Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1μm/min) and the long intracellular distances (>100 μm) impose. ...Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central to the hyphal mode of growth.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
This review describes how systematic studies on RAB proteins in Aspergilllus nidulans, complemented with contributions from other model filamentous fungi, have served to delineate the composition and physiological roles of the fungal RABOME
Cytoplasmic dynein transports membranous cargoes along microtubules, but the mechanism of dynein-cargo interaction is unclear. From a genetic screen, we identified a homologue of human Hook proteins, ...HookA, as a factor required for dynein-mediated early endosome movement in the filamentous fungus Aspergillus nidulans. HookA contains a putative N-terminal microtubule-binding domain followed by coiled-coil domains and a C-terminal cargo-binding domain, an organization reminiscent of cytoplasmic linker proteins. HookA-early endosome interaction occurs independently of dynein-early endosome interaction and requires the C-terminal domain. Importantly, HookA interacts with dynein and dynactin independently of HookA-early endosome interaction but dependent on the N-terminal part of HookA. Both dynein and the p25 subunit of dynactin are required for the interaction between HookA and dynein-dynactin, and loss of HookA significantly weakens dynein-early endosome interaction, causing a virtually complete absence of early endosome movement. Thus, HookA is a novel linker important for dynein-early endosome interaction in vivo.
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
TRAPPII can be assembled by addition of a TRAPPII-specific subcomplex onto core TRAPP prompted us to investigate the possibility that TRAPPI and/or 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 (the homolog of metazoan TRAPPC9) is recruited to existing trans-Golgi network (TGN) cisternae slightly before RAB11 arrives, and resides for ∼45 s on them before cisternae tear off into RAB11 secretory carriers. Notably, the core TRAPP reporter Bet3 (the homolog of metazoan TRAPPC3) was not detectable in early Golgi cisternae, being instead recruited to TGN cisternae simultaneously with Trs120, indicating
recruitment of TRAPPII to the Golgi and arguing strongly against the TRAPP maturation model.
Hyphal tip cells of the fungus
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
by anterograde transport involving cooperation of kinesin-1 with myosin-5 and can move away from the apex powered by dynein.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The mechanism(s) by which proteins traverse and exit the Golgi are incompletely understood. Using Aspergillus nidulans hyphae, we show that late Golgi cisternae undergo changes in composition to ...gradually lose Golgi identity while acquiring post-Golgi RabE(RAB11) identity. This behavior of late Golgi cisternae is consistent with the cisternal maturation model. Post-Golgi RabE(RAB11) carriers travel to, and accumulate at, the apex, indicating that fusion is rate limiting for exocytosis. These carriers, which are loaded with kinesin, dynein, and MyoE(MYO5), move on a microtubule-based bidirectional conveyor belt relaying them to actin, which ultimately focuses exocytosis at the apex. Dynein drags RabE(RAB11) carriers away if engagement of MyoE(MYO5) to actin cables fails. Microtubules seemingly cooperating with F-actin capture can sustain secretion if MyoE(MYO5) is absent. Thus, filamentous fungal secretion involving post-Golgi carriers is remarkably similar, mechanistically, to the transport of melanosomes in melanocyte dendrites, even though melanosome biogenesis involves lysosomes rather than Golgi.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Using affinity chromatography we identified the Aspergillus nidulans F‐BAR‐and‐PH domain‐containing protein BapH as a RabERAB11 effector. BapH localizes to the Spitzenkörper (SPK) in an ...F‐actin‐ and Sec7‐dependent manner, becoming cytosolic after inactivation of Trs120 in TRAPPII, the oligomeric GEF for RabERAB11. Therefore, RabERAB11 contributes to the recruitment of BapH to secretory vesicles in vivo. BapH has a close homologue, SlmA, which is related to yeast Slm1p/Slm2p, localizes to eisosomes and does not bind RabERAB11. bapHΔ, slmAΔ and double bapHΔ slmAΔ mutations do not affect growth, although slmAΔ results in myriocin hypersensitivity. Both the PH and the F‐BAR domain in BapH are necessary to recruit the protein to membranes, whereas its C‐terminal moiety negatively regulates localization to the SPK. Strong overexpression of full‐length BapH or of BapH lacking the C‐terminal moiety impairs growth. The tandemly duplicated PHBapH domain is recruited to the plasma membrane in a manner dependent on critical Lys residues in its ‘noncanonical’ lipid binding pocket, suggesting that it binds to biological membranes containing PtdIns(4,5)P2. Ablation of BapH, or deletion of the PH or BAR domains critical for the SPK localization increases autophagy under nitrogen‐replete conditions. Therefore, BapH localizing to SPK vesicles influences basal levels of autophagy.
RabE/RAB11 is a key regulator of exocytosis in fungal hyphae. The effectors that this GTPase recruits to play its physiological roles are unknown. Here, we report on one such effector that appears to connect secretory membranes in the Spitzenkörper with autophagy.
Aspergillus nidulans hyphae grow exclusively by apical extension. Golgi equivalents (GEs) labeled with mRFP-tagged PH(OSBP) domain form a markedly polarized, dynamic network of ring-shaped and ...fenestrated cisternae that remains intact during "closed" mitosis. mRFP-PH(OSBP) GEs advance associated with the growing apex where secretion predominates but do not undergo long-distance movement toward the tip that could account for their polarization. mRFP-PH(OSBP) GEs overlap with the trans-Golgi resident Sec7 but do not colocalize with also polarized accretions of the early Golgi marker GrhA(Grh1)-GFP, indicating that early and late Golgi membranes segregate spatially. AnSec23-GFP ER exit sites (ERES) are numerous, relatively static foci localizing across the entire cell. However, their density is greatest near the tip, correlating with predominance of early and trans-Golgi elements in this region. Whereas GrhA-GFP structures and ERES reach the apical dome, mRFP-PH(OSBP) GEs are excluded from this region, which contains the endosome dynein loading zone. After latrunculin-mediated F-actin disruption, mRFP-PH(OSBP) GEs fragment and, like AnSec23-GFP ERES, depolarize. Brefeldin A transiently collapses late and early GEs into distinct aggregates containing Sec7/mRFP-PH(OSBP) and GrhA-GFP, respectively, temporarily arresting apical extension. Rapid growth reinitiates after washout, correlating with reacquisition of the normal Golgi organization that, we conclude, is required for apical extension.
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