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Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Genetic studies placed Uso1 paradoxically acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S in the GHD that rescued lethality of rab1-deficient Aspergillus nidulans. The mutations are phenotypically additive, their combination suppressing the complete absence of RAB1, which emphasizes the key physiological role of the GHD. In living hyphae Uso1 recurs on puncta (60 sec half-life) colocalizing partially with the Golgi markers RAB1, Sed5 and GeaA/Gea1/Gea2, and totally with the retrograde cargo receptor Rer1, consistent with Uso1 dwelling in a very early Golgi compartment from which ER residents reaching the Golgi recycled back to the ER. Localization of Uso1, but not of Uso1E6K/G540S, to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. That Uso1 delocalization correlates with a decrease in the number of Gea1 cisternae supports that Uso1-and-Rer1-containing puncta are where the protein exerts its physiological role. In S-tag-coprecipitation experiments Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Using purified proteins, we show that Bos1 and Bet1 bind the Uso1 GHD directly. However, Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. G540S alone markedly increases GHD binding to Bos1, whereas E6K causes a weaker effect, correlating with their phenotypic contributions. AlphaFold2 predicts that G540S increases binding of the GHD to the Bos1 Habc domain. In contrast, E6K lies i

Hyphal tip growth allows filamentous fungi to colonize space, reproduce, or infect. It features remarkable morphogenetic plasticity including unusually fast elongation rates, tip turning, branching, or bulging. These shape changes are all driven from the expansion of a protective cell wall (CW) secreted from apical pools of exocytic vesicles. How CW secretion, remodeling, and deformation are modulated in concert to support rapid tip growth and morphogenesis while ensuring surface integrity remains poorly understood. We implemented subresolution imaging to map the dynamics of CW thickness and secretory vesicles in Aspergillus nidulans. We found that tip growth is associated with balanced rates of CW secretion and expansion, which limit temporal fluctuations in CW thickness, elongation speed, and vesicle amount, to less than 10% to 20%. Affecting this balance through modulations of growth or trafficking yield to near-immediate changes in CW thickness, mechanics, and shape. We developed a model with mechanical feedback that accounts for steady states of hyphal growth as well as rapid adaptation of CW mechanics and vesicle recruitment to different perturbations. These data provide unprecedented details on how CW dynamics emerges from material secretion and expansion, to stabilize fungal tip growth as well as promote its morphogenetic plasticity.

Filamentous fungi have been used for studying long-distance transport of cargoes driven by cytoplasmic dynein. Aspergillus nidulans is a well-established genetic model organism used for studying dynein function and regulation in vivo. Here, we describe how we grow A. nidulans strains for live-cell imaging and how we observe the dynein-mediated distribution of early endosomes and secretory vesicles. Using an on-stage incubator and culture chambers for inverted microscopes, we can image fungal hyphae that naturally attach to the bottom of the chambers, using wide-field epifluorescence microscopes or the new Zeiss LSM 980 (with Airyscan 2) microscope. In addition to methods for preparing cells for imaging, a procedure for A. nidulans transformation is also described.

In the apex-directed RAB11 exocytic pathway of Aspergillus nidulans, kinesin-1/KinA conveys secretory vesicles (SVs) to the hyphal tip, where they are transferred to the type V myosin MyoE. MyoE concentrates SVs at an apical store located underneath the PM resembling the presynaptic active zone. A rod-shaped RAB11 effector, UDS1, and the intrinsically-disordered and coiled-coil HMSV associate with MyoE in a stable HUM (HMSV-UDS1-MyoE) complex recruited by RAB11 to SVs through an interaction network involving RAB11 and HUM components, with the MyoE globular tail domain (GTD) binding both HMSV and RAB11-GTP, and RAB11-GTP binding both the MyoE-GTD and UDS1. UDS1 bridges RAB11-GTP to HMSV, an avid interactor of the MyoE-GTD. The interaction between the UDS1-HMSV sub-complex and RAB11-GTP can be reconstituted in vitro. Ablating UDS1 or HMSV impairs actomyosin-mediated transport of SVs to the apex, resulting in spreading of RAB11 SVs across the apical dome as KinA/microtubule-dependent transport gains prominence.

Los hongos filamentosos, tales como el organismo modelo Aspergillus nidulans, crecen casi exclusivamente por extensión apical. La velocidad de crecimiento es rápida, de alrededor de una micra por minuto, lo que implica la necesidad de que el aporte de lípidos y proteínas modificadores de la pared celular sea eficiente. Por tanto, el último paso de la ruta secretora, la salida de vesículas derivadas del aparato de Golgi con destino a la membrana plasmática, es crítico para mantener la extensión apical. En el denominado Golgi tardío o trans Golgi Network (TGN) confluyen la ruta secretora procedente del retículo endoplásmico y la ruta de reciclaje endocítico procedente de la membrana plasmática. La transición entre Golgi y post-Golgi está mediada por la GTPasa RAB11, denominada Ypt31 en Saccharomyces cerevisiae. Cuando las membranas del TGN maduran (= adquieren un nivel suficiente de RAB11), las cisternas del TGN se deflecan en vesículas de secreción. Por tanto, el verdadero semáforo de salida del TGN es el factor de intercambio del nucleótido de guanosina (GEF) que media el reclutamiento de la RAB. Mi charla se centrará en la caracterización genética, bioquímica y subcelular de TRAPPII, el complejo de > 2 MDa que regula la salida del TGN en los eucariotas.

Contrary to the opinion recently offered by Dimou et al., our previously published biochemical, subcellular and genetic data supported our contention that AN11127 corresponds to the A. nidulans gene encoding Sec12, which is the guanine nucleotide exchange factor (GEF) specific for SAR1. We add here additional bioinformatics evidence that fully disprove the otherwise negative evidence reported by Dimou et al., highlighting the dangers associated with the lax interpretation of genomic data. On the positive side, we establish guidelines for the identification of this key secretory gene in other species of Ascomycota and Basidiomycota, including species of medical and applied interest.

RAB GTPases are major determinants of membrane identity that have been exploited as highly specific reporters to study intracellular traffic in vivo. A score of fungal papers have considered individual RABs, but systematic, integrated studies on the localization and physiological role of these regulators and their effectors have been performed only with Aspergillus nidulans. These studies have influenced the intracellular trafficking field beyond fungal specialists, leading to findings such as the maturation of trans‐Golgi (TGN) cisternae into post‐Golgi RAB11 secretory vesicles, the concept that these RAB11 secretory carriers are loaded with three molecular nanomotors, the understanding of the role of endocytic recycling mediated by RAB6 and RAB11 in determining the hyphal mode of life, the discovery that early endosome maturation and the ESCRT pathway are essential, the identification of specific adaptors of dynein‐dynactin to RAB5 endosomes, the exquisite dependence that autophagy displays on RAB1 activity, the role of TRAPPII as a GEF for RAB11, or the conclusion that the RAB1‐to‐RAB11 transition is not mediated by TRAPP maturation. A remarkable finding was that the A. nidulans Spitzenkörper contains four RABs: RAB11, Sec4, RAB6, and RAB1. How these RABs cooperate during exocytosis represents an as yet outstanding question.

[EN] The secretory pathway transports proteins and lipids from the Endoplasmatic Reticulum (ER) to the plasma membrane and extracellular millieu, or to the membrane-bound compartments of the endosomal-lysosomal system. The first step of this pathway is the incorporation to vesicles bound for the Golgi of the proteins synthetized by the ER-associated ribosomes, once these have been properly folded and the necessary posttranslational modifications added [1, 2]. In their journey throughout the Golgi cisternae, these proteins, collectively known as cargo, are subjected to further modifications in their N- and O-glycosilation patterns [3, 4]. Finally, cargo arrives to the Trans-Golgi Network (TGN), where is sorted and concentrated into specialized domains, and subsequently exported by membrane carriers bound for the PM or the endosomal-lysosomal system [5]. Transit through the different pathways of intracellular trafficking is regulated at the molecular level by several mechanisms that, on the one hand, secure that cargo reaches its destination, and on the other, maintain the identity of the different compartments that constitute the endomembrane system [6, 7]. Small GTPases of the Rab and Arf families play a fundamental role as molecular switches, recruiting peripheral membrane proteins to the specific location where they are needed [8]. The loading/hydrolysis cycle of a GTPase is governed by a set of regulators, the most crucial of whom are the Guanine nucleotide Exchange Factors (GEFs) and the GTPase activating Proteins (GAPs), which are responsible for the loading of GTP and its hydrolysis, respectively[11, 12]. The main focus of this doctoral thesis is the study of certain regulatory aspects of ER to early Golgi trafficking. This process begins with the activation of the Sar1 GTPase by its cognate GEF, Sec12 [13, 14]. The GTP-bound form of Sar1 gets anchored to the ER membrane and starts the recruitment of the Coat Protein Complex II (COPII) subunits, firstly, the i, [ES] La ruta secretora permite el transporte de proteínas y lípidos desde el retículo endoplasmático (RE) a la membrana plasmática (MP) y al exterior celular (exocitosis), o bien a los orgánulos del sistema endosomal-vacuolar. Esta ruta comienza con la exportación en vesículas, con destino al aparato de Golgi, de las proteínas sintetizadas por los ribosomas asociados al RE, una vez éstas han sido plegadas correctamente y han incorporado las modificaciones post-traduccionales adecuadas [1, 2]. En su tránsito a través de las cisternas del aparato de Golgi estas proteínas o cargo sufren posteriores modificaciones en sus patrones de N- y O-glicosilación [3, 4]. Finalmente, el cargo llega al Golgi tardío o Trans-Golgi Network (TGN), donde es seleccionado y concentrado para ser posteriormente exportado en vesículas cuyo destino final es la fusión con la MP o con el sistema endosomal-vacuolar [5]. El tránsito de cargo por las rutas de tráfico intracelular se encuentra regulado a nivel molecular por mecanismos que permiten, por un lado, asegurar que llegue al destino correcto y por otro, mantener la identidad de los diferentes compartimentos que constituyen el sistema de endomembranas [6, 7]. En este sentido las GTPasas o proteínas G monoméricas de las familias Rab y Arf desempeñan un papel fundamental como interruptores moleculares, reclutando proteínas periféricas de membrana de forma específica allá donde sea necesaria su función [8-10]. El ciclo de carga/hidrólisis de una GTPasa se encuentra gobernado por una serie de proteínas reguladoras, siendo las principales los factores de intercambio de nucleótido de guanina (GEF) y las proteínas estimuladoras de la actividad GTPasa (GAP), responsables de la activación o carga con GTP y de la hidrólisis del mismo, respectivamente [11, 12]. Esta tesis doctoral se centra en el estudio de determinados aspectos de la regulación del tráfico entre el RE y el Golgi temprano. Este proceso se inicia con la activación de la GTPasa Sar1 por

Secretion of the GPI‐anchored protein (GPI‐AP) EglC was investigated in the filamentous fungus Aspergillus nidulans, exploiting a sucrose‐inducible promoter to conditionally express the protein in cells blocked at different steps of exocytosis. EglC is delivered to the cell surface in a polarized fashion, but appears to redistribute rapidly towards apico‐distal regions. Inactivation of SarASar1 mediating COPII vesicle biogenesis resulted in the accumulation of EglC in the ER but, rather than concentrating in ER‐exit‐sites, the reporter labeled the ER uniformly. Abnormal posttranslational modifications of EglC were detected in sarAts and sed5ts mutants, suggesting that blocking COPII biogenesis or traffic in the ER/Golgi interface might affect GPI remodeling. EglC delivery to the PM requires, besides Golgi function, the TRAPPII complex mediating the biogenesis of RAB11 secretory vesicles at the TGN, but is unaffected by the absence of RAB5, the key regulator of early endosome biogenesis/maturation. Thus, unlike the soluble extracellular enzyme inulinase, EglC is directly delivered from the TGN to the plasma membrane without involvement of endosomes. We conclude that in A. nidulans GPI‐APs follow a direct secretory pathway from the ER to the plasma membrane.

TRAnsport Protein Particle (TRAPP) complexes regulate membrane traffic. TRAPPII and TRAPPIII share a core hetero-heptamer, also denoted TRAPPI. In fungi TRAPPIII and TRAPPII mediate GDP exchange on RAB1 and RAB11, respectively, regulating traffic across the Golgi, with TRAPPIII also activating RAB1 in autophagosomes. Our finding that Aspergillus nidulans TRAPPII can be assembled by addition of a TRAPPII-specific subcomplex onto core TRAPP prompted us to investigate the possibility that TRAPPI/TRAPPIII already residing in the Golgi matures into TRAPPII to determine a RAB1-to-RAB11 conversion as Golgi cisternae progress from early Golgi to TGN identity. By time-resolved microscopy we determine that the TRAPPII reporter Trs120/TRAPPC9 is recruited to existing TGN cisternae slightly before RAB11 arrives, and resides for∼45 sec on them before cisternae tear off into RAB11 secretory carriers. Notably, the core TRAPP reporter Bet3/TRAPPC3 was not detectable in early Golgi cisternae, being instead recruited to TGN cisternae simultaneously with Trs120/TRAPPC9, indicating en bloc recruitment of TRAPPII to the Golgi and arguing strongly against the TRAPP maturation model.

TRAnsport Protein Particle complexes (TRAPPs) are ubiquitous regulators of membrane traffic mediating nucleotide exchange on the Golgi regulatory GTPases RAB1 and RAB11. In S. cerevisiae and metazoans TRAPPs consist of two large oligomeric complexes: RAB11-activating TRAPPII and RAB1-activating TRAPPIII. These share a common core TRAPPI hetero-heptamer, absent in metazoans but detected in minor proportions in yeast, likely originating from in vitro-destabilized TRAPPII/III. Despite overall TRAPP conservation, the budding yeast genome has undergone extensive loss of genes, and lacks homologues of some metazoan TRAPP subunits. With nearly twice the total number of genes of S. cerevisiae, another ascomycete Aspergillus nidulans has also been used for studies on TRAPPs. We combined size-fractionation chromatography with single-step purification coupled to mass-spectrometry and negative-stain electron microscopy to establish the relative abundance, composition and architecture of Aspergillus TRAPPs, which consist of TRAPPII and TRAPPIII in a 2:1 proportion, plus a minor amount of TRAPPI. We show that Aspergillus TRAPPIII contains homologues of metazoan TRAPPC11, TRAPPC12 and TRAPPC13 subunits, absent in S. cerevisiae, and establish that these subunits are recruited to the complex by Tca17/TRAPPC2L, which itself binds to the ‘Trs33 side’ of the complex. Thus Aspergillus TRAPPs compositionally resemble mammalian TRAPPs to a greater extent than those in budding yeast. Exploiting the ability of constitutively-active (GEF-independent, due to accelerated GDP release) RAB1* and RAB11* alleles to rescue viability of null mutants lacking essential TRAPP subunits, we establish that the only essential role of TRAPPs is activating RAB1 and RAB11, and genetically classify each essential subunit according to their role(s) in TRAPPII (TRAPPII-specific subunits) or TRAPPII and TRAPPIII (core TRAPP subunits). Constitutively-active RAB mutant combinations allowed examination of TRAPP comp

The pal/RIM ambient pH signalling pathway is crucial for the ability of pathogenic fungi to infect hosts. The Aspergillus nidulans 7‐TMD receptor PalH senses alkaline pH, subsequently facilitating ubiquitination of the arrestin PalF. Ubiquitinated PalF triggers downstream signalling events. The mechanism(s) by which PalH transduces the alkaline pH signal to PalF is poorly understood. We show that PalH is phosphorylated in a signal dependent manner, resembling mammalian GPCRs, although PalH phosphorylation, in contrast to mammalian GPCRs, is arrestin dependent. A genetic screen revealed that an ambient‐exposed region comprising the extracellular loop connecting TM4‐TM5 and ambient‐proximal residues within TM5 is required for signalling. In contrast, substitution by alanines of four aromatic residues within TM6 and TM7 results in a weak ‘constitutive’ activation of the pathway. Our data support the hypothesis that PalH mechanistically resembles mammalian GPCRs that signal via arrestins, such that the relative positions of individual helices within the heptahelical bundle determines the Pro316‐dependent transition between inactive and active PalH conformations, governed by an ambient‐exposed region including critical Tyr259 that potentially represents an agonist binding site. These findings open the possibility of screening for agonist compounds stabilizing the inactive conformation of PalH, which might act as antifungal drugs against ascomycetes.

[EN]The secretory pathway is a cellular process that is used to package certain protein molecules that are synthesized inside of the cell and are shipped out of the cell, to the plasma membrane (PM) or to the endosomes. Initially, newly synthesized proteins enter the endoplasmic reticulum (ER) by a process known as translocation. In the ER, cargo proteins are properly folded and some of them undergo N-glycosylation [1] or acquire a covalently attached glycosylphosphatidylinositol (GPI) anchor [2]. Next, cargoes exit the ER in COPII (coat protein complex-II) vesicles bound for the Golgi apparatus [3]. The initial interaction of this vesicle with a nascent cisterna of the Golgi is mediated by tethering factors and membrane fusion is facilitated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Once in the Golgi the N-glycans bound to proteins are modified and extended [4, 5]. The Golgi complex consists of a network of compartments or cisternae that organize intracellular traffic. The identity and functions of these cisternae are determined by small GTPases; mainly members of the Rab [6] family or the Arf/Sar family [7, 8]. These enzymes are recruited to the cytosolic face of the Golgi membrane and thus activated upon GTP binding, and become soluble and inactive by hydrolysis of the GTP. GTP hydrolysis is stimulated by interaction with a GTPase-activating protein (GAP) whereas GDP to GTP exchange is stimulated by GTPase exchange factors (GEFs). Each GTPase recruits specific effectors to the Golgi apparatus, such as tethering factors, coat complexes and GEF or GAP proteins, each of them performing diverse functions. Cargo molecules traverse the Golgi eventually reaching the late Golgi (also known as trans-Golgi, TGN) where proteins and lipids are sorted into distinct transport carriers (such as tubules or vesicles) that are targeted to various destinations, such as the endosomes or the PM. Secretory vesicles can be delivered to s, Cytoskeleton plays an important role in the secretory pathway. In mammals and filamentous fungi microtubules and actin cables cooperate to facilitate intracellular movement of transport carriers. However, yeast cells do not seem to use microtubules for trafficking functions, as they only require actin cables for efficient exocytosis [14]. Secretory vesicles are attached to cellular motors (dynein and kinesin-1 for microtubule interactions and myosin-5 to interact with actin) by Rabs, directly or through their effectors, [14, 15]. The filamentous fungus Aspergillus nidulans is a well-suited model organism to study the secretory pathway [16, 17]. Hyphal growth is driven exclusively by apical extension. Cell wall synthesizing enzymes and the membrane required for cell expansion are delivered to the apex by exocytosis. This mode of growth and its heavy dependence on exocytosis represent an important experimental advantage, because mutations impairing exocytosis delay or preclude polarity establishment or result in a tip swelling [18]., [ES]La ruta secretora es un proceso celular por el que determinadas proteínas maduran postraduccionalmente y son enviadas a los endosomas, al exterior celular o distribuidas en la membrana plasmática. Esta ruta comienza con la entrada de las proteínas recién sintetizadas al retículo endoplasmático (RE), donde se pliegan y sufren modificaciones postraduccionales, como la N-glicosilación [1] o el anclaje a glicosil fosfatidilinositol (GPI) [2]. A continuación las proteínas (cargos) viajan desde el RE al aparato de Golgi en vesículas recubiertas por el complejo proteico COPII (coat protein complex-II) [3]. Los llamados factores de anclaje o tethering las acercan a las cisternas en formación y la fusión de las vesículas con las cisternas ocurre gracias a la función de proteínas SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor). En el Golgi las cadenas de N-oligosacáridos se modifican y extienden [4, 5]. El aparato de Golgi es un conjunto de cisternas que organizan el tráfico intracelular, cuya identidad y funciones subyacentes están controladas por GTPasas pequeñas de la familia Rab [6] y de la familia Arf / Sar [7, 8]. Estas enzimas se reclutan a la cara citosólica de las membranas del Golgi al cargarse con GTP y se inactivan y liberan de dicha membranas al hidrolizar el GTP. Estos dos eventos de carga e hidrólisis están regulados por los factores intercambiadores de nucleótidos de guanina (GEF) y por las proteínas estimuladoras de la actividad GTPásica (GAP), respectivamente. Cada GTPasa recluta un conjunto de proteínas efectoras (factores de tethering, proteínas de cubierta y proteínas GEFs y GAPs, entre otros) a través de los cuales se ejecutan diversas funciones. Después de pasar por las cisternas del Golgi temprano o cis-Golgi, el cargo llega al Golgi tardío o trans-Golgi (TGN). Desde el TGN, el cargo, posiblemente concentrado en subdominios especializados [9], es reconocido y enviado a su destino final (los endosomas o la membrana plas, Se trata de un hongo filamentoso que crece exclusivamente por extensión apical gracias a la fusión con el ápice de sus hifas de vesículas de secreción, lo que aporta las membranas requeridas para el crecimiento celular y las enzimas necesarias para la síntesis de pared. El modo de crecimiento polarizado de las hifas y su dependencia de la exocitosis representan una ventaja experimental, ya que las mutaciones que perturban la exocitosis alteran el establecimiento de la polaridad de crecimiento o lo imposibilitan, lo que provoca una alteración morfológica en la región apical de las hifas [18].

Aspergillus nidulans (Pal) ambient pH signaling takes place in cortical structures containing components of the ESCRT pathway, which are hijacked by the alkaline pH-activated, ubiquitin-modified version of the arrestin-like protein PalF and taken to the plasma membrane. There, ESCRTs scaffold the assembly of dedicated Pal proteins acting downstream. The molecular details of this pathway, which results in the two-step proteolytic processing of the transcription factor PacC, have received considerable attention due to the key role that it plays in fungal pathogenicity. While current evidence strongly indicates that the pH signaling role of ESCRT complexes is limited to plasma membrane-associated structures where PacC proteolysis would take place, the localization of the PalB protease, which almost certainly catalyzes the first and only pH-regulated proteolytic step, had not been investigated. In view of ESCRT participation, this formally leaves open the possibility that PalB activation requires endocytic internalization. As endocytosis is essential for hyphal growth, nonlethal endocytic mutations are predicted to cause an incomplete block. We used a SynA internalization assay to measure the extent to which any given mutation prevents endocytosis. We show that none of the tested mutations impairing endocytosis to different degrees, including slaB1, conditionally causing a complete block, have any effect on the activation of the pathway. We further show that PalB, like PalA and PalC, localizes to cortical structures in an alkaline pH-dependent manner. Therefore, signaling through the Pal pathway does not involve endocytosis.

The study of fungal cells is of great interest due to their importance as pathogens and as fermenting fungi and for their appropriateness as model organisms. The differential pressure between the hyphal cytoplasm and the bordering medium is essential for the growth process, because the pressure is correlated with the growth rate. Notably, during the invasion of tissues, the external pressure at the tip of the hypha may be different from the pressure in the surrounding medium. We report the use of a method, based on the micropipette-aspiration technique, to study the influence of this external pressure at the hyphal tip. Moreover, this technique makes it possible to study hyphal growth mechanics in the case of very thin hyphae, not accessible to turgor pressure probes.We found a correlation between the local pressure at the tip and the growth rate for the species Arpergillus nidulans. Importantly, the proposed method allows one to measure the pressure at the tip required to arrest the hyphal growth. Determining that pressure could be useful to develop newmedical treatments for fungal infections.Finally, we provide a mechanical model for these experiments, taking into account the cytoplasm flow and the wall deformation.

Microtubule-targeting agents (MTAs) are some of the clinically most successful anti-cancer drugs. Unfortunately, instances of multidrug resistances to MTA have been reported, which highlights the need for developing MTAs with different mechanistic properties. One less explored class of MTAs are [1,2,4]triazolo[1,5-a]pyrimidines (TPs). These cytotoxic compounds are microtubule-stabilizing agents that inexplicably bind to vinblastine binding site on tubulin, which is typically targeted by microtubule-destabilizing agents. Here we used cellular, biochemical, and structural biology approaches to address this apparent discrepancy. Our results establish TPs as vinca-site microtubule-stabilizing agents that promote longitudinal tubulin contacts in microtubules, in contrast to classical microtubule-stabilizing agents that primarily promote lateral contacts. Additionally we observe that TPs studied here are not affected by p-glycoprotein overexpression, and suggest that TPs are promising ligands against multidrug-resistant cancer cells.

Hyphal tip cells of the fungus Aspergillus nidulans are useful for studying long-range intracellular traffic. Post-Golgi secretory vesicles (SVs) containing the RAB11 orthologue RabE engage myosin-5 as well as plus end– and minus end–directed microtubule motors, providing an experimental system with which to investigate the interplay between microtubule and actin motors acting on the same cargo. By exploiting the fact that depolymerization of F-actin unleashes SVs focused at the apex by myosin-5 to microtubule-dependent motors, we establish that the minus end–directed transport of SVs requires the dynein/dynactin supercomplex. This minus end–directed transport is largely unaffected by genetic ablation of the Hook complex adapting early endosomes (EEs) to dynein but absolutely requires p25 in dynactin. Thus dynein recruitment to two different membranous cargoes, namely EEs and SVs, requires p25, highlighting the importance of the dynactin pointed-end complex to scaffold cargoes. Finally, by studying the behavior of SVs and EEs in null and rigor mutants of kinesin-3 and kinesin-1 (UncA and KinA, respectively), we demonstrate that KinA is the major kinesin mediating the anterograde transport of SVs. Therefore SVs arrive at the apex of A. nidulans by anterograde transport involving cooperation of kinesin-1 with myosin-5 and can move away from the apex powered by dynein.

Aspergillus nidulans utilizes phenylacetate as a carbon source via homogentisate, which is degraded to fumarate and acetoacetate. Mutational evidence strongly suggested that phenylacetate is converted to homogentisate through two sequential hydroxylating reactions in positions 2 and 5 of the aromatic ring. Using cDNA substraction techniques, we have characterized a gene, denoted phacA, whose transcription is strongly induced by phenylacetate and which putatively encodes a cytochrome P450 protein. A disrupted phacA strain does not grow on phenylacetate but grows on 2-hydroxy- or 2, 5-dihydroxyphenylacetate. Microsomal extracts of the disrupted strain are deficient in the NADPH-dependent conversion of phenylacetate to 2-hydroxyphenylacetate. We conclude that PhacA catalyzes the ortho-hydroxylation of phenylacetate, the first step of A. nidulans phenylacetate catabolism. The involvement of a P450 enzyme in the ortho-hydroxylation of a monoaromatic compound has no precedent. In addition, PhacA shows substantial sequence divergence with known cytochromes P450 and defines a new family of these enzymes, suggesting that saprophytic fungi may represent a source of novel cytochromes P450. Phenylacetate is a precursor for benzylpenicillin production. phacA disruption increases penicillin production 3-5-fold, indicating that catabolism competes with antibiotic biosynthesis for phenylacetate and strongly suggesting strategies for Penicillium chrysogenum strain improvement by reverse genetics.

We have previously used Aspergillus nidulans as a fungal model for human phenylalanine catabolism. This model was crucial for our characterization of the human gene involved in alcaptonuria. We use here an identical approach to characterize at the cDNA level the human gene for maleylacetoacetate isomerase (MAAI, EC 5.2.1.2), the only as yet unidentified structural gene of the phenylalanine catabolic pathway. We report here the first characterization of a gene encoding a MAAI enzyme from any organism, the A. nidulans maiA gene. maiA disruption prevents growth on phenylalanine (Phe) and phenylacetate and results in the absence of MAAI activity in vitro and Phe toxicity. The MaiA protein shows strong amino acid sequence identity to glutathione S-transferases and has MAAI activity when expressed in Escherichia coli. maiA is clustered with fahA and hmgA, the genes encoding the two other enzymes of the common part of the Phe/phenylacetate pathways. Based on the high amino acid sequence conservation existing between other homologous A. nidulans and human enzymes of this pathway, we used the MaiA sequence in data base searches to identify human expressed sequence tags encoding its putative homologues. Four such cDNAs were sequenced and shown to be encoded by the same gene. They encode a protein with 45% sequence identity to MaiA, which showed MAAI activity when expressed in E. coli. Human MAAI deficiency would presumably cause tyrosinemia that would be characterized by the absence of succinylacetone, the diagnostic compound resulting from fumarylacetoacetate hydrolase deficiency in humans and fungi. Culture supernatants of an A. nidulans strain disrupted for maiA are succinylacetone-negative but specifically contain cis and/or trans isomers of 2, 4-dioxohept-2-enoic acid. We suggest that this compound(s) might be diagnostic for human MAAI deficiency.

The isopenicillin N synthase (ipnA) gene, encoding a key penicillin biosynthetic enzyme in Aspergillus nidulans, represents a prototype of an alkaline-expressed gene. ipnA is under ambient pH regulation, and its promoter (ipnAp) contains binding sites for the zinc-finger transcription factor PacC. We show here that three of these sites, denoted ipnA2, ipnA3, and ipnA4AB, are efficiently recognized by the protein in an isolated sequence context. Single, double, and triple inactivation of these sites in any possible combination reduced promoter activity under alkaline conditions but had no effect under acidic conditions (under which promoter activity was low), as measured by the expression of wild-type and mutant ipnAp::lacZ fusion genes integrated in single copy into a common chromosomal location. This establishes a physiological role for these PacC binding sites and demonstrates a direct role for PacC in ambient pH regulation of ipnA gene expression. In addition, this confirms our previous proposal that PacC is an activator for alkaline-expressed genes. Notably, our experiments show that ipnA2, the highest affinity site for PacC in the ipnAp, contributes relatively modestly to PacC-mediated activation. By contrast, the lower affinity sites ipnA3 and ipnA4AB contribute more substantially to regulation by ambient pH. Inactivation of these three binding sites reduced promoter activity under alkaline conditions to that observed under acidic conditions, showing that these three PacC sites at ipnAp are sufficient to account for its activation by alkaline ambient pH.

We report here the first characterization of a gene encoding a homogentisate dioxygenase, the Aspergillus nidulans hmgA gene. The HmgA protein catalyzes an essential step in phenylalanine catabolism, and disruption of the gene results in accumulation of homogentisate in broths containing phenylalanine. hmgA putatively encodes a 448-residue polypeptide (Mr = 50,168) containing 21 histidine and 23 tyrosine residues. This polypeptide has been expressed in Escherichia coli as a fusion to glutathione S-transferase, and the affinity-purified protein has homogentisate dioxygenase activity. A. nidulans, an ascomycete amenable to classical and reverse genetic analysis, is a good metabolic model to study inborn errors in human Phe catabolism. One such disease, alkaptonuria, was the first human inborn error recognized (Garrod, A. E. (1902) Lancet 2, 1616-1620) and results from loss of homogentisate dioxygenase. Here we take advantage of the high degree of conservation between the amino acid sequences of the fungal and higher eukaryote enzymes of this pathway to identify expressed sequence tags encoding human and plant homologues of HmgA. This is a significant advance in characterizing the genetic defect(s) of alkaptonuria and illustrates the usefulness of our fungal model.

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.

Summary of the report entitled “Aspergillus nidulans alkaline ambient perception: the receptor-transducer complex” for Doctorate in Science of MSc. Daniel Lucena Agell. A.1-INTRODUCTION Living organisms are able to sense, respond and adapt to different environmental stimuli. In the same way, cells have developed homeostatic regulation systems to adapt to different external stimuli. The signal transduction, the manner that cells sense and external stimulus and transduce it inside them in a comprehensive signal, was first defined by Professor Martin Rodbell [1]. This system is always formed by a receptor, a transducer and an effector [1,2]. Over the wide type of receptors that cells have, there exists a superfamily of receptors called GPCRs which have approximately 800 members that are able to activate themselves to different external stimuli, from small molecules, cations and hormones, to light [3,4]. The main feature of this sort of receptors is the seven transmembrane &-helices that they have in their structure. GCPRs undergo a rearrangement in their structure upon ligand binding that ends in receptor activation. This activation allows the recognition of specific sites in the intracellular side of the receptor by a heterotrimeric G protein, which then dissociates in its subunits $ and B,Y that in turn, are able to activate through second messengers different signalling pathways inside the cell [4]. To avoid over-signalling by GPCRs, a desensitization system exists, based on the phosphorylation of the activated receptors by the GRKs (G protein coupled receptors kinases) or the PkcC or PkcA kinases which in turn, allows the recruitment of B-arrestins [5]., The binding of B-arrestin upon activated receptors blocks the interaction with a G protein and promotes the internalization of the complex receptor-arrestin. Recently, a new paradigm of signalling has emerged upon receptor-arrestin binding. After its internalization, this complex through the arrestin is able to interact with different proteins and activate different signalling pathways [5,6]. Fungi are able to grow over wide environmental conditions. pH is one of these conditions and fungi are able to adapt over a wide range. This is possible thanks to a signalling pathway called rim/pal that has extensively studied in Aspergillus nidulans. This pathway is formed by the product of the six pal genes: PalA, PalB, PalC, PalF, PalH and PalI and by the zinc transcription factor PacC [7-72 14]. PacC is activated after two processing steps. Firstly, PacC inactive form, PacC , is processed to 53 PacC in a pH and pal dependent manner. The second processing step that leads to PacC active form, 27 27 PacC , is mediated by the proteasome. PacC is able to enter to the nucleus and then activate genes that are required in alkaline conditions and repress those required in acidic conditions [15-18]. PalH and PalI are both membrane proteins [13]. PalH is the putative pH-sensing receptor which contains 7 predicted transmembrane helices [11], thus resembling GPCRs. Upon alkalinisation, PalF, the arrestin-like protein of the pH signalling pathway is recruited by PalH and then, is phosphorylated and ubiquitinated [19]. Ubiquitination is crucial to activate pH signalling [20] and mediates the recruitment of Endosomal Sorting Complex Required for Transport (ESCRT)-I protein, Vps23, to plasma membrane [21]. Immediately, ESCRT-II and -III proteins arrive at the plasma membrane. In turn, ESCRT-III protein, Vps32, recruits PalC and PalA to cortical sites [14,21,22]., PalC arrives to the plasma membrane before PalA as Galindo et al. [21], demonstrated. PalA is able to 72 bind to YPXL/I motifs in PacC and recruit it [23]. On the other hand, Vps24 is able to recruit calpain-like protease PalB through its MIT motif that interacts with MIM motif of Vps24 [24]. Then, PalB 72 53 27 processes PacC to PacC which releases from PalA and is able to be processed to PacC by the proteasome. A.2-MAIN OBJECTIVES AND RESULTS The main goals of this thesis are: -To demonstrate that PalH is the pH-sensing receptor. -To study the role of PalH phosphorylation in the pH signalling context. -To demonstrate whether PalH endocytosis is required or not for pH signalling activation. -To study PalF subcellular localisation and the influence of PalF and PalI in PalH traffic. -To examine the long term response at alkaline pH in terms of PacC processing and PalH and PalF levels. The results of this thesis are divided into three different chapters: Chapter One, The first chapter has been focused in PalH. Overexpression analyses of PalH and PalF have demonstrated that PalF has a crucial role upon activation but PalH is necessary for pH signalling. In order to develop different studies based in site-directed mutagenesis of PalH, a triple HA (hemaglutinin) was used to tag palH. This construct was then re-inserted in palH locus by gene replacement. This tag allowed discovering that PalH is phosphorylated exclusively under alkaline conditions. Site-directed mutagenesis of palH::ha3 permitted the identification of critical residues for pH signalling in the second extracellular loop. Moreover, two substitutions located in TM6 and TM7 in PalH lead to the first alkalinity mutant ever found in a pal protein. Both substituted residues (Phe319 and Trp332), together with a conserved proline located at TM6 resembles a molecular switch found in some GPCRs which is named rotamer toggle [25]. On the other hand, deletion analyses of conserved regions in PalH sequence have allowed identifying a phosphorylation region within PalH. Studies focused in this region ruled out the GPCR-B-arrestin recruitment paradigm in which phosphorylation is required. In this way, PalH phosphorylation is not necessary for PalF recruitment, thus not affecting pH signalling activation. Deletion of the region contained between the PalF binding sites I and II (this sites are necessary for PalH-PalF interaction [19]) within PalH cytosolic tail (the C-terminal region of PalH) has demonstrated that both sites are sufficient to activate PacC processing. To finish this chapter, analyses of the endocytosis requirement in order to activate pH signalling have been carried out, using different endocytic-defective mutants. Studies with these mutants have shown that overexpressed PalH::GFP is located at the plasma membrane instead of the endovacuolar system as it occurs in the wild-type. Furthermore, PacC processing analyses in these defective mutants are not different of the, Chapter Two The second chapter has been focused in PalF localisation. Using a mCherry tagging in the N-terminal region of arrestin-like protein, first-time microscopy of PalF has been carried out. PalF, although is prominent at the citosol, is able to localise at the plasma membrane without PalH necessity. Moreover, fusion of two PLC domains (which have high affinity for the phosphatydilinositol (4,5)-bisphosphate of the plasma membrane), to the N-terminal region of PalF has redirected it to the plasma membrane. In these conditions, PalF is able to signalling without PalH more efficiently that PalF with ubiquitin fusion [20], demonstrating that plasma membrane localisation of PalF is important for signalling. On the other hand, studies of overexpressed PalH::GFP and mCherry::PalF mutual localisation have been made. Both proteins mutually cooperate to localise at the plasma membrane more efficiently. Furthermore, the same analysis was carried out using a reproduction of two single substitutions that break PalH-PalF interaction [19], into the PalH and PalF fluorescent proteins, showing that Ser86Pro affects PalF sole localisation (is excluded from plasma membrane) and its interaction with PalH. However, studies of PalF and PalI localisation have been shown that they don’t directly interact and cooperate in their mutual plasma membrane localisation. Finally, microscopy analyses of the co-overexpression of PalH::HA3, PalI::GFP and mCherry::PalF have been made, giving an idea of PalF and PalI trafficking upon ambient alkalinisation., Chapter three The last chapter has been centred in the study of the steady-state of PacC processing. Different short-time and long-time PacC processing assays have been carried out. PacC activation requires minutes after the ambient alkalinisation whereas it arrives at equilibrium between its different forms at 150-180 minutes after the pH shift. In the same manner, PalH and PalF arrive to equilibrium between their different forms. Moreover, return to the basal state after acidification of the environment in a strain which has previously grown in alkaline conditions, requires hours. Finally, studies using a loss of function mutant of pacX, a zinc finger protein, have shown that negatively regulates PacC levels. These results together with the fact that PacX localises in a single focus inside the nucleus allow thinking that PacX is a transcription factor which repress pacC. A.3-CONCLUSIONS These are the main conclusions of this thesis: 1-PalH is the environmental pH sensing receptor. 2-The Phe319 and Trp332 residues in PalH play a main role in its activation. nd 3-There are some residues located in and around the 2 extracellular loop of PalH which are critical for pH signalling. 4-PalH is phosphorylated exclusively under alkaline conditions. 5-PalH phosphorylation is not implicated in PalF recruitment and is not necessary fo pH signalling. 6-PalH endocytosis is not necessary for pH signalling. 7-PalF localises at the plasma membrane without PalH necessity. 8-Plasma membrane sole localisation of PalF activates the pH signalling. 9-PalF and PalH mutually cooperate to localise at the plasma membrane.

The genetically amenable fungus Aspergillus nidulans is well suited for cell biology studies involving the secretory pathway and its relationship with hyphal tip growth by apical extension. We exploited live-cell epifluorescence microscopy of the ER labeled with the translocon component Sec63, endogenously tagged with GFP, to study the organization of 'secretory' ER domains. The Sec63 A. nidulans ER network includes brightly fluorescent peripheral strands and more faintly labeled nuclear envelopes. In hyphae, the most abundant peripheral ER structures correspond to plasma membrane-associated strands that are polarized, but do not invade the hyphal tip dome, at least in part because the subapical collar of endocytic actin patches constrict the cortical strands in this region. Thus the subapical endocytic ring might provide an attachment for ER strands, thereby ensuring that the growing tip remains 'loaded' with secretory ER. Acute disruption of secretory ER function by reductive stress-mediated induction of the unfolded protein response results in the reversible aggregation of ER strands, cessation of exocytosis and swelling of the hyphal tips. The secretory ER is insensitive to brefeldin A treatment and does not undergo changes during mitosis, in agreement with the reports that apical extension continues at normal rates during this period. © 2013 Markina-Iñarrairaegui et al.

We exploit the ease with which highly motile early endosomes are distinguished from static late endosomes in order to study Aspergillus nidulans endosomal traffic. RabSRab7 mediates homotypic fusion of late endosomes/vacuoles in a homotypic fusion- and vacuole protein sorting/Vps41–dependent manner. Progression across the endocytic pathway involves endosomal maturation because the end products of the pathway in the absence of RabSRab7 are minivacuoles that are competent in multivesicular body sorting and cargo degradation but retain early endosomal features, such as the ability to undergo long-distance movement and propensity to accumulate in the tip region if dynein function is impaired. Without RabSRab7, early endosomal Rab5s—RabA and RabB—reach minivacuoles, in agreement with the view that Rab7 homologues facilitate the release of Rab5 homologues from endosomes. RabSRab7 is recruited to membranes already at the stage of late endosomes still lacking vacuolar morphology, but the transition between early and late endosomes is sharp, as only in a minor proportion of examples are RabA/RabB and RabSRab7 detectable in the same—frequently the less motile—structures. This early-to-late endosome/vacuole transition is coupled to dynein-dependent movement away from the tip, resembling the periphery-to-center traffic of endosomes accompanying mammalian cell endosomal maturation. Genetic studies establish that endosomal maturation is essential, whereas homotypic vacuolar fusion is not

Aspergillus nidulans (Emericella nidulans) es un hongo filamentoso ascomiceto perteneciente a la familia Trichocomaceae (Harris et al., 2005;Thom and Raper, 1945). Las células vegetativas, también denominadas hifas, son tubulares y crecen de forma hiperpolarizada por extensión apical y ramificación lateral (Harris et al., 2005). Estas hifas se entrecruzan y fusionan (anastomosis) formando una red entramada denominada micelio la cual se expande simétricamente formando colonias de forma radial. Aspergillus es un organismo cenocítico, en el cual las células vegetativas son sincitios multinucleados, generalmente haploides, y están divididas por septos perforados que permiten el intercambio selectivo de nutrientes y efectores entre los compartimentos celulares. Generalmente en una colonia determinada todos los núcleos en cada hifa contienen el mismo material genético y por ello a estas cepas se las denomina homocariontes. En los heterocariontes conviven en un mismo citoplasma núcleos con dotación genética diferente. Pueden distinguirse cuatro fases en el ciclo de vida de Aspergillus nidulans (recientemente revisado en (Todd et al., 2007)): fase de crecimiento vegetativo, reproducción asexual, reproducción sexual y ciclo parasexual. El ciclo vegetativo del hongo comienza tras el inicio de la germinación de la conidiospora o espora de reproducción asexual con el establecimiento de la polaridad de dicha célula, (Harris and Momany, 2004). A un determinado punto de la superficie del conidio se recluta una maquinaria molecular específica del hongo que le permite depositar componentes de la membrana plasmática y pared celular en dicho punto. Esta continua deposición de material da lugar a que la espora emita el denominado tubo germinativo (estadío germínula) y se continúe el crecimiento en dicha dirección formándose así las células típicas de este hongo, las denominadas hifas. A este tipo de crecimiento le denominamos polarizado ya que se produce solo en los ápices de estas cél, El cuerpo fructífero donde se encuentra el conjunto de ascas recubiertas por una pared es una estructuras esférica denomina cleistotecio. Cuando el cleistotecio se rompe, las 10000 ascosporas que puede llegar a contener (Todd et al., 2007) se liberan y germinan comenzando así un nuevo ciclo de vida vegetativo (Pontecorvo et al., 1953). El ciclo parasexual se caracteriza por la fusión de hifas vegetativas y el intercambio de material celular por anastomosis, dando lugar a individuos heterocarióticos que poseen núcleos haploides de distinta dotación genética. Adicionalmente se puede producir la fusión de los núcleos haploides para dar lugar a estirpes diploides. La dotación cromosómica n se reestablece por haploidización (Pontecorvo et al., 1953). Aspergillus nidulans es un organismo modelo en el que se realizan estudios de genética y biología celular para comprender como se desarrollan procesos celulares en eucariotas. Las técnicas de biología molecular aplicables en este organismo permiten realizar diferentes abordajes experimentales como: etiquetados, reemplazamientos génicos (Nayak et al., 2006), análisis de localización subcelular de proteínas y análisis proteómicos (Yang et al., 2004). Debido a su naturaleza homotálica y a su capacidad de oryzae o A. flavus, o clínico como A. fumigatus. A esta característica se une su enorme versatilidad metabólica, que ha permitido identificar numerosas rutas del metabolismo primario y secundario. Además este hongo ha servido como modelo para comprender la base molecular de ciertas enfermedades metabólicas de origen genético en humanos mediante de la identificación de los genes implicados en dichas rutas en Aspergillus (Peñalva, 2001). Por último la diferente organización celular de Aspergillus frente a otros hongos levaduriformes ascomicetos como S. cerevisiae ofrece un nuevo sistema donde estudiar procesos de diferenciación, distribución celular, organización nuclear y de procesos de transporte inter- e intra-compartimentos c

Cytoplasmic dynein transports various cellular cargoes including early endosomes, but how dynein is linked to early endosomes is unclear. We find that the Aspergillus nidulans orthologue of the p25 subunit of dynactin is critical for dynein-mediated early endosome movement but not for dynein-mediated nuclear distribution. In the absence of NUDF/LIS1, p25 deletion abolished the localization of dynein-dynactin to the hyphal tip where early endosomes abnormally accumulate but did not prevent dynein-dynactin localization to microtubule plus ends. Within the dynactin complex, p25 locates at the pointed end of the Arp1 filament with Arp11 and p62, and our data suggest that Arp11 but not p62 is important for p25-dynactin association. Loss of either Arp1 or p25 significantly weakened the physical interaction between dynein and early endosomes, although loss of p25 did not apparently affect the integrity of the Arp1 filament. These results indicate that p25, in conjunction with the rest of the dynactin complex, is important for dynein-early endosome interaction.