Francisco Lázaro-Diéguez

Mutant huntingtin impairs post-Golgi trafficking to lysosomes by delocalizing optineurin/Rab8 complex from the Golgi apparatus.

Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function.

Dynamics of an F-actin aggresome generated by the actin-stabilizing toxin jasplakinolide

In this study, we report the formation of several cytoplasmic inclusion bodies composed of filamentous actin (F-actin) and generated by experimental treatments using depolymerizing or stabilizing actin toxins in neuronal and non-neuronal mammalian cell lines. The actin-stabilizing toxin jasplakinolide (Jpk) induced, in a microtubule-dependent manner, a single, large F-actin aggregate, which contained β- and γ-actin, ADF/cofilin, cortactin, and the actin nucleator Arp2/3. This aggregate was tightly associated with the Golgi complex and mitochondria, and was surrounded by vimentin intermediate filaments, microtubules and MAP4. Therefore, the Jpk-induced single, large F-actin aggregate fits the established criteria for being considered an aggresome. Lysosomes and/or autophagic vacuoles, proteasomes and microtubules were found to directly participate in the dissolution of this F-actin aggresome. Finally, the model reported here is simple, highly reproducible and reversible, and it provides an opportunity to test pharmacological agents that interfere with the formation, maintenance and/or disappearance of F-actin-enriched pathological inclusion bodies.

Variable actin dynamics requirement for the exit of different cargo from the trans-Golgi network

Efficient post‐Golgi trafficking depends on microtubules, but actin filaments and actin‐associated proteins are also postulated. Here we examined, by inverse fluorescence recovery after photobleaching, the role of actin dynamics in the exit from the TGN of fluorescent‐tagged apical or basolateral and raft or non‐raft‐associated cargoes. Either the actin‐stabilizing jasplakinolide or the actin‐depolymerising latrunculin B variably but significantly inhibited post‐Golgi traffic of non‐raft associated apical p75NTR and basolateral VSV‐G cargoes. The TGN‐exit of the apical‐destined VSV‐G mutant was impaired only by latrunculin B. Strikingly, the raft‐associated GPI‐anchor protein was not affected by either actin toxin. Results indicate that actin dynamics participates in the TGN egress of both apical‐ and basolateral‐targeted proteins but is not needed for apical raft‐associated cargo.

Protective effects of lysophosphatidic acid (LPA) on chronic ethanol-induced injuries to the cytoskeleton and on glucose uptake in rat astrocytes

Ethanol induces severe alterations in membrane trafficking in hepatocytes and astrocytes, the molecular basis of which is unclear. One of the main candidates is the cytoskeleton and the molecular components that regulate its organization and dynamics. Here, we examine the effect of chronic exposure to ethanol on the organization and dynamics of actin and microtubule cytoskeletons and glucose uptake in rat astrocytes. Ethanol‐treated cells cultured in either the presence or absence of fetal calf serum showed a significant increase in 2‐deoxyglucose uptake. Ethanol also caused alterations in actin organization, consisting of the dissolution of stress fibres and the appearance of circular filaments beneath the plasma membrane. When lysophosphatidic acid (LPA), which is a normal constituent of serum and a potent intercellular lipid mediator with growth factor and actin rearrangement activities, was added to ethanol‐treated astrocytes cultured without fetal calf serum, it induced the re‐appearance of actin stress fibres and the normalization of 2‐deoxyglucose uptake. Furthermore, ethanol also perturbed the microtubule dynamics, which delayed the recovery of the normal microtubule organization following removal of the microtubule‐disrupting agent nocodazole. Again, pre‐treatment with LPA prevented this alteration. Ethanol‐treated rodent fibroblast NIH3T3 cells that constitutively express an activated Rho mutant protein (GTP‐bound form) were insensitive to ethanol, as they showed no alteration either in actin stress‐fibre organization or in 2‐deoxyglucose uptake. We discuss the putative signalling targets by which ethanol could alter the cytoskeleton and hexose uptake and the cytoprotective effect of LPA against ethanol‐induced damages. The latter opens the possibility that LPA or a similar non‐hydrolysable lipid derivative could be used as a cytoprotective agent against the noxious effects of ethanol.

Vacuole Membrane Protein 1 Is an Endoplasmic Reticulum Protein Required for Organelle Biogenesis, Protein Secretion, and Development

Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1(-) Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1(-) cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised.

Phospholipid Synthesis Participates in the Regulation of Diacylglycerol Required for Membrane Trafficking at the Golgi Complex

The lipid metabolite diacylglycerol (DAG) is required for transport carrier biogenesis at the Golgi, although how cells regulate its levels is not well understood. Phospholipid synthesis involves highly regulated pathways that consume DAG and can contribute to its regulation. Here we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the post-Golgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking.

Lysophosphatidic acid rescues RhoA activation and phosphoinositides levels in astrocytes exposed to ethanol

Long‐term ethanol treatment substantially impairs glycosylation and membrane trafficking in primary cultures of rat astrocytes. Our previous studies indicated that these effects were attributable to a primary alteration in the dynamics and organization of the actin cytoskeleton, although the molecular mechanism(s) remains to be elucidated. As small Rho GTPases and phosphoinositides are involved in the actin cytoskeleton organization, we now explore the effects of chronic ethanol treatment on these pathways. We show that chronic ethanol treatment of rat astrocytes specifically reduced endogenous levels of active RhoA as a result of the increase of in the RhoGAP activity. Furthermore, ethanol‐treated astrocytes showed reduced phosphoinositides levels. When lysophosphatidic acid was added to ethanol‐treated astrocytes, it rapidly reverted actin cytoskeleton reorganization and raised active RhoA levels and phosphoinositides content to those observed in untreated astrocytes. Overall, our results indicate that the harmful effects of chronic exposure to ethanol on a variety of actin dynamics‐associated cellular events are primarily because of alterations of activated RhoA and phosphoinositides pools.

Fluorescent analogues of plasma membrane sphingolipids are sorted to different intracellular compartments in astrocytes; Harmful effects of chronic ethanol exposure on sphingolipid trafficking and metabolism.

Sphingolipids are basic constituents of cellular membranes and are essential for numerous functions such as intracellular signalling. They are transported along the exocytic and endocytic pathways in eukaryotic cells. After endocytosis, fluorescent‐labelled sphingolipids are sorted to distinct intracellular organelles prior to recycling (via early/recycling endosomes) or degradation (late endosomes/lysosomes). Here we examine, in primary cultures of rat astrocytes, the internalisation routes followed by C6‐NBD‐glucosylceramide (NBD‐GlcCer) and C6‐NBD‐sphingomyelin (NBD‐SM) and the effects of ethanol on their endocytic trafficking. Endocytosed plasma membrane NBD‐GlcCer and NBD‐SM are diverted to the Golgi apparatus and lysosomes, respectively. These different internalisation pathways are maintained regardless of the differentiation stage of astrocytes. Chronic ethanol exposure did not alter this endocytic sorting, but delayed the internalisation of both NBD‐sphingolipids. Moreover, ethanol also stimulated the in situ metabolism of NBD‐ceramide to NBD‐GlcCer and NBD‐SM. We conclude that in rat astrocytes internalised plasma membrane NBD‐sphingolipids are sorted to different subcellular compartments. The exposure to chronic ethanol perturbed the lipid endocytic process and stimulated the de novo synthesis of NBD‐sphingolipids, shifting the balance of sphingolipid metabolism in favour of the sphingomyelin pathway.

Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway

Summary The inhibition of phosphatidic acid phosphatase (PAP) activity by propanolol indicates that diacylglycerol (DAG) is required for the formation of transport carriers at the Golgi and for retrograde trafficking to the ER. Here we report that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER–Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER–Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.

Clearance of a hirano body-like F-actin aggresome generated by jasplakinolide

We have reported in a variety of mammalian cells the reversible formation of a filamentous actin (F-actin)-enriched aggresome generated by the actin toxin jasplakinolide (Lázaro-Diéguez et al., J Cell Sci 2008; 121:1415-25). Notably, this F-actin aggresome (FAG) resembles in many aspects the pathological Hirano body, which frequently appears in some diseases such as Alzheimer’s and alcoholism. Using selective inhibitors, we examined the molecular and subcellular mechanisms that participate in the clearance of the FAG. Chaperones, microtubules, proteasomes and autophagosomes all actively participate to eliminate the FAG. Here we compile and compare these results and discuss the involvement of each process. Because of itssimplicity and high reproducibility, our cellular model could help to test pharmacological agents designed to interfere with the mechanisms involved in the clearance of intracellular bodies and, in particular, of those enriched in F-actin. Addendum to: Lázaro-Diéguez F, Aguado C, Mato E, Sánchez-Ruíz Y, Esteban I, Alberch J, Knecht E, Egea G. Dynamics of an F-actin aggresome generated by the actin-stabilizing toxin jasplakinolide.J Cell Sci 2008; 121:1415-25.