Biology subjects

Penalva, M. A.

Publications and source records attributed to Penalva, M. A..

3 recordsLinked to original sources

bioRxiv2025

The actin module of endocytic internalization in Aspergillus nidulans: a critical role of the WISH/DIP/SPIN90 family protein Dip1

Using fluorescent protein-tagged F-actin reporters we studied the actin cytoskeleton in Aspergillus nidulans. F-actin probes labeled endocytic patches, contractile actin rings and the Spitzenkorper (SPK), but not exocytic cables generated by the SPK-associated formin, illuminated only by tropomyosin. The SPK actin mesh contains tropomyosin and capping protein, but not fimbrin or Arp2/3, showing that it does not involve branched actin. Arp2/3 and fimbrin are recruited to endocytic patches at the end of their lifecycle, staying in them for 12-14 sec, coinciding with the burst of branched actin polymerization that powers vesicle internalization, whereas verprolin stays only during the first half of this actin phase. Hyphal growth requires endocytic recycling, which we exploited to assess the efficiency of endocytosis following genetic interventions. Ablation of SlaBSla2, Arp2/3, cofilin and fimbrin is lethal, whereas that of Srv2, verprolin and capping protein are debilitating, with the lifetime of actin in mutant patches roughly correlating with the extent of growth and endocytic defects. An outstanding problem is the origin of seed filaments required to prime Arp2/3 during endocytosis. Actin patches associate with cortical cables that give rise to long distance-moving "actin worms" that are different from tropomyosin-containing cables emanating from the SPK. Cables and worms are dependent on formin, yet inactivation of formin does not affect the F-actin patch lifecycle, arguing against formin playing an endocytic role. Ablation of the WISH/DIP/SPIN90 protein Dip1 priming Arp2/3 for the synthesis of linear actin delocalizes the endocytic machinery and severely impairs, but does not preclude, endocytosis. This establishes the existence of Dip1-independent mechanism(s) that synthesize seed filaments. Our data negate the possibility that this alternative mechanism results from a priming role of formin that is unmasked in dip1{Delta} cells, but do not exclude that cofilin-mediated filament severing could produce seed microfilaments for Arp2/3, as suggested for Schizosaccharomyces pombe. Authors summaryFilamentous fungi have a deep impact in our lives as friends and foes. Certain species are used as cell factories for the production of proteins or biopharmaceuticals. In contrast, phytopathogenic species, cause important losses in crops whereas those able to infect humans represent serious risks for global health. Filamentous fungi form tubular cells, denoted hyphae, that grow by apical extension. This requires the coupling between exocytosis and endocytosis in the so-called endocytic recycling pathway, which is needed, for example, to maintain the polarization of enzymes which synthesize the cell wall as growth proceeds. Remarkably, detailed studies on endocytosis in filamentous fungi are wanting. Here we report the characterization of the endocytic pathway in the genetic model Aspergillus nidulans, a filamentous ascomycete which is well-suited for genetic manipulation and in vivo fluorescence microscopy. Our study demonstrates that endocytosis is essential for filamentous fungal life and provides significant insight on how F-actin powers the internalization of endocytic vesicles, including an important physiological role of Dip1, a protein required to provide seed filaments for the formation of F-actin branching networks. Importantly, as yet unidentified Dip1-independent mechanisms that synthesize these seeds must exist, opening new avenues for future research.

cell biology↗

bioRxiv2022

Tethering by Uso1 is dispensable: The Uso1 monomeric globular head domain interacts with SNAREs to maintain viability.

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. Paradoxically, genetic studies placed Uso1 acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S substitutions in the GHD permitting growth of otherwise inviable rab1-deficient Aspergillus nidulans. Remarkably, the double mutant suppresses the complete absence of RAB1. Full-length Uso1 and CTR{Delta} proteins are dimeric and the GHD lacking the CC/CTR is monomeric irrespective of whether they carry or not E6K/G540S. Microscopy showed recurrence of Uso1 on puncta (60 sec half-life) colocalizing with RAB1 and less so with early Golgi markers Sed5 and GeaA/Gea1/Gea2. Localization of Uso1 but not of Uso1E6K/G540S to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. By S-tag-coprecipitation we demonstrate that Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Bos1 and Bet1 bind the Uso1 GHD directly, but Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. AlphaFold2 predicts that G540S actually increases binding of GHD to the Bos1 Habc domain. In contrast, E6K seemingly increases membrane targeting of an N-terminal amphipathic -helix, explaining phenotypic additivity. Overexpression of E6K/G540S and wild-type GHD complemented uso1{Delta}. Thus, a GHD monomer provides the essential Uso1 functions, demonstrating that long-range tethering activity is dispensable. Therefore, when enhanced by E6K/G540S, Uso1 binding to Bos1/Bet1 required to regulate SNAREs bypasses both the contribution of RAB1 to Uso1 recruitment and the reported role of RAB1 in SNARE complex formation (Lupashin and Waters, 1997), suggesting that the latter is consequence of the former.

cell biology↗

bioRxiv2021

A novel RAB11-containing adaptor complex anchoring myosin-5 to secretory vesicles

Hyphal fungi grow rapidly by apical extension, providing a notorious example of polarized growth. The continuous supply of secretory vesicles necessary to meet the demands of the extending tip and the long intracellular distances existing between the tip and the basal septum, often localized > 100 {micro}m away from the former, impose the need of efficient networks of intracellular traffic involving exquisite cooperation between microtubule- and actin-mediated transport. In Aspergillus nidulans kinesin-1 conveys secretory vesicles to the hyphal tip, where they are transferred to myosin-5, which focuses them at the growing apex, thereby determining cell shape. This relay mechanism and the central role played by myosin-5 in hyphal morphogenesis suggested that the mechanisms anchoring secretory vesicles to this motor should involve specific adaptor(s) ensuring the robustness of actomyosin-dependent transport. Secretory vesicles are charged with RAB11, a regulatory GTPase that determines the Golgi to post-Golgi identity transition. By using a combination of shotgun proteomics, GST-RAB pull-down assays, in vitro reconstitution experiments, targeted reverse genetics and multidimensional fluorescence microscopy with endogenously tagged proteins we show that RAB11, the master regulator of fungal exocytosis, mediates myosin-5 engagement both by contacting the motor and by recruiting UDS1, a homologue of an as yet uncharacterized Schizosaccharomyces protein upregulated during mitosis, which we demonstrate to be a novel RAB11 effector. Analytical ultracentrifugation determined that UDS1 is an elongated dimer and negative-stain electron microscopy showed that, in agreement, UDS1 is rod-shaped. UDS1 does not contact myosin-5 directly, but rather recruits the coiled-coil HMSV, which bridges RAB11/UDS1 to myosin-5. An HMSV-scaffolded complex containing UDS1 and myosin-5 is present in cells, and a RAB11-UDS1-HMSV complex can be reconstituted in vitro in a RAB nucleotide state-dependent manner. In the absence of UDS1/HMSV the steady state levels of myosin-5 at the apical vesicle supply center diminish markedly, such that microtubule-dependent transport spreading vesicles across the apical dome predominates over apex-focused actin-mediated transport. As a consequence, RAB11 and chitin-synthase B (a cargo of the RAB11 pathway) are not focused at the apex, being distributed instead across the apical dome. Therefore, the RAB11 effector UDS1/HMSV cooperates with the GTPase to adapt secretory vesicles to myosin-5, which is required for the apical targeting of RAB11 cargoes and thus for the normal morphology of the hyphae.

cell biology↗