Visualizing the Cisternal Organization of Golgi Ministacks in HeLa Cells by Side-averaging.

The mammalian Golgi complex consists of laterally connected Golgi stacks, each comprising close-packed and flattened membrane sacks called cisternae. However, the convoluted spatial organization of Golgi stacks and limited resolution of light microscopy prevent us from resolving the cisternal organization of the Golgi. Here, we describe our recently developed side-averaging approach coupled with Airyscan microscopy to visualize the cisternal organization of nocodazole-induced Golgi ministacks. First, the nocodazole treatment greatly simplifies the organization of Golgi stacks by spatially separating the crowded and amorphous Golgi complex into individual disk-shaped ministacks. The treatment also makes it possible to identify en face and side-views of Golgi ministacks. Next, after manually selecting the side-view Golgi ministack images, they are transformed and aligned. Finally, the resulting images are averaged to enhance the common structural features and suppress the morphological variations among individual Golgi ministacks. This protocol describes how to image and analyze the intra-Golgi localization of giantin, GalT-mCherry, GM130, and GFP-OSBP in HeLa cells by side-averaging. Graphical abstract.

Studying the Organization of the Golgi by Super-Resolution Microscopy.

The Golgi complex is essential for protein transport and posttranslational modification in mammalian cells. It is critical to know the cisternal distribution of Golgi proteins to understand Golgi functions. The cis-to-trans or axial localization of a Golgi protein can be obtained using our previously developed method, Golgi protein localization by imaging centers of mass (GLIM), in nocodazole-induced Golgi ministacks (hereafter referred to as ministacks). However, there is no effective light microscopic method to reveal the lateral localization of a Golgi protein, which is the distribution within the Golgi cisternae. The challenge is partially caused by the random orientations and the tight congregation of Golgi stacks at the perinuclear region. Here, we summarize our method to identify en face and side views of ministacks. It takes advantage of the characteristic ring and double-punctum staining patterns exhibited by cisternal rim-localized proteins. After averaging multiple en face views, the resulting image reveals the intrinsic organization of cisternae in a non-biased manner.

Arl15 upregulates the TGFß family signaling by promoting the assembly of the Smad-complex.

The hallmark event of the canonical transforming growth factor ß (TGFß) family signaling is the assembly of the Smad-complex, consisting of the common Smad, Smad4, and phosphorylated receptor-regulated Smads. How the Smad-complex is assembled and regulated is still unclear. Here, we report that active Arl15, an Arf-like small G protein, specifically binds to the MH2 domain of Smad4 and colocalizes with Smad4 at the endolysosome. The binding relieves the autoinhibition of Smad4, which is imposed by the intramolecular interaction between its MH1 and MH2 domains. Activated Smad4 subsequently interacts with phosphorylated receptor-regulated Smads, forming the Smad-complex. Our observations suggest that Smad4 functions as an effector and a GTPase activating protein (GAP) of Arl15. Assembly of the Smad-complex enhances the GAP activity of Smad4 toward Arl15, therefore dissociating Arl15 before the nuclear translocation of the Smad-complex. Our data further demonstrate that Arl15 positively regulates the TGFß family signaling.

Visualizing intra-Golgi localization and transport by side-averaging Golgi ministacks.

The mammalian Golgi comprises tightly adjacent and flattened membrane sacs called cisternae. We still do not understand the molecular organization of the Golgi and intra-Golgi transport of cargos. One of the most significant challenges to studying the Golgi is resolving Golgi proteins at the cisternal level under light microscopy. We have developed a side-averaging approach to visualize the cisternal organization and intra-Golgi transport in nocodazole-induced Golgi ministacks. Side-view images of ministacks acquired from Airyscan microscopy are transformed and aligned before intensity normalization and averaging. From side-average images of >30 Golgi proteins, we uncovered the organization of the pre-Golgi, cis, medial, trans, and trans-Golgi network membrane with an unprecedented spatial resolution. We observed the progressive transition of a synchronized cargo wave from the cis to the trans-side of the Golgi. Our data support our previous finding, in which constitutive cargos exit at the trans-Golgi while the secretory targeting to the trans-Golgi network is signal dependent.

Glycans function as a Golgi export signal to promote the constitutive exocytic trafficking.

Most proteins in the secretory pathway are glycosylated. However, the role of glycans in membrane trafficking is still unclear. Here, we discovered that transmembrane secretory cargos, such as interleukin 2 receptor α subunit or Tac, transferrin receptor, and cluster of differentiation 8a, unexpectedly displayed substantial Golgi localization when their O-glycosylation was compromised. By quantitatively measuring their Golgi residence times, we found that the observed Golgi localization of O-glycan-deficient cargos is due to their slow Golgi export. Using a superresolution microscopy method that we previously developed, we revealed that O-glycan-deficient Tac chimeras localize at the interior of the trans-Golgi cisternae. O-Glycans were observed to be both necessary and sufficient for the efficient Golgi export of Tac chimeras. By sequentially introducing O-glycosylation sites to ST6GAL1, we demonstrated that O-glycan's effect on Golgi export is probably additive. Finally, the finding that N-glycosylated GFP substantially reduces the Golgi residence time of a Tac chimera suggests that N-glycans might have a similar effect. Therefore, both O- and N-glycans might function as a generic Golgi export signal at the trans-Golgi to promote the constitutive exocytic trafficking.

Dopey1-Mon2 complex binds to dual-lipids and recruits kinesin-1 for membrane trafficking.

Proteins are transported among eukaryotic organelles along the cytoskeleton in membrane carriers. The mechanism regarding the motility of carriers and the positioning of organelles is a fundamental question in cell biology that remains incompletely understood. Here, we find that Dopey1 and Mon2 assemble into a complex and localize to the Golgi, endolysosome and endoplasmic reticulum exit site. The Golgi localization of Dopey1 and Mon2 requires their binding to phosphatidylinositol-4-phosphate and phosphatidic acid, respectively, two lipids known for the biogenesis of membrane carriers and the specification of organelle identities. The N-terminus of Dopey1 further interacts with kinesin-1, a plus-end or centrifugal-direction microtubule motor. Dopey1-Mon2 complex functions as a dual-lipid-regulated cargo-adaptor to recruit kinesin-1 to secretory and endocytic organelles or membrane carriers for centrifugally biased bidirectional transport. Dopey1-Mon2 complex therefore provides an important missing link to coordinate the budding of a membrane carrier and subsequent bidirectional transport along the microtubule.

The spatial separation of processing and transport functions to the interior and periphery of the Golgi stack.

It is unclear how the two principal functions of the Golgi complex, processing and transport, are spatially organized. Studying such spatial organization by optical imaging is challenging, partially due to the dense packing of stochastically oriented Golgi stacks. Using super-resolution microscopy and markers such as Giantin, we developed a method to identify en face and side views of individual nocodazole-induced Golgi mini-stacks. Our imaging uncovered that Golgi enzymes preferentially localize to the cisternal interior, appearing as a central disk or inner-ring, whereas components of the trafficking machinery reside at the periphery of the stack, including the cisternal rim. Interestingly, conventional secretory cargos appeared at the cisternal interior during their intra-Golgi trafficking and transiently localized to the cisternal rim before exiting the Golgi. In contrast, bulky cargos were found only at the rim. Our study therefore directly demonstrates the spatial separation of processing and transport functions within the Golgi complex.

Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5.

The endosome-to-Golgi or endocytic retrograde trafficking pathway is an important post-Golgi recycling route. Here we show that amino acids (AAs) can stimulate the retrograde trafficking and regulate the cell surface localization of certain Golgi membrane proteins. By testing components of the AA-stimulated mTORC1 signaling pathway, we demonstrate that SLC38A9, v-ATPase and Ragulator, but not Rag GTPases and mTORC1, are essential for the AA-stimulated trafficking. Arl5, an ARF-like family small GTPase, interacts with Ragulator in an AA-regulated manner and both Arl5 and its effector, the Golgi-associated retrograde protein complex (GARP), are required for the AA-stimulated trafficking. We have therefore identified a mechanistic connection between the nutrient signaling and the retrograde trafficking pathway, whereby SLC38A9 and v-ATPase sense AA-sufficiency and Ragulator might function as a guanine nucleotide exchange factor to activate Arl5, which, together with GARP, a tethering factor, probably facilitates the endosome-to-Golgi trafficking.

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 development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex.

We report here an image-based method to quantify the stoichiometry of diffraction-limited sub-cellular protein complexes in vivo under spinning disk confocal microscopy. A GFP single molecule fluorescence standard was first established by immobilizing His-tagged GFP molecules onto the glass surface via nickel nitrilotriacetic acid functionalized polyethylene glycol. When endogenous nucleoporins were knocked down and replaced by the exogenously expressed and knockdown-resistant GFP-nucleoporins, the stoichiometry of the nucleoporin was estimated by the ratio of its fluorescence intensity to that of the GFP single molecules. Our measured stoichiometry of Nup35, Nup93, Nup133 and Nup88 is 23, 18, 14 and 9 and there are possibly16 copies of Nup107-160 complex per nuclear pore complex.

A novel imaging method for quantitative Golgi localization reveals differential intra-Golgi trafficking of secretory cargoes.

Cellular functions of the Golgi are determined by the unique distribution of its resident proteins. Currently, electron microscopy is required for the localization of a Golgi protein at the sub-Golgi level. We developed a quantitative sub-Golgi localization method based on centers of fluorescence masses of nocodazole-induced Golgi ministacks under conventional optical microscopy. Our method is rapid, convenient, and quantitative, and it yields a practical localization resolution of ∼ 30 nm. The method was validated by the previous electron microscopy data. We quantitatively studied the intra-Golgi trafficking of synchronized secretory membrane cargoes and directly demonstrated the cisternal progression of cargoes from the cis- to the trans-Golgi. Our data suggest that the constitutive efflux of secretory cargoes could be restricted at the Golgi stack, and the entry of the trans-Golgi network in secretory pathway could be signal dependent.