Riconosciamo consigli utili da Mingzhai Sole su cellule vincolanti alle superfici. Ringraziamo Ned Wingreen e zemer Gitai per le discussioni importanti. Questa ricerca è stata sostenuta dal National Institutes of Health concedere P50GM07150, National Science Foundation riconoscimento alla CARRIERA PHY-0844466 e la Fondazione Alfred P. Sloan.

<ol><li> Crescere le cellule di E. coli in brodo Luria-Beltrani (LB), medio-fase esponenziale (OD = 0,2-0,4). </li><li> Far crescere la coltura in LB integrata con 50 mg / ml di cefalexina per 15 minuti per indurre la crescita filamentosa, e poi concentrare la cultura di 5 volte tramite centrifugazione. </li><li> Fai PEI rivestite da copri scorre 1% polyethylenimine diluito in acqua in una camera di flusso, e lavare con acqua dopo 5 minuti di incubazione. </li><li> Flusso della cultura concentrato di cellule nella camera, e lavato con una miscela di LB e cefalexina (50μg/mL) dopo 3 min per rimuovere le cellule senza legami. </li><li> Incubare la camera a 37 ° C per 30 min-1 ora per far crescere le cellule attaccato prima di posizionarlo nello strumento ottico cattura. </li><li> Fai polilisina rivestite perle incubando 0,5 micron di diametro perle di polistirene (Labs Bangs) nel 0,1% polilisina diluito in acqua per 30 min. Quindi lavare le perline 3 volte e risospendere in acqua. </li><li> Diluire il tallone di un fattore due in LB con cefalexina (50μg/mL), e aggiungerlo nella camera di flusso. </li><li> Otticamente intercettare un galleggiante tallone e toccare alla punta libera di una cella. (Usiamo un Mad City Labs fase piezoelettrici per il controllo del moto del campione.) Quando una combinazione adatta perla / cellula viene trovato, lavare la camera con cefalexina in LB (50μg/mL) per rimuovere perline separati. </li><li> Eseguire programma appositamente scritto LabView di applicare forze di piegatura alla cella e registrare forza-spostamento dei dati. Dettagli del programma <ol class="lalpha"><li> Calibrare la risposta del rivelatore da raster scansione del tallone allegato all&#39;interno del raggio laser di rilevamento e la registrazione di segnali in tensione 3D PSD. </li><li> Identificare asse lungo cellula immagine al microscopio. </li><li> Spostare celle a passi in una direzione perpendicolare all&#39;asse lunga cella. </li><li> Registrare la distanza percorsa e lo spostamento dalla trappola ottica. </li><li> Conversione spostamento di forza applicata con la rigidità trappola precedentemente misurati e salvare spostamento punta e forza in un file. </li></ol></li></ol> Segreti di successo: Nel passaggio 8), si ha la necessità di trovare una cella con un ben definito fine bloccato. Alcune cellule sono semplicemente bloccati in una sola punta, e la forza di piega alla punta che conduce a un insieme di cellule girevole, piuttosto che piegarsi. Una coppia adatta si trova piegando ogni cella rapidamente a mano utilizzando il joystick controllato movimento palco. Rappresentante dei risultati: <img src="/files/ftp_upload/2012/2012fig1.jpg" alt="Figura 1" /> Figura 1. La figura mostra forza-spostamento di dati per una singola cella. La pendenza di questa linea è la rigidezza flessionale della cellula.

<ol>
	<li>Janmey, P. A., McCulloch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics:+integrating+cell+responses+to+mechanical+stimuli.">Cell mechanics: integrating cell responses to mechanical stimuli.</a> Annu Rev Biomed Eng. 9, 1-34 (2007).</li><li>Morris, D. M., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+biomechanical+understanding+of+whole+bacterial+cells.">Toward a biomechanical understanding of whole bacterial cells.</a> Annu Rev Biochem. 77, 583-613 (2008).</li></ol>

La produzione video di questo articolo è stato sponsorizzato da Mad Città Labs, che produce strumenti utilizzati in questi studi.

Il protocollo presentato qui è progettato per misurare quantitativamente la piegatura delle cellule batteriche. Il setup esperimento può essere applicato a qualsiasi forma di bastoncello cella che può essere fatto per crescere filamentously. Abbiamo usato con successo questa configurazione per sondare gli effetti dei filamenti del citoscheletro sulla rigidità alla flessione di E. coli cellule. Questa stessa tecnica può essere usata per valutare i ruoli di pressione, rigidità della parete cellulare e gli altri componenti intracellulari nella determinazione complessiva rigidità alla flessione delle cellule.

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<td>cephalexin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4895-5G</td>
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<td>polyethylenimine</td>
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<td>0.5-&mu;m-diameter polystyrene beads</td>
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<td>Bangs Laboratory</td>
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<td>Nano-LP Series nanopositioning system</td>
<td>&nbsp;</td>
<td>Mad City Labs</td>
<td>NanoLP series</td>
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measuring the bending stiffness of bacterial cells using an optical trap

Abbiamo sviluppato un protocollo per misurare la rigidità flessione di filamentose a forma di bastoncello batteri. Le forze sono applicate con una trappola ottica, un microscopico tridimensionale primavera fatta di luce che si forma quando ad alta intensità del fascio laser viene focalizzato in un punto molto piccolo da lente dell&#39;obiettivo di un microscopio. Per piegare una cella, per prima cosa si legano batteri vivi ad un trattato chimicamente coprioggetto. Poiché queste cellule crescono, la metà delle cellule rimane vincolato alla coprioggetto, ma le estremità in crescita sono liberi di questa restrizione. Inducendo crescita filamentosa con la cefalexina droga, siamo in grado di identificare le cellule in cui è stato bloccato un&#39;estremità della cellula per la superficie, mentre l&#39;altra estremità è rimasto libero e suscettibile alle forze di flessione. A forza di piega viene poi applicata con una trappola ottica di associazione di un polilisina rivestita tallone alla punta di una cellula in crescita. Sia la forza e lo spostamento del tallone sono registrati e la rigidezza flessionale della cellula è la pendenza di questa relazione.

Watch this Scientific Journal Video about Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap at JoVE.com

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

Vi presentiamo un protocollo per la piegatura di cellule batteriche filamentose attaccato ad una copertura antiscivolo della superficie di un dispositivo ottico per misurare la rigidità cellulare flessione.

Misurare la rigidezza flessionale di cellule batteriche Utilizzando una trappola ottica

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

measuring-bending-stiffness-bacterial-cells-using-an-optical

Research

JoVE Journal

Biology

12.0K Views.  Princeton University. Vi presentiamo un protocollo per la piegatura di cellule batteriche filamentose attaccato ad una copertura antiscivolo della superficie di un dispositivo ottico per misurare la rigidità cellulare flessione.

Video: Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Manutenzione e pulizia adeguata del microscopio

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Ricerca

Biologia

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

I componenti principali del microscopio ottico

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

Cryotomography elettrone di cellule batteriche

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Studiare gli effetti di rigidità Matrix sulla funzione cellulare utilizzando acrilamide a base di idrogeli

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Un test per misurare l&#39;attività di Escherichia coli Inducibile Lisina Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

Misurazione della Cinetica di Trascrizione mRNA nelle cellule viventi singolo

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Misurare gli effetti dei batteri su<em&gt; C. Elegans</em&gt; Comportamento utilizzando un saggio di ritenzione dell&#39;uovo

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Misurare il osmotica dell&#39;acqua Permeabilità Coefficiente (P<sub&gt; F</sub&gt;) Di celle sferiche: protoplasti impianto isolato come un esempio

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens&#39; optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens&#39; ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

Sequenziale Applicazione di vetrini di vetro per valutare la compressione Rigidità della lente Mouse: Strain e morfometriche analisi

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Misurare la rigidità<em&gt; Ex Vivo</em&gt; Mouse aorte Utilizzando microscopia a forza atomica

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...