This work was supported by NIH R01 NS042228 to M.C.H. The Olympus FV1000 confocal was acquired with an NIH shared instrumentation grant S10RR023717 to the UW Zoology Department (PI Bill Bement).
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this paper.

1. Assembly of injection slides and imaging slides
Injection slides:
<ol>
	<li>Prepare Sylgard silicone elastomer according to manufacturer's instructions.</li>
<li>Use the Sylgard to bond three standard glass microscope slides together by stacking one slide on top of the intersection of the other two slides, which are arranged side-by-side at the long edges. The top slide creates a right-angled corner or "wall" between the top and bottom slides.</li>
<li>Let Sylgard set overnight. These slides are reusable.</li>
</ol>
Imaging slide:
<ol>
	<li>We use stainless steel rectangles cut to the same size of a standard glass microscope slide (75mm X 25mm X 1mm) so the chamber fits the slide holder on the microscope stage. A 1.5 cm hole is cut in the center of the rectangle.</li>
	<li>Affix a 22 mm2 coverslip to the metal rectangle with Sylgard. Use just enough Sylgard to thoroughly seal the coverslip. To image on an inverted microscope, embryos will be mounted on this coverslip and imaged from the bottom.</li>
	<li>Let Sylgard set overnight. These slides are reusable and cleaned with 70% ethanol between uses.</li>
</ol>
2. Preparation of DNA constructs
To express a transgene in a tissue-specific manner, the gene of interest is cloned behind a cell-specific promoter. We use a cis-regulatory element of the zebrafish neurogenin1 gene (-3.1ngn1)2 or the sox10 gene (-4.9sox10)3 to drive expression in sensory neurons or neural crest cells, respectively. To visualize cell and membrane dynamics, we express cytoplasmic or membrane-localized fluorophores. To image F-actin distribution, we express DNA encoding the calponin homology domain of utrophin fused to mCherry (UtrCH-mCherry). The UtrCH-mCherry biosensor probe allows visualization of F-actin without interfering with actin dynamics1. We generate these DNA constructs using the Invitrogen Gateway recombination-based cloning strategy4 and vectors provided in the zebrafish Tol2kit5.
<ol>
	<li>Generate expression vector(s) using Multisite-Gateway Technology and vectors from the zebrafish Tol2kit5. Plasmids contain the Tol2 backbone from pT2KXIGDin.</li>
	<li>Purify plasmid DNA with QIAfilter Plasmid Midi Prep kit (QIAGEN Inc.) and elute in sterile water. DNA does not need to be linearized for injection.</li>
</ol>
3. Injection of DNA constructs
<ol>
 <li>Dilute the DNA to a final concentration of 10-50 &mu;g/ml DNA in 0.2% phenol red (for visualization during injection) in water.</li>
 <li>Fill and calibrate needle: Back-fill a pulled capillary needle with approximately 1 &mu;l DNA solution and insert it into the needle holder of the injection apparatus (we use a Picospritzer). Using a fine forceps, break the tip off the needle such that the tip opening is approximately 10 mm in diameter. Measure the diameter of the droplet in mineral oil with an ocular micrometer. Adjust pressure duration setting to get a droplet of approximately 1 nl volume.</li>
 <li>Collect 1-cell stage embryos in E3 embryo medium (5mM NaCl, 0.17mM KCl, 0.33mM CaCl2, 0.33mM MgSO4*7H2O, pH 7.0).</li>
 <li>Transfer approximately 30-60 embryos to the injection slide and position the embryos in the right angle corner formed between the top and bottom slides. Remove most of the E3 so that the embryos are held to the slide by surface tension.</li>
 <li>Use a bent dissecting pin to rotate embryos so the cell is against the wall of the injection slide.</li>
 <li>Use a micromanipulator to guide the needle through the embryo chorion, through the yolk and into the single cell of the embryo. Inject 0.5-1 nl DNA solution into the cell.</li>
 <li>Transfer the embryos into Petri dishes in E3 and incubate at 28.5&deg;C. If necessary, development can be slowed by incubating embryos at lower temperature.</li>
</ol>
4. Mounting embryos for imaging
<ol>
	<li>Remove the chorions from the embryos with fine forceps about an hour before embryos reach the desired age for imaging (approximately 14-17 hours post-fertilization for our purposes).</li>
 <li>Select embryos with labeled cells using a dissecting microscope equipped with fluorescence. We use a 4X objective (40X magnification) on a Nikon AZ100 scope because of its combination of long working distance and high magnification.</li>
 <li>Place an embryo into a microfuge tube with approximately 50 &mu;l E3 containing 0.2% Tricaine anesthetic (10X concentration).</li>
 <li>Add 500 &mu;l 1% low melting point agarose in E3 with 10 mM HEPES (kept at 42&deg;C), diluting the Tricaine to a final concentration of 0.02%. </li>
 <li>Immediately transfer the embryo in an agarose drop to the coverslip of the imaging slide using a wide bore glass pipette. </li>
 <li>Before the agarose hardens, position the embryo so the region with labeled cells is facing down against the bottom coverslip (dorsal down for neural crest cells and dorsolateral down for spinal sensory neurons).</li>
 <li>Assemble imaging chamber: Coat one side of a plastic ring (2 cm outer diameter, 3 mm high) with silicone vacuum grease and affix to the metal imaging slide. Coat the other side of the ring with grease. </li>
 <li>Fill the chamber with E3 containing 10 mM HEPES and 0.02% Tricaine. Seal the chamber top by affixing a 22 mm2 coverslip to the top greased surface of the ring.</li>
</ol>
5. 4D imaging of cell behaviors on Olympus FV1000 confocal with IX81 microscope
The details of image acquisition will depend on the specifics of your microscope system. Here we describe the time-lapse acquisition process for the Olympus FV1000 confocal microscope. See previous JoVE protocol for time-lapse acquisition with the Zeiss LSM 510 confocal system6.
<ol>
 <li>Place the imaging chamber into the slide holder on the microscope stage and focus on the embryo using a 10X or 20X objective under transmitted light. </li>
 <li>Switch to a 60X objective (N.A. of 1.2 or higher) and focus on labeled cell with epifluorescence. We use either an oil immersion (UPLSAPO 60xO N.A. 1.35) or water immersion (UPLSAPO 60xW N.A. 1.20) lens.</li>
 <li>In the FV software (Ver 1.7c), click on XY repeat to begin laser scanning. </li>
 <li>Adjust acquisition settings and image acquisition controls (laser output, scan speed, HV, gain, and offset levels) to optimize signal while keeping laser power at a minimum (25% or lower) to prevent tissue damage and reduce photobleaching. </li>
 <li>To acquire a Z-series, set the upper and lower limits of the z-stack, along with step size. Make sure the Depth icon is engaged so that the XY Sweep icon has XY and Z highlighted. Start acquisition by clicking the XYZ Sweep icon.</li>
	<li>To acquire a time-lapse series, set the time interval and number of time points under the TimeScan heading in the Acquisition Settings window. Click the Time Icon in the Image Acquisition Control Window so that the XY Sweep icon has XY(Z) and T highlighted. Start acquisition by clicking the XY(Z)T Sweep icon.</li>
	<li>Alternatively, the TimeController can be used to set up a time-lapse. This programming is useful when generating large data sets, as it does not exceed memory allocation. Under Device, open TimeController window. Create a new Imaging Task. The image parameter window will open. Click the FV Status button to update acquisition settings. Make sure Save and Delete are checked under the Terminate heading. Set output file name and click OK to save settings and close the window. Use the Loop function to set the desired time interval and number of time points. Click Ready and Start to begin acquisition. The Pause and Z-shift functions can be used to refocus while imaging.</li>
</ol>
6. Representative Results
The figures and movies depict examples of sensory neurons and neural crest cells labeled with membrane-targeted fluorophores or the actin biosensor probe.
<img src="/files/ftp_upload/1726/1726fig1.jpg" alt="Figure 1" /> Figure 1. Confocal images (z-projections) of a Tg(ngn1:gfp-caax) transgenic embryo injected with 25 pg ngn1:mCherry-caax DNA. The mCherry-CAAX labels one neuron red (A) in a background with all Rohon-Beard sensory neurons labeled green (B). C) Merged image of both green and red channels. Scale bar = 50 &mu;m.
<img src="/files/ftp_upload/1726/1726fig2.jpg" alt="Figure 2" /> Figure 2. Confocal z-projections of neurons in embryos injected with ~20 pg ngn1:mCherry-UtrCH DNA. A) Rohon-Beard sensory neuron in 16.5 hpf embryo. F-actin signal is strong in growth cones (arrowheads) and in protrusions along newly formed axons. B) Neuron in 20 hpf embryo showing strong F-actin signal in growth cones of branched peripheral axon (arrowheads) and weaker signal in more mature central axons (arrows). Scale bar = 50 &mu;m.

<ol>
	<li>Burkel, B. M., von Dassow, G., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+fluorescent+probes+for+actin+filaments+based+on+the+actin-binding+domain+of+utrophin.">Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.</a> Cell motility and the cytoskeleton. 64, 822-832 (2007).</li><li>Blader, P., Plessy, C., Strahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+regulatory+elements+with+spatially+and+temporally+distinct+activities+control+neurogenin1+expression+in+primary+neurons+of+the+zebrafish+embryo.">Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo.</a> Mech Dev. 120, 211-218 (2003).</li><li>Wada, N., Javidan, Y., Nelson, S., Carney, T. J., Kelsh, R. N., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132, 3977-3988 (2005).</li><li>Cheo, D. L., Titus, S. A., Byrd, D. R., Hartley, J. L., Temple, G. F., Brasch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+assembly+and+cloning+of+multiple+DNA+segments+using+in+vitro+site-specific+recombination:+functional+analysis+of+multi-segment+expression+clones.">Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones.</a> Genome Res. 14, 2111-2120 (2004).</li><li>Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E., Campbell, D. S., Parant, J. M., Yost, H. J., Kanki, J. P., Chien, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev Dyn. 236, 3088-3099 (2007).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J Vis Exp. 24, (2009).</li><li>Berndt, J. D., Clay, M. R., Langenberg, T., Halloran, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-kinase+and+myosin+II+affect+dynamic+neural+crest+cell+behaviors+during+epithelial+to+mesenchymal+transition+in+vivo.">Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo.</a> Dev Biol. 324, 236-244 (2008).</li></ol>

The optimal concentration of injected DNA will vary depending on size and strength of the promoter construct and should be determined empirically. Injection of too much DNA can lead to unhealthy embryos with extensive cell death, while too little will result in a very small proportion of injected embryos expressing the transgene. The DNA expression level correlates with the strength of the fluorophore signal, which varies from cell to cell. While sorting embryos under epifluorescence, exclude those with extremely high le...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5040-250G</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard Silicone Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corporation</td>
<td>184</td>
<td>&nbsp;</td>
<tr>
<td>QIAfilter Plasmid Midi Kit</td>
<td>&nbsp;</td>
<td>Qiagen Inc.</td>
<td>12243</td>
<td>&nbsp;</td>
<tr>
<td>Low melting point agarose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15517-014</td>
<td>&nbsp;</td>
<tr>
<td>Picospritzer</td>
<td>&nbsp;</td>
<td>Parker Instrumentation, General Valve Division</td>
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live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

Watch this Scientific Journal Video about Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos at JoVE.com

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

live-imaging-cell-motility-actin-cytoskeleton-individual-neurons

Research

JoVE Journal

Biology

13.3K 조회수.  University of Wisconsin-Madison. 이 프로토콜은 zebrafish 배아를 생활에서 개별 뉴런 또는 신경 능선 세포의 이미징을 설명합니다. 이 방법은 형광 공촛점 시간 경과 현미경을 사용하여 셀룰러 행동과 굴지의 현지화을 검사하는 데 사용됩니다.

비디오: Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

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).

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 ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...