Photo-convertible tagging for localization and dynamic analyses of low-expression proteins in filamentous fungi

Perez-de-Nanclares-Arregi, Elixabet; Etxebeste, Oier

doi:10.1016/j.fgb.2014.06.006

Cited by 12 publications

(14 citation statements)

References 25 publications

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“…The above results suggested a tip‐to‐nucleus retrograde movement of FlbB. To analyze this possibility, we used a strain expressing a gpdA mini ‐driven chimera of FlbB fused to the green‐to‐red photo‐convertible fluorescent protein Dendra2 (Chudakov et al ., ; Perez‐de‐Nanclares‐Arregi and Etxebeste, ). Green fluorescence was detected at the tip and all nuclei before photo‐conversion while red fluorescence was undetectable (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…After photo‐conversion, the tip‐to‐nuclei redistribution of the red fluorescence of FlbB :: D endra2 was studied. Photo‐conversion was achieved by reducing UV light intensity to 80% of the maximum and using 2500 ms expositions ( P erez‐de‐ N anclares‐ A rregi and E txebeste, ). White arrows and arrowheads indicate the tip and nuclei respectively.…”

Section: Resultsmentioning

confidence: 99%

“…The usefulness of the gpdA mini promoter (Pantazopoulou and Peñalva, 2009) for the constitutive expression of flbB had previously been verified by us (Perez-de-Nanclares-Arregi and Etxebeste, 2014). Construction of pgpdA mini ::GFP::FlbB plasmid was based on pgpd003 ( Fig.…”

Section: Oligonucleotides Plasmids Strains and Culture Conditionsmentioning

confidence: 86%

“…FlbB accumulates at the tip of hyphae at the very beginning of germ‐tube formation and continues in this location as long as polarized cell growth is maintained (Etxebeste et al ., ; Perez‐de‐Nanclares‐Arregi and Etxebeste, ). Actin filaments play a central role in the transport of FlbB from the subapex to the apex, whereas the microtubule cytoskeleton seems not to be essential for the apical accumulation (number 1 in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The association of FlbB localization to different cytoskeletal networks was analyzed FlbB domains and conidiation signaling 621 essentially as described in Araújo-Bazán et al (2008), using 100 μM Latrunculin B (Calbiochem) and/or 2.4 μg ml −1 Benomyl (Aldrich) concentrations. Cultures were observed as previously described using a Leica DMI-6000b or a Zeiss Axio Observer Z1 inverted microscopes (Hernandez-Ortiz and Espeso, 2013; Perez-de-Nanclares-Arregi and Etxebeste, 2014). Images were acquired after 200, 100 or 50 ms of exposure times for strains carrying a single, double or multiple copies of the pgpdA mini ::GFP::FlbB vector respectively.…”

Section: Microscopymentioning

confidence: 99%

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Tip‐to‐nucleus migration dynamics of the asexual development regulator FlbB in vegetative cells

Herrero-García

Perez-de-Nanclares-Arregi

Cortese

et al. 2015

Molecular Microbiology

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SummaryIn Aspergillus nidulans, asexual differentiation requires the presence of the transcription factor FlbB at the cell tip and apical nuclei. Understanding the relationship between these two pools is crucial for elucidating the biochemical processes mediating conidia production. Tip-to-nucleus communication was demonstrated by photo-convertible FlbB::Dendra2 visualization. Tip localization of FlbB depends on Cys382 in the C-terminus and the bZIP DNA-binding domain in the N-terminus. FlbE, a critical FlbB interactor, binds the bZIP domain. Furthermore, the absence of FlbE results in loss of tip localization but not nuclear accumulation. flbE deletion also abrogates transcriptional activity indicating that FlbB gains transcriptional competence from interactions with FlbE at the tip. Finally, a bipartite nuclear localization signal is required for nuclear localization of FlbB. Those motifs of FlbB may play various roles in the sequence of events necessary for the distribution and activation of this transcriptionally active developmental factor. The tip accumulation, FlbE-dependent activation, transport and nuclear import sketch out a process of relaying an environmentally triggered signal from the tip to the nuclei. As the first known instance of transcription factor-mediated tip-to-nucleus communication in filamentous fungi, this provides a general framework for analyses focused on elucidating the set of molecular mechanisms coupling apical signals to transcriptional events.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Oligonucleotides Plasmids Strains and Culture Conditionsmentioning

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Microscopymentioning

confidence: 99%

See 3 more Smart Citations

Tip‐to‐nucleus migration dynamics of the asexual development regulator FlbB in vegetative cells

Herrero-García

Perez-de-Nanclares-Arregi

Cortese

et al. 2015

Molecular Microbiology

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Fluorescence-Based Methods for the Study of Protein Localization, Interaction, and Dynamics in Filamentous Fungi

Etxebeste

Takeshita

2015

Fungal Biology

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Developmental regulators FlbE/D orchestrate the polarity site-to-nucleus dynamics of the fungal bZIP transcription factor FlbB

Otamendi

Perez-de-Nanclares-Arregi

Oiartzabal-Arano

et al. 2019

Cell. Mol. Life Sci.

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Permanently polarized cells have developed transduction mechanisms linking polarity-sites with gene regulation in the nucleus. In neurons, one mechanism is based on long-distance retrograde migration of transcription factors (TFs). Aspergillus nidulans FlbB is the only known fungal TF shown to migrate retrogradely to nuclei from the polarized region of fungal cells known as hyphae. There, FlbB controls developmental transitions by triggering the production of asexual multicellular structures. FlbB dynamics in hyphae is orchestrated by regulators FlbE and FlbD. At least three FlbE domains are involved in the acropetal transport of FlbB, with a final MyoE/actin filament-dependent step from the subapex to the apex. Experiments employing a T2A viral peptide-containing chimera (FlbE::mRFP::T2A::FlbB::GFP) suggest that apical FlbB/FlbE interaction is inhibited in order to initiate a dynein-dependent FlbB transport to nuclei. FlbD controls the nuclear accumulation of FlbB through a cMyb domain and a C-terminal LxxLL motif. Overall, results elucidate a highly dynamic pattern of FlbB interactions, which enable timely developmental induction. Furthermore, this system establishes a reference for TF-based long-distance signaling in permanently polarized cells..

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Photo-convertible tagging for localization and dynamic analyses of low-expression proteins in filamentous fungi

Cited by 12 publications

References 25 publications

Tip‐to‐nucleus migration dynamics of the asexual development regulator FlbB in vegetative cells

Tip‐to‐nucleus migration dynamics of the asexual development regulator FlbB in vegetative cells

Fluorescence-Based Methods for the Study of Protein Localization, Interaction, and Dynamics in Filamentous Fungi

Developmental regulators FlbE/D orchestrate the polarity site-to-nucleus dynamics of the fungal bZIP transcription factor FlbB

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