Name: multi-color localization microscopy of single membrane proteins in organelles of live mammalian cells
Uploaded: 2018-06-30T13:30:00
Description: Watch this Scientific Journal Video about Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells at JoVE.com

The authors would like to thank the Biophysics group and Jacob Piehler at the University of Osnabr&#252;ck for continuous support, Wladislaw Kohl for technical assistance and preparation of material, and the CellNanOs board for providing microscopes for use. The project was funded by the SFB 944.

The following protocol follows the guidelines of the local institution research ethics committee.
1. Methods
<ol>
	<li>Cell culture
	<ol>
		<li>Cultivate cells, for example HeLa cells (human cervix carcinoma), in a T25 cell culture flask containing 5 mL of growth medium at 37 &#176;C and 5% CO2. 
		NOTE: For imaging, split the cells onto prepared coverslips (see steps 1.3 and 1.4) and keep in imaging medium.</li>
	</ol>
	</li>
	<li>Cell transfection</...

<ol>
	<li>Appelhans, T., Richter, C., Wilkens, V., Hess, S., Piehler, J., Busch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+organization+of+mitochondrial+microcompartments+revealed+by+combining+tracking+and+localization+microscopy.">Nanoscale organization of mitochondrial microcompartments revealed by combining tracking and localization microscopy.</a> Nano Letters. 12 (2), 610-616 (2012).</li><li>Appelhans, T., Busch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Molecule+Tracking+and+Localization+of+Mitochondrial+Protein+Complexes+in+Live+Cells.">Single Molecule Tracking and Localization of Mitochondrial Protein Complexes in Live Cells.</a> Methods Mol Biol. 1567, 273-291 (2017).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat Methods. 3 (10), 793-795 (2006).</li><li>Gould, T. J., Verkhusha, V. V., Hess, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+biological+structures+with+fluorescence+photoactivation+localization+microscopy.">Imaging biological structures with fluorescence photoactivation localization microscopy.</a> Nat Protoc. 4 (3), 291-308 (2009).</li><li>Pennacchietti, F., Gould, T. J., Hess, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Probe+Photophysics+in+Localization-Based+Superresolution+Microscopy.">The Role of Probe Photophysics in Localization-Based Superresolution Microscopy.</a> Biophys J. 113 (9), 2037-2054 (2017).</li><li>Wegel, E., G&#246;hler, A., Lagerholm, B. C., Wainman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cellular+structures+in+super-resolution+with+SIM,+STED+and+Localisation+Microscopy:+A+practical+comparison.">Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.</a> Scientific reports. , (2016).</li><li>Pellett, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+STED+microscopy+in+living+cells.">Two-color STED microscopy in living cells.</a> Biomedical Optics Express. 2 (8), (2011).</li><li>Ishigaki, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+super-resolution+imaging+of+mitochondria+labeled+with+TMRM+in+living+cells.">STED super-resolution imaging of mitochondria labeled with TMRM in living cells.</a> Mitochondrion. 28, 79 (2016).</li><li>Liss, V., Barlag, B., Nietschke, M., Hensel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-labelling+enzymes+as+universal+tags+for+fluorescence+microscopy,+super-resolution+microscopy+and+electron+microscopy.">Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy.</a> Scientific Reports. 5, 17740 (2015).</li><li>Serg&#233;, A., Bertaux, N., Rigneault, H., Marguet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+multiple-target+tracing+to+probe+spatiotemporal+cartography+of+cell+membranes.">Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes.</a> Nat Methods. 5 (8), (2008).</li><li>Appelhans, T., Busch, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+mitochondrial+membrane+proteins+in+specific+sub-organelle+membrane+locations.">Dynamic imaging of mitochondrial membrane proteins in specific sub-organelle membrane locations.</a> Biophysical reviews. 9 (4), 345-352 (2017).</li><li>Wilmes, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triple-color+super-resolution+imaging+of+live+cells:+resolving+submicroscopic+receptor+organization+in+the+plasma+membrane.">Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane.</a> Angewandte Chemie. 51 (20), 4868-4871 (2012).</li><li>Niewidok, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+imaging+reveals+dynamic+biphasic+partition+of+RNA-binding+proteins+in+stress+granules.">Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules.</a> J Cell Biol. , (2018).</li><li>Wurm, C. A., Jakobs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+protein+distributions+define+two+sub-compartments+of+the+mitochondrial+inner+membrane+in+yeast.">Differential protein distributions define two sub-compartments of the mitochondrial inner membrane in yeast.</a> FEBS Lett. 580 (24), 5628-5634 (2006).</li><li>Schmidt, R., Wurm, C. A., Punge, A., Egner, A., Jakobs, S., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+cristae+revealed+with+focused+light.">Mitochondrial cristae revealed with focused light.</a> Nano Lett. 9 (6), 2508-2510 (2009).</li><li>Kukat, C., Wurm, C. A., Spahr, H., Falkenberg, M., Larsson, N. G., Jakobs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+reveals+that+mammalian+mitochondrial+nucleoids+have+a+uniform+size+and+frequently+contain+a+single+copy+of+mtDNA.">Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (33), 13534-13539 (2011).</li><li>Beinlich, F., Drees, C., Piehler, J., Busch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shuttling+of+PINK1+between+Mitochondrial+Microcompartments+Resolved+by+Triple-Color+Superresolution+Microscopy.">Shuttling of PINK1 between Mitochondrial Microcompartments Resolved by Triple-Color Superresolution Microscopy.</a> ACS chemical biology. 10 (9), 1970-1976 (2015).</li><li>Shim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+fluorescence+imaging+of+organelles+in+live+cells+with+photoswitchable+membrane+probes.">Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes.</a> Proceedings of the National Academy of Sciences. 109 (35), 13978-13983 (2012).</li><li>Sbalzarini, I., Mezzacasa, A., Helenius, A., Koumoutsakos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Organelle+Shape+on+Fluorescence+Recovery+after+Photobleaching.">Effects of Organelle Shape on Fluorescence Recovery after Photobleaching.</a> Biophysical Journal. 89 (3), 1482-1492 (2005).</li><li>Reits, E., Neefjes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+fixed+to+FRAP:+measuring+protein+mobility+and+activity+in+living+cells.">From fixed to FRAP: measuring protein mobility and activity in living cells.</a> Nature Cell Biology. 3 (6), E145-E147 (2001).</li><li>Goehring, N., Chowdhury, D., Hyman, A., Grill, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+Analysis+of+Membrane-Associated+Proteins:+Lateral+Diffusion+and+Membrane-Cytoplasmic+Exchange.">FRAP Analysis of Membrane-Associated Proteins: Lateral Diffusion and Membrane-Cytoplasmic Exchange.</a> Biophysical Journal. 99 (8), 2443-2452 (2010).</li><li>Bacia, K., Haustein, E., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+correlation+spectroscopy:+principles+and+applications.">Fluorescence correlation spectroscopy: principles and applications.</a> Cold Spring Harbor protocols. 2014 (7), 709-725 (2014).</li><li>Sukhorukov, V., Dikov, D., Busch, K., Strecker, V., Wittig, I., Bereiter-Hahn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+protein+mobility+in+mitochondrial+membranes+of+living+cells.">Determination of protein mobility in mitochondrial membranes of living cells.</a> Biochimica et biophysica acta. 1798 (11), 2022-2032 (2010).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Anal Bioanal Chem. 397 (8), 3173-3178 (2010).</li><li>Graham, F. L., van der Eb, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+assay+of+infectivity+of+human+adenovirus+5+DNA.">A new technique for the assay of infectivity of human adenovirus 5 DNA.</a> Virology. 52 (2), 456-467 (1973).</li><li>Wedeking, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+Controlled+Reorganization+of+Signaling+Complexes+in+the+Plasma+Membrane+of+Living+Cells.">Spatiotemporally Controlled Reorganization of Signaling Complexes in the Plasma Membrane of Living Cells.</a> Small. 11 (44), 5912-5918 (2015).</li><li>Mironov, S. L., Ivannikov, M. V., Johansson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Ca2+]i+signaling+between+mitochondria+and+endoplasmic+reticulum+in+neurons+is+regulated+by+microtubules.+From+mitochondrial+permeability+transition+pore+to+Ca2+-induced+Ca2++release.">[Ca2+]i signaling between mitochondria and endoplasmic reticulum in neurons is regulated by microtubules. From mitochondrial permeability transition pore to Ca2+-induced Ca2+ release.</a> J Biol Chem. 280 (1), 715-721 (2005).</li><li>Poot, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+morphology+and+function+with+novel+fixable+fluorescent+stains.">Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.</a> J Histochem Cytochem. 44 (12), 1363-1372 (1996).</li><li>Tokunaga, M., Imamoto, N., Sakata-Sogawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+inclined+thin+illumination+enables+clear+single-molecule+imaging+in+cells+(vol+5,+pg+159).">Highly inclined thin illumination enables clear single-molecule imaging in cells (vol 5, pg 159).</a> Nature Methods. 5 (5), 455 (2008).</li><li>Elmokadem, A., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+Drift+Correction+for+Superresolution+Localization+Microscopy+with+Bayesian+Inference.">Optimal Drift Correction for Superresolution Localization Microscopy with Bayesian Inference.</a> Biophys J. 109 (9), 1772-1780 (2015).</li><li>Barlag, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+super-resolution+imaging+of+proteins+in+living+Salmonella+enterica+using+self-labelling+enzymes.">Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes.</a> Sci Rep. 6, 31601 (2016).</li><li>Tokunaga, M., Imamoto, N., Sakata-Sogawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+inclined+thin+illumination+enables+clear+single-molecule+imaging+in+cells.">Highly inclined thin illumination enables clear single-molecule imaging in cells.</a> Nature Methods. 5 (2), 159-161 (2008).</li><li>Mortensen, K. I., Churchman, L. S., Spudich, J. A., Flyvbjerg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+localization+analysis+for+single-molecule+tracking+and+super-resolution+microscopy.">Optimized localization analysis for single-molecule tracking and super-resolution microscopy.</a> Nat Methods. 7 (5), 377-381 (2010).</li><li>Jin, S., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1-+and+Parkin-Mediated+Mitophagy+at+a+Glance.">PINK1- and Parkin-Mediated Mitophagy at a Glance.</a> Journal of Cell Science. 125, 795-799 (2013).</li><li>Yamano, K., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1+is+degraded+through+the+N-end+rule+pathway.">PINK1 is degraded through the N-end rule pathway.</a> Autophagy. 9 (11), 1758-1758 (2013).</li><li>Wiedemann, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machinery+for+protein+sorting+and+assembly+in+the+mitochondrial+outer+membrane.">Machinery for protein sorting and assembly in the mitochondrial outer membrane.</a> Nature. 424 (6948), 565-571 (2003).</li><li>Thompson, R., Larson, D., Webb, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+Nanometer+Localization+Analysis+for+Individual+Fluorescent+Probes.">Precise Nanometer Localization Analysis for Individual Fluorescent Probes.</a> Biophysical Journal. 82 (5), 2775-2783 (2002).</li></ol>

Here, a technique for dual color single molecule localization of mobile membrane proteins was presented. Following the protocol, membrane proteins are fused to self-labeling proteins that react with the rhodamine dyes TMR and SiR conjugated to their respective substrates. Rhodamine dyes are bright and photostable and thus allow for repetitive imaging1. For successful performance, several conditions and critical topics have to be kept in mind.
First, it is important to c...

The goal of this protocol is to provide an imaging method to localize and track single membrane proteins inside live cells. We call this method Tracking and Localization Microscopy (TALM)1,2. Like Stochastic Optical Reconstruction Microscopy (STORM)3 and Fluorescence Photoactivation Localization Microscopy ((F)PALM)4,5, TALM is a single molecule-based fluorescence localization technique. However, it is distinct in the way that the mobility of membrane proteins in combination with repetitive imaging of the same labeled molecule at different positions reveals the microcompartment that is accessible for the mobile protein. In other words, the possible localizations of the protein are set by the architecture of the organelle and by the mobility of the protein1. The method is complementary to various other super-resolution techniques6,7,8 because it reveals localization and trajectory maps by imaging mobile proteins. The labeling is based on using genetically engineered fusion proteins that are per se non-fluorescent. These fusion proteins are self-labeling enzymes that react covalently with a substrate conjugated to a dye. This procedure has the advantage that the labeling degree can be controlled by the amount of added substrate. Furthermore, it allows to vary the color of fluorescence, depending on the chosen conjugated dye. Several self-labeling enzyme-tags are available9. Another advantage of using self-labeling enzyme-tags is, that the conjugated dyes usually are more stable and brighter than fluorescent proteins1 and individual proteins therefore can be recorded longer and more precisely until they are bleached. This allows for the recording of trajectories of mobile proteins and the extraction of diffusion coefficients10,11.
Here, we demonstrate the feasibility of TALM with mitochondrial membrane proteins, but it can also be applied for other intra- and extra-cellular membrane proteins, including different cell types12,13. We show that multi-color TALM further allows for the simultaneous distinction of proteins in different subcompartments in complementation to existing super-resolution fluorescence microscopy techniques14,15,16. TALM is compatible with live cell imaging17. The photo-physics of the chosen rhodamines Tetramethylrhodamine (TMR) and Silicon-Rhodamien (SiR), in particular their brightness and stability, allows to record single membrane proteins over multiple frames providing localization (and trajectory) maps. However, TALM is limited for the localization of soluble proteins with high diffusion coefficients since the motion blur is too high and the collected photons per frame are too low for proper localization. Besides, TALM requires less excitation power than for example STORM or Stimulated Emission Depletion (STED) microscopy6,7, reducing phototoxic effects. This is important, since phototoxic stress often affects organellar morphology18&#160;and thus mobility analysis19. In sum, we present multi-color TALM in living cells as a technique that fills a gap between the localization microscopy methods STORM/STED/(F)PALM and techniques that analyze protein mobility such as fluorescence recovery after photobleaching (FRAP)20,21, fluorescence correlation spectroscopy (FCS)22, and fluorescence cross correlation spectroscopy (FCCS)11,23.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >(2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid, 1 M) (HEPES)</td>
			<td >Biochrom</td>
			<td >#1104E</td>
			<td ></td>
		</tr>
		<tr >
			<td >DC1: Dichroid beam splitter&#160;</td>
			<td>Chroma</td>
			<td>640 dcxr</td>
			<td>NC506031</td>
		</tr>
		<tr >
			<td >DC2: Polychroic Mirror, beamsplitter</td>
			<td >Chroma</td>
			<td>zt405/488/561/640rpc</td>
			<td>discontinued</td>
		</tr>
		<tr >
			<td >Dulbecco&#180;s Phosphate-Buffered Saline (PBS) 1x (w/o Ca &#38; Mg)</td>
			<td >Sigma-Aldrich &#38; Co.</td>
			<td >#RNBF8311</td>
			<td></td>
		</tr>
		<tr >
			<td >Earle&#180;s MEM without phenol red, without L-Glutamine and without NaHCO3 containing 1% FBS, 0.1% HEPES, 0.1% NEAA, 0.1% Alanyl-L-Glutamine and 34.78% sodium hydrogen carbonate (NaHCO3 0.75g/l)</td>
			<td ></td>
			<td ></td>
			<td>Imaging medium</td>
		</tr>
		<tr >
			<td >Earle&#180;s minimum essential medium (MEM) with phenol red, containing 1% Fetal Bovine Serum Superior (FBS), 0.1% HEPES (2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid, 1 M), and 0.1% non-essential amino acids (NEAA)</td>
			<td ></td>
			<td ></td>
			<td>Growth medium</td>
		</tr>
		<tr >
			<td >EF: Emission filter quadbandpass</td>
			<td>AHF analysentechnik</td>
			<td>F72-866</td>
			<td>Brightline HC 446 nm/523 nm/600 nm/677 nm</td>
		</tr>
		<tr >
			<td >EMCCD camera</td>
			<td >Andor</td>
			<td>Andor iXON 897</td>
			<td>EMCCD camera</td>
		</tr>
		<tr >
			<td >Emission filter QuadView filter cubes, orange</td>
			<td>AHF analysentechnik</td>
			<td>F39-637</td>
			<td>bandpass 582 - 619 nm</td>
		</tr>
		<tr >
			<td >Emission filter QuadView filter cubes, red</td>
			<td>Chroma</td>
			<td></td>
			<td>bandpass 655 - 725 nm (HQ 690/70)</td>
		</tr>
		<tr >
			<td >FBS (Fetal bovine serum) superior</td>
			<td >Biochrom</td>
			<td >S0615</td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescent beads: TetraSpeck&#8482; Microspheres, 0.1 &#181;m, fluorescent blue/green/orange/dark red</td>
			<td >Thermo Fisher Scientific</td>
			<td >T7279</td>
			<td>fluorescent microspheres</td>
		</tr>
		<tr >
			<td >Glutamine</td>
			<td >Biochrom</td>
			<td >#0951C</td>
			<td></td>
		</tr>
		<tr >
			<td >HeLa cells&#160;</td>
			<td >DSMZ</td>
			<td>ACC-57</td>
			<td>Cervical carcinoma cells from patient Henrietta Lacks</td>
		</tr>
		<tr >
			<td >Hela cells CI::paGFP, stable</td>
			<td >Muster et al., PLOSOne 2010</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Hela cells CV g::Halo7-Tag, stable</td>
			<td >Appelhans et al., NanoLett 2012</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Hela cells Tom20::Halo7-Tag, stable</td>
			<td >Appelhans et al., NanoLett 2012</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Image splitter</td>
			<td >Photometrics</td>
			<td >Dual-View QV2</td>
			<td>image splitter emission</td>
		</tr>
		<tr >
			<td >Imaging processing software&#160;</td>
			<td >ImageJ2 / Fiji</td>
			<td >freeware</td>
			<td></td>
		</tr>
		<tr >
			<td >Immersion Oil - ImmersolTM 518 F (ne = 1.518, ve = 45)</td>
			<td >Carl Zeiss Jena GmbH</td>
			<td >444960-0000-000</td>
			<td></td>
		</tr>
		<tr >
			<td >Inverted epifluorescence microscope</td>
			<td >Olympus IX-71/73/83</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Laser 561 nm, 200 mW</td>
			<td >CrystaLaser</td>
			<td>CL-561-200</td>
			<td>561 nm emission</td>
		</tr>
		<tr >
			<td >Laser 642 nm, 140 mW</td>
			<td >Omicron</td>
			<td >Luxx-642-140</td>
			<td>642 nm emission</td>
		</tr>
		<tr >
			<td >MATLAB</td>
			<td >MathWorks</td>
			<td >version R2013a</td>
			<td></td>
		</tr>
		<tr >
			<td >MEM with Earle&#39;s Balanced Salt Solution 2.2 g/L NaHCO3, stable glutamine w/o PR</td>
			<td >Biochrom</td>
			<td >FG-0385</td>
			<td></td>
		</tr>
		<tr >
			<td >MEM with Earle&#39;s Balanced Salt Solution with 2.2 g/L NaHCO3, stable glutamine, Phenolred</td>
			<td >Biochrom</td>
			<td >FG-0325</td>
			<td></td>
		</tr>
		<tr >
			<td >MitoTracker&#174; Deep Red FM</td>
			<td >Thermo Fisher Scientific</td>
			<td >M22426</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >MitoTracker&#174; Green FM</td>
			<td >Thermo Fisher Scientific</td>
			<td >M7514</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >Multi-mode-optical polarization maintaining monomode fiber</td>
			<td >Pointsource/Qioptiq</td>
			<td></td>
			<td>KineFLEX</td>
		</tr>
		<tr >
			<td >NHS-PEG-MAL, Rapp Polymer&#160;</td>
			<td >Rapp Polymere GmbH T&#252;bingen</td>
			<td ></td>
			<td>coverslip coating</td>
		</tr>
		<tr >
			<td >non-essential amino acids (NEAA)</td>
			<td >Biochrom</td>
			<td >#0802E</td>
			<td></td>
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		<tr >
			<td >PEG 800 (Polyethylene glycol) 10 %</td>
			<td >Carl Roth GmbH</td>
			<td >Art No. 0263.1</td>
			<td>coverslip coating</td>
		</tr>
		<tr >
			<td >Penicillin/Streptomycin</td>
			<td >Biochrom</td>
			<td >#0122E</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasmid for PINK1-Halo7-Tag expression</td>
			<td >Beinlich et al., ACS Chemical Biology 2015</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Poly-L-lysine (1.2 mg/ml)</td>
			<td >Sigma-Aldrich &#38; Co.</td>
			<td >Cat. No.P9155</td>
			<td>coverslip coating</td>
		</tr>
		<tr >
			<td >RGD Peptide (Ac-CGRGDS-COOH)</td>
			<td >Coring System Diagnostix GmbH, Gernsheim</td>
			<td ></td>
			<td>coverslip coating / Intergrin receptor motif</td>
		</tr>
		<tr >
			<td >Silicon Rhodamine linked to HaloTag&#174;-Ligand (SiRHTL)&#160;</td>
			<td >personal gift from Kai Johnson</td>
			<td ></td>
			<td>dye</td>
		</tr>
		<tr >
			<td >Software analysis plugin</td>
			<td >self-written C. P. Richter, Biophysik Osnabr&#252;ck</td>
			<td >SLIMFAST 16g</td>
			<td></td>
		</tr>
		<tr >
			<td >Tetramethylrhodamine / SNAP-Cell&#174; TMR-Star linked to SNAP-Ligand (TMRstar)&#160;</td>
			<td >New England Biolab&#174;</td>
			<td >S9105S</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >Tetramethylrhodamine linked to HaloTag&#174;-Ligand (TMRHTL)&#160;</td>
			<td >Promega</td>
			<td >G8251</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >TIRF condensor</td>
			<td >Olympus</td>
			<td>Cell^TIRF MITICO System</td>
			<td>TIRF condensor</td>
		</tr>
		<tr >
			<td >TIRF microscope controlling software</td>
			<td >Olympus cellSens 1.12</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >TIRF objective</td>
			<td>Olympus</td>
			<td>150x oil objective (N.A. 1.45; Olympus UAPO)</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin/EDTA 10x</td>
			<td >Biochrom</td>
			<td >#0266</td>
			<td></td>
		</tr>
		<tr >
			<td >Water H2O 99,5 % Rotipuran&#174; Low organic&#160;</td>
			<td >Carl Roth GmbH</td>
			<td >Art. No. HN57.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

multi-color localization microscopy of single membrane proteins in organelles of live mammalian cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Multi-color imaging and colocalization analysis can help to determine the sub-organellar localization&#160;of proteins. We demonstrated this earlier with the cytosolic phosphatase and tensin homologue, PINK1, that has different sub-mitochondrial locations due to its processing by mitochondrial proteases17. PINK1 is an important factor guaranteeing mitochondrial functionality34,35. To determine the localizat...

Watch this Scientific Journal Video about Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells at JoVE.com

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

This technique can answer key questions in regulatory processes such as which role protein dynamics and organization play in functional adaptation. The main advantage of this technique is that it can be done in living cells, thereby revealing protein dynamics on the single-molecule level. Though this method was originally implemented to provide insights into the spatiotemporal organization of mitochondrial membrane proteins, it can also be applied for other organelles and other membrane proteins.Demonstrating the technique will be Timo Appelhans, a grad student from my lab. After transfecting and preparing cell samples and the microscope according to the text protocol, mount the prepared sample with fluorescent beads in the sample holder between the PTFE ring and the red rubber ring. Start the microscope, all hardware components, and all software needed for microscopy.Use a fresh, lint-free tissue sprayed with isopropanol to clean the objective lens and the bottom of the cover slip. Then place a droplet of immersion oil on the pupil of the objective lens. Next, place the sample holder with the bead sample onto the microscope stage so that the bottom of the cover slip contacts the oil.Then focus on the beads by using transmission light or a laser line. Adjust the power of the two excitation lasers to achieve similar signal density in the two fluorescence channels. Then search for an area with many distinct fluorescent signals.Generate a merged view of the fluorescent channels by using the camera control software. Then use the screws at the image splitter to manually tilt the internal optical image splitter to achieve the best overlay of the signals from the two fluorescent channels. Start the TIRF microscope-controlling software and choose to display individual channels in live stream mode.Take a snapshot image, exciting fluorescents in all channels. Then use this image to produce the transformation matrix. Start the software analysis plugin and load the previously-recorded dual-color images of fluorescent beads into the software.Choose the used orientation of the fluorescent channels. Click Yes when asked for calibrate images, and select the previously-taken snapshot. Open the unit manager to define unit conversion factors, then open the localization manager.To determine the point spread function, or PSF, press the button PSF Radius. In the PSF Estimator window that opens, define the numerical aperture and the emission maximum. Then click Estimate PSF Radius.Next, accept the obtained experimental PSF. Define the evaluation box, number of deflation loops, and how many cores of the computer are used for calculation. Then press Localize to start fitting the intensity distribution of single particles, using a 2D symmetric Gaussian function.Open the calibration manager. In the rendered, merged image of the two channels, the original signal and the localized centers will be shown. Choose Affine mode.Then manually connect the corresponding pairs of localized centers in the two channels, that have originated from the same fluorescent bead, by drawing a connection line. Connect the corresponding signals distributed over the field of view. Then press Accept, and save the transformation matrix.To carry out single molecular imaging of mitochondrial membrane proteins, mount the specimen cover slip between the rubber and PTFE rings. Then fill the chamber with 0.5 to 0.8 milliliters of imaging medium. Clean the objective lens and bottom of the cover slip.Then use transmission light or a laser line to focus on the cells, after adding a drop of oil to the lens. Then adjust the power of the two excitation lasers to achieve a similar intensity, and search for an area with many fluorescent signals. Adjust the illumination angle in the TIRF microscope-controlling software to create an incident angle that is smaller than the critical angle, or the TIR-mode, to excite the specific region of interest via a HILO sheet.Adapting the illumination of the TIRF microscope to excite cell organelles using the HILO illumination requires quick judgment of the signal-to-background ratio and experience in TIRF microscopy, as well as a knowledge of cell morphology. Set the EM gain and choose an exposure time suitable for the experiment that collects sufficient photons per frame. Then adjust the laser power in order to achieve a high signal-to-noise ratio, as this ratio directly corresponds to the localization precision.Find an area in the cell periphery with non-overlapping, elongated mitochondria and single-molecule signals. If no single signals are visible in one or both channels, wait until bleaching results in the appearance of single-molecule signals. In the case only one channel needs to be bleached, switch the laser of the other channel off, to avoid bleaching, before recording this channel.Record until the number of signals is too low for reasonable continuation. Start the imaging-processing software and check for mitochondrial structures by generating a cumulative rendered-sum image of at least 1, 000 recorded frames. Start the software analysis plugin.To carry out data processing for localization and tracking, load the raw data. Check multicolor. Choose the correct orientation of both channels.When asked for Calibrate images, load the transformation matrix. The channels will be separately displayed. Open the raw data from the image container by clicking Show.Then open unit manager in the top tab, and define unit conversion factors in each channel. Open the localization manager in the top tab, and define the evaluation box and number of deflation loops. Click on PSF Radius to open the PSF estimator.Set numerical aperture and emission maximum to get the theoretical PSF value. Press Accept. Repeat this for all channels.Then press Localize to start the single-particle localization for the recorded signals in both channels. Wait for the software to finish. Now, a super-resolved image for each channel is obtained in the image container of the software analysis plugin.PINK1 is an important factor guaranteeing mitochondrial functionality. To determine the localization of PINK1 relative to Tom20 in functional and dysfunctional mitochondria, a time series of 1, 000 frames was recorded. The cumulative image of all signals over time from one channel showed that PINK1 was localized in the cytosol, at the outer membrane, and inside mitochondria under normal conditions, but preferentially in the outer membrane of depolarized mitochondria.As the summed images of localized particles show, PINK1 is distributed mainly inside polarized mitochondria, while Tom20 is localized in the outer mitochondrial membrane. This becomes even clearer in the merged image of localized Tom20 and PINK1 particles, which shows that the distribution of the outer membrane protein Tom20 is much broader. To verify the localization of PINK1 inside mitochondria, the 30 kilodalton subunit of respiratory complex one was fused to photoactivable GFP and recorded by fluorescence photoactivation localization microscopy.The cumulative triple-color image and the cross-section distribution show the overlay of complex one and PINK1, confirming the import of full-length PINK1. These data demonstrate that subpopulations of PINK1 occupy several mitochondrial micro-compartments. Once mastered, this technique can be done in 20 hours, including recording and post-processing of data, if it's done properly.While attempting this procedure, it is important to remember to test for organelle movement. Following this procedure, the relative localization of proteins can be determined. After its development, this technique paved the way for researchers in the field of single-particle tracking of mitochondrial proteins to observe their behavior in Z2, and different metabolic conditions, and under stress.After watching this video, you should have a good understanding of how to perform dual-column microscopy of mitochondrial proteins to reveal their sub-organelle position and trafficking.

multi-color-localization-microscopy-single-membrane-proteins

Research

JoVE Journal

Biology

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...