Editorial: Nck has a knack for T cell differentiation

Peer Reviewe

Modification of cysteine residues by cyclopentenone prostaglandins: Interplay with redox regulation of protein function

40 p.-5 fig.-1 tab.Cyclopentenone prostaglandins (cyPG) are endogenous lipid mediators involved in the resolution of inflammation and the regulation of cell proliferation and cellular redox status. Upon exogenous administration they have shown beneficial effects in models of inflammation and tissue injury, as well as potential antitumoral actions, which have raised a considerable interest in their study for the development of therapeutic tools. Due to their electrophilic nature, the best-known mechanism of action of these mediators is the covalent modification of proteins at cysteine residues through Michael addition. Identification of cyPG targets through proteomic approaches, including MS/MS analysis to pinpoint the modified residues, is proving critical to characterize their mechanisms of action. Among the targets of cyPG are proinflammatory transcription factors, proteins involved in cell defense, such as the regulator of the antioxidant response Keap1 and detoxifying enzymes like GST, and key signaling proteins like Ras proteins. Moreover, cyPG may interact with redox-active small molecules, such as glutathione and hydrogen sulfide. Much has been learned about cyPG in the past few years and this knowledge has also contributed to clarify both pharmacological actions and signaling mechanisms of these and other electrophilic lipids. Given the fact that many cyPG targets are involved in or are targets for redox regulation, there is a complex interplay with redox-induced modifications. Here we address the modification of protein cysteine residues by cyPG elucidated by proteomic studies, paying special attention to the interplay with redox signaling.Work in the authors’ laboratory is supported by grants from MINECO SAF2009-11642 and SAF2012-36519, RETIC RIRAAF RD07/0064/0007 and RD12/0013/0008 from ISCIII and COST action CM1001. CLO is the recipient of a FPI fellowship from MINECO.Peer reviewe

Vimentin filament organization and stress sensing depend on its single cysteine residue and zinc binding

17 pág., 10 figs.The vimentin filament network plays a key role in cell architecture and signalling, as well as in epithelial–mesenchymal transition. Vimentin C328 is targeted by various oxidative modifications, but its role in vimentin organization is not known. Here we show that C328 is essential for vimentin network reorganization in response to oxidants and electrophiles, and is required for optimal vimentin performance in network expansion, lysosomal distribution and aggresome formation. C328 may fulfil these roles through interaction with zinc. In vitro, micromolar zinc protects vimentin from iodoacetamide modification and elicits vimentin polymerization into optically detectable structures; in cells, zinc closely associates with vimentin and its depletion causes reversible filament disassembly. Finally, zinc transportdeficient human fibroblasts show increased vimentin solubility and susceptibility to disruption, which are restored by zinc supplementation. These results unveil a critical role of C328 in vimentin organization and open new perspectives for the regulation of intermediate filaments by zinc. DOI: 10.1038/ncomms8287 OPEN 1This work was supported by grants SAF2012–36519, MINECO, Spain and RD12/0013/0008, ISCIII to D.P.-S., and CTQ2012–32025, MINECO, y CAM MHIT S2010/BMD-2353 to F.J.C. C.L.O. and B.G. have been recipients of fellowships BES-2010–033718 and BES-2007–15806, respectively (FPI, MINECO). We acknowledge support from COST Action CM1001.Peer reviewe

An isoprenylation and palmitoylation motif promotes intraluminal vesicle delivery of proteins in cells from distant species

12 p.-7 fig.The C-terminal ends of small GTPases contain hypervariable sequences which may be posttranslationally modified by defined lipid moieties. The diverse structural motifs generated direct proteins towards specific cellular membranes or organelles. However, knowledge on the factors that determine these selective associations is limited. Here we show, using advanced microscopy, that the isoprenylation and palmitoylation motif of human RhoB (–CINCCKVL) targets chimeric proteins to intraluminal vesicles of endolysosomes in human cells, displaying preferential co-localization with components of the late endocytic pathway. Moreover, this distribution is conserved in distant species, including cells from amphibians, insects and fungi. Blocking lipidic modifications results in accumulation of CINCCKVL chimeras in the cytosol, from where they can reach endolysosomes upon release of this block. Remarkably, CINCCKVL constructs are sorted to intraluminal vesicles in a cholesterol-dependent process. In the lower species, neither the C-terminal sequence of RhoB, nor the endosomal distribution of its homologs are conserved; in spite of this, CINCCKVL constructs also reach endolysosomes in Xenopus laevis and insect cells. Strikingly, this behavior is prominent in the filamentous ascomycete fungus Aspergillus nidulans, in which GFP-CINCCKVL is sorted into endosomes and vacuoles in a lipidation-dependent manner and allows monitoring endosomal movement in live fungi. In summary, the isoprenylated and palmitoylated CINCCKVL sequence constitutes a specific structure which delineates an endolysosomal sorting strategy operative in phylogenetically diverse organisms.This work was supported by grants SAF2009-11642 and SAF2012-36519 from MINECO and RETIC RD12/0013/0008 to DPS, grants BIO2012-30695 fromcMINECO and S2010/BMD-2414 from Comunidad de Madrid to MAP, and grants from the Norwegian Cancer Society and the South-Eastern Norway Regional Health Authority to HS. CLO is supported by the FPI program from MINECO (BES-2010-033718). The stay of CLO at HS laboratory was supported by the short stay grant EEBB-I-12-04482 from MINECO.Peer reviewe

A hotspot mutation targeting the R-RAS2 GTPase acts as a potent oncogenic driver in a wide spectrum of tumors

A missense change in RRAS2 (Gln to Leu), analogous to the Gln-to-Leu mutation of RAS oncoproteins, has been identified as a long-tail hotspot mutation in cancer and Noonan syndrome. However, the relevance of this mutation for in vivo tumorigenesis remains understudied. Here we show, using an inducible knockin mouse model, that R-Ras2 triggers rapid development of a wide spectrum of tumors when somatically expressed in adult tissues. These tumors show limited overlap with those originated by classical Ras oncogenes. R-Ras2-driven tumors can be classified into different subtypes according to therapeutic susceptibility. Importantly, the most relevant R-Ras2-driven tumors are dependent on mTORC1 but independent of phosphatidylinositol 3-kinase-, MEK-, and Ral guanosine diphosphate (GDP) dissociation stimulator. This pharmacological vulnerability is due to the extensive rewiring by R-Ras2 of pathways that orthogonally stimulate mTORC1 signaling. These findings demonstrate that RRAS2 is a bona fide oncogenic driver and unveil therapeutic strategies for patients with cancer and Noonan syndrome bearing RRAS2 mutations.We thank M. Blázquez and the personnel of the CIC Flow Cytometry, Microscopy, Pathology, and Genomics Units for expert technical work. X.R.B.’s project leading to these results has received funding from the Spanish Association against Cancer (GC16173472GARC), the Castilla-León government (CSI252P18, CSI145P20, and CLC-2017-01), the RTI2018-096481-B-100 grant cofounded by MCIN/AEI/10.13039/501100011033 and the European Research Development Fund “A way of making Europe” of the European Union, and “la Caixa” Banking Foundation (HR20-00164). X.R.B.’s institution is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Castilla-León government (CLC-2017-01). J.R.-V. received funding from the Carlos III Health Institute (PI20/01724). J.R.-V.’s contract is supported by a senior postdoctoral contract of the Spanish Association against Cancer. L.C.’s contract was supported by contracts from the Spanish Association against Cancer and the CLC-2017-01 grant. L.F.L.-M.’s contract was mostly supported by funding from the Spanish Ministry of Education, Culture and Sports (FPU13/02923) and, subsequently, by the CLC-2017-01 grant. R.C. was supported by a predoctoral contract from the MSI (BES-2016-0077909) and the CLC-2017-01 grant

Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia

[Background]: Chronic lymphocytic leukemia (CLL) is the most frequent, and still incurable, form of leukemia in the Western World. It is widely accepted that cancer results from an evolutionary process shaped by the acquisition of driver mutations which confer selective growth advantage to cells that harbor them. Clear examples are missense mutations in classic RAS genes (KRAS, HRAS and NRAS) that underlie the development of approximately 13% of human cancers. Although autonomous B cell antigen receptor (BCR) signaling is involved and mutations in many tumor suppressor genes and oncogenes have been identified, an oncogenic driver gene has not still been identified for CLL. [Methods]: Conditional knock-in mice were generated to overexpress wild type RRAS2 and prove its driver role. RT-qPCR analysis of a human CLL sample cohort was carried out to measure RRAS2 transcriptional expression. Sanger DNA sequencing was used to identify a SNP in the 3’UTR region of RRAS2 in human CLL samples. RNAseq of murine CLL was carried out to identify activated pathways, molecular mechanisms and to pinpoint somatic mutations accompanying RRAS2 overexpression. Flow cytometry was used for phenotypic characterization and shRNA techniques to knockdown RRAS2 expression in human CLL. [Results]: RRAS2 mRNA is found overexpressed in its wild type form in 82% of the human CLL samples analyzed (n = 178, mean and median = 5-fold) as well as in the explored metadata. A single nucleotide polymorphism (rs8570) in the 3’UTR of the RRAS2 mRNA has been identified in CLL patients, linking higher expression of RRAS2 with more aggressive disease. Deliberate overexpression of wild type RRAS2 in mice, but not an oncogenic Q72L mutation in the coding sequence, provokes the development of CLL. Overexpression of wild type RRAS2 in mice is accompanied by a strong convergent selection of somatic mutations in genes that have been identified in human CLL. R-RAS2 protein is physically bound to the BCR and mediates BCR signals in CLL. [Conclusions]: The results indicate that overexpression of wild type RRAS2 is behind the development of CLL.This work was supported by grants from the Spanish Association against Cancer (GC16173472GARC), PID2019-104935RB-I00 from the ‘Comision Interministerial de Ciencia y Tecnología’, the ‘Fundación Ramón Areces’, and by the European Research Council ERC 2013-Advanced Grant 334763 “NOVARIPP”, Instituto de Salud Carlos III (ISCIII) (CIBERONC – groups CB16/12/00233, CB16/12/00351), the Health Council of the Junta de Castilla y León (GRS 2036/A/19) and private Gilead (GLD15/00348). Juan de la Cierva (FJCI-2016-28756)

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Modificaciones postraduccionales de GTPasas endosomales implicaciones en localización y tráfico intracelular

173 p.-53 fig.-4 tab.[EN] Small GTPases of the Ras superfamily are key to cellular processes such as differentiation, proliferation or regulation of the cytoskeleton (Takai et al., 2001). These proteins undergo lipid modifications that elicit their attachment to membranes and are crucial to their activity. Specifically, Ras protein isoprenylation serves as an anchor to the membrane, and Ras proteins are further amenable to reversible palmitoylation, which contributes to their dynamic localization at distinct subcellular compartments (Hancock et al., 1989). Lipid modifications take place at cysteine residues in the Cterminal region. Furthermore, these cysteines can undergo oxidation or addition of electrophilic compounds, e.g. cyclopentenone prostaglandin binding. These compounds are formed by non-enzymatic oxidation of arachidonic acid or from dehydration of other prostaglandins, which endows them with electrophilic carbons (Funk, 2001). Rho proteins are a group of Ras superfamily proteins that are crucial to cell adhesion and migration, hence modulating cardiovascular pathophysiology and tumorigenic processes (Ridley, 2001). RhoB is a member of this family with roles invesicular trafficking, tumor suppression and localization of signaling proteins. RhoB lipid modifications (isoprenylation and double palmitoylation) are necessary for its localization to endolysosomes and its degradation through this pathway (Pérez-Sala et al., 2009; Stamatakis et al., 2002). The last eight amino acids of RhoB, which correspond to the lipidation motif, constitute an endolysosomal localization and degradation sequence for chimeric proteins to which this extension is added at their C-terminal end (Pérez-Sala et al., 2009).In the context of this dissertation, in vitro and cell models have been used to study mechanistic and structural aspects of Ras and Rho protein modification, particularly that of RhoB, and its influence on localization and trafficking of these proteins or their chimeras modified by lipids and other compounds. Though lipid modifications of Ras proteins have been described as important regulatory factors, the involvement of lipidation and its interplay with other structural determinants in subcellular targeting of GTPases has not been fully elucidated. Therefore, the role of lipid modifications of RhoB cysteine residues and other important residues at the hypervariable region on its intracellular sorting has been analyzed. Furthermore, studies were carried out to determine whether the RhoB lipidation motif per se (CINCCKVL) is able to mimic the full-length protein. Cell models from different species were used to evaluate whether the latent sorting mechanisms for CINCCKVL proteins are conserved in these model organisms. Using this C-terminal construct in parallel to the full-length protein, the potential involvement of key molecular machineries, i.e. ESCRT complexes, CD63 or lipid dynamics, in their sorting was assessed. Considering the potential alteration of subcellular localization of Ras proteins by modification, targeting of lipidation sequences by other structurally diverse moieties, including reactive species arising in pathological scenarios, was also explored. In addition, assays were carried out to study the potential alteration of endosomal GTPase localization in genetic models of lysosomal disease.In order to characterize the precise subcellular localization of RhoB, constructs of fluorescent proteins fused to this protein were compared to those of other small GTPases used as markers of endocytic vesicles. Co-localization with fusion proteins of Rab7, Rab9 or Lamp and negligible overlap with early endosome markers, i.e. Rab5 fusion proteins, or the Golgi marker, giantin, underscore RhoB localization within the endolysosomal lumen. These studies also include assays with autophagy probes, with which RhoB or its chimeras show negligible co-localization. The small GTPase, TC10, contains a C-terminal sequence that is very similar to that of RhoB, though a basic amino acid patch upstream of the lipidation sequence elicits retention at the limiting membrane of endolysosomes. Insertion of this basic patch into the RhoB sequence hinders its entry into endolysosomes, highlighting that exchange of structural determinants between these proteins elicits interconversion of their subcellular localization patterns. Furthermore, it has been determined that chimeras bearing the RhoB C-terminal sequence, CINCCKVL, are also internalized into endolysosomes. It was also described that chimeric proteins fused to the C-terminal sequence of RhoB (CINCCKVL) enter into the endolysosomal lumen. Therefore, studies were carried out to assess whether the CINCCKVL sequence can travel to endolysosomes in cells from diverse species, which in some cases lack an endogenous RhoB homolog sequence. The universality of endolysosomal localization of chimeras bearing the RhoB C-terminus was set forth by detection of these proteins in endolysosomes of cells as phylogenetically distant as amphibians, insects and fungi.It has been previously described that RhoB is involved in diverse pathologies such as tumorigenic processes and cardiovascular malfunction (Prendergast, 2001). The results obtained showing RhoB localization to endolysosomal membranes warranted studies in models of lysosomal diseases such as Chediak-Higashi or Niemann-Pick. These disorders result in oculocutaneous albinism, immunodeficiency and cognitive impairment of varying degree (Huizing et al., 2008). Comparing RhoB to other endosomal GTPases in cells of patients suffering from these diseases, confocal laser microscopy shows that RhoB is localized to the dilated endolysosomes typical of these diseases. In many pathological scenarios, proteins suffer modifications due to alterations in overall cellular redox status. By means of electrophoretic assays (SDS-PAGE) or proteomic approaches (MALDI-TOF), it was shown that proteins of the Ras superfamily, particularly H-Ras and RhoB, could be modified in vitro at their Cterminal cysteine residues by structurally diverse electrophilic lipids. Specifically, dienone cyclopentenone prostaglandins, derived from non-enzymatic oxidation of arachidonic acid or dehydration of other prostaglandins, bind to these residues.[ES] Las GTPasas de bajo peso molecular comprendidas en la superfamilia Ras son proteínas fundamentales para procesos celulares como la diferenciación celular, la regulación del citoesqueleto y la proliferación (Takai et al., 2001). Estas proteínas sufren modificaciones lipídicas que les permiten asociarse con las membranas celulares y son clave para su actividad. En concreto, las proteínas Ras están isopreniladas para anclarse a la membrana, además de ser susceptibles de palmitoilación reversible, lo cual contribuye a su localización dinámica en distintos compartimentos subcelulares (Hancock et al., 1989). Estas modificaciones lipídicas tienen lugar sobre residuos de cisteína en la región carboxilo-terminal (C-terminal). Además, estas cisteínas pueden sufrir oxidaciones o adición de compuestos de naturaleza electrofílica, como por ejemplo, la unión de prostaglandinas ciclopentenonas (Oeste and Pérez-Sala, 2014). Estos compuestos se forman por oxidación no enzimática del ácido araquidónico o por deshidratación de otras prostaglandinas, lo cual las dota de carbonos electrófilos (Funk, 2001). Las proteínas Rho juegan papeles cruciales en la adhesión y migración celular que repercuten sobre la fisiopatología cardiovascular y procesos tumorigénicos (Ridley, 2001). RhoB es un miembro de esta familia con funciones reguladoras del tráfico vesicular, la supresión de tumores y la localización de proteínas señalizadoras. Las modificaciones lipídicas de RhoB (isoprenilación y doble palmitoilación) son necesarias para su localización endolisosomal y posterior degradación por esta vía (Pérez-Sala et al., 2009; Stamatakis et al., 2002). Los últimos ocho amino ácidos de RhoB, zona donde se encuentran las cisteínas que se lipidan, comprenden una secuencia de localización y degradación endolisosomal para proteínas quiméricas a las que se les añade esta extensión en su extremo carboxilo-terminal (Pérez-Sala et al., 2009). En el contexto de esta tesis se han estudiado, tanto in vitro como en modelos celulares, aspectos mecanísticos y estructurales de la modificación de proteínas Ras y Rho, en concreto RhoB, y su repercusión sobre la localización y tráfico de estas proteínas o sus quimeras modificadas por lípidos y otros compuestos.2. Objetivos: Las GTPasas de bajo peso molecular ejercen diversas funciones celulares desde localizaciones de membrana específicas. Entre los factores que las regulan, la modificación en secuencias C-terminales juegan un papel crucial, aunque la participación de la lipidación y su interacción con otros determinantes estructurales en la localización subcelular de las GTPasas no ha sido completamente dilucidado. Por ello, el trabajo presentado en esta tesis se centra en analizar el papel de las modificaciones lipídicas en residuos de cisteína de RhoB sobre su tráfico intracelular y determinar si el motivo de lipidación per se es capaz de reproducir el comportamiento de la proteína completa. Además, la posibilidad de que las secuencias de lipidación puedan ser dianas de modificación por otros compuestos de estructura diversa, incluidas especies reactivas que se producen en situaciones patológicas, no se ha explorado. Por tanto, los siguientes objetivos fueron planteados: ! Estudiar el papel de las secuencias C-terminales de GTPasas endosomales en su localización subcelular. ! Valorar la importancia de la isoprenilación y la palmitoilación en la asociación de estas proteínas a vesículas intracelulares. ! Determinar la localización subcelular de quimeras que contienen la secuencia de lipidación del C-terminal de RhoB. ! Evaluar si los mecanismos latentes de localización del C-terminal de RhoB están conservados en células de diversas especies. ! Explorar el posible rol de maquinarias moleculares clave, tales como los complejos ESCRT, CD63 o la dinámica lipídica, en la localización de RhoB y quimeras relacionadas. ! Asentar las bases para estudiar posibles alteraciones en la localización y modificación de GTPasas endosomales en modelos experimentales de enfermedad.3. Resultados: Se ha llevado a cabo una caracterización de RhoB fusionada a proteínas fluorescentes en el contexto de las endomembranas al comparar su localización y degradación con las de otras GTPasas pequeñas. Su colocalización con proteínas fluorescentes fusionadas a Rab7, Rab9 o Lamp y falta de coincidencia con marcadores de endosomas tempranos (GFP-Rab5) o aparato de Golgi (giantin) atestiguan su localización en el lumen endolisosomal. Los estudios aquí presentados incluyen también la utilización de sondas de la vía autofagocítica, con las cuales ni RhoB ni sus proteínas quimera colocalizan de forma detectable. La GTPasa de bajo peso molecular, TC10, cuya secuencia C-terminal es muy similar a la de RhoB, contiene unos amino ácidos básicos que sin embargo la retienen en la membrana limitante de dichas vesículas. La inserción de la secuencia polibásica de TC10 en la secuencia de RhoB impide su entrada a endolisosomas, lo cual pone de manifiesto que hay determinantes estructurales que al intercambiarse entre estas proteínas dan lugar a patrones subcelulares específicos. Se describió además que las proteínas quimera fusionadas a la secuencia Cterminal de RhoB (CINCCKVL) también acceden al lumen endolisosomal. Por lo tanto, se exploró si la secuencia CINCCKVL accede a los endolisosomas no sólo en células de mamíferos, sino en células de organismos que pueden contener o no una secuencia endógena para proteínas homólogas a RhoB. La universalidad de la localización endolisosomal de quimeras con el C-terminal de RhoB se pone de manifiesto al detectarse en endolisosomas de células de organismos muy alejados entre sí en la escala filogenética, tales como anfibios, insectos y hongos.Para explorar los posibles mecanismos de localización de RhoB, se emplearon técnicas de microscopía láser avanzada confocal y de superresolución. Por medio de estas potentes herramientas se ha podido establecer la localización inequívoca de RhoB y quimeras CINCCKVL en vesículas intraluminales de cuerpos multivesiculares. Se transfectaron células con RNA de interferencia para así silenciar componentes de la maquinaria ESCRT, responsable del tráfico endolisosomal y la formación de cuerpos multivesiculares (Raiborg and Stenmark, 2009). Al bloquear la función de algunos de estos componentes, RhoB no se internaliza en los endolisosomas, lo cual refleja un posible papel de las proteínas ESCRT en el tráfico endosomal de RhoB. Sin embargo, las quimeras CINCCKVL no se ven igualmente afectadas por el bloqueo de componentes ESCRT. Se estudiaron pues otros componentes proteicos y lipídicos de los cuerpos multivesiculares que podrían jugar también un papel en la localización de proteínas CINCCKVL, como por ejemplo la tetraspanina CD63. Esta proteína presenta una alta colocalización con RhoB y quimeras relacionadas en cuerpos multivesiculares. Curiosamente, se observó que la sobreexpresión de construcciones fluorescentes de CD63 cambian el patrón de localización subcelular de quimeras CINCCKVL, posiblemente hacia destinos extracelulares, pero no así el de RhoB. Para explorar el papel de la dinámica lipídica en la localización de estas proteínas, se realizaron ensayos de tratamiento celular con compuestos que alteran la dinámica de colesterol en las membranas. En estas células, se redujo la internalización de quimeras del C-terminal de RhoB hacia el lumen de cuerpos multivesiculares, mientras que la proteína completa siguió su camino hacia el interior de estos compartimentos. Además, el tratamiento con ceramida indujo cambios en la morfología celular que afectan la localización de RhoB y proteínas relacionadas. En conjunto, estos resultados sugieren que las quimeras CINCCKVL pueden seguir un destino subcelular mediado por maquinarias distintas a las responsables del transporte de RhoB completa, lo cual sugiere que RhoB contiene determinantes estructurales de localización o interacción más allá de los que aparecen en su secuencia de lipidación.Programa de becas (BES-2010-033718), incluido el programa "Estancias Breves" (EEBB-I-12-04482), del MINECO en el marco de las subvenciones SAF2009-11642 Y SAF2012-36519, y por RETIC RIRAAF de ISCIII (RD07 / 0064/0007 yRD12 / 0013/0008). El financiamiento del programa JAE-Intro de CSIC así como el FEBS. También se reconoce el programa de becas de verano.Peer reviewe

Taking a lipidation-dependent path toward endolysosomes

We recently reported that the isoprenylation and palmitoylation motif present at the C-terminus of human RhoB protein promotes intraluminal vesicle delivery of proteins in cells from organisms as phylogenetically apart as fungi and humans. Here we build on these observations by showing that chimeras of fluorescent proteins bearing this sequence, namely, CINCCKVL, which become isoprenylated and palmitoylated in cells, may be used to mark endolysosomes while preserving their morphology. Indeed, these chimeric proteins are devoid of the effects derived from overexpression of fluorescent constructs of full-length, active proteins widely used as endolysosomal markers, such as Lamp1 or Rab7, which cause lysosomal enlargement, or RhoB, which induces actin stress fibers. Moreover, the fact that lipidation-dependent endolysosomal localization of CINCCKVL chimeras can be ascertained in a wide variety of cells indicates that they follow a path toward endolysosomes that is conserved in diverse species. Therefore, CINCCKVL chimeras serve as robust tools to mark these late endocytic compartments.MINECO and RETIC RIRAAF (RD12/0013/0008) from ISCIII, Spain. C.L.O. has been the recipient of fellowship BES-2010–033718 from FPI, MINECOPeer Reviewe