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). We identify GM130 as a membrane-bound RNA-binding protein, which directly recruits RNA and associated RNA-binding proteins to the Golgi membrane. Acute degradation of RNA or GM130 in cells disrupts the ribbon. Under stress conditions, RNA dissociates from GM130 and the ribbon is disjointed, but after the cells recover from stress the ribbon is restored. When overexpressed in cells, GM130 forms RNA-dependent liquid-like condensates. GM130 contains an intrinsically disordered domain at its amino terminus, which binds RNA to induce liquid-liquid phase separation. These co-condensates are sufficient to link purified Golgi membranes, reconstructing lateral linking of stacks into a ribbon-like structure. Together, these studies show that RNA acts as a structural biopolymer that together with GM130 maintains the integrity of the Golgi ribbon.

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. We find that Oral-Facial-Digital syndrome 1 functions as a class II Nucleation promoting factor to promote Arp2/3 complex-mediated actin branching. Perturbation of actin branching promotes OFD1 degradation and inactivation via liquid-to-gel transition. Elimination of OFD1 or disruption of OFD1-Arp2/3 interaction drives proliferating, non-transformed cells into quiescence with ciliogenesis by an RB-dependent mechanism, while it leads oncogene-transformed/cancer cells to incomplete cytokinesis and irreversible mitotic catastrophe via actomyosin ring malformation. Inhibition of OFD1 leads to suppression of multiple cancer cell growth in mouse xenograft models. Thus, targeting OFD1-mediated actin filament branching surveillance system provides a direction for cancer therapy.

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. Recently, we showed that PM-to-ER transport of LDL cholesterol requires phosphatidylserine (PS). Others showed that PM-to-ER transport of cholesterol derived from other sources requires Asters (also called GRAMD1s), a family of three ER proteins that bridge between the ER and PM by binding to PS. Here, we use a cholesterol esterification assay and other measures of ER cholesterol delivery to demonstrate that Asters participate in PM-to-ER transport of LDL cholesterol in Chinese hamster ovary cells. Knockout of the gene encoding PTDSS1, the major PS-synthesizing enzyme, lowered LDL-stimulated cholesterol esterification by 85%, whereas knockout of all three Aster genes lowered esterification by 65%. The reduction was even greater (94%) when the genes encoding PTDSS1 and the three Asters were knocked out simultaneously. We conclude that Asters participate in LDL cholesterol delivery from PM to ER, and their action depends in large part, but not exclusively, on PS. The data also indicate that PS participates in another delivery pathway, so far undefined, that is independent of Asters.

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. TPX2 in the spindle pole area is activated by GM130 (GOLGA2) on Golgi membranes to promote astral microtubule growth. GM130 relieves TPX2 inhibition by competing for importin α1 (KPNA2) binding. Mitotic phosphorylation of importin α at serine 62 (S62) by CDK1 switches its substrate preference from TPX2 to GM130, thereby enabling competition-based activation. Importin α S62A mutation impedes local TPX2 activation and compromises astral microtubule formation, ultimately resulting in misoriented spindles. Blocking the GM130-importin α-TPX2 pathway impairs astral microtubule growth. Our results reveal a novel role for TPX2 in the organization of astral microtubules. Furthermore, we show that the substrate preference of the important mitotic modulator importin α is regulated by CDK1-mediated phosphorylation.

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. We show that acute depletion of either GRASP55 or GRASP65 does not affect the Golgi ribbon, while chronic degradation of GRASP55 disrupts lateral connectivity of the ribbon. Acute double depletion of both GRASPs coincides with the loss of the vesicle tethering proteins GM130, p115, and Golgin-45 from the Golgi and compromises ribbon linking. Furthermore, GRASP55 and/or GRASP65 is not required for maintaining stacks or de novo assembly of stacked cisternae at the end of mitosis. These results demonstrate that both GRASPs are dispensable for Golgi stacking but are involved in maintaining the integrity of the Golgi ribbon together with GM130 and Golgin-45.

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. We identified the gene encoding PTDSS1, an enzyme that synthesizes phosphatidylserine (PS), a phospholipid constituent of the inner layer of the plasma membrane (PM). In PTDSS1-deficient cells where PS is low, LDL cholesterol leaves lysosomes but fails to reach the ER, instead accumulating in the PM. The addition of PS restores cholesterol transport to the ER. We conclude that LDL cholesterol normally moves from lysosomes to the PM. When the PM cholesterol exceeds a threshold, excess cholesterol moves to the ER in a process requiring PS. In the ER, excess cholesterol acts to reduce cholesterol uptake, preventing toxic cholesterol accumulation. These studies reveal that one lipid-PS-controls the movement of another lipid-cholesterol-between cell membranes. We relate these findings to recent evidence indicating that PM-to-ER cholesterol transport is mediated by GRAMD1/Aster proteins that bind PS and cholesterol.

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. When co-expressed, the two NPC1 fragments reconstitute cholesterol transport, indicating that the NTD has the flexibility to interact with the remaining parts of NPC1 even when not covalently linked. We also show that cholesterol can be transferred from the NTD of one full-length NPC1 to another NPC1 molecule that lacks the NTD. These data support the hypothesis that cholesterol is transported through interactions between two or more NPC1 molecules.

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. PROCEDURES: The ME's surface epitopes, contrast-producing paramagnetic magnetosomal iron, and genome were studied using immunocytochemistry (ICC), Fe and MRI contrast measurements, and quantitative polymerase chain reaction (qPCR), respectively. These assays, coupled with other common assays, enabled validation of ME cell labeling and dissection of ME subcellular processing. RESULTS: The assays mentioned above provide qualitative and quantitative assessments of cell labeling, the subcellular localization and the fate of MEs. ICC results, with an ME-specific antibody, qualitatively shows homogenous labeling with MEs. The ferrozine assay shows that MEs have an average of 7 fg Fe/ME, ∼30 % of which contributes to MRI contrast and ME-labeled MDA-MB-231 (MDA-231) cells generally have 2.4 pg Fe/cell, implying ∼350 MEs/cell. Adjusting the concentration of Fe in the ME growth media reduces the concentration of non-MRI contrast-producing Fe. Results from the qPCR assay, which quantifies ME genomes in labeled cells, shows that processing of MEs begins within 24 h in MDA-231 cells. ICC results suggest this intracellular digestion of MEs occurs by the lysosomal degradation pathway. MEs coated with listeriolysin O (LLO) are able to escape the primary phagosome, but subsequently co-localize with LC3, an autophagy-associated molecule, and are processed for digestion. In embryos, where autophagy is transiently suppressed, MEs show an increased capacity for survival and even replication. Finally, transmission electron microscopy (TEM) of ME-labeled MDA-231 cells confirms that the magnetosomes (the MRI contrast-producing particles) remain intact and enable in vivo cell tracking. CONCLUSIONS: MEs are used to label mammalian cells for the purpose of cell tracking in vivo, with MRI. Various assays described herein (ICC, ferrozine, and qPCR) allow qualitative and quantitative assessments of labeling efficiency and provide a detailed understanding of subcellular processing of MEs. In some cell types, MEs are digested, but the MRI-producing particles remain. Coating with LLO allows MEs to escape the primary phagosome, enhances retention slightly, and confirms that MEs are ultimately processed by autophagy. Numerous intracellular bacteria and all endosymbiotically derived organelles have evolved molecular mechanisms to avoid intracellular clearance, and identification of the specific processes involved in ME clearance provides a framework on which to develop MEs with enhanced retention in mammalian cells.

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. Here, we report a crystal structure at 3.3 Å resolution of NPC1* (residues 314-1,278), which-in contrast to previous lower resolution structures-features the entire CTD well resolved. Notably, all eight cysteines of the CTD form four disulfide bonds, one of which (C909-C914) enforces a specific loop that in turn mediates an interaction with a loop of the N-terminal domain (NTD). Importantly, this loop and its interaction with the NTD were not observed in any previous structures due to the lower resolution. Our mutagenesis experiments highlight the physiological relevance of the CTD-NTD interaction, which might function to keep the NTD in the proper orientation for receiving cholesterol from NPC2. Additionally, this structure allows us to more precisely map all of the disease-causing mutations, allowing future molecular insights into the pathogenesis of NPC disease.

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. However, the ribbon can undergo distinct disassembly processes that reflect the cellular state or environmental demands and stress. For instance, its most dramatic change takes place in mitosis when the ribbon is efficiently disassembled into vesicles through a combination of ribbon unlinking, cisternal unstacking and vesiculation. Furthermore, the ribbon can also be detached and positioned at specific cellular locations to gain additional functionalities during differentiation, or fragmented to different degrees along disease progression or upon cell death. Here, we describe the major morphological alterations of Golgi ribbon disassembly under physiological and pathological conditions and discuss the underlying mechanisms that drive these changes.

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. The DAB polymer does not affect interphase cell viability, but inhibits Golgi fragmentation by nocodazole and brefeldin A and also halts cells in early mitosis. The arrest is Golgi specific and does not occur when DAB is polymerized in the endosomes. Cells with a DAB polymer in the Golgi enter mitosis normally but arrest with an intact Golgi clustered at a monopolar spindle and an active spindle assembly checkpoint (SAC). Mitotic progression is restored upon centrosome depletion by the Polo-like kinase 4 inhibitor, centrinone, indicating that the link between the Golgi and the centrosomes must be dissolved to reach metaphase. These results demonstrate that Golgi disassembly is required for mitotic progression because failure to vesiculate the Golgi activates the canonical SAC. This requirement suggests that cells actively monitor Golgi integrity in mitosis.

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. The addition to cells of geranylgeraniol (GGOH), which can become converted to GGpp, triggers release of UBIAD1 from reductase, allowing for its maximal degradation and permitting ER-to-Golgi transport of UBIAD1. Here, we further characterize geranylgeranyl-regulated transport of UBIAD1. Results of this characterization support a model in which UBIAD1 continuously cycles between the ER and medial-trans Golgi of isoprenoid-replete cells. Upon sensing a decline of GGpp in ER membranes, UBIAD1 becomes trapped in the organelle where it inhibits reductase degradation. Mutant forms of UBIAD1 associated with Schnyder corneal dystrophy (SCD), a human eye disease characterized by corneal accumulation of cholesterol, are sequestered in the ER and block reductase degradation. Collectively, these findings disclose a novel sensing mechanism that allows for stringent metabolic control of intracellular trafficking of UBIAD1, which directly modulates reductase degradation and becomes disrupted in SCD.

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. However, the reduced levels of Sec13 in vivo yielded specific immunological defects. In particular, these Sec13 mutant mice showed low levels of MHC I and II expressed by macrophages, low levels of INF-γ and IL-6 expressed by stimulated T cells, and low frequencies of splenic IFN-γ+CD8+ T cells. In contrast, the levels of soluble and membrane-bound TGF-ß as well as serum immunoglobulin production are high in these mice. Furthermore, frequencies of CD19+CD5-CD95+ and CD19+CD5-IL-4+ B cells were diminished in Sec13(H/-) mice. Upon stimulation or immunization, some of the defects observed in the naïve mutant mice were compensated. However, TGF-ß expression remained high suggesting that Sec13 is a negative modulator of TGF-ß expression and of its immunosuppressive functions on certain immune cells. In sum, Sec13 regulates specific expression of immune factors with key functions in inflammation.

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. Upon mitotic entry, the Golgi matrix protein GM130 interacts with importin α via a classical nuclear localization signal that recruits importin α to the Golgi membranes. Sequestration of importin α by GM130 liberates the spindle assembly factor TPX2, which activates Aurora-A kinase and stimulates local microtubule nucleation. Upon filament assembly, nascent microtubules are further captured by GM130, thus linking Golgi membranes to the spindle. Our results reveal an active role for the Golgi in regulating spindle formation to ensure faithful organelle inheritance.

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. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

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. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion and thus membrane extraction of reductase were tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference-mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane-extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19 S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19 S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates.

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. In cancer cells, BMVC escapes lysosomal retention and localizes to the mitochondria or to the nucleus, where DNA-binding dramatically increases BMVC fluorescence intensity, allowing it to light up only cancer cells. Structure-function analyses of BMVC derivatives show that hydrogen-bonding capacity is a key determinant of lysosomal retention in normal cells, whereas lipophilicity directs these derivatives to the mitochondria or the nucleus in cancer cells. In addition, drug-resistant cancer cells preferentially retain BMVC in their lysosomes compared to drug-sensitive cancer cells, and BMVC can be released from drug-resistant lysosomes using lysosomotropic agents. Our results further our understanding of how properties of cellular organelles differ between normal and cancer cells, which can be exploited for diagnostic and/or therapeutic use. We also provide physiochemical design principles for selective targeting of small molecules to different organelles. Moreover, our results suggest that agents which can increase lysosomal membrane permeability may re-sensitize drug-resistant cancer cells to chemotherapeutic agents.

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. Cholesterol binding to Loop 1 alters the configuration of Loop 6, precluding COPII binding and preventing the exit of Scap from the ER. Here, we create a point mutation (Y640S) in luminal Loop 7 that prevents Scap movement to Golgi. Trypsin cleavage assays show that Loop 6 of Scap(Y640S) is always in the configuration that precludes COPII binding, even in the absence of cholesterol. When expressed separately by co-transfection, the NH2-terminal portion of Scap (containing TM helices 1-6, including Loop 1) binds to the COOH-terminal portion (containing TM helices 7-8 and Loop 7) as determined by co-immunoprecipitation. This binding does not occur when Loop 7 contains the Y640S mutation. Co-immunoprecipitation is also abolished by a point mutation in Loop 1 (Y234A) that also prevents Scap movement. These data suggest that Scap Loop 1 must interact with Loop 7 to maintain Loop 6 in the configuration that permits COPII binding. These results help explain the operation of Scap as a sterol sensor.

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. Linking cisternae into stacks that are further connected through tubules into a continuous Golgi ribbon greatly increases its efficiency and expands its repertoire of functions. During cell division, the different modules of the Golgi are inherited by different mechanisms to maintain its functional and morphological composition.