis:+what+have+we+learned.">Recent outbreaks of atypical contact lens-related keratitis: what have we learned.</a> Am J Ophthalmol. 150 (5), 602-608 (2010).</li><li>International Organization for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISO+14729:2001/A1.+Ophthalmic+optics-Contact+lens+care+products-Microbiological+requirements+and+test+methods+for+products+and+regimens+for+hygienic+management+of+contact+lenses.">ISO 14729:2001/A1. Ophthalmic optics-Contact lens care products-Microbiological requirements and test methods for products and regimens for hygienic management of contact lenses.</a> International Organization for Standardization. , (2010).</li><li>. ASC Z80, Parent Committee Meeting Available from: <a target="_blank" href="https://www.thevisioncouncil.org/sites/default/files/ASCZ80_ParentCommitteeMinutes_February_27_2018_FINALMar19-2018.pdf">https://www.thevisioncouncil.org/sites/default/files/ASCZ80_ParentCommitteeMinutes_February_27_2018_FINALMar19-2018.pdf</a> (2023)</li><li>Rayamajhee, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba,+an+environmental+phagocyte+enhancing+survival+and+transmission+of+human+pathogens.">Acanthamoeba, an environmental phagocyte enhancing survival and transmission of human pathogens.</a> Trends Parasitol. 38 (11), 975-990 (2022).</li><li>Reverey, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superdiffusion+dominates+intracellular+particle+motion+in+the+supercrowded+cytoplasm+of+pathogenic+Acanthamoeba+castellanii.">Superdiffusion dominates intracellular particle motion in the supercrowded cytoplasm of pathogenic Acanthamoeba castellanii.</a> Sci Rep. 5, 11690 (2015).</li><li>Thapa, S., Lukat, N., Selhuber-Unkel, C., Cherstvy, A. G., Metzler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+superdiffusion+of+polydisperse+vacuoles+in+highly+motile+amoeboid+cells.">Transient superdiffusion of polydisperse vacuoles in highly motile amoeboid cells.</a> J Chem Phys. 150 (14), 144901 (2019).</li><li>Rudell, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba+migration+in+an+electric+field.">Acanthamoeba migration in an electric field.</a> Invest Ophthalmol Vis Sci. 54 (6), 4225-4233 (2013).</li><li>Fukaya, S., Aoki, K., Kobayashi, M., Takemura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+the+motility+of+giant+virus-infected+amoebae+using+phase-contrast+microscopic+images.">Kinetic analysis of the motility of giant virus-infected amoebae using phase-contrast microscopic images.</a> Front Microbiol. 10, 3014 (2019).</li><li>Fukaya, S., Takemura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+Acanthamoeba+castellanii+infected+with+giant+viruses+quantitatively+revealed+process+of+morphological+and+behavioral+changes+in+host+cells.">Kinetic analysis of Acanthamoeba castellanii infected with giant viruses quantitatively revealed process of morphological and behavioral changes in host cells.</a> Microbiol Spectr. 9 (1), 0036821 (2021).</li><li>Cheng, H. J., Hsu, C. H., Hung, C. L., Lin, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+for+cell+and+particle+tracking+on+microscopy+images+using+algorithms+and+deep+learning+technologies.">A review for cell and particle tracking on microscopy images using algorithms and deep learning technologies.</a> Biomed J. 45 (3), 465-471 (2022).</li><li>Mathieu, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+lens-free+imaging+of+cell+migration+in+diverse+physical+microenvironments.">Time-lapse lens-free imaging of cell migration in diverse physical microenvironments.</a> Lab Chip. 16 (17), 3304-3316 (2016).</li><li>Svensson, C. M., Medyukhina, A., Belyaev, I., Al-Zaben, N., Figge, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untangling+cell+tracks:+Quantifying+cell+migration+by+time+lapse+image+data+analysis.">Untangling cell tracks: Quantifying cell migration by time lapse image data analysis.</a> Cytometry A. 93 (3), 357-370 (2018).</li><li>Piltti, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+time-lapse+imaging+and+single-cell+tracking+of+in+vitro+cultured+neural+stem+cells+-+Tools+for+analyzing+dynamics+of+cell+cycle,+migration,+and+lineage+selection.">Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells - Tools for analyzing dynamics of cell cycle, migration, and lineage selection.</a> Methods. 133, 81-90 (2018).</li><li>Campolo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+real-time+motility+analysis+of+Acanthamoeba+reveals+sustained+movement+in+absence+of+nutrients.">Continuous real-time motility analysis of Acanthamoeba reveals sustained movement in absence of nutrients.</a> Pathogens. 10 (8), 995 (2021).</li><li>Campolo, A., Patterson, B., Lara, E., Shannon, P., Crary, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+recovery+of+Acanthamoeba+motility+among+surviving+organisms+after+contact+lens+care+disinfection.">Complete recovery of Acanthamoeba motility among surviving organisms after contact lens care disinfection.</a> Microorganisms. 11 (2), 299 (2023).</li><li>Crary, M. J., Walters, R., Shannon, P., Gabriel, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variables+affecting+the+recovery+of+Acanthamoeba+trophozoites.">Variables affecting the recovery of Acanthamoeba trophozoites.</a> Pathogens. 10 (2), 221 (2021).</li><li>. imagej Available from: <a target="_blank" href="https://imagej.net/plugins/trackmate/tutorials/getting-started">https://imagej.net/plugins/trackmate/tutorials/getting-started</a> (2024)</li><li>Cherstvy, A. G., Nagel, O., Beta, C., Metzler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-Gaussianity,+population+heterogeneity,+and+transient+superdiffusion+in+the+spreading+dynamics+of+amoeboid+cells.">Non-Gaussianity, population heterogeneity, and transient superdiffusion in the spreading dynamics of amoeboid cells.</a> Phys Chem Chem Phys. 20 (35), 23034-23054 (2018).</li></ol>

<table><tbody><tr><td>&amp;frac14; Ringer&amp;rsquo;s solution</td><td>ThermoFisher Scientific</td><td>BR0052G</td><td>Oxoid Ringers Solution Tablets. Follow directions to make one-quarter strength instead of full strength Ringers.&amp;nbsp;</td></tr><tr><td>10 &amp;micro;L pipette</td><td>Eppendorf Research</td><td>3123000039</td><td>2 &amp;micro;L-20 &amp;micro;L single channel</td></tr><tr><td>10 &amp;micro;L pipette tips</td><td>Neptune Scientific, San Diego, CA, USA</td><td>BT10.N</td><td>10 &amp;micro;L Universal Barrier Tip</td></tr><tr><td>48 well plate</td><td>Millipore Sigma,&amp;nbsp;</td><td>CLS3548</td><td>Corning Costar TC-Treated Multiple Well Plate: polystyrene plate, flat bottom wells, sterile, with lid</td></tr><tr><td>50 mL conical tubes (1 for each sample, 1 for each pass 2 sample)</td><td>Fisher Scientific</td><td>Falcon 352098</td><td>Falcon 50 mL High Clarity Conical Centrifuge Tubes, polypropylene</td></tr><tr><td>96-well plate</td><td>Millipore Sigma, Burlington, MA, USA</td><td>CLS3596</td><td>Corning Costar TC-Treated Multiple Well Plate: polystyrene plate, flat bottom wells, sterile, with lid</td></tr><tr><td>&lt;em&gt;Acanthamoeba&amp;nbsp;&lt;/em&gt;</td><td>American Type Culture Collection, Manassas, VA, USA</td><td>ATCC 30461</td><td>This protocol has been verified for ATCC 50655, 30872, 50702, 30010, 30461, 50370, 50703, 30137, PRA-115, and PRA-411</td></tr><tr><td>Axenic culture media (AC6)</td><td>Made in house</td><td>n/a</td><td>Containing 20 g biosate peptone, 5 g glucose, 0.3 g KH2PO4, 10 &amp;micro;g vitamin B12, and 1 glass5 mg&amp;nbsp;l-methionine per liter of distilled deionized water. Adjust pH to 6.6-6.95 with 1 M NaOH and autoclave at 121 &amp;deg;C for 20 min, store at room temperature for up to 3 months.&amp;nbsp;</td></tr><tr><td>Centrifuge and appropriate rotor</td><td>Thermo Scientific, Waltham, MA, USA</td><td>Sorvall ST 40R</td><td>Any equivalent centrifuge and rotor are acceptable so long as it can spin 50 mL conical tubes at 500 x g for 5 min</td></tr><tr><td>Chemotaxis and Migration Tool</td><td></td><td></td><td>Free software based on ImageJ, available at https://ibidi.com/chemotaxis-analysis/171-chemotaxis-and-migration-tool.html</td></tr><tr><td>Disposable hemocytometer</td><td>Bulldog Bio, Portsmouth, NH, USA</td><td>DHC-N01</td><td>Neubauer Improved 2-Chip Disposable Hemocytometer, Individually packaged, Nonpyrogenic</td></tr><tr><td>ImageJ software with Trackmate plugin (this protocol written with Trackmate version 6.0.2.)</td><td></td><td></td><td>Free software developed by the National Institutes of Health, available at imagej.net. Trackmate plugin available at https://imagej.net/plugins/trackmate/</td></tr><tr><td>Microscope, preferably with automated moveable stage</td><td>Nikon, Tokyo, Japan</td><td></td><td>A Nikon Eclipse Ti-U Microscope was used in this study and the automated moveable stage was utilized to be able to record images in multiple wells at a time.&amp;nbsp;</td></tr><tr><td>NIS-Elements software</td><td>Nikon</td><td></td><td></td></tr><tr><td>Serological pipette</td><td>Fisher Scientific, Hampton, NH, USA</td><td>BrandTech 26331</td><td>BrandTech accu-jet pro Pipet Controller</td></tr><tr><td>Serological pipette tips</td><td>VWR</td><td>5 mL: 76201-710&lt;br/&gt; 10 mL: 170356&lt;br/&gt; 25 mL: 89130-900&lt;br/&gt; 50 mL: 75816-088</td><td>VWR Serological Pipette, Non-Pyrogenic</td></tr><tr><td>Sterile aluminum transmission flow cell</td><td>Biosurface Technologies Corporation, Bozeman, MT, USA</td><td>FC 81-AL</td><td>Anodized aluminum single channel transmission flow cell with 96-well plate footprint for use with an inverted microscope</td></tr><tr><td>T75 Flasks</td><td>VWR, Radnor, PA, USA</td><td>734-2316</td><td>VWR Tissue Culture Flask, 182.5 cm, Surface treated, Plug seal cap, Sterile</td></tr></tbody></table>

quantification of acanthamoeba spp. motility

<meta charset="utf8"></head><body>作者聚焦：研究 棘阿米巴 的行为以制定预防 棘阿米巴 角膜炎的针对性策略

棘阿米巴属的定量运动性

Acanthamoeba keratitis is a severe ocular infection that poses treatment challenges and can lead to blindness. Despite its ubiquity and potential ...

quantification-of-acanthamoeba-spp-motility

Research

JoVE Journal

Biology

Quantification of Acanthamoeba spp. Motility

2.5K 次观看.  Alcon Research, LLC. 我们的研究深入研究了棘阿米巴的自然行为，因为当人类和棘阿米巴不可避免地相遇时，它在自然界中是如此。从这个基础上，我们可以推断出棘阿米巴如何在各种环境和表面（无论是隐形眼镜还是角膜）中相互作用的见解，使我们能够制定有针对性的策略来预防棘阿米巴角膜炎和改善隐形眼镜卫生习惯。从历史上看，棘阿米巴领域在采用遗传学和?...

视频: 作者聚焦：研究 棘阿米巴 的行为以制定预防 棘阿米巴 角膜炎的针对性策略

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

科研

免疫与感染

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

量化诱导剂梯度板上细菌表面聚集运动

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...

生物化学

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor impairment that worsens with time. While considerable advances have been made in determining genetic drivers of ALS for a subset of patients, the majority of cases have an unknown etiology. Further, the mechanisms underlying motor neuron dysfunction and degeneration are not well understood; therefore, there is an ongoing need to develop and characterize representative models to study these processes. Caenorhabditis elegans can adapt their movement to the physical constraints of their surroundings, with two primary movement paradigms studied in a laboratory environment- crawling on a solid surface and swimming in liquid. These represent a complex interplay between sensation, motor neurons, and muscles. C. elegans models of ALS can exhibit impairment in one or both of these movement paradigms. This protocol describes two sensitive assays for evaluating motility in C. elegans: an optimized radial locomotion assay measuring crawling on a solid surface and an automated method for tracking and analyzing swimming in liquid (thrashing). In addition to the characterization of baseline motor impairment of ALS models, these assays can detect suppression or enhancement of the phenotypes from genetic or small molecule interventions. Thus, these methods have utility for studying ALS&#160;models and any C. elegans strain that exhibits altered motility.

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

This protocol describes two sensitive assays for discriminating among mild, moderate, and severe motor impairment in C. elegans models of amyotrophic lateral sclerosis, with general utility for C. elegans strains, with altered motility.

秀丽隐杆线虫运动障碍评估 肌萎缩性侧索硬化症模型

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor ...

神经科学

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

准备，成像和定量的细菌表面运动性测定

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

生物学

Biophysical Characterization of Flagellar Motor Functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

鞭毛马达功能的生物物理表征

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

通过在系列中应用三种既定技术，调查大 肠杆菌 的弗拉盖拉驱动的动性

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes&#8212;many not associated with the oral apparatus (OA)&#8212;that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

We present a protocol for the characterization of motility and behavior of a population of hundred micron- to millimeter-sized cells using brightfield microscopy and cell tracking. This assay reveals that Stentor coeruleus transitions through four behaviorally distinct phases when regenerating a lost oral apparatus.

使用细胞追踪分析口腔器械再生期间和之后的张力模式

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds ...

Protocol for Analysis and Consolidation of TrackMate Outputs for Measuring Two-Dimensional Cell Motility using Nuclear Tracking

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To understand the regulation of cell motility, we must be able to measure it easily and consistently under different conditions. Here we describe a method for measuring and quantifying single-cell and bulk motility of HaCaT keratinocytes using a nuclear stain. This method includes a MATLAB script for analyzing TrackMate output files to calculate displacements, motility rates, and trajectory angles in single cells and in bulk for an imaging site. This motility analysis script allows for quick, straightforward, and scalable analysis of cell motility rates from TrackMate data and could be broadly used to identify and study the regulation of motility in epithelial cells. We also provide a MATLAB script for reorganizing microscopy videos collected on a microscope and converting them to TIF stacks, which can be analyzed using the ImageJ TrackMate plugin in bulk. Using this methodology to explore the roles of adherens junctions and actin cytoskeletal dynamics in regulating cell motility in HaCaT keratinocytes, we demonstrate evidence that Arp2/3 activity is required for the elevated motility seen after &#945;-catenin depletion in HaCaT keratinocytes.

This protocol for high-throughput measurement of cell motility in HaCaT keratinocytes describes methods for collecting and processing images of cell nuclei and performing particle tracking using the ImageJ plugin TrackMate.

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To ...

棘阿米巴属的定量运动性

副本

摘要

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此视频中的章节

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

量化诱导剂梯度板上细菌表面聚集运动

从酿酒酵母细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

秀丽隐杆线虫运动障碍评估肌萎缩性侧索硬化症模型

准备，成像和定量的细菌表面运动性测定

鞭毛马达功能的生物物理表征

通过在系列中应用三种既定技术，调查大肠杆菌的弗拉盖拉驱动的动性

使用细胞追踪分析口腔器械再生期间和之后的张力模式

Protocol for Analysis and Consolidation of TrackMate Outputs for Measuring Two-Dimensional Cell Motility using Nuclear Tracking

棘阿米巴属的定量运动性

副本

摘要

探索更多视频

此视频中的章节

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

量化诱导剂梯度板上细菌表面聚集运动

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

秀丽隐杆线虫运动障碍评估 肌萎缩性侧索硬化症模型

准备，成像和定量的细菌表面运动性测定

鞭毛马达功能的生物物理表征

通过在系列中应用三种既定技术，调查大 肠杆菌 的弗拉盖拉驱动的动性

使用细胞追踪分析口腔器械再生期间和之后的张力模式

Protocol for Analysis and Consolidation of TrackMate Outputs for Measuring Two-Dimensional Cell Motility using Nuclear Tracking

从酿酒酵母细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

秀丽隐杆线虫运动障碍评估肌萎缩性侧索硬化症模型

通过在系列中应用三种既定技术，调查大肠杆菌的弗拉盖拉驱动的动性