Name: Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass (Video) | JoVE Journal
Uploaded: 2017-08-10T13:00:00.000+00:00
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Description: Learn to quantitatively localize Golgi proteins at nanometer resolution using conventional light microscopy and software analysis in HeLa cells. This method bridges conventional and super-resolution imaging for cell biology research.

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We would like to thank D. Stephens (University of Bristol, Bristol, United Kingdom) for the TPST1-EGFP DNA plasmid, as well as Lakshmi Narasimhan Govindarajan for helping with the software optimization. This work was supported by grants from the National Medical Research Council (NMRC/CBRG/007/2012), Ministry of Education (AcRF Tier1 RG 18/11, RG 48/13 and RG132/15 and AcRF Tier2 MOE2015-T2-2-073) to L.L.

<ol><li>Glick, B. S., Luini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Models+for+Golgi+traffic%3A+a+critical+assessment%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Biol%22%5BJournal%5D)">Models for Golgi traffic: a critical assessment.</a> Cold Spring Harb Perspect Biol. 3 (11), 005215(2011).</li>
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<li>Trucco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Secretory+traffic+triggers+the+formation+of+tubular+continuities+across+Golgi+sub-compartments%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments.</a> Nat Cell Biol. 6 (11), 1071-1081 (2004).</li>
<li>Cole, N. B., Sciaky, N., Marotta, A., Song, J., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Golgi+dispersal+during+microtubule+disruption%3A+regeneration+of+Golgi+stacks+at+peripheral+endoplasmic+reticulum+exit+sites%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.</a> Mol Biol Cell. 7 (4), 631-650 (1996).</li>
<li>Rogalski, A. A., Bergmann, J. E., Singer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+microtubule+assembly+status+on+the+intracellular+processing+and+surface+expression+of+an+integral+protein+of+the+plasma+membrane%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.</a> J Cell Biol. 99 (3), 1101-1109 (1984).</li>
<li>Van De Moortele, S., Picart, R., Tixier-Vidal, A., Tougard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nocodazole+and+taxol+affect+subcellular+compartments+but+not+secretory+activity+of+GH3B6+prolactin+cells%5BTitle%5D)+AND+%22Eur+J+Cell+Biol%22%5BJournal%5D)">Nocodazole and taxol affect subcellular compartments but not secretory activity of GH3B6 prolactin cells.</a> Eur J Cell Biol. 60 (2), 217-227 (1993).</li>
<li>Nakamura, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+cis-Golgi+matrix+protein%2C+GM130%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Characterization of a cis-Golgi matrix protein, GM130.</a> J Cell Biol. 131, 6 Pt 2 1715-1726 (1995).</li>
<li>Roth, J., Berger, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunocytochemical+localization+of+galactosyltransferase+in+HeLa+cells%3A+codistribution+with+thiamine+pyrophosphatase+in+trans-Golgi+cisternae%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae.</a> J Cell Biol. 93 (1), 223-229 (1982).</li>
<li>Baeuerle, P. A., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tyrosine+sulfation+is+a+trans-Golgi-specific+protein+modification%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Tyrosine sulfation is a trans-Golgi-specific protein modification.</a> J Cell Biol. 105 (6), Pt 1 2655-2664 (1987).</li>
<li>Honda, A., Al-Awar, O. S., Hay, J. C., Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+of+Arf-1+to+the+early+Golgi+by+membrin%2C+an+ER-Golgi+SNARE%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE.</a> J Cell Biol. 168 (7), 1039-1051 (2005).</li>
<li>Lavieu, G., Zheng, H., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stapled+Golgi+cisternae+remain+in+place+as+cargo+passes+through+the+stack%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Stapled Golgi cisternae remain in place as cargo passes through the stack.</a> Elife. 2, 00558(2013).</li>
<li>Zhao, X., Lasell, T. K., Melancon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization+of+large+ADP-ribosylation+factor-guanine+nucleotide+exchange+factors+to+different+Golgi+compartments%3A+evidence+for+distinct+functions+in+protein+traffic%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Localization of large ADP-ribosylation factor-guanine nucleotide exchange factors to different Golgi compartments: evidence for distinct functions in protein traffic.</a> Mol Biol Cell. 13 (1), 119-133 (2002).</li>
<li>Dejgaard, S. Y., Murshid, A., Dee, K. M., Presley, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Confocal+microscopy-based+linescan+methodologies+for+intra-Golgi+localization+of+proteins%5BTitle%5D)+AND+%22J+Histochem+Cytochem%22%5BJournal%5D)">Confocal microscopy-based linescan methodologies for intra-Golgi localization of proteins.</a> J Histochem Cytochem. 55 (7), 709-719 (2007).</li>
<li>Fourriere, L., Divoux, S., Roceri, M., Perez, F., Boncompain, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microtubule-independent+secretion+requires+functional+maturation+of+Golgi+elements%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Microtubule-independent secretion requires functional maturation of Golgi elements.</a> J Cell Sci. 129 (17), 3238-3250 (2016).</li>
<li>Volchuk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Countercurrent+distribution+of+two+distinct+SNARE+complexes+mediating+transport+within+the+Golgi+stack%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack.</a> Mol Biol Cell. 15 (4), 1506-1518 (2004).</li>
<li>Popoff, V., Adolf, F., Brugger, B., Wieland, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((COPI+budding+within+the+Golgi+stack%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Biol%22%5BJournal%5D)">COPI budding within the Golgi stack.</a> Cold Spring Harb Perspect Biol. 3 (11), 005231(2011).</li>
</ol>

The authors declare that they have no competing financial interests.

Previously, the localization of a Golgi protein under the light microscopy was mainly quantified by the degree of correlation or overlapping of the image of the protein with the image of a Golgi marker of known localization15,16,17. The resulting correlation or overlapping coefficient reflects how close the testing protein is to the Golgi marker spatially. There are at least three caveats for this approach. First, the correlatio...

The Golgi complex plays essential roles in secretory/endocytic trafficking of proteins and lipids (hereafter cargos) in mammalian cells1,2,3. At the Golgi, cargos are not only sorted to various sub-cellular compartments but also modified by diverse types of glycosylation. The mammalian Golgi complex comprises numerous laterally connected Golgi stacks, which typically consists of 4 - 11 tightly adjacent and flat membrane sacs called cisternae. The serially stacked Golgi cisternae are further categorized, from one end to the other, as cis, medial and trans-cisternae. At the trans-side of a Golgi stack, the trans-most membrane sac develops into a tubular and reticulum membrane network called the trans-Golgi network (TGN)4. In the secretory pathway, cargos derived from the endoplasmic reticulum (ER) enter a Golgi stack at its cis-side and then sequentially pass through medial and trans-cisternae. Cargos eventually exit the Golgi at the trans-Golgi or TGN destining to the plasma membrane, endosomes or secretory granules.
The molecular and cellular mechanisms of how cargos transit a Golgi stack and how the Golgi maintains its cisternal organization remain mysterious and are currently still under a heated debate1. One of difficulties in this field is that Golgi cisternae can only be resolved under the electron microscopy (EM) since the resolution of an optical microscope (~ 200 nm) is insufficient to resolve individual Golgi cisternae (&#60; 100 nm in both cisternal thickness and distance). Therefore, the sub-Golgi localization of resident proteins and transiting cargos are conventionally determined by the immuno-gold EM. However, the immuno-gold EM is very technically demanding and it is beyond the capability of most cell biology labs. Although the resolution of the EM can be sub-nanometer, the resolution afforded by the immuno-gold EM is greatly hampered by the size of the antibody complex (primary plus the secondary antibody) and the gold particle, and it can be worse than 20 nm. Furthermore, EM images are obtained from 2D thin-sections instead of a 3D global view of the Golgi, which can result in erroneous conclusions depending on the relative position and orientation of the 2D section5. For example, studying an EM single-section is unable to reliably differentiate a vesicle from the orthogonal view of a tubule since both can display identical round membrane profiles. The recent advent of super-resolution microscopy techniques, such as 3D-structured illumination microscopy (3D-SIM), stimulated emission depletion (STED), photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), makes it possible to resolve sub-Golgi structures under light microscopes6. However, there are at least four drawbacks that can significantly limit their uses in the cell biological study of the Golgi. 1) Current super-resolution techniques require expensive and special hardware configuration which is beyond most cell biology labs. 2) Special fluorescence labeling protocols are needed for some super-resolution techniques. 3) Although, under the best condition, these techniques claim 20-110 nm in spatial resolution, the practical resolution obtained in real samples can be much worse. 4) In comparison to conventional microscopy, these super-resolution techniques still have difficulties in conducting multicolor, 3D or live cell imaging, either singly or in combination. Probably most importantly, both immuno-gold EM and the super-resolution microscopy techniques yield qualitative instead of quantitative localization data.
Attempting to partially solve problems mentioned above, we have recently developed a conventional light microscopy based method, which is named Golgi protein localization by imaging centers of mass (GLIM), to systematically and quantitatively localize a Golgi protein at a resolution equivalent to that of the immuno-gold EM7. In this method, the Golgi in cultured mammalian cells is dispersed as Golgi mini-stacks by the treatment of nocodazole, a microtubule depolymerizing drug. Extensive studies have demonstrated that nocodazole-induced Golgi mini-stacks (hereafter Golgi mini-stacks) closely resemble native Golgi stacks in both organization and cellular functions8,9,10,11. The localization quotient (LQ) of a test protein can be acquired through GLIM and it denotes the quantitative sub-Golgi localization. The numerical values of LQs can be compared and a LQ database of more than 25 Golgi markers has been available.
In GLIM, Golgi mini-stacks are triple-labeled by endogenous or exogenously expressed GM130, GalT-mCherry and the test protein (x). GM130 and GalT-mCherry, cis- and trans-Golgi markers respectively12,13, provide reference points. The triple fluorescence, red (R), green (G) and far-red (B), are artificially displayed as red, green and blue, respectively. Center of fluorescence mass (hereafter center) is adopted to achieve sub-pixel resolution. The Golgi axis is defined as the vector from the center of GM130 to that of GalT-mCherry. The Golgi mini-stack is modeled as a cylindrical structure with infinite rotational symmetry around the Golgi axis. Therefore, a Golgi mini-stack can be further modeled as an one-dimensional structure along the Golgi axis. The LQ of the test protein x is defined as dx/d1, in which dx is the distance from the center of x to that of GM130, while d1 is the distance from the center of GalT-mCherry to that of GM130. If the center of x is off-axis, its projection axial distance is used for the calculation. The variables, including Golgi axis, axial angle, dx, d1, angle &#945; and angle &#946;, for GLIM are schematically illustrated in Figure 1. LQ is independent of the Golgi axial angle though Golgi mini-stacks orient randomly in a cell.
Golgi mini-stacks appear inhomogeneous in images. We developed three criteria to select analyzable Golgi mini-stacks for GLIM. 1) The signal-to-noise ratio criterion, in which the ratio of the total intensity of a Golgi mini-stack to the standard deviation (SD) of the background is &#8805; 30 in each channel. This criterion is to ensure the positioning accuracy of the center of mass, which depends on the signal-to-noise ratios of Golgi mini-stacks. 2) The axial angle or distance criterion, which requires d1&#8805; 70 nm. d1 decreases with the increase of the Golgi axial angle. When the axial angle is approaching 90&#176; or vertical, the mini-stack becomes non-resolvable as d1 is approaching 0. d1&#8805; 70 nm can effectively exclude near vertical Golgi mini-stacks. 3) The co-linearity criterion, in which either |tan &#945;| or |tan &#946;| is &#8804; 0.3. This criterion ensures that the three centers of a mini-stack are sufficiently co-linear for our one-dimensional model of the Golgi mini-stack. All light microscopes suffer from chromatic aberration which can seriously distort the relative positions of red, green and far-red fluorescence centers. Chromatic aberration of microscope systems is experimentally calibrated by imaging 110 nm beads, which are triple-labeled by red, green and far-red fluorescence. For each bead image, the center of red is defined as the true position of the bead and chromatic-shifts of green and far-red centers are fitted by first-order polynomial functions. Centers of Golgi mini-stacks are subjected to the polynomial functions to correct the chromatic-shifts in green and far-red channels.
Through GLIM we can achieve a resolution of ~ 30 nm along the Golgi axis under standard conditions. Importantly, it provides a systematical method to quantitatively map any Golgi protein. GLIM can be performed by conventional microscopes, such as wide-field or confocal microscopes, using common fluorescence labeling protocols. The imaging and data processing can take as short as an hour. Through GLIM, we have directly demonstrated the progressive transition of the secretory cargo from the cis- to trans-side of the Golgi7.

<table><tbody><tr><td>fluorescence beads. Commercial name: TetraSpeck beads</td><td>Invitrogen</td><td>T7279</td><td>As multi-color beads to calibrate chromatic-shift of the microscope</td></tr><tr><td>Glass coverslip &amp;Phi; 12 mm (No. 1.5)</td><td>Menzel</td><td>CB00120RAC</td><td></td></tr><tr><td>Glass coverslip &amp;Phi; 25 mm (No. 1.5)</td><td>Menzel</td><td></td><td></td></tr><tr><td>DMEM</td><td>Capricon</td><td>DMEM-HPA-P50</td><td></td></tr><tr><td>Trypsin-EDTA</td><td></td><td></td><td></td></tr><tr><td>FBS</td><td>GE Hyclone</td><td>SV30160.03</td><td></td></tr><tr><td>Nocodazole</td><td>Merck</td><td>487928</td><td></td></tr><tr><td>transfection reagent. Commercial name: Lipofectamine 2000</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>transfection medium. Commercial name: OptiMEM</td><td>Invitrogen</td><td>31985070</td><td></td></tr><tr><td>TPST1-EGFP</td><td>Addgene</td><td>66617</td><td>A gift from D. Stephens (University of Bristol, Bristol, United Kingdom)</td></tr><tr><td>GalT-mCherry</td><td></td><td></td><td>Made in our lab</td></tr><tr><td>paraformaldehyde</td><td>Merck</td><td>1.04005.1000</td><td></td></tr><tr><td>saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>poly(vinyl alcohol) (Mw ~31,000). Commercial name: Mowiol-488</td><td>CALBIOCHEM</td><td>475904</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9647</td><td></td></tr><tr><td>Mouse anti-GM130</td><td>BD Biosciences</td><td>610823</td><td>Primary antibody for human GM130</td></tr><tr><td>far-red fluorescence conjugated goat anti-mouse IgG. Commercial name: Alexa Fluor 647 conjugated goat anti-mouse IgG</td><td>Invitrogen</td><td>A-21235</td><td>Far red fluorescence conjugated secondary antibody</td></tr></tbody></table>

quantitative localization of a golgi protein by imaging its center of fluorescence mass

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The modern research grade light microscope equipped with a plan apochromatic lens, such as the one used in our lab, shows minimal chromatic aberration (Figure 2A). However, a careful examination of the multi-color fluorescent bead image can reveal the shift of different color images of the same bead (Figure 2B). We define that the red channel is free of chromatic aberration and therefore centers ...

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

The overall goal of this imaging-based method is to quantitatively localize a golgi protein at nanometer resolution. This method can help to answer the key questions in the study of the golgi apparatus such as the golgi's organization and how it works. The main advantage of this method is that it uses a conventional light microscope and software tools.To begin, culture HeLa cells in a T25 flask in DMEM supplemented with 10%FBS and incubate the culture at 37 degrees Celsius and 5%carbon dioxide. When cells reach approximately 80%confluency, aspirate the culture medium and add one milliliter of 25%Trypsin EDTA to the flask. Incubate the cells at 37 degrees Celsius for two minutes.Remove the cells from the incubator and add one milliliter of complete medium to gently flush the cells off the wall of the flask. Transfer the detached cells to a sterile centrifugation tube and spin the tube at 500 g's for two minutes to pellet the cells. After aspirating the supernatant, use one milliliter of complete medium to suspend the cell pellet.Remove the medium from the well of a 24-well plate containing a sterilized glass cover slip and seed approximately 100, 000 cells into the well. Use complet

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Learn to quantitatively localize Golgi proteins at nanometer resolution using conventional light microscopy and software analysis in HeLa cells. This method bridges conventional and super-resolution imaging for cell biology research.

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

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