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.

The fate of PrP GPI-anchor signal peptide is modulated by P238S pathogenic mutation.

Glycosylphosphatidylinositol (GPI)-anchored proteins are localized to the plasma membrane via a C-terminally linked GPI anchor. The GPI anchor is added concomitantly to the cleavage of the carboxy-terminal GPI-anchor signal sequence, thereby causing the release of a C-terminal hydrophobic peptide, whose fate has not yet been investigated. Here we followed the fate of the GPI-attachment signal of the prion protein (PrP), a protein implicated in various types of transmissible neurodegenerative spongiform encephalopathies (TSE). The PrP GPI-anchor signal sequence shows a remarkable and unusual degree of conservation across the species and contains two point mutations (M232R/T and P238S) that are responsible for genetic forms of prion disorders. We show that the PrP GPI-anchor signal peptide (SP), but not the one from an unrelated GPI-anchored protein (folate receptor), undergoes degradation via the proteasome. Moreover, the P238S point mutation partially protects the PrP GPI-anchor SP from degradation. Our data provide the first attempt to address the fate of a GPI-anchor SP and identify a role for the P238S mutation, suggesting the possibility that the PrP GPI-anchor SP could play a role in neurodegenerative prion diseases.

A-ring oxygenation modulates the chemistry and bioactivity of caged Garcinia xanthones.

Natural products of the caged Garcinia xanthones (CGX) family are characterized by a unique chemical structure, potent bioactivities and promising pharmacological profiles. We have developed a Claisen/Diels-Alder reaction cascade that, in combination with a Pd(0)-catalyzed reverse prenylation, provides rapid and efficient access to the CGX pharmacophore, represented by the structure of cluvenone. To further explore this pharmacophore, we have synthesized various A-ring oxygenated analogues of cluvenone and have evaluated their bioactivities in terms of growth inhibition, mitochondrial fragmentation, induction of mitochondrial-dependent cell death and Hsp90 client inhibition. We found that installation of an oxygen functionality at various positions of the A-ring influences significantly both the site-selectivity of the Claisen/Diels-Alder reaction and the bioactivity of these compounds, due to remote electronic effects.

Subcellular localization and activity of gambogic acid.

The natural product gambogic acid (GA) has shown significant potential as an anticancer agent as it is able to induce apoptosis in multiple tumor cell lines, including multidrug-resistant cell lines, as well as displaying antitumor activity in animal models. Despite the fact that GA has entered phase I clinical trials, the primary cellular target and mode of action of this compound remain unclear, although many proteins have been shown to be affected by it. By thorough analysis of several cellular organelles, at both the morphological and functional levels, we demonstrate that the primary effect of GA is at the mitochondria. We found that GA induces mitochondrial damage within minutes of incubation at low-micromolar concentrations. Moreover, a fluorescent derivative of GA was able to localize specifically to the mitochondria and was displaced from these organelles after competition with unlabeled GA. These findings indicate that GA directly targets the mitochondria to induce the intrinsic pathway of apoptosis, and thus represents a new member of the mitocans.

Cluvenone induces apoptosis via a direct target in mitochondria: a possible mechanism to circumvent chemo-resistance?

The synthetic caged Garcinia xanthone, cluvenone, has potent and selective cytotoxicity against numerous cancer cell lines including those that are multi-drug resistant. The direct target of this structurally and functionally unique agent is unknown and that of the parent natural product, gambogic acid (GA), presently in clinical trials, is not yet entirely clear. For the first time, using fluorescently labeled GA (GA-Bodipy), we determined that GA-Bodipy localized in mitochondria and was effectively displaced by cluvenone in competition experiments indicating that the direct target of cluvenone resided in mitochondria and was shared by GA. In agreement with these findings, treatment of HeLa cells with cluvenone or GA resulted in disruption of mitochondrial morphology within 4 h. Furthermore, experiments using the potential sensitive JC-1 dye demonstrated that cells treated with 1 µM cluvenone for 1 h had significant loss of MMP compared to control cells. Examination of Cyt c levels in leukemia cells treated with 1 µM cluvenone resulted in a 4-fold increase in levels of both cytosolic and mitochondrial Cyt c. In agreement with Cyt c release, caspase 9 activity was increased 2.6-fold after treatment of cells for 5 h with 1 µM cluvenone. Remarkably, the caspase-9 inhibitor, Z-LEHD-FMK, blocked cluvenone-induced apoptosis in a dose-dependent manner with apoptosis being completely blocked by 10 µM of the inhibitor. In conclusion, cluvenone, an agent with potent cytotoxicity against multi-drug resistant tumor cells, has direct targets in mitochondria thus setting precedence for drug discovery efforts against these targets in the treatment of refractory cancers.

Self-calibrating viscosity probes: design and subcellular localization.

We describe the design, synthesis and fluorescence profiles of new self-calibrating viscosity dyes in which a coumarin (reference fluorophore) has been covalently linked with a molecular rotor (viscosity sensor). Characterization of their fluorescence properties was made with separate excitation of the units and through resonance energy transfer from the reference to the sensor dye. We have modified the linker and the substitution of the rotor in order to change the hydrophilicity of these probes thereby altering their subcellular localization. For instance, hydrophilic dye 12 shows a homogeneous distribution inside the cell and represents a suitable probe for viscosity measurements in the cytoplasm.

Functional genomics reveals genes involved in protein secretion and Golgi organization.

Yeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum.

Chemical biology studies on norrisolide.

The cellular activity of norrisolide (7), a novel Golgi-vesiculating agent, was dissected as function of its chemical structure. This natural product induces irreversible vesiculation of the Golgi membranes and blocks protein transport at the level of the Golgi. The Golgi localization and fragmentation effects of 7 depend on the presence of the perhydroindane core, while the irreversibility of fragmentation depends on the acetyl group of 7. We show that fluorescent derivatives of norrisolide are able to localize to the Golgi apparatus and represent important tools for the study of the Golgi structure and function.

An optimized immunoaffinity fluorescent method for natural product target elucidation.

Understanding the mode of action of small molecules is an integral facet of drug discovery. We report an optimized immunoaffinity fluorescent method that allows one to conduct parallel studies at both the cellular and molecular level using a single probe construct. Viability of the method has been evaluated analytically and applied using glycyrrhetic acid as a model.

Expression of T cell receptor alpha gene (TCRA) in human rhabdomyosarcoma and other musculo-skeletal sarcomas.

Expression of the T cell receptor (TCR) genes is not restricted to T lymphocytes. Human prostate and breast express a truncated TCR gamma transcript. In the mouse, TCR alpha (TCRA) and beta partial transcripts are expressed by mesenchymal cells and TCRA transcripts by epithelial cells of the kidney. We show now that TCRA constant region expression is common in normal and neoplastic human cells of mesenchymal and neuroectodermal origin. TCR transcripts are derived from an unrearranged TCRA locus. Moreover, rhabdomyosarcoma cells highly expressed a specific J49-C splicing product deriving from the assembly of J49 segment and constant region. TCRA ectopic transcripts/proteins negatively regulate rhabdomyosarcoma cell growth as suggested by TCRA gene expression downmodulation effects using a specific duplex small interfering RNA.

Cytosolically expressed PrP GPI-signal peptide interacts with mitochondria.

We previously reported that PrP GPI-anchor signal peptide (GPI-SP) is specifically degraded by the proteasome. Additionally, we showed that the point mutation P238S, responsible for a genetic form of prion diseases, while not affecting the GPI-anchoring process, results in the accumulation of PrP GPI-SP, suggesting the possibility that PrP GPI-anchor signal peptide could play a role in neurodegenerative prion diseases. We now show that PrP GPI-SP, when expressed as a cytosolic peptide, is able to localize to the mitochondria and to induce mitochondrial fragmentation and vacuolarization, followed by loss in mitochondrial membrane potential, ultimately resulting in apoptosis. Our results identify the GPI-SP of PrP as a novel candidate responsible for the impairment in mitochondrial function involved in the synaptic pathology observed in prion diseases, establishing a link between PrP GPI-SP accumulation and neuronal death.

Trifunctional norrisolide probes for the study of Golgi vesiculation.

Inspired by the effect of norrisolide on the Golgi complex, we synthesized norrisolide probes that contain: the perhydroindane core of the parent natural product for Golgi localization, a crosslinking unit (aryl azide or epoxide) for covalent binding to the target, and a tag (biotin or iodine) for subsequent target purification. We found that biotin-containing probes 14, 20 and 24 induced inefficient Golgi vesiculation. However, the iodinated probe 25 induced extensive and irreversible Golgi fragmentation. This probe can be used for the isolation of the cellular target of norrisolide.

The Golgi-associated protein GRASP is required for unconventional protein secretion during development.

During Dictyostelium development, prespore cells secrete acyl-CoA binding protein (AcbA). Upon release, AcbA is processed to generate a peptide called spore differentiation factor-2 (SDF-2), which triggers terminal differentiation of spore cells. We have found that cells lacking Golgi reassembly stacking protein (GRASP), a protein attached peripherally to the cytoplasmic surface of Golgi membranes, fail to secrete AcbA and, thus, produce inviable spores. Surprisingly, AcbA lacks a signal sequence and is not secreted via the conventional secretory pathway (endoplasmic reticulum-Golgi-cell surface). GRASP is not required for conventional protein secretion, growth, and the viability of vegetative cells. Our findings reveal a physiological role of GRASP and provide a means to understand unconventional secretion and its role in development.

Chemical analysis of norrisolide-induced Golgi vesiculation.

The chemical origin of the norrisolide-induced irreversible Golgi vesiculation was studied using a variety of norrisolide probes. This natural product was found to bind to a receptor on the Golgi membranes using the perhydroindane core fragment as the recognition element. The acetylated gamma-lactol-gamma-lactone side chain of norrisolide is essential for the irreversible Golgi vesiculation and can be replaced by other electrophilic motifs without loss of biological function. In particular, compound 10 reproduces the cellular phenotype of the natural product.

Fragmentation of Golgi membranes by norrisolide and designed analogues.

The effect of norrisolide (4) and designed analogues on the Golgi membranes is presented. We found that 4 is the first compound known to induce an irreversible vesiculation of these membranes. To investigate the chemical origins of this new effect we synthesized and evaluated a series of norrisolide analogues in which the chemical functionalities present in the parent structure were altered. Such structure/function studies suggest that the perhydroindane core of 4 is critical for binding to the target protein, while the C21 acetate unit is essential for the irreversible vesiculation of the Golgi membranes.