Rab8 and TNPO1 are ciliary transport adaptors for GTPase Arl13b by interacting with its RVEP motif containing ciliary targeting sequence.

Arl13b, an ARF/Arl-family GTPase, is highly enriched in the cilium. Recent studies have established Arl13b as one of the most crucial regulators for ciliary organization, trafficking, and signaling. The ciliary localization of Arl13b is known to require the RVEP motif. However, its cognate ciliary transport adaptor has been elusive. Here, by imaging the ciliary localization of truncation and point mutations, we defined the ciliary targeting sequence (CTS) of Arl13b as a C-terminal stretch of 17 amino acids containing the RVEP motif. We found Rab8-GDP, but not Rab8-GTP, and TNPO1 simultaneously and directly bind to the CTS of Arl13b in pull-down assays using cell lysates or purified recombinant proteins. Furthermore, Rab8-GDP substantially enhances the interaction between TNPO1 and CTS. Additionally, we determined that the RVEP motif is an essential element as its mutation abolishes the interaction of the CTS with Rab8-GDP and TNPO1 in pull-down and TurboID-based proximity ligation assays. Finally, the knockdown of endogenous Rab8 or TNPO1 decreases the ciliary localization of endogenous Arl13b. Therefore, our results suggest Rab8 and TNPO1 might function together as a ciliary transport adaptor for Arl13b by interacting with its RVEP-containing CTS.

A quantitative study of the Golgi retention of glycosyltransferases.

How Golgi glycosyltransferases and glycosidases (hereafter glycosyltransferases) localize to the Golgi is still unclear. Here, we first investigated the post-Golgi trafficking of glycosyltransferases. We found that glycosyltransferases can escape the Golgi to the plasma membrane, where they are subsequently endocytosed to the endolysosome. Post-Golgi glycosyltransferases are probably degraded by ectodomain shedding. We discovered that most glycosyltransferases are not retrieved from post-Golgi sites, indicating that retention rather than retrieval is the primary mechanism for their Golgi localization. We therefore used the Golgi residence time to study Golgi retention of glycosyltransferases quantitatively and systematically. Quantitative analysis of chimeras of ST6GAL1 and either transferrin receptor or tumor necrosis factor α revealed the contributions of three regions of ST6GAL1, namely the N-terminal cytosolic tail, the transmembrane domain and the ectodomain, to Golgi retention. We found that each of the three regions is sufficient for Golgi retention in an additive manner. N-terminal cytosolic tail length negatively affects the Golgi retention of ST6GAL1, similar to effects observed for the transmembrane domain. Therefore, the long N-terminal cytosolic tail and transmembrane domain could act as Golgi export signals for transmembrane secretory cargos. This article has an associated First Person interview with the first author of the paper.

Versatile Types of DNA-Based Nanobiosensors for Specific Detection of Cancer Biomarker FEN1 in Living Cells and Cell-Free Systems.

Flap structure-specific endonuclease 1 (FEN1) is overexpressed in various types of human cancer cells and has been recognized as a promising biomarker for cancer diagnosis in the recent years. In order to specifically detect the abundance and activity of this cancer-overexpressed enzyme, different types of DNA-based nanodevices were created during our investigations. It is shown in our studies that these newly designed biosensors are highly sensitive and specific for FEN1 in living cells as well as in cell-free systems. It is expected that these nanoprobes could be useful for monitoring FEN1 activity in human cancer cells, and also for cell-based screening of FEN1 inhibitors as new anticancer drugs.

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.

Regulation of cellular cholesterol distribution via non-vesicular lipid transport at ER-Golgi contact sites.

Abnormal distribution of cellular cholesterol is associated with numerous diseases, including cardiovascular and neurodegenerative diseases. Regulated transport of cholesterol is critical for maintaining its proper distribution in the cell, yet the underlying mechanisms remain unclear. Here, we show that lipid transfer proteins, namely ORP9, OSBP, and GRAMD1s/Asters (GRAMD1a/GRAMD1b/GRAMD1c), control non-vesicular cholesterol transport at points of contact between the ER and the trans-Golgi network (TGN), thereby maintaining cellular cholesterol distribution. ORP9 localizes to the TGN via interaction between its tandem α-helices and ORP10/ORP11. ORP9 extracts PI4P from the TGN to prevent its overaccumulation and suppresses OSBP-mediated PI4P-driven cholesterol transport to the Golgi. By contrast, GRAMD1s transport excess cholesterol from the Golgi to the ER, thereby preventing its build-up. Cells lacking ORP9 exhibit accumulation of cholesterol at the Golgi, which is further enhanced by additional depletion of GRAMD1s with major accumulation in the plasma membrane. This is accompanied by chronic activation of the SREBP-2 signalling pathway. Our findings reveal the importance of regulated lipid transport at ER-Golgi contacts for maintaining cellular cholesterol distribution and homeostasis.

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.

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.

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.

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.

Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase.

Arl1 is a member of Arf family small GTPases that is essential for the organization and function of Golgi complex. Mon2/Ysl2, which shares significant homology with Sec7 family Arf guanine nucleotide exchange factors, was poorly characterized in mammalian cells. Here, we report the first in depth characterization of mammalian Mon2. We found that Mon2 localized to trans-Golgi network which was dependent on both its N and C termini. The depletion of Mon2 did not affect the Golgi localized or cellular active form of Arl1. Furthermore, our in vitro assay demonstrated that recombinant Mon2 did not promote guanine nucleotide exchange of Arl1. Therefore, our results suggest that Mon2 could be neither necessary nor sufficient for the guanine nucleotide exchange of Arl1. We demonstrated that Mon2 was involved in endosome-to-Golgi trafficking as its depletion accelerated the delivery of furin and CI-M6PR to Golgi after endocytosis.