scholarly journals FHIP and FTS proteins are critical for dynein-mediated transport of early endosomes in Aspergillus

2014 ◽  
Vol 25 (14) ◽  
pp. 2181-2189 ◽  
Author(s):  
Xuanli Yao ◽  
Xiangfeng Wang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Protein Level ◽  
Cytoplasmic Dynein ◽  
Early Endosome ◽  
Human Homologue ◽  
Genome Wide ◽  
A Genome ◽  
Early Endosomes ◽  
Microtubule Motor

The minus end–directed microtubule motor cytoplasmic dynein transports various cellular cargoes, including early endosomes, but how dynein binds to its cargo remains unclear. Recently fungal Hook homologues were found to link dynein to early endosomes for their transport. Here we identified FhipA in Aspergillus nidulans as a key player for HookA (A. nidulans Hook) function via a genome-wide screen for mutants defective in early-endosome distribution. The human homologue of FhipA, FHIP, is a protein in the previously discovered FTS/Hook/FHIP (FHF) complex, which contains, besides FHIP and Hook proteins, Fused Toes (FTS). Although this complex was not previously shown to be involved in dynein-mediated transport, we show here that loss of either FhipA or FtsA (A. nidulans FTS homologue) disrupts HookA–early endosome association and inhibits early endosome movement. Both FhipA and FtsA associate with early endosomes, and interestingly, while FtsA–early endosome association requires FhipA and HookA, FhipA–early endosome association is independent of HookA and FtsA. Thus FhipA is more directly linked to early endosomes than HookA and FtsA. However, in the absence of HookA or FtsA, FhipA protein level is significantly reduced. Our results indicate that all three proteins in the FtsA/HookA/FhipA complex are important for dynein-mediated early endosome movement.

scholarly journals The p25 subunit of the dynactin complex is required for dynein–early endosome interaction

2011 ◽  
Vol 193 (7) ◽  
pp. 1245-1255 ◽  
Author(s):  
Jun Zhang ◽  
Xuanli Yao ◽  
Lauren Fischer ◽  
Juan F. Abenza ◽  
Miguel A. Peñalva ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Cytoplasmic Dynein ◽  
Early Endosome ◽  
Physical Interaction ◽  
Nuclear Distribution ◽  
Early Endosomes ◽  
Pointed End ◽  
Dynactin Complex ◽  
Hyphal Tip

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.

scholarly journals Discovery of a vezatin-like protein for dynein-mediated early endosome transport

2015 ◽  
Vol 26 (21) ◽  
pp. 3816-3827 ◽  
Author(s):  
Xuanli Yao ◽  
Herbert N. Arst ◽  
Xiangfeng Wang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Genetic Study ◽  
Cytoplasmic Dynein ◽  
Early Endosome ◽  
Dependent Manner ◽  
C Terminus ◽  
Early Endosomes ◽  
Abnormal Accumulation ◽  
Hyphal Tip

Early endosomes are transported bidirectionally by cytoplasmic dynein and kinesin-3, but how the movements are regulated in vivo remains unclear. Here our forward genetic study led to the discovery of VezA, a vezatin-like protein in Aspergillus nidulans, as a factor critical for early endosome distribution. Loss of vezA causes an abnormal accumulation of early endosomes at the hyphal tip, where microtubule plus ends are located. This abnormal accumulation depends on kinesin-3 and is due to a decrease in the frequency but not the speed of dynein-mediated early endosome movement. VezA-GFP signals are enriched at the hypha tip in an actin-dependent manner but are not obviously associated with early endosomes, thus differing from the early endosome association of the cargo adapter HookA (Hook in A. nidulans). On loss of VezA, HookA associates normally with early endosomes, but the interaction between dynein-dynactin and the early-endosome-bound HookA is significantly decreased. However, VezA is not required for linking dynein-dynactin to the cytosolic ∆C-HookA, lacking the cargo-binding C-terminus. These results identify VezA as a novel regulator required for the interaction between dynein and the Hook-bound early endosomes in vivo.

scholarly journals A genome-wide polyketide synthase deletion library uncovers novel genetic links to polyketides and meroterpenoids in Aspergillus nidulans

2011 ◽  
Vol 321 (2) ◽  
pp. 157-166 ◽  
Author(s):  
Michael L. Nielsen ◽  
Jakob B. Nielsen ◽  
Christian Rank ◽  
Marie L. Klejnstrup ◽  
Dorte K. Holm ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Polyketide Synthase ◽  
Genome Wide ◽  
A Genome ◽  
Deletion Library

scholarly journals LIS1 regulates cargo-adapter–mediated activation of dynein by overcoming its autoinhibition in vivo

2019 ◽  
Vol 218 (11) ◽  
pp. 3630-3646 ◽  
Author(s):  
Rongde Qiu ◽  
Jun Zhang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Mechanism Of Action ◽  
Developmental Disorder ◽  
Model Organism ◽  
Cytoplasmic Dynein ◽  
Binding Domain ◽  
Significant Extent ◽  
Experimental Systems ◽  
Microtubule Motor

Deficiency of the LIS1 protein causes lissencephaly, a brain developmental disorder. Although LIS1 binds the microtubule motor cytoplasmic dynein and has been linked to dynein function in many experimental systems, its mechanism of action remains unclear. Here, we revealed its function in cargo-adapter–mediated dynein activation in the model organism Aspergillus nidulans. Specifically, we found that overexpressed cargo adapter HookA (Hook in A. nidulans) missing its cargo-binding domain (ΔC-HookA) causes dynein and its regulator dynactin to relocate from the microtubule plus ends to the minus ends, and this relocation requires LIS1 and its binding protein, NudE. Astonishingly, the requirement for LIS1 or NudE can be bypassed to a significant extent by mutations that prohibit dynein from forming an autoinhibited conformation in which the motor domains of the dynein dimer are held close together. Our results suggest a novel mechanism of LIS1 action that promotes the switch of dynein from the autoinhibited state to an open state to facilitate dynein activation.

scholarly journals Three sorting nexins drive the degradation of apoptotic cells in response to PtdIns(3)P signaling

2011 ◽  
Vol 22 (3) ◽  
pp. 354-374 ◽  
Author(s):  
Nan Lu ◽  
Qian Shen ◽  
Timothy R. Mahoney ◽  
Xianghua Liu ◽  
Zheng Zhou
Keyword(s):  
Specific Protein ◽  
Apoptotic Cells ◽  
Phagosome Maturation ◽  
Sorting Nexins ◽  
Bar Domain ◽  
Protein Protein Interaction ◽  
Genome Wide ◽  
A Genome ◽  
Early Endosomes ◽  
Intracellular Vesicles

Apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Phagosome maturation requires phosphatidylinositol 3-phosphate [PtdIns(3)P], yet how PtdIns(3)P triggers phagosome maturation remains largely unknown. Through a genome-wide PtdIns(3)P effector screen in the nematode Caenorhabditis elegans, we identified LST-4/SNX9, SNX-1, and SNX-6, three BAR domain-containing sorting nexins, that act in two parallel pathways to drive PtdIns(3)P-mediated degradation of apoptotic cells. We found that these proteins were enriched on phagosomal surfaces through association with PtdIns(3)P and through specific protein–protein interaction, and they promoted the fusion of early endosomes and lysosomes to phagosomes, events essential for phagosome maturation. Specifically, LST-4 interacts with DYN-1 (dynamin), an essential phagosome maturation initiator, to strengthen DYN-1’s association to phagosomal surfaces, and facilitates the maintenance of the RAB-7 GTPase on phagosomal surfaces. Furthermore, both LST-4 and SNX-1 promote the extension of phagosomal tubules to facilitate the docking and fusion of intracellular vesicles. Our findings identify the critical and differential functions of two groups of sorting nexins in phagosome maturation and reveal a signaling cascade initiated by phagocytic receptor CED-1, mediated by PtdIns(3)P, and executed through these sorting nexins to degrade apoptotic cells.

scholarly journals A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning

2020 ◽  
Vol 104 (22) ◽  
pp. 9801-9822
Author(s):  
Andreas Schüller ◽  
Lisa Wolansky ◽  
Harald Berger ◽  
Lena Studt ◽  
Agnieszka Gacek-Matthews ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Target Genes ◽  
Negative Impact ◽  
Constitutive Expression ◽  
Gene Clusters ◽  
Regulation System ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Genome Wide ◽  
A Genome

Abstract Programmable transcriptional regulation is a powerful tool to study gene functions. Current methods to selectively regulate target genes are mainly based on promoter exchange or on overexpressing transcriptional activators. To expand the discovery toolbox, we designed a dCas9-based RNA-guided synthetic transcription activation system for Aspergillus nidulans that uses enzymatically disabled “dead” Cas9 fused to three consecutive activation domains (VPR-dCas9). The dCas9-encoding gene is under the control of an estrogen-responsive promoter to allow induction timing and to avoid possible negative effects by strong constitutive expression of the highly active VPR domains. Especially in silent genomic regions, facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to targeted promoters, we have created a genome-wide nucleosome map from actively growing cells and stationary cultures to identify the cognate nucleosome-free regions (NFRs). Based on these maps, different single-guide RNAs (sgRNAs) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to regulate several genes in parallel and, depending on the VPR-dCas9 positioning, expression can be pushed to very high levels. We have used the system to turn on individual genes within two different biosynthetic gene clusters (BGCs) which are silent under normal growth conditions. This method also opens opportunities to stepwise activate individual genes in a cluster to decipher the correlated biosynthetic pathway. Keypoints • An inducible RNA-guided transcriptional regulator based on VPR-dCas9 was established in Aspergillus nidulans. • Genome-wide nucleosome positioning maps were created that facilitate sgRNA positioning. • The system was successfully applied to activate genes within two silent biosynthetic gene clusters.

scholarly journals miR-106b-responsive gene landscape identifies regulation of Kruppel-like factor family

2016 ◽  
Author(s):  
Cody J. Wehrkamp ◽  
Sathish Kumar Natarajan ◽  
Ashley M. Mohr ◽  
Mary Anne Phillippi ◽  
Justin L. Mott
Keyword(s):  
Protein Level ◽  
Mrna Level ◽  
Tumor Biology ◽  
Rna Seq ◽  
Data Set ◽  
Genome Wide ◽  
A Genome ◽  
Mrna Analysis ◽  
Novel Targets ◽  
Microrna Dysregulation

MicroRNA dysregulation is a common feature of cancer and due to the promiscuity of microRNA binding this can result in a wide array of genes whose expression is altered. miR-106b is an oncomiR overexpressed in cholangiocarcinoma and its upregulation in this and other cancers often leads to repression of anti-tumorigenic targets. The goal of this study was to identify the miR-106b-regulated gene landscape in cholangiocarcinoma cells using a genome-wide, unbiased mRNA analysis. Through RNA-Seq we found 112 mRNAs significantly repressed by miR-106b. The majority of these genes contain the specific miR-106b seed-binding site. We have validated 11 genes from this set at the mRNA level and demonstrated regulation by miR-106b of five proteins. Combined analysis of our miR-106b-regulated mRNA data set plus published reports indicate that miR-106b binding is anchored by G:C pairing in and near the seed. Novel targets Kruppel-like factor 2 (KLF2) and KLF6 were verified both at the mRNA and at the protein level. Further investigation showed regulation of four other KLF family members by miR-106b. We have discovered coordinated repression of several members of the KLF family by miR-106b that may play a role in cholangiocarcinoma tumor biology.

scholarly journals Insights into LIS1 function in cargo-adapter-mediated dynein activation in vivo

2019 ◽  
Author(s):  
Rongde Qiu ◽  
Jun Zhang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Mechanism Of Action ◽  
Developmental Disorder ◽  
Model Organism ◽  
Cytoplasmic Dynein ◽  
Significant Extent ◽  
Experimental Systems ◽  
Microtubule Motor

AbstractDeficiency of the LIS1 protein causes lissencephaly, a brain developmental disorder. Although LIS1 binds the microtubule motor cytoplasmic dynein and has been linked to dynein function in many experimental systems, its mechanism of action remains unclear. Here we revealed the function of LIS1 in cargo-adapter-mediated dynein activation in the model organism Aspergillus nidulans. Specifically, we found that overexpressed cargo adapter HookA (Hook in A. nidulans) missing its cargo-binding domain (ΔC-HookA) causes dynein and its regulator dynactin to relocate from the microtubule plus ends to the minus ends, and this dramatic relocation requires LIS1 and its binding protein NudE. Astonishingly, the requirement for LIS1 or NudE can be bypassed to a significant extent by specific mutations that open the auto-inhibited “phi-dynein” in which the motor domains of the dynein dimer are held close together. Our results suggest a novel mechanism of LIS1 action: it promotes the switch of dynein from the auto-inhibited state to an open state to facilitate dynein activation.SummaryThis study reveals the role of Lissencephaly 1 (LIS1) in cargo-adapter-mediated dynein activation. Furthermore, it discovers a novel mechanism of LIS1 action involving a switch of dynein from an auto-inhibited state to an active state.

scholarly journals HookA is a novel dynein–early endosome linker critical for cargo movement in vivo

2014 ◽  
Vol 204 (6) ◽  
pp. 1009-1026 ◽  
Author(s):  
Jun Zhang ◽  
Rongde Qiu ◽  
Herbert N. Arst ◽  
Miguel A. Peñalva ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungus ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Early Endosome ◽  
Genetic Screen ◽  
Binding Domain ◽  
Terminal Part ◽  
Microtubule Binding Domain

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.

scholarly journals Tamoxifen blocks retrograde trafficking of Shiga toxin 1 and 2 and protects against lethal toxicosis

2019 ◽  
Vol 2 (3) ◽  
pp. e201900439 ◽  
Author(s):  
Andrey S Selyunin ◽  
Steven Hutchens ◽  
Stanton F McHardy ◽  
Somshuvra Mukhopadhyay
Keyword(s):  
Shiga Toxin ◽  
Lethal Dose ◽  
Early Endosome ◽  
Base Property ◽  
Retrograde Trafficking ◽  
Golgi Transport ◽  
Late Endosomes ◽  
Genome Wide ◽  
A Genome ◽  
Using Data

Shiga toxin 1 (STx1) and 2 (STx2), produced by Shiga toxin–producingEscherichia coli, cause lethal untreatable disease. The toxins invade cells via retrograde trafficking. Direct early endosome-to-Golgi transport allows the toxins to evade degradative late endosomes. Blocking toxin trafficking, particularly at the early endosome-to-Golgi step, is appealing, but transport mechanisms of the more disease-relevant STx2 are unclear. Using data from a genome-wide siRNA screen, we discovered that disruption of the fusion of late endosomes, but not autophagosomes, with lysosomes blocked the early endosome-to-Golgi transport of STx2. A subsequent screen of clinically approved lysosome-targeting drugs identified tamoxifen (TAM) to be a potent inhibitor of the trafficking and toxicity of STx1 and STx2 in cells. The protective effect was independent of estrogen receptors but dependent on the weak base property of TAM, which allowed TAM to increase endolysosomal pH and alter endosomal dynamics. Importantly, TAM treatment enhanced survival of mice injected with a lethal dose of STx1 or STx2. Thus, it may be possible to repurpose TAM for treating Shiga toxin–producingE. coliinfections.

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