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An endoplasmic reticulum domain is associated with the polarized growing cells of Podospora anserina hyphae
Journal Pre-proofs An endoplasmic reticulum domain is associated with the polarized growing cells of Podospora anserina hyphae Antonio de Jesús López-Fuentes, Aldo Meizoso-Huesca, Leonardo PerazaReyes PII: DOI: Reference:
S1087-1845(19)30303-2 https://doi.org/10.1016/j.fgb.2020.103338 YFGBI 103338
To appear in:
Fungal Genetics and Biology
Received Date: Revised Date: Accepted Date:
7 October 2019 28 November 2019 20 January 2020
Please cite this article as: de Jesús López-Fuentes, A., Meizoso-Huesca, A., Peraza-Reyes, L., An endoplasmic reticulum domain is associated with the polarized growing cells of Podospora anserina hyphae, Fungal Genetics and Biology (2020), doi: https://doi.org/10.1016/j.fgb.2020.103338
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Inc.
Video Article: Research An endoplasmic reticulum domain is associated with the polarized growing cells of Podospora anserina hyphae
Antonio de Jesús López-Fuentes, Aldo Meizoso-Huesca#, Leonardo Peraza-Reyes* Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, Ciudad de México 04510, Mexico * Corresponding author: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México Ciudad de México 04510, Mexico. Tel. (52) (55) 5622 5628 E-mail address: [email protected] #Present
address:
School of Biomedical Sciences, University of Queensland, Brisbane, QLD, 4072, Australia.
Keywords: Endoplasmic reticulum / dynamics / hyphal growth Word count: 1,265 Character count: 8,537
1
Abstract The endoplasmic reticulum (ER) is composed of distinct structural domains that perform diverse essential functions, including the synthesis of membrane lipids and proteins of the cell endomembrane system. The polarized growth of fungal hyphal cells depends on a polarized secretory system, which delivers vesicles to the hyphal apex for localized cell expansion, and that involves a polarized distribution of the secretory compartments, including the ER. Here we show that, additionally, the ER of the ascomycete Podospora anserina possesses a peripheral ER domain consisting of highly dynamic pleomorphic ER sub-compartments, which are specifically associated with the polarized growing apical hyphal cells.
2
Introduction The endoplasmic reticulum (ER) is composed of different structural domains that perform diverse essential functions, including the synthesis, processing and transport of membrane lipids and proteins of the cell endomembrane system (Schwarz and Blower, 2016; Westrate et al., 2015). In addition, the ER participates in organelle biogenesis (Joshi et al., 2017) and interacts with most other organelles (Wu et al., 2018), which contributes to the regulation of intracellular dynamics. The ER is composed of two major domains, the nuclear envelope and the peripheral ER. The peripheral ER consists of a reticular network of tubules and cisternal sheets that extends throughout the cell and that comprises diverse structural subdomains, whose arrangement is adapted to their specialized functions (Westrate et al., 2015). The polarized growth mode of fungal hyphae is determined by a secretory system, which drives vesicles to the hyphal tip to deliver membrane lipids and proteins for localized cell surface extension. This process relies on vesicles that transport enzymes involved in cell wall assembly, and is coupled to enzyme endocytic recycling (Hernandez-Gonzalez et al., 2018; Riquelme, 2013; Riquelme et al., 2018; Shaw et al., 2011). Polarized vesicle traffic orchestration involves the Spitzenkörper, a complex structure located in the apex of growing hyphae that is proposed to act as a vesicle supply center that concentrates vesicles for their subsequent delivery to the foremost apical membrane (Riquelme et al., 2018; Riquelme and Sanchez-Leon, 2014). Additionally, the secretory compartments –namely the Golgi cisternae (Pantazopoulou and Penalva, 2009) and the ER (Kimura et al., 2010; Markina-Inarrairaegui et al., 2013; Maruyama et al., 2006; Wedlich-Söldner et al., 2002)– display a polarized distribution along hyphae. Here we show that, in addition, a dynamic peripheral ER domain is specifically associated with the growing apical hyphal cells of the ascomycete Podospora anserina.
3
Results and Discussion We analyzed the P. anserina ER by studying the localization of an ectopically-expressed ER-targeted GFP (ER-GFP) containing the ER targeting and retention signals of the putative P. anserina ER chaperone BiP/Kar2, a protein that localizes to the ER in filamentous ascomycetes (Maruyama et al., 2006). Consistent with ER localization, ERGFP localized to a large network of interconnected strands extending throughout hyphae, similar to the ER arrangement of other filamentous fungi (Kimura et al., 2010; MarkinaInarrairaegui et al., 2013; Maruyama et al., 2006; Rico-Ramirez et al., 2018; WedlichSöldner et al., 2002). Also, like other mycelial fungi, this network exhibited a polarized distribution with increasing abundance towards the hyphal tip. In addition, we found that the ER of the apical cells of growing leading hyphae contained a number of pleomorphic patches, which were interconnected with the peripheral ER strands (Video 1). These apical ER sub-compartments typically ranged between 1-4 μm in length (1-1.5 μm in width), but longer patches stretching up to 9 μm were observed. These compartments were confined to the first 15μm extending behind the tip (i.e, in average, 12.9 2.09μm behind the tip, n=30), but they were not observed to reach the apex (Video 1) or the Spitzenkörper (Videos 2 and 3). Of note, these apical compartments were also detected with a GFP-tagged version of the endogenous ER membrane protein Sec61 (not shown).
Video 1
4
We found that the apical ER compartments were highly dynamic and frequently converged to produce larger patches (e.g., Video 3, yellow arrows), or were separated into smaller patches (Video 1, green arrows). Moreover, we also discovered that the apical ER patches displayed dynamic displacements, which were associated with their cell location and with their fusion and division events. In general, we observed two main classes of apical ER patches based on their distribution and behavior. The first one consisted of ER patches closely apposed to the cell cortex (e.g., Video 3, open arrows). These patches were situated at a relatively constant distance from the hyphal tip and some exhibited displacements, mostly towards the hyphal apex (acropetal). The second group was mainly present in the hypha middle plane and typically exhibited basipetal displacements (backward from the apex). A number of these medial patches were relatively static relative to the subapical cell surface (Video 1, open arrows) and thus separated away from the extending cell tip with a velocity (av. 70 17 nm/s, n=30) comparable to the hyphal growth rate (i.e, av. 73 6nm/s, n=30). In addition, some of these compartments were engaged in faster, mostly basipetal movements (Videos 1-3). Overall, these displacements occurred with an average velocity of 205 87nm/s (range 107–428 nm/s, n=53), but two main categories of displacements with different velocities were observed, with average velocities of 293 53nm/s (n=23, e.g., Video 2, arrowhead) and 138 23nm/s (n=30, e.g., Videos 13, arrows), respectively.
Video 2
5
The medial apical ER compartments moving basipetally emerged from the apical hyphal region behind the Spitzenkörper, frequently from the cortical cell region (e.g., Video 3, arrowheads). Some medial compartments seemed to be derived from cortical tipward-moving patches, suggesting an acropetal-basipetal flow of cortical to medial compartments (e.g., Video 2). In addition, patches derived from opposite sites of the hypha frequently merged following their emergence (e.g., Video 3, yellow arrows).
Video 3 The morphology of the apical ER patches suggests that they represent ER cisternae, however, higher resolution structural analyses are required to ascertain their precise structure. Likewise, the specific functions of these compartments need to be established. However, their specific apical localization suggests that they are associated with the process of polarized hyphal growth. Equivalent apical ER domains could be present in other mycelial fungi. For example, Aspergillus nidulans possesses a finger-like ER protrusion capping the ER apical region (Markina-Inarrairaegui et al., 2013), although this domain seems to protrude from the ER network towards the apex rather than being immersed in the peripheral ER. Although further comparative analyses of different fungi and of distinct hyphal types are required, these observations suggest different configurations of the apical ER adapted to specific hyphal growth habits.
6
Methods P. anserina methods, sequences. P. anserina methods and media composition can be consulted at http://podospora.i2bc.paris-saclay.fr. BiP (Pa_4_6420) gene sequence was obtained from the P. anserina genome sequence (Espagne et al., 2008) and is available under GenBank accession number CDP28607.1. BiP signal peptide sequence was predicted on SignalP 4.1 (Nielsen, 2017). Construction and analysis of ER-targeted GFP. Fusion PCR was used to generate a construct coding for GFP flanked by the ER targeting and retention signals of the potential P. anserina ER chaperone BiP (ER-GFP), preceded by the A. nidulans gpdA promoter. This construct consisted of the fusion of: (i) 892bp of gpdA promoter, (ii) a 105bp sequence encoding BiP signal peptide, (iii) EGFP gene, and (iv) 21bp encompassing BiP C-terminal HDEL-coding sequence and stop codon. This construct was cloned into plasmid pPable (Coppin and Debuchy, 2000) to yield pAM01, which was verified by sequencing and used to transform P. anserina protoplasts. Three independent transformants were selected and purified after sexual crosses with the wild type. Confocal microscopy analysis of ER-GFP showed that the ER arrangement and dynamics in these three strains were alike. For the quantitative analysis of ER dynamics, 30 leading hyphae of one of these strains issued from 3 biological replicates (10 hyphae/replicate) were analyzed. Microscopy. Imaging was performed on a Zeiss LSM-800 inverted laser scanning confocal microscope equipped with a temperature chamber, using a Plan-Apochromat 63x/1.4 oil immersion objective and a 488 nm laser line. Imaging was done on whole individual P. anserina colonies growing at 27C, as previously described (Suaste-Olmos et al., 2018). Micrographs were processed using ZEN 2012 software (Carl Zeiss, Jena, Germany) and ImageJ (NIH, Bethesda, USA). The Spitzenkörper was stained with FM4-64 (N-(3triethylammoniumpropyl)-4-(6-(4-(diethylamino)
phenyl)
hexatrienyl)
pyridinium
dibromide, 7 μM) (Molecular Probes, Eugene, OR).
7
Acknowledgements We are grateful to Fernando Suaste Olmos for technical assistance throughout this research. We thank Sara Schroder for her careful review of the manuscript, and the IFC Imaging Facility for assistance on microscopy. This work was supported by CONACYT [CONACYT (FONCICYT)-DFG 277869, and by PAPIIT-DGAPA, UNAM [IA203317, IV200519]. AJLF was supported by a scholarship from CONACYT. References Coppin, E., Debuchy, R., 2000. Co-expression of the mating-type genes involved in internuclear recognition is lethal in Podospora anserina. Genetics. 155, 65769. Espagne, E., et al., 2008. The genome sequence of the model ascomycete fungus Podospora anserina. Genome Biol. 9, R77. Hernandez-Gonzalez, M., et al., 2018. Endocytic recycling via the TGN underlies the polarized hyphal mode of life. PLoS Genet. 14, e1007291. Joshi, A. S., et al., 2017. Organelle biogenesis in the endoplasmic reticulum. Nat Cell Biol. 19, 876-882. Kimura, S., et al., 2010. In vivo imaging of endoplasmic reticulum and distribution of mutant alpha-amylase in Aspergillus oryzae. Fungal Genet Biol. 47, 1044-54. Markina-Inarrairaegui, A., et al., 2013. The Aspergillus nidulans peripheral ER: disorganization by ER stress and persistence during mitosis. PLoS One. 8, e67154. Maruyama, J., et al., 2006. Differential distribution of the endoplasmic reticulum network as visualized by the BipA-EGFP fusion protein in hyphal compartments across the septum of the filamentous fungus, Aspergillus oryzae. Fungal Genet Biol. 43, 642-54. Nielsen, H., 2017. Predicting Secretory Proteins with SignalP. Protein Function Prediction: Methods and Protocols. 1611, 59-73. Pantazopoulou, A., Peñalva, M. A., 2009. Organization and dynamics of the Aspergillus nidulans Golgi during apical extension and mitosis. Mol Biol Cell. 20, 4335-47. 8
Rico-Ramirez, A. M., et al., 2018. Imaging the secretory compartments involved in the intracellular traffic of CHS-4, a class IV chitin synthase, in Neurospora crassa. Fungal Genet Biol. 117, 30-42. Riquelme, M., 2013. Tip growth in filamentous fungi: a road trip to the apex. Annu Rev Microbiol. 67, 587-609. Riquelme, M., et al., 2018. Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures. Microbiol Mol Biol Rev. 82. Riquelme, M., Sanchez-Leon, E., 2014. The Spitzenkörper: a choreographer of fungal growth and morphogenesis. Curr Opin Microbiol. 20, 27-33. Schwarz, D. S., Blower, M. D., 2016. The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci. 73, 79-94. Shaw, B. D., et al., 2011. A role for endocytic recycling in hyphal growth. Fungal Biol. 115, 541-6. Suaste-Olmos, F., et al., 2018. Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery. Biochim Biophys Acta. 1865, 572-586. Wedlich-Söldner, R., et al., 2002. Dynein supports motility of endoplasmic reticulum in the fungus Ustilago maydis. Mol Biol Cell. 13, 965-77. Westrate, L. M., et al., 2015. Form follows function: the importance of endoplasmic reticulum shape. Annu Rev Biochem. 84, 791-811. Wu, H., et al., 2018. Here, there, and everywhere: The importance of ER membrane contact sites. Science. 361.
9
Video Still Image legends Video 1. ER dynamics in a P. anserina growing hypha. Live-cell imaging of a growing hypha expressing ER-GFP (top). Video 1 shows the presence of dynamic pleomorphic ER sub-compartments in the apical region of a growing hypha. Middle: bright field; bottom: merge. Time scale is in min:sec. Video 2. ER and Spitzenkörper dynamics in a growing hypha. ER and Spitzenkörper dynamics in a growing hypha expressing ER-GFP and stained with FM4-64. Video 2 shows the dynamics of the apical ER compartments compared to the localization of the Spitzenkörper stained with FM4-64. Panels (from top to bottom): (i) ER-GFP, (ii) FM464, (iii) ER-GFP/FM4-64 merge, (iv) FM4-64/bright-field merge. Time scale is in min:sec. Video 3. ER dynamics in the apical hyphal region. Details of ER and Spitzenkörper dynamics in a growing hypha expressing ER-GFP and stained with FM4-64. Video 3 shows the dynamics of the apical ER compartments in the apical hyphal region compared to the localization of the FM4-64-stained Spitzenkörper. Panels (from top to bottom): (i) ER-GFP, (ii) FM4-64, (iii) ER-GFP/FM4-64 merge, (iv) FM4-64/bright-field merge. Time scale is in min:sec. Video legends Video 1. ER dynamics in a P. anserina growing hypha. Live-cell imaging of a growing hypha expressing ER-GFP (top). Middle: bright field; bottom: merge. In the second part of the video arrows point to medial sub-compartments moving basipetally, open arrows to low motility sub-compartments and green arrows to sub-compartments dividing. Frames were taken every 2.2 sec and are displayed at 12 fps. Time scale is in min:sec. Video 2. ER and Spitzenkörper dynamics in a growing hypha. ER and Spitzenkörper dynamics in a growing hypha expressing ER-GFP and stained with FM4-64. Panels (from top to bottom): (i) ER-GFP, (ii) FM4-64, (iii) ER-GFP/FM4-64 merge, (iv) FM4-64/brightfield merge. In the second part of the video arrows and arrowheads point to medial sub-
10
compartments moving basipetally with different velocities, respectively. S, Spitzenkörper. Images were acquired every 2.7 sec and are displayed at 10 fps. Time scale is in min:sec. Video 3. ER dynamics in the apical hyphal region. Details of ER and Spitzenkörper dynamics in a growing hypha expressing ER-GFP and stained with FM4-64. Panels (from top to bottom): (i) ER-GFP, (ii) FM4-64, (iii) ER-GFP/FM4-64 merge, (iv) FM4-64/brightfield merge. In the second part of the video arrows indicate medial sub-compartments moving basipetally and open arrows cortical sub-compartments. Arrowheads point to subcompartments emerging from the subapical cortical hyphal region, and yellow arrows to sub-compartments converging after emerging. S, Spitzenkörper. Images were taken every 2.4 sec and are displayed at 11 fps. Time scale is in min:sec.
11
Video Article: Research An endoplasmic reticulum domain is associated with the polarized growing cells of Podospora anserina hyphae Antonio de Jesús López-Fuentes, Aldo Meizoso-Huesca , Leonardo Peraza-Reyes* Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, Ciudad de México 04510, Mexico
Highlights
The P. anserina peripheral ER possesses an apical domain associated with the polarized growing apical hyphal cells.
The apical ER domain consists of pleomorphic ER sub-compartments confined to the first 15μm extending behind the apex.
The apical ER compartments are highly dynamic and undergo fusion and division events.
The apical ER compartments display acropetal and basipetal displacements, which are associated with their location across the hypha.
12
S1087-1845(19)30303-2 https://doi.org/10.1016/j.fgb.2020.103338 YFGBI 103338
To appear in:
Fungal Genetics and Biology
Received Date: Revised Date: Accepted Date:
7 October 2019 28 November 2019 20 January 2020
Please cite this article as: de Jesús López-Fuentes, A., Meizoso-Huesca, A., Peraza-Reyes, L., An endoplasmic reticulum domain is associated with the polarized growing cells of Podospora anserina hyphae, Fungal Genetics and Biology (2020), doi: https://doi.org/10.1016/j.fgb.2020.103338
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Inc.
Video Article: Research An endoplasmic reticulum domain is associated with the polarized growing cells of Podospora anserina hyphae
Antonio de Jesús López-Fuentes, Aldo Meizoso-Huesca#, Leonardo Peraza-Reyes* Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, Ciudad de México 04510, Mexico * Corresponding author: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México Ciudad de México 04510, Mexico. Tel. (52) (55) 5622 5628 E-mail address: [email protected] #Present
address:
School of Biomedical Sciences, University of Queensland, Brisbane, QLD, 4072, Australia.
Keywords: Endoplasmic reticulum / dynamics / hyphal growth Word count: 1,265 Character count: 8,537
1
Abstract The endoplasmic reticulum (ER) is composed of distinct structural domains that perform diverse essential functions, including the synthesis of membrane lipids and proteins of the cell endomembrane system. The polarized growth of fungal hyphal cells depends on a polarized secretory system, which delivers vesicles to the hyphal apex for localized cell expansion, and that involves a polarized distribution of the secretory compartments, including the ER. Here we show that, additionally, the ER of the ascomycete Podospora anserina possesses a peripheral ER domain consisting of highly dynamic pleomorphic ER sub-compartments, which are specifically associated with the polarized growing apical hyphal cells.
2
Introduction The endoplasmic reticulum (ER) is composed of different structural domains that perform diverse essential functions, including the synthesis, processing and transport of membrane lipids and proteins of the cell endomembrane system (Schwarz and Blower, 2016; Westrate et al., 2015). In addition, the ER participates in organelle biogenesis (Joshi et al., 2017) and interacts with most other organelles (Wu et al., 2018), which contributes to the regulation of intracellular dynamics. The ER is composed of two major domains, the nuclear envelope and the peripheral ER. The peripheral ER consists of a reticular network of tubules and cisternal sheets that extends throughout the cell and that comprises diverse structural subdomains, whose arrangement is adapted to their specialized functions (Westrate et al., 2015). The polarized growth mode of fungal hyphae is determined by a secretory system, which drives vesicles to the hyphal tip to deliver membrane lipids and proteins for localized cell surface extension. This process relies on vesicles that transport enzymes involved in cell wall assembly, and is coupled to enzyme endocytic recycling (Hernandez-Gonzalez et al., 2018; Riquelme, 2013; Riquelme et al., 2018; Shaw et al., 2011). Polarized vesicle traffic orchestration involves the Spitzenkörper, a complex structure located in the apex of growing hyphae that is proposed to act as a vesicle supply center that concentrates vesicles for their subsequent delivery to the foremost apical membrane (Riquelme et al., 2018; Riquelme and Sanchez-Leon, 2014). Additionally, the secretory compartments –namely the Golgi cisternae (Pantazopoulou and Penalva, 2009) and the ER (Kimura et al., 2010; Markina-Inarrairaegui et al., 2013; Maruyama et al., 2006; Wedlich-Söldner et al., 2002)– display a polarized distribution along hyphae. Here we show that, in addition, a dynamic peripheral ER domain is specifically associated with the growing apical hyphal cells of the ascomycete Podospora anserina.
3
Results and Discussion We analyzed the P. anserina ER by studying the localization of an ectopically-expressed ER-targeted GFP (ER-GFP) containing the ER targeting and retention signals of the putative P. anserina ER chaperone BiP/Kar2, a protein that localizes to the ER in filamentous ascomycetes (Maruyama et al., 2006). Consistent with ER localization, ERGFP localized to a large network of interconnected strands extending throughout hyphae, similar to the ER arrangement of other filamentous fungi (Kimura et al., 2010; MarkinaInarrairaegui et al., 2013; Maruyama et al., 2006; Rico-Ramirez et al., 2018; WedlichSöldner et al., 2002). Also, like other mycelial fungi, this network exhibited a polarized distribution with increasing abundance towards the hyphal tip. In addition, we found that the ER of the apical cells of growing leading hyphae contained a number of pleomorphic patches, which were interconnected with the peripheral ER strands (Video 1). These apical ER sub-compartments typically ranged between 1-4 μm in length (1-1.5 μm in width), but longer patches stretching up to 9 μm were observed. These compartments were confined to the first 15μm extending behind the tip (i.e, in average, 12.9 2.09μm behind the tip, n=30), but they were not observed to reach the apex (Video 1) or the Spitzenkörper (Videos 2 and 3). Of note, these apical compartments were also detected with a GFP-tagged version of the endogenous ER membrane protein Sec61 (not shown).
Video 1
4
We found that the apical ER compartments were highly dynamic and frequently converged to produce larger patches (e.g., Video 3, yellow arrows), or were separated into smaller patches (Video 1, green arrows). Moreover, we also discovered that the apical ER patches displayed dynamic displacements, which were associated with their cell location and with their fusion and division events. In general, we observed two main classes of apical ER patches based on their distribution and behavior. The first one consisted of ER patches closely apposed to the cell cortex (e.g., Video 3, open arrows). These patches were situated at a relatively constant distance from the hyphal tip and some exhibited displacements, mostly towards the hyphal apex (acropetal). The second group was mainly present in the hypha middle plane and typically exhibited basipetal displacements (backward from the apex). A number of these medial patches were relatively static relative to the subapical cell surface (Video 1, open arrows) and thus separated away from the extending cell tip with a velocity (av. 70 17 nm/s, n=30) comparable to the hyphal growth rate (i.e, av. 73 6nm/s, n=30). In addition, some of these compartments were engaged in faster, mostly basipetal movements (Videos 1-3). Overall, these displacements occurred with an average velocity of 205 87nm/s (range 107–428 nm/s, n=53), but two main categories of displacements with different velocities were observed, with average velocities of 293 53nm/s (n=23, e.g., Video 2, arrowhead) and 138 23nm/s (n=30, e.g., Videos 13, arrows), respectively.
Video 2
5
The medial apical ER compartments moving basipetally emerged from the apical hyphal region behind the Spitzenkörper, frequently from the cortical cell region (e.g., Video 3, arrowheads). Some medial compartments seemed to be derived from cortical tipward-moving patches, suggesting an acropetal-basipetal flow of cortical to medial compartments (e.g., Video 2). In addition, patches derived from opposite sites of the hypha frequently merged following their emergence (e.g., Video 3, yellow arrows).
Video 3 The morphology of the apical ER patches suggests that they represent ER cisternae, however, higher resolution structural analyses are required to ascertain their precise structure. Likewise, the specific functions of these compartments need to be established. However, their specific apical localization suggests that they are associated with the process of polarized hyphal growth. Equivalent apical ER domains could be present in other mycelial fungi. For example, Aspergillus nidulans possesses a finger-like ER protrusion capping the ER apical region (Markina-Inarrairaegui et al., 2013), although this domain seems to protrude from the ER network towards the apex rather than being immersed in the peripheral ER. Although further comparative analyses of different fungi and of distinct hyphal types are required, these observations suggest different configurations of the apical ER adapted to specific hyphal growth habits.
6
Methods P. anserina methods, sequences. P. anserina methods and media composition can be consulted at http://podospora.i2bc.paris-saclay.fr. BiP (Pa_4_6420) gene sequence was obtained from the P. anserina genome sequence (Espagne et al., 2008) and is available under GenBank accession number CDP28607.1. BiP signal peptide sequence was predicted on SignalP 4.1 (Nielsen, 2017). Construction and analysis of ER-targeted GFP. Fusion PCR was used to generate a construct coding for GFP flanked by the ER targeting and retention signals of the potential P. anserina ER chaperone BiP (ER-GFP), preceded by the A. nidulans gpdA promoter. This construct consisted of the fusion of: (i) 892bp of gpdA promoter, (ii) a 105bp sequence encoding BiP signal peptide, (iii) EGFP gene, and (iv) 21bp encompassing BiP C-terminal HDEL-coding sequence and stop codon. This construct was cloned into plasmid pPable (Coppin and Debuchy, 2000) to yield pAM01, which was verified by sequencing and used to transform P. anserina protoplasts. Three independent transformants were selected and purified after sexual crosses with the wild type. Confocal microscopy analysis of ER-GFP showed that the ER arrangement and dynamics in these three strains were alike. For the quantitative analysis of ER dynamics, 30 leading hyphae of one of these strains issued from 3 biological replicates (10 hyphae/replicate) were analyzed. Microscopy. Imaging was performed on a Zeiss LSM-800 inverted laser scanning confocal microscope equipped with a temperature chamber, using a Plan-Apochromat 63x/1.4 oil immersion objective and a 488 nm laser line. Imaging was done on whole individual P. anserina colonies growing at 27C, as previously described (Suaste-Olmos et al., 2018). Micrographs were processed using ZEN 2012 software (Carl Zeiss, Jena, Germany) and ImageJ (NIH, Bethesda, USA). The Spitzenkörper was stained with FM4-64 (N-(3triethylammoniumpropyl)-4-(6-(4-(diethylamino)
phenyl)
hexatrienyl)
pyridinium
dibromide, 7 μM) (Molecular Probes, Eugene, OR).
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Acknowledgements We are grateful to Fernando Suaste Olmos for technical assistance throughout this research. We thank Sara Schroder for her careful review of the manuscript, and the IFC Imaging Facility for assistance on microscopy. This work was supported by CONACYT [CONACYT (FONCICYT)-DFG 277869, and by PAPIIT-DGAPA, UNAM [IA203317, IV200519]. AJLF was supported by a scholarship from CONACYT. References Coppin, E., Debuchy, R., 2000. Co-expression of the mating-type genes involved in internuclear recognition is lethal in Podospora anserina. Genetics. 155, 65769. Espagne, E., et al., 2008. The genome sequence of the model ascomycete fungus Podospora anserina. Genome Biol. 9, R77. Hernandez-Gonzalez, M., et al., 2018. Endocytic recycling via the TGN underlies the polarized hyphal mode of life. PLoS Genet. 14, e1007291. Joshi, A. S., et al., 2017. Organelle biogenesis in the endoplasmic reticulum. Nat Cell Biol. 19, 876-882. Kimura, S., et al., 2010. In vivo imaging of endoplasmic reticulum and distribution of mutant alpha-amylase in Aspergillus oryzae. Fungal Genet Biol. 47, 1044-54. Markina-Inarrairaegui, A., et al., 2013. The Aspergillus nidulans peripheral ER: disorganization by ER stress and persistence during mitosis. PLoS One. 8, e67154. Maruyama, J., et al., 2006. Differential distribution of the endoplasmic reticulum network as visualized by the BipA-EGFP fusion protein in hyphal compartments across the septum of the filamentous fungus, Aspergillus oryzae. Fungal Genet Biol. 43, 642-54. Nielsen, H., 2017. Predicting Secretory Proteins with SignalP. Protein Function Prediction: Methods and Protocols. 1611, 59-73. Pantazopoulou, A., Peñalva, M. A., 2009. Organization and dynamics of the Aspergillus nidulans Golgi during apical extension and mitosis. Mol Biol Cell. 20, 4335-47. 8
Rico-Ramirez, A. M., et al., 2018. Imaging the secretory compartments involved in the intracellular traffic of CHS-4, a class IV chitin synthase, in Neurospora crassa. Fungal Genet Biol. 117, 30-42. Riquelme, M., 2013. Tip growth in filamentous fungi: a road trip to the apex. Annu Rev Microbiol. 67, 587-609. Riquelme, M., et al., 2018. Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures. Microbiol Mol Biol Rev. 82. Riquelme, M., Sanchez-Leon, E., 2014. The Spitzenkörper: a choreographer of fungal growth and morphogenesis. Curr Opin Microbiol. 20, 27-33. Schwarz, D. S., Blower, M. D., 2016. The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci. 73, 79-94. Shaw, B. D., et al., 2011. A role for endocytic recycling in hyphal growth. Fungal Biol. 115, 541-6. Suaste-Olmos, F., et al., 2018. Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery. Biochim Biophys Acta. 1865, 572-586. Wedlich-Söldner, R., et al., 2002. Dynein supports motility of endoplasmic reticulum in the fungus Ustilago maydis. Mol Biol Cell. 13, 965-77. Westrate, L. M., et al., 2015. Form follows function: the importance of endoplasmic reticulum shape. Annu Rev Biochem. 84, 791-811. Wu, H., et al., 2018. Here, there, and everywhere: The importance of ER membrane contact sites. Science. 361.
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Video Still Image legends Video 1. ER dynamics in a P. anserina growing hypha. Live-cell imaging of a growing hypha expressing ER-GFP (top). Video 1 shows the presence of dynamic pleomorphic ER sub-compartments in the apical region of a growing hypha. Middle: bright field; bottom: merge. Time scale is in min:sec. Video 2. ER and Spitzenkörper dynamics in a growing hypha. ER and Spitzenkörper dynamics in a growing hypha expressing ER-GFP and stained with FM4-64. Video 2 shows the dynamics of the apical ER compartments compared to the localization of the Spitzenkörper stained with FM4-64. Panels (from top to bottom): (i) ER-GFP, (ii) FM464, (iii) ER-GFP/FM4-64 merge, (iv) FM4-64/bright-field merge. Time scale is in min:sec. Video 3. ER dynamics in the apical hyphal region. Details of ER and Spitzenkörper dynamics in a growing hypha expressing ER-GFP and stained with FM4-64. Video 3 shows the dynamics of the apical ER compartments in the apical hyphal region compared to the localization of the FM4-64-stained Spitzenkörper. Panels (from top to bottom): (i) ER-GFP, (ii) FM4-64, (iii) ER-GFP/FM4-64 merge, (iv) FM4-64/bright-field merge. Time scale is in min:sec. Video legends Video 1. ER dynamics in a P. anserina growing hypha. Live-cell imaging of a growing hypha expressing ER-GFP (top). Middle: bright field; bottom: merge. In the second part of the video arrows point to medial sub-compartments moving basipetally, open arrows to low motility sub-compartments and green arrows to sub-compartments dividing. Frames were taken every 2.2 sec and are displayed at 12 fps. Time scale is in min:sec. Video 2. ER and Spitzenkörper dynamics in a growing hypha. ER and Spitzenkörper dynamics in a growing hypha expressing ER-GFP and stained with FM4-64. Panels (from top to bottom): (i) ER-GFP, (ii) FM4-64, (iii) ER-GFP/FM4-64 merge, (iv) FM4-64/brightfield merge. In the second part of the video arrows and arrowheads point to medial sub-
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compartments moving basipetally with different velocities, respectively. S, Spitzenkörper. Images were acquired every 2.7 sec and are displayed at 10 fps. Time scale is in min:sec. Video 3. ER dynamics in the apical hyphal region. Details of ER and Spitzenkörper dynamics in a growing hypha expressing ER-GFP and stained with FM4-64. Panels (from top to bottom): (i) ER-GFP, (ii) FM4-64, (iii) ER-GFP/FM4-64 merge, (iv) FM4-64/brightfield merge. In the second part of the video arrows indicate medial sub-compartments moving basipetally and open arrows cortical sub-compartments. Arrowheads point to subcompartments emerging from the subapical cortical hyphal region, and yellow arrows to sub-compartments converging after emerging. S, Spitzenkörper. Images were taken every 2.4 sec and are displayed at 11 fps. Time scale is in min:sec.
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Video Article: Research An endoplasmic reticulum domain is associated with the polarized growing cells of Podospora anserina hyphae Antonio de Jesús López-Fuentes, Aldo Meizoso-Huesca , Leonardo Peraza-Reyes* Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, Ciudad de México 04510, Mexico
Highlights
The P. anserina peripheral ER possesses an apical domain associated with the polarized growing apical hyphal cells.
The apical ER domain consists of pleomorphic ER sub-compartments confined to the first 15μm extending behind the apex.
The apical ER compartments are highly dynamic and undergo fusion and division events.
The apical ER compartments display acropetal and basipetal displacements, which are associated with their location across the hypha.
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