RNA scaffolds the Golgi ribbon by forming condensates with GM130.

The mammalian Golgi is composed of stacks that are laterally connected into a continuous ribbon-like structure. The integrity and function of the ribbon is disrupted under stress conditions, but the molecular mechanisms remain unclear. Here we show that the ribbon is maintained by biomolecular condensates of RNA and the Golgi matrix protein GM130 (GOLGA2).

An actin filament branching surveillance system regulates cell cycle progression, cytokinesis and primary ciliogenesis.

Dysfunction of cell cycle control and defects of primary ciliogenesis are two features of many cancers. Whether these events are interconnected and the driving mechanism coordinating them remains elusive. Here, we identify an actin filament branching surveillance system that alerts cells of actin branching insufficiency and regulates cell cycle progression, cytokinesis and primary ciliogenesis.

Interplay between Asters/GRAMD1s and phosphatidylserine in intermembrane transport of LDL cholesterol.

Low-density lipoprotein (LDL) delivers cholesterol to mammalian cells through receptor-mediated endocytosis. The LDL cholesterol is liberated in lysosomes and transported to the plasma membrane (PM) and from there to the endoplasmic reticulum (ER). Excess ER cholesterol is esterified with a fatty acid for storage as cholesteryl esters.

Importin α phosphorylation promotes TPX2 activation by GM130 to control astral microtubules and spindle orientation.

Spindle orientation is important in multiple developmental processes as it determines cell fate and function. The orientation of the spindle depends on the assembly of a proper astral microtubule network. Here, we report that the spindle assembly factor TPX2 regulates astral microtubules.

Rapid degradation of GRASP55 and GRASP65 reveals their immediate impact on the Golgi structure.

GRASP55 and GRASP65 have been implicated in stacking of Golgi cisternae and lateral linking of stacks within the Golgi ribbon. However, RNAi or gene knockout approaches to dissect their respective roles have often resulted in conflicting conclusions. Here, we gene-edited GRASP55 and/or GRASP65 with a degron tag in human fibroblasts, allowing for induced rapid degradation by the proteasome.

Last step in the path of LDL cholesterol from lysosome to plasma membrane to ER is governed by phosphatidylserine.

Animal cells acquire cholesterol from receptor-mediated uptake of low-density lipoprotein (LDL), which releases cholesterol in lysosomes. The cholesterol moves to the endoplasmic reticulum (ER), where it inhibits production of LDL receptors, completing a feedback loop. Here we performed a CRISPR-Cas9 screen in human SV589 cells for genes required for LDL-derived cholesterol to reach the ER.

Lysosomal cholesterol export reconstituted from fragments of Niemann-Pick C1.

Niemann-Pick C1 (NPC1) is a polytopic membrane protein with 13 transmembrane helices that exports LDL-derived cholesterol from lysosomes by carrying it through the 80 Å glycocalyx and the 40 Å lipid bilayer. Transport begins when cholesterol binds to the N-terminal domain (NTD) of NPC1, which projects to the surface of the glycocalyx. Here, we reconstitute cholesterol transport by expressing the NTD as a fragment separate from the remaining portion of NPC1.

3.3 Å structure of Niemann-Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport.

Niemann-Pick C1 (NPC1) and NPC2 proteins are indispensable for the export of LDL-derived cholesterol from late endosomes. Mutations in these proteins result in Niemann-Pick type C disease, a lysosomal storage disease. Despite recent reports of the NPC1 structure depicting its overall architecture, the function of its C-terminal luminal domain (CTD) remains poorly understood even though 45% of NPC disease-causing mutations are in this domain.

Cell Labeling with Magneto-Endosymbionts and the Dissection of the Subcellular Location, Fate, and Host Cell Interactions.

Purpose: The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments.

Golgi ribbon disassembly during mitosis, differentiation and disease progression.

The Golgi apparatus is tightly integrated into the cellular system where it plays essential roles required for a variety of cellular processes. Its vital functions include not only processing and sorting of proteins and lipids, but also serving as a signaling hub and a microtubule-organizing center. Golgi stacks in mammalian cells are interconnected into a compact ribbon in the perinuclear region.

Mitotic Golgi disassembly is required for bipolar spindle formation and mitotic progression.

During mitosis, the mammalian Golgi vesiculates and, upon partitioning, reassembles in each daughter cell; however, it is not clear whether the disassembly process per se is important for partitioning or is merely an outcome of mitotic entry. Here, we show that Golgi vesiculation is required for progression to metaphase. To prevent Golgi disassembly, we expressed HRP linked to a Golgi-resident protein and acutely triggered the polymerization of 3,3'-diaminobenzidine (DAB) in the Golgi lumen.

Geranylgeranyl-regulated transport of the prenyltransferase UBIAD1 between membranes of the ER and Golgi.

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. Previously, we found that sterols trigger binding of UBIAD1 to endoplasmic reticulum (ER)-localized HMG-CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids, including GGpp. This binding inhibits sterol-accelerated degradation of reductase, which contributes to feedback regulation of the enzyme.

Sec13 Regulates Expression of Specific Immune Factors Involved in Inflammation In Vivo.

The Sec13 protein functions in various intracellular compartments including the nuclear pore complex, COPII-coated vesicles, and inside the nucleus as a transcription regulator. Here we developed a mouse model that expresses low levels of Sec13 (Sec13(H/-)) to assess its functions in vivo, as Sec13 knockout is lethal. These Sec13 mutant mice did not present gross defects in anatomy and physiology.

GM130 Regulates Golgi-Derived Spindle Assembly by Activating TPX2 and Capturing Microtubules.

Spindle assembly requires the coordinated action of multiple cellular structures to nucleate and organize microtubules in a precise spatiotemporal manner. Among them, the contributions of centrosomes, chromosomes, and microtubules have been well studied, yet the involvement of membrane-bound organelles remains largely elusive. Here, we provide mechanistic evidence for a membrane-based, Golgi-derived microtubule assembly pathway in mitosis.

The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism.

Sequential actions of the AAA-ATPase valosin-containing protein (VCP)/p97 and the proteasome 19 S regulatory particle in sterol-accelerated, endoplasmic reticulum (ER)-associated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Accelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26 S proteasomes through poorly defined reactions mediated by the AAA-ATPase valosin-containing protein (VCP)/p97 and augmented by the nonsterol isoprenoid geranylgeraniol.

Chemical principles for the design of a novel fluorescent probe with high cancer-targeting selectivity and sensitivity.

Understanding of principles governing selective and sensitive cancer targeting is critical for development of chemicals for cancer diagnostics and treatment. We determined the underlying mechanisms of how a novel fluorescent small organic molecule, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), selectively labels cancer cells but not normal cells. We show that BMVC is retained in the lysosomes of normal cells.

Point mutation in luminal loop 7 of Scap protein blocks interaction with loop 1 and abolishes movement to Golgi.

Scap is a polytopic protein of the endoplasmic reticulum (ER) that controls cholesterol homeostasis by transporting sterol regulatory element-binding proteins (SREBPs) from the ER to the Golgi complex. Scap has eight transmembrane helices (TM) joined by four small hydrophilic loops and three large loops. Two of the large loops (Loops 1 and 7) are in the ER lumen, and the other large loop (Loop 6) faces the cytosol where it binds COPII proteins that initiate transport to Golgi.

Modular organization of the mammalian Golgi apparatus.

The Golgi apparatus is essential for post-translational modifications and sorting of proteins in the secretory pathway. In addition, it further performs a broad range of specialized functions. This functional diversity is achieved by combining basic morphological modules of cisternae into higher ordered structures.

The membrane-bound transcription factor CREB3L1 is activated in response to virus infection to inhibit proliferation of virus-infected cells.

CREB3L1/OASIS is a cellular transcription factor synthesized as a membrane-bound precursor and activated by regulated intramembrane proteolysis in response to stimuli like ER stress. Comparing gene expression between Huh7 subclones that are permissive for hepatitis C virus (HCV) replication versus the nonpermissive parental Huh7 cells, we identified CREB3L1 as a host factor that inhibits proliferation of virus-infected cells. Upon infection with diverse DNA and RNA viruses, including murine γ-herpesvirus 68, HCV, West Nile virus (WNV), and Sendai virus, CREB3L1 was proteolytically cleaved, allowing its NH(2) terminus to enter the nucleus and induce multiple genes encoding inhibitors of the cell cycle to block cell proliferation.

Golgi biogenesis.

The Golgi is an essential membrane-bound organelle in the secretary pathway of eukaryotic cells. In mammalian cells, the Golgi stacks are integrated into a continuous perinuclear ribbon, which poses a challenge for the daughter cells to inherit this membrane organelle during cell division. To facilitate proper partitioning, the mammalian Golgi ribbon is disassembled into vesicles in early mitosis.

Identification of luminal Loop 1 of Scap protein as the sterol sensor that maintains cholesterol homeostasis.

Cellular cholesterol homeostasis is maintained by Scap, an endoplasmic reticulum (ER) protein with eight transmembrane helices. In cholesterol-depleted cells, Scap transports sterol regulatory element-binding proteins (SREBPs) to the Golgi, where the active fragment of SREBP is liberated by proteases so that it can activate genes for cholesterol synthesis. When ER cholesterol increases, Scap binds cholesterol, and this changes the conformation of cytosolic Loop 6, which contains the binding site for COPII proteins.

Nakiterpiosin targets tubulin and triggers mitotic catastrophe in human cancer cells.

Agents that interfere with mitotic progression by perturbing microtubule dynamics are commonly used for cancer chemotherapy. Here, we identify nakiterpiosin as a novel antimitotic drug that targets microtubules. Nakiterpiosin induces mitotic arrest and triggers mitotic catastrophe in human cancer cells by impairing bipolar spindle assembly.

Unraveling the Golgi ribbon.

The Golgi apparatus lies at the heart of the secretory pathway where it receives, modifies and sorts protein cargo to the proper intracellular or extracellular location. Although this secretory function is highly conserved throughout the eukaryotic kingdom, the structure of the Golgi complex is arranged very differently among species. In particular, Golgi membranes in vertebrate cells are integrated into a single compact entity termed the Golgi ribbon that is normally localized in the perinuclear area and in close vicinity to the centrosomes.