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.

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<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>
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<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>
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<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 correlation or overlapping coefficient is nonlinear and it does not directly indicate spatial distance. Second, the degree of correlation is critically dependent on the resolution of the microscopic system. Therefore, the coefficient between two Golgi proteins is not a system-independent constant. Third, two Golgi proteins with the same axial localization but different lateral distribution along cisternae can have distinct coefficients. Hence, the correlation or overlapping coefficient does not indicate the axial localization of a Golgi protein. In an alternative method proposed by Dejgaard et al., cis, trans-Golgi markers and the protein of interest are triple-labeled in nocodazole treated Golgi mini-stacks, similar to GLIM. Within each Golgi mini-stack, positions of the three maximum intensity pixels are acquired and the relative position of the test protein is calculated as a distance ratio18. However, their method is unable to achieve sub-pixel resolution, which greatly limits its application. Compared to previous quantitative methods, GLIM, which was independently developed but bears a similar concept to that of Dejgaard et al., is able to quantify the axial localization of a Golgi protein with unprecedented accuracy and consistency.
We presented the protocol of GLIM to acquire the LQ of exogenously expressed GFP-tagged protein - TPST1-EGFP. The LQ of endogenous protein can be determined if its antibody is available. Depending on the species of the primary antibody, mouse or rabbit, then a rabbit or mouse anti-GM130 antibody, respectively, can be used in the triple-labeling protocol. Both rabbit and mouse anti-GM130 antibodies for immunofluorescence labeling are commercially available. We have previously demonstrated that rabbit and mouse anti-GM130 antibodies give the same results in GLIM7. If the test protein is intrinsically red in fluorescence emission, such as mCherry fusion, a GFP-tagged Golgi marker protein with known LQ in combination with GM130 antibody labeling can be used to triple-label cells and indirectly deduce the LQ of the test protein. Assuming the marker protein&#39;s LQ is LQm and the distance from the center of the marker protein to that of GM130 is dm, the LQ of the test protein x can be calculated as (dx/dm)&#215;LQm. One of the greatest advantages of GLIM in comparison to super-resolution microscopy is that it can easily be applied to the live cell imaging in conventional microscopic setups. We have tried a combination of three fluorescence proteins, GFP-Golgin84, mCherry-GM130 and GalT-iRFP670, for dynamically monitoring the LQ of GFP-Golgin84 in single Golgi mini-stacks7.
In GLIM, the most time-consuming step is the post-acquisition analysis, especially on the selection of analyzable Golgi mini-stacks. As Golgi mini-stacks are heterogeneous in both size and molecular composition19, it is unclear if the distribution of LQs (Figure 3F) represents the heterogeneous architecture of Golgi mini-stacks or the uncertainty of our calculation of the LQ. Regardless of the causes, we found that it is important to have a large number of analyzable Golgi mini-stacks (n) to ensure the accuracy of the LQ, which is compromised when n is small. Typically, data with n &#8805; 100 from multiple cells yields reliable LQs. Therefore, GLIM gives ensemble-averaged localization data, obscuring the individuality of Golgi mini-stacks. Another limitation of GLIM is that it assumes that all Golgi proteins have a narrow distribution around a single center. Some Golgi proteins, such as COPI subunits, ARF1 and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNAREs)20,21, have been reported to distribute broadly from the cis to the trans-Golgi and the TGN. It is probably inappropriate to study their Golgi localization by the LQ. As GLIM currently involves manual selection of the Golgi mini-stacks, which is both laborious and subjective, the future development of GLIM will be to implement a software tool to automatically select Golgi mini-stacks with minimal human interference.
What is the spatial resolution of GLIM? Since the LQ is a ratio, which is unitless, it does not indicate a spatial distance. Here, we attempt to give a very rough estimation. The spatial resolution of GLIM can be defined as the smallest axial distance between two resolvable Golgi proteins. Examining LQs of various Golgi proteins7, we found that SEMs of datasets with n &#8805; 100 range around 0.03. Assuming two Golgi proteins have the same n = 100 and SEM = 0.03, the two Golgi proteins have significant different localization by t-test (p &#60; 0.05), i.e., resolvable, if the difference between their LQs is &#8805; 3 &#215; SEM = 0.09. From the EM data, we can estimate that the axial length of the Golgi mini-stack, which is also the distance from GM130 to GalT-mCherry in GLIM, is ~ 300 nm8. Hence, the resolution of GLIM is estimated to be 0.09 &#215; 300 = 30 nm along the Golgi axis. In GLIM, a larger n generally yields smaller SEM, which in turn results in higher resolution.
It is probably inappropriate to directly compare the resolution of GLIM to that of the immuno-gold EM since the latter technique does not directly yield quantitative localization data. Similar to GLIM, the resolution of the immuno-gold EM along the Golgi axis can be defined as the smallest distance between two resolvable Golgi markers. To measure its resolution requires the study of dual-immuno-gold labeling of two Golgi proteins that are closely adjacent to each other along the Golgi axis. Such systemic study is probably unavailable according to our knowledge. As discussed in the introduction, one of the limiting factors in the spatial resolution of the immuno-gold EM is the large size of the antibody complex, which makes the resolution to be worse than ~ 20 nm. Another important limiting factor of the image resolution is the labeling density of antigen molecules. According to the Nyquist sampling theory, there must be at least two gold particles per resolution unit. In most immuno-gold EM studies, the distances between neighbor gold particles are not &#60; 15 nm due to the very low labeling efficiency; this makes it difficult for this technique to achieve an image resolution less than 30 nm. From this point of view, we estimate that the resolution of GLIM is at least comparable to that of the immuno-gold EM.
GLIM is a robust method that gives highly consistent results. It is independent of the type of microscope used. We have tested that wide-field and spinning disk confocal microscopes yielded the same results. LQs of the same Golgi proteins acquired at different batches of experiments were also in good agreement with each other. A LQ database consisting of a diverse range of Golgi resident proteins has been generated for comparison and interpretation. We expect that more usage of GLIM in the research community will significantly expand the database. Consequently, a more complete database quantitatively describing the localization of a large number of Golgi proteins will greatly help in understanding the organization and function of the Golgi complex.

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

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 of red fluorescence are the true positions of beads. The relative chromatic-shifts of green and far-red fluorescence can be represented by green and blue vectors originating from the center of red fluorescence (Figure 2C). The chromatic-shift within the imaging-plane is empirically illustrated by the green and blue vector for each bead (Figure 2D). For our microscope, the chromatic-shifts are not uniform as both magnitudes and directions of shifts change according to x and y positions. The chromatic-shift can significantly affect the accuracy of GLIM as the shift of far-red centers can be as much as 50 nm. Therefore chromatic-shift must be corrected. Once centers of beads are acquired, we employ first-order polynomial fitting to calibrate and subsequently correct the chromatic-shifts of centers. The chromatic-shift aberration is greatly improved by this approach (Figure 2 D-G).
In mammalian cells, the Golgi complex is aggregated at the perinuclear area which is usually not resolvable under a conventional optical microscope (Figure 3A). After nocodazole treatment for 3 hours, the perinuclear Golgi complex disappears and dozens of Golgi mini-stacks assemble at the endoplasmic reticulum exit sites throughout the cytoplasm (Figure 3B). Large chunks of Golgi membrane and aggregated Golgi mini-stacks are present and they are not selected for analysis. An example image illustrating the selection of Golgi mini-stacks is shown in Figure 3C. Using the &#34;Macro-Golgi ROI inspection&#34; tool, 40 selected Golgi mini-stacks are listed in Figure 3D. After applying the three criteria, 21 Golgi mini-stacks were analyzable and chosen for the calculation of LQs of TPST1-EGFP (Figure 3D; labeled as &#34;L&#34;), with their statistics shown in Figure 3E. In total, 111 analyzable Golgi mini-stacks were selected from 12 cells and the LQ of TPST1-EGFP was measured to be 0.76 0.04 (mean &#177; SEM; n = 111) (Figure 3F). The LQ of TPST1-EGFP positions it between giantin (LQ = 0.59) and EGFP-Rab6 (LQ = 1.04)7. It is near GS15 (LQ = 0.83) and ST6GalT1-AcGFP1 (LQ = 0.82). We have defined the sub-Golgi regions according to LQs by considering qualitative localization data from literature: the ERES/ERGIC, &#60; -0.25; cis, 0.00 &#177; 0.25; medial, 0.50 &#177; 0.25; trans-Golgi, 1.00 &#177; 0.25 and TGN, 1.25 - 2.00. The LQ of TPST1-EGFP therefore indicates its trans-Golgi localization, which is in agreement with a previous study14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55996/55996fig1.jpg" /> 
Figure 1. Schematic Illustrations Showing Variables Used in the Calculation of GLIM. (A) xz view of a Golgi mini-stack that has co-axial centers of GM130, GalT-mCherry and protein x fluorescence showing Golgi axis, axial angle, dx and d1. The image of the Golgi mini-stack is its projection onto the image plane. (B) xy view of a Golgi mini-stack with off-axis center of protein x showing angle &#945;, angle &#946; and projection axial distance dx. A and B are adapted from Figure 1E and Figure 1F of our previous publication7, respectively. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55996/55996fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55996/55996fig2.jpg" /> 
Figure 2. Correction of the Chromatic-shift. (A) The three-color merged image of 110 nm multi-color beads acquired by a wide-field microscope. Each bead emits green, red, and far-red (artificially colored as blue) fluorescence. Scale bar, 10 &#181;m. (B) Enlarged view of a merged single-bead image with a noticeable chromatic-shift. Scale bar, 200 nm. (C) A schematic diagram showing that shifts of green and far-red bead images can be represented by vectors from the center of the red bead image (0,0) to centers of green (green vector) and far-red (blue vector) bead images, respectively. (D, E) The effect of the chromatic-shift correction. Within the same field of image, green and blue vectors are plotted before and after shift correction. To visualize the tiny shift, the magnitude of each vector is amplified 200-fold. (F, G) Scatter plots showing centers of green (green dots) and far-red (blue dots) beads before and after shift correction. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55996/55996fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55996/55996fig3.jpg" /> 
Figure 3. A Typical Example of GLIM, Here Acquiring the LQ of TPST1-EGFP. A HeLa cell expressing TPST1-EGFP (green) and GalT-mCherry (red) were stained for endogenous GM130 (blue). (A) A typical cell. (B, C, D, E) A cell treated with nocodazole was imaged (B). From the image, analyzable Golgi mini-stacks were selected as shown in C. Selected Golgi mini-stacks are labeled by identification numbers. Images of these masked Golgi mini-stacks are displayed below in D with their identification numbers. &#34;L&#34; denotes that the Golgi mini-stack is valid for the calculation of the LQ (analyzable Golgi mini-stacks). Histogram of LQs of 21 mini-stacks from the cell is shown in E. (F) Histogram of LQs of 111 mini-stacks from 12 cells. The value shown in the histogram is mean &#177; SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55996/55996fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
SUPPLEMENTAL MATERIALS: Macro-Golgi ROI inspection.ijm, Macro-Output 3 channels data.ijm, Matlab codes, My_train.exe, My_test.exe, and Spreadsheet template.opj.

Watch this Scientific Journal Video about Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass at JoVE.com

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

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, ...

quantitative-localization-golgi-protein-imaging-its-center

Nanyang Technological University

Research

JoVE Journal

Biology

10.8K Views.  Nanyang Technological University. 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.

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

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biochemistry

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Neuroscience

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 ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Immunology and Infection

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

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.

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 ...

Investigating ATG9A Localization in Cells

Imaging ATG9A, a Multi-Spanning Membrane Protein

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive pathological conditions. A set of proteins, called ATG proteins, comprise the core autophagy machinery and work together in a defined hierarchy. Studies in recent years have improved our knowledge of the autophagy pathway. Most recently, it has been proposed that ATG9A vesicles are at the heart of autophagy, as they control the rapid de novo synthesis of an organelle called the phagophore. The study of ATG9A has proven challenging, since ATG9A is a transmembrane protein, and it is present in different membrane compartments. As such, understanding its trafficking is an important element for understanding autophagy. Here, detailed methods are presented that can be used to study ATG9A and, in particular, its localization using immunofluorescence techniques, which can be assessed and quantified. The pitfalls of transient overexpression are also addressed. The correct characterization of ATG9A function and the standardization of techniques to analyze its trafficking are crucial to further characterize the events governing autophagy initiation.

Author Spotlight: Imaging ATG9A, a Multi-Spanning Membrane Protein  

This protocol describes various methods that can help in the study of ATG9A biology, including immunofluorescence followed by image analysis, transient overexpression considerations, and investigating the ATG9A glycosylation status using western blot.

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive ...

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

Summary

Explore More Videos

Quantitative Immunofluorescence to Measure Global Localized Translation

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Automated Quantification of Synaptic Fluorescence in C. elegans

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

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Ground State Depletion Super-resolution Imaging in Mammalian Cells

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

Imaging ATG9A, a Multi-Spanning Membrane Protein