I am grateful to Dr. Masaru Fujimoto for his technical suggestions for VAEM. I am also grateful to Prof. Koh Iba and Dr. Mimi Hashimoto-Sugimoto for providing GFP-PATROL1 transgenic plants, and discussions about PATROL1. I thank Prof. Seiichiro Hasezawa for his continuing support of my work. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 25711017.

1. Preparazione di Giovani pianta <ol><li> Sterilizzare i semi. <ol><li> Preparare la soluzione di sterilizzazione aggiungendo 500 microlitri NaClO (cloro disponibile: 5.0%) e 1 ml 10% Triton X-100 a 500 microlitri di acqua sterile. </li><li> Posto circa 10 transgenico A. thaliana semi trasportano GFP-PATROL1 8 in una provetta da 1,5 ml. </li><li> Aggiungere la soluzione di etanolo 1 ml di 70%, e mescolare bene capovolgendo cinque volte. Lasciare agire per 1 min. </li><li> Osservare i semi si depositano sul fondo della provetta. In una cappa a flusso laminare pulito, rimuovere delicatamente la etanolo al 70% con una micropipetta, e aggiungere 1 ml di soluzione di sterilizzazione. Mescolare bene capovolgendo cinque volte e lasciar riposare per 5 minuti. </li><li> Lavare i semi. Ancora lavorando sotto condizioni asettiche su un banco pulito, rimuovere delicatamente la soluzione con una micropipetta, e aggiungere 1 ml di acqua sterile. Ripetere questa operazione cinque volte. I semi sterilizzati possono essere conservati in acqua sterile a 4 ° C per 2 giorni. </li></ol></li><li> Cosìw i semi sterili su un 0,5% gellano gomma-solidificato 1/2 Murashige e Skoog piatto (pH 5,8) 9. Nastro il coperchio sulla piastra utilizzando due strati di nastro chirurgico. </li><li> Incubare la piastra al buio a 4 ° C con una cella frigorifera, O / N. </li><li> Trasferire la lastra in una camera di crescita fissato a 23,5 ° C con un / 12 hr-chiaro-scuro ciclo di 12 ore con 100 mmol m -2 s -1 luci bianche, e incubare per 7 giorni. Piantine con cotiledoni di circa 1 mm di lunghezza possono essere raccolte. </li></ol> 2. Cadutalibera Montaggio di Cotyledon campioni NOTA: Un fattore importante nella preparazione dei campioni per l&#39;osservazione VAEM è di evitare l&#39;inclusione di bolle d&#39;aria tra il campione e il vetro di copertura. Bolle riducono notevolmente la qualità delle immagini di VAEM provocando differenze nell&#39;indice di rifrazione. Un metodo semplice, che abbiamo chiamato &#39;cielo drop&#39; di montaggio, può essere utilizzato per evitare le bolletra la A. cotiledoni thaliana e il vetro di copertura. Questo dovrebbe essere fatto immediatamente prima osservazione. <ol><li> Mettere 30 ml di tampone basale [5 mM 2- (N -morpholino) -ethanesulfonic acid-Tris (MES-Tris) a pH 6,5, 50 mM KCl, 100 mM CaCl 2] al centro di un vetrino (dimensioni: 76 × 26 mm, spessore: 1,0-1,2 mm). </li><li> Rimuovere un cotiledone da una piantina 7 giorni di vita con le forbici dissezione. Float cotiledone con il lato rivolto verso l&#39;alto di osservazione sulla goccia basale tampone (Figura 1, fase 1). </li><li> Mettere 30 ml di tampone basale al centro di un vetro di copertura (dimensioni: 18 × 18 mm, spessore: 0,12-0,17 mm) (Figura 1, fase 2). Ruotare il vetro di copertura a testa in giù con delicatezza. La tensione superficiale impedisce la caduta di tampone di cadere. Tenere il vetro di copertura con una pinzetta su un bordo. Posizionare il bordo opposto sul vetrino modo che la goccia buffer è approssimativamente sopra cotiledone. </li><li>Con il bordo del vetro di copertura ancora sul vetrino, e ancora tenendo il bordo opposto con pinzette, regolare la posizione della goccia tampone sotto il vetro di copertura in modo che sia direttamente sopra il campione cotiledone (Figura 1, fase 3). Lasciate andare il vetro di copertura. Montare una goccia sul campione risultante in un preparato senza bolle d&#39;aria. </li><li> Pulire il tampone in eccesso utilizzando tessuti privo di lanugine. Osservare immediatamente la preparazione (vedi punto 3). NOTA: Non è necessario sigillare i campioni. </li></ol> 3. VAEM Osservazione e Movie acquisizione NOTA: Il sistema di microscopia TIRF 9 utilizzati nel presente studio è descritto come segue: Un microscopio invertito è dotato di una unità TIRF e una lente obiettivo TIRF con un&#39;apertura numerica di 1,49. Per il controllo computerizzato del dell&#39;angolo di ingresso del laser, viene utilizzata una scatola di controllo. Proteina fluorescente verde (GFP) è eccitato con un laser a semiconduttore 488 nm pompaggio ottico, e tegli fluorescenza viene rilevata attraverso un filtro passa-banda 510-550 nm per impedire autofluorescenza da cloroplasti. Il valore massimo misurato della potenza di uscita fibra è 13,0-13,5 mW. Per il rilevamento, un elettrone moltiplicando charge-coupled device (EM-CCD) Sistema di testa macchina fotografica e di un C-mount unità cambiamento di ingrandimento della fotocamera sono utilizzati. <ol><li> Calibrare il laser centratura e messa a fuoco in base alle istruzioni del produttore. NOTA: Questo passaggio è fondamentale per precise osservazioni VAEM. Si raccomanda vivamente che i controlli periodici delle procedure di regolazione percorso laser, appropriata al microscopio, vengono effettuati. <ol><li> Determinare la posizione centrale illuminato con una luce obiettivo percorso senza lente sul soffitto della stanza microscopia. Per segnare la posizione centrale, mettere un sigillo cerchio colorato sul soffitto (figura 2, punto 1, 2). </li><li> Illuminare il soffitto con una lente obiettivo (Figura 2, fase 3, 4). Spostare l&#39;illuminated regione sulla posizione centrale (figura 2, punto 5). Mettere a fuoco il laser (figura 2, punto 6). Mettere a punto la posizione del laser focalizzato al centro (Figura 2, punto 7). </li></ol></li><li> Impostare un preparato sul tavolino del microscopio e selezionare le celle di osservazione in illuminazione campo chiaro. </li><li> Verificare che la proteina fluorescente può essere osservato nelle cellule, e impostare la posizione z -axis sulla superficie cellulare mediante illuminazione epifluorescenza (Figura 3A). </li><li> Eseguire osservazioni VAEM. <ol><li> Inclinare l&#39;angolo di ingresso del fascio laser gradualmente con la scatola del regolatore. Allo stesso tempo, controllare l&#39;immagine inquadrata accuratamente. Inizialmente, l&#39;immagine sarà offuscata (Figura 3B). </li><li> Quando l&#39;angolo laser aumenta, l&#39;immagine VAEM diventerà meno offuscata, fino a produrre una chiara immagine (Figura 3C e D). A questo punto, interrompere Increacantare l&#39;angolo del laser. Se il segnale di fluorescenza viene perso, ridurre ad un angolo poco profondo. </li><li> Ottimizzare l&#39;angolo di ingresso del laser per ottenere una migliore immagine. Messa a punto della posizione z -axis può anche migliorare l&#39;immagine. Se necessario, regolare i parametri ottici (potenza del laser, lunghezza d&#39;onda e filtro scelto) e parametri del sensore immagine [dimensioni dell&#39;immagine, tempo di esposizione, guadagno, digitizer e-elettroni moltiplicando (EM) di guadagno]. NOTA: Nel caso delle apparecchiature microscopio TIRF descritto sopra, i parametri rappresentativi sono i seguenti. Ingrandimento dell&#39;obiettivo proprio di fronte alla macchina da presa: 2X; Uscita laser: 1,0 mW; Dimensione immagine: 512 × 512 pixel; Tempo di esposizione: 100 msec; Guadagno: 5 ×; Digitizer: 11 MHz; e EM guadagno: 100. </li></ol></li><li> Acquisire un filmato come file TIFF immagine di più pagine tramite il software del microscopio commerciale. Qui, il film è di punti GFP-etichettata lampeggianti sulla superficie delle cellule stomatici. NOTA: Le condizioni di acquisizione di immagini rappresentative sono come folbassi. Modalità acquisizione: Flusso di RAM; Numero di fotogrammi: 600; e la dimensione del file TIFF multi-page: ~ 302 MB. </li></ol> 4. Analisi chimografo per la quantificazione di GFP-etichettata Dot Residence tempo usando Fiji Software <ol><li> Installare Fiji (&#39;Fiji è solo ImageJ&#39;) software 10 utilizzando le istruzioni degli autori (http://fiji.sc/Fiji). </li><li> Eseguire Fiji e aprire il file TIFF a più pagine acquisite tramite il menu Fiji &quot;File-Open&quot;. </li><li> Posizionare una linea sul sito di interesse, utilizzando il menu barra degli strumenti Fiji &quot;Straight Line Selection Tool&quot;. Facoltativamente, utilizzare la funzione &quot;segmentato Linea Selection Tool&quot; o &quot;a mano libera linea strumento di selezione&quot;. Nei risultati presentati qui, una linea retta di 100 pixel (corrispondenti a 8,0 micron) è stato posto su una cella controllata (Figura 4A). </li><li> Fare una immagine chimografo usando il menu Fiji &quot;Immagine-Stack-Dynamic reslice </em&gt; &quot;(http://fiji.sc/Dynamic_Reslice) (Figura 4B). Opzionalmente, &quot;Rifletti verticalmente&quot; e &quot;ruotare di 90 gradi&quot; in una finestra di casella di controllo sono disponibili (non utilizzato nei risultati presentati qui). La x e y l&#39;immagine chimografo nella indicano la posizione della linea (100 pixel corrispondenti a anche 8.0 micron) e tempo di osservazione (600 pixel corrispondenti a 60 sec), rispettivamente. Se l&#39;immagine chimografo non è soddisfacente, la posizione e il tipo (dritto, segmentati e mano libera) della linea sull&#39;immagine più pagine possono essere modificate. Per quanto i cambiamenti di linea, l&#39;immagine cambia in modo dinamico chimografo. Salvare una immagine chimografo come file TIFF utilizzando il menu Fiji &quot;File-Salva con nome-Tiff&quot;. </li><li> Misurare la lunghezza del blob nell&#39;orientamento dell&#39;asse tempo. <ol><li> Per eseguire la riduzione del rumore, applicare un filtro gaussiano alle immagini chimografo utilizzando il menu Fiji &quot;Process-Filtri-Controllo sfocatura&quot;. La &quot;Sigma (Radius) &quot;parametro è regolabile (1 raggio pixel viene utilizzato per i risultati presentati). </li><li> Segmento le regioni del segnale di soglia, utilizzando il menu Fiji &quot;Immagine-Adjust-soglia&quot;. NOTA: Ci sono diversi algoritmi di thresholding tra cui scegliere. Nei risultati presentati, l&#39;algoritmo &quot;Yen&quot; è stato scelto (Figura 4C). </li><li> Preparatevi a misurare il tempo (y) -axis lunghezza del blob utilizzando il menu &quot;Analizza-Set misure&quot;. Controllare il &quot;rettangolo di delimitazione&quot; nella finestra &quot;Imposta misure&quot;. Idealmente il set area dovrebbe essere il più piccolo possibile, escludere rumore. Qui, la superficie minima è stata fissata a 50 pixel. Misurare il tempo (y) -axis lunghezza del blob utilizzando il menu &quot;Analizza Analizza-particelle&quot;. Per ottenere i valori dei risultati e dimostrare la credibilità delle regioni di misura, controllare i &quot;Risultati&quot; e &quot;Aggiungi a Gestione &lt;/ em&gt; &quot;scatole. </li><li> Valori nella colonna &quot;Altezza&quot; sono il tempo (y) -axis lunghezze per il blob misurato (Figura 4D). Salvare i valori utilizzando il menu tabella dei risultati &quot;File-Salva con nome&quot; e calcolare ed elaborare il set di dati di durate immagine blob utilizzando un pacchetto statistico e / o software foglio di calcolo (Figura 4E). </li></ol></li></ol>

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L&#39;autore non ha nulla da rivelare.

In questo articolo video, protocolli sono dati per monitorare e misurare il comportamento dinamico di punti GFP-PATROL1 sul complesso stomatico di Arabidopsis thaliana. Come mostrato qui, l&#39;osservazione VAEM è uno strumento potente per l&#39;imaging dal vivo di superficie delle cellule delle piante. Nelle condizioni sperimentali utilizzate qui per il monitoraggio GFP-PATROL1, c&#39;era molto poco photobleaching fluorescenza nel campione utilizzato per 1 min di cattura video, perché altamente sensibile EM-...

The plant cell surface, including the plasma membrane and its immediately adjacent cytoplasm, is the main region of a plant cell&#8217;s perception and integration of biotic and abiotic cues from the extracellular environment. In response to these cues, cell surface components including plasma membrane proteins and the cortical cytoskeleton undergo dynamic changes, on a time scale of seconds to minutes1-4. Thus, real-time and high-resolution imaging of fluorescent proteins on the cell surface can illuminate a plant&#8217;s responses to environmental cues at the molecular level.
Confocal laser scanning microscopy is a powerful tool for determination of fluorescently-tagged protein localization3, however, it is often difficult to monitor the real-time protein dynamics because of its relatively long capturing times. An emerging technique for real-time monitoring of proteins in the plant cell is variable angle epifluorescence microscopy (VAEM), which is an adaptation of equipment usually used for total internal reflection fluorescence (TIRF) microscopy. In TIRF microscopy, the fluorescence-excitation light source is an evanescent light field that is generated when the entry angle of the laser is shallow enough to totally internally reflect light at the glass&#8211;water interface. The penetration depth of the evanescent light field is around 100 nm. TIRF microscopy is an outstanding tool for single molecule imaging, such as the detection of exocytosis in animal cells5. However, evanescent light cannot reach plasma membranes or the cortical cytoplasm in plant cells, because they have thick cell walls. Recently, TIRF microscopy equipment has been adapted by plant cell biologists, observing that a laser, if angled slightly more deeply than when being used to induce total internal reflection phenomena, could excite the surface of plant cell samples, resulting in high-quality plant cell imaging6,7. The excitation illumination depth is varied by adjusting the entry angle of the laser; therefore, this technique is described as VAEM. This optical system is also called variable angle TIRF microscopy (VA-TIRFM) because there is a possibility that total reflection may take place at the cell wall-periplasm interface7, however, the term VAEM is used in this article, as per the first report in plants6.
The goal of this protocol is to demonstrate practical procedures for using VAEM to visualize fluorescently-tagged protein dynamics on plant cell surfaces. Additionally, an image analysis protocol to quantify the residence time (duration of presence) of molecules is described for VAEM movie analysis. GFP-PATROL1 dot blinking on stomatal complex cells in Arabidopsis thaliana cotyledons is used as an example. PATROL1 was identified by forward genetic approaches as a causal gene of a stomatal response defect mutant in A. thaliana8. PATROL1 is a plant homolog of MUNC-13, which is a priming factor in synapse vesicle exocytosis8. In response to environmental cues, such as light or humidity, it is thought that PATROL1 reversibly regulates the delivery of a proton pump to plasma membranes in the stomatal complex. Stomatal complexes each comprise a pair of guard cells8 and subsidiary cells9, and they require a proton pump for stomatal movement. In these cells, GFP-tagged PATROL1 localizes to dot-like structures that remain on the plasma membrane for less than 1 min9.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX-73</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TIRF unit</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX3-RFAEVAW</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TIRF objective lens&#160;</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">UAPON 100 &#215; OTIRF&#160;</td>
			<td>NA = 1.49</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser angle control box</td>
			<td style="width:71px;">Chuo Seiki</td>
			<td style="width:119px;">QT-AK</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Optically pumped semiconductor laser</td>
			<td style="width:71px;">Coherent</td>
			<td style="width:119px;">SapphireTM LP USB 488-20 CDRH Laser</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">510&#8211;550 nm band-pass filter</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">U-FBNA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EM CCD camera</td>
			<td style="width:71px;">Hamamatsu Photonics</td>
			<td style="width:119px;">ImagEM C9100-13</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C-mount camera magnification change unit&#160;</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">U-TVCAC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MetaMorph software</td>
			<td style="width:71px;">Molecular Devices</td>
			<td style="width:119px;">MetaMorph version 7.7.11.0</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">TIRF microscopy manual</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">AX7385</td>
			<td style="width:167px;">Instructions: Total Internal Reflection Illumination System (Printed in Japan on August 24, 2012)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Immersion oil</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">Immersion Oil Typr-F</td>
			<td>ne = 1.518 (23 degrees)</td>
		</tr>
	</tbody>
</table>

real-time imaging of plant cell surface dynamics with variable-angle epifluorescence microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

In questo articolo il video, i protocolli per l&#39;osservazione VAEM di GFP-PATROL1 in A. sono forniti cellule complesse thaliana cotyledon stomatiche. Montaggio Sky goccia è un semplice metodo di preparazione che può aiutare a ridurre la comparsa di bolle d&#39;aria in preparazioni VAEM di A. cotiledoni thaliana (Figura 1). Un&#39;inclinazione eccessiva del laser ingresso e / o Z-posizionamento di campioni per VAEM fornirà un&#39;immagine chiara. Se ciò accade, ...

Watch this Scientific Journal Video about Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy at JoVE.com

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

L&#39;obiettivo di questo protocollo è quello di dimostrare come monitorare la dinamica delle proteine ​​fluorescenti-taggato sulla superficie delle cellule di piante con variabile angolo epifluorescenza, mostrando punti di GFP-tagged PATROL1, una proteina del traffico di membrana a lampeggiare, nella corteccia cella del complesso stomi in Arabidopsis thaliana.

In tempo reale Imaging di Pianta Dynamics cellulare con superficie variabile angolo epifluorescenza Microscopia

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

real-time-imaging-plant-cell-surface-dynamics-with-variable-angle

Research

JoVE Journal

Biology

8.9K Views.  The University of Tokyo.  L'obiettivo di questo protocollo è quello di dimostrare come monitorare la dinamica delle proteine ​​fluorescenti-taggato sulla superficie delle cellule di piante con variabile angolo epifluorescenza, mostrando punti di GFP-tagged PATROL1, una proteina del traffico di membrana a lampeggiare, nella corteccia cella del complesso stomi in Arabidopsis thaliana. 

Video: Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Imaging cellulare dal vivo di F-actina Dynamics tramite microscopia a fluorescenza Speckle (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Ricerca

Biologia

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Assemblaggio, carico, allineamento e di una cella analitica del campione Ultracentrifuga

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Avidità-based Screening Interaction extracellulare (AVEXIS) per il rilevamento scalabile di bassa affinità extracellulare del recettore-ligando interazioni

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

Tandem congelamento ad alta pressione e congelamento rapido Sostituzione di tessuti vegetali per la microscopia elettronica a trasmissione

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Le misurazioni in tempo reale delle Membrane Protein: recettore interazioni Uso risonanza plasmonica di superficie (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Real Time Analisi di profilo metabolico in<em&gt; Ex Vivo</em&gt; Culture del mouse intestinale Cripta organoide

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizzazione Frequenza Stromule con microscopia a fluorescenza

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

Luce Foglio microscopia a fluorescenza delle radici delle piante che crescono sulle superficie di un gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

Flash-e-Freeze: una tecnica innovativa per l&#39;acquisizione di membrana Dynamics con microscopia elettronica

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Microscopia confocale rivela l'aggregazione di recettore di superficie delle cellule tramite spettroscopia di correlazione di immagine

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...