A bird’s-eye view of the endoplasmic reticulum in filamentous fungi

SUMMARY

The endoplasmic reticulum (ER) is one of the most extensive organelles in eukaryotic cells. It performs crucial roles in protein and lipid synthesis and Ca²⁺ homeostasis. Most information on ER types, functions, organization, and domains comes from studies in uninucleate animal, plant, and yeast cells. In contrast, there is limited information on the multinucleate cells of filamentous fungi, i.e., hyphae. We provide an analytical review of existing literature to categorize different types of ER described in filamentous fungi while emphasizing the research techniques and markers used. Additionally, we identify the knowledge gaps that need to be resolved better to understand the structure-function correlation of ER in filamentous fungi. Finally, advanced technologies that can provide breakthroughs in understanding the ER in filamentous fungi are discussed.

INTRODUCTION

The endoplasmic reticulum (ER) is a conserved organelle consisting of an interconnected membranous network that, in general, forms a series of flattened sheets with ribosomes associated with the cytosolic surface of the membrane, i.e., rough ER (rER), and tube-like structures, smooth ER (sER), without associated ribosomes. The combined use of fluorescence and electron microscopy techniques has identified the ER as the most extensive and highly complex eukaryotic organelle (1, 2). The first account of a filamentous subcellular structure corresponding to the ER, coined the ergastoplasm at that time, appears in the light microscopy observations of Charles Garnier in exocrine cells of salivary glands and pancreas (3). Later, Keith et al. observed a lace-like reticulum by transmission electron microscopy (TEM) in tissue culture cells (4). It was not until a few years later that he coined the term “endoplasmic reticulum” (5). Following those studies, George E. Palade made outstanding contributions to characterize the general volumetric disposition of the ER in diverse mammalian cell types and to recognize the role of the rER in the synthesis of secretory proteins (6). In filamentous fungi, the first evidence regarding ER organization and structure came from the elegant and meticulous TEM works of Girbardt (Fig. 1A) (7) and Grove and Bracker (8). Using traditional fixation methods, the ER was identified as tubular/pleomorphic structures of sER and as sheet-like cisternae of rER. Thereafter, cryofixation methods significantly improved the preservation of endomembranes and other cytoplasmic components in fungal hyphae and germ tubes (Fig. 1B) (9–13). The subsequent development of tools for fluorescence microscopy, including markers and improved hardware and software, has significantly contributed to understanding the ER’s structure, function, and behavior in filamentous fungi (14–19). Recent bioimaging data of mammalian cells have revealed new levels of structural complexity of the ER, e.g., polygonal networks (20), tubular matrices (2), and nanoholes within ER cisternae (21).

Fig 1.

The ER in filamentous fungi. (A) A colored 3D hypha model of Polystictus versicolor based on TEM serial section reconstruction (reproduced from reference 7 with permission from Springer Nature) and (B) a 2D TEM image of Athelia rolfsii corresponding to hyphal region II (reproduced from reference 12 with permission from Springer Nature). (C) Number of ER publications in filamentous fungi over the past 20 years. (D) Stacked bars represent the frequency of rER/sER reported in 2D ultrastructure studies (n = 15) from 1963 to 2018. Scale bars in A and B: 1 µm.

Although the number of publications on the ER in filamentous fungi has increased over the past few decades (Fig. 1C), there remains a paucity of information concerning the diversity of ER structure in fungal hyphae. Based on cytoplasmic organization, growing hyphae are divided into three distinct regions (22). Apical region I (2–5 µm) is referred to as the hyphal apex, and it contains the Spitzenkörper (SPK) and mitochondria (23). Subapical region II extends 10–20 µm behind region I and includes a significant number of mitochondria (23), and subapical region III, extending 30–40 µm behind region II, is occupied by nuclei among other organelles (15). Here, we carry out a systematic review of ER structure and function in hyphae of various fungi, including members of the Ascomycota, Basidiomycota, Mucoromycota, and Chitridiomycota to identify unique features and shared patterns of the ER across the fungal Kingdom (7, 11, 12, 15, 23–33).

THE MANY SHAPES OF THE FUNGAL ER

Rough and smooth ER

Commonly, the rER is involved in protein synthesis, whereas the sER has a role in lipid synthesis and Ca²⁺ homeostasis (34, 35). Cell size and morphology affect internal cellular architecture (36). Accordingly, hyphae from filamentous fungi would require a particular arrangement of ER membranes distinct from yeast cells.

An analysis of 2D TEM images of cryofixed and freeze-substituted fungal cells indicates that rER is more abundant than sER (11, 12, 15, 23–25, 27, 28, 30–33) (Fig. 1D). In filamentous fungi, the rER is observed as elongated, flattened sheets found in high abundance near nuclei (Fig. 2A), while the sER morphology is characteristically tubular and pleomorphic (Fig. 2B). Noticeably, rER sheets can be observed within hyphae and germ tubes of the ascomycete Neurospora crassa and the basidiomycetes Athelia rolfsii, formerly Sclerotium rolfsii, Uromyces phaseoli var. typical, and Pisolithus tinctorius (11, 12, 15, 37). Smooth ER is often more difficult to identify in fungal hyphae. In A. rolfsii, smooth membranous elements identified as sER were described in apical and subapical regions (Fig. 2B) (12).

Fig 2.

Representative TEM micrographs of many ER shapes in 2D. (A) Longitudinal median section of Neurospora crassa (photograph by R. W. Roberson). Note the rER as elongated sheets. Ribosomes associated with cytoplasmic surfaces are indicated with arrows. Membranes of ER have low electron contrast. (B) Near median section of Athelia rolfsii (photograph by R. W. Roberson). The sER is observed as pleomorphic tubules (arrows). (C and D) Whorls/lamellar-like structures are other arrangements referred to as organized smooth ER (OSER) when there is an induction of mycotoxin biosynthesis in vitro and in planta of Fusarium graminearum. (Reproduced from reference 24.) Scale bars in A: 100 nm; B: 250 nm; and C and D: 500 nm.

In yeast and mammalian cells, the sER can be found in other diverse arrangements referred to as OSER (38). While it remains unclear as to the roles of some OSER arrangements, it is suggested that they may serve to concentrate sER-associated proteins and as lipid reservoirs (38, 39). In hyphae of Fusarium graminearum (Ascomycota), OSER in the form of whorls (Fig. 2C) and lamellar-like structures (Fig. 2D) have been identified during induction of mycotoxin biosynthesis (24).

ER sheets

In filamentous fungi, ER sheets (also described as ER cisternae) consist of flattened sacs and constitute the most abundant type of ER observed in published ultrastructural studies (Fig. 3A through C). ER sheets are generally restricted to rER (12). The luminal width within the rER sheets ranges between 20 and 40 nm (Fig. 3B) (12, 15, 24, 27, 28, 30, 33). In comparison, values in yeast are ~30 nm (40, 41) and those in mammalian cells are ~50 nm (42, 43). ER sheets are typically viewed as discrete, solitary membranous segments distributed throughout the cytoplasm in TEM thin sections of fungal cells. However, light microscopy evidence suggests they are likely a single interconnected unit (24, 44). High-resolution 3D imaging by electron tomography and advanced fluorescent microscopy are needed to confirm whether interconnections exist between the ER sheets. In neuronal cell bodies and acinar cells, the ER near the nuclear envelope (NE) forms stacked sheets connected by helicoidal membrane motifs (45, 46). A similar distribution has been documented with 3D reconstructions in cryofixed cells of the budding yeast Saccharomyces cerevisiae (Ascomycota), where ER sheets are connected with the NE through tubules that branch out from the NE (47). Using TEM, stacked ER sheets have been observed in conidia of Magnaporthe oryzae (previously Magnaporthe grisea; Ascomycota) and hyphae of F. graminearum without evidence of connections (24, 25). Stacked ER sheets have also been observed connected by helicoidal motifs in germ tubes of Candida albicans (Ascomycota) in 3D reconstructions by focused ion beam-scaninng EM (FIB-SEM) (48). In Sebacina vermifera and Trichosporon sporotrichoides (Basidiomycota), SEM analysis showed ER sheets in the form of plates connected with a tubular membranous structure (49, 50) (Fig. 3C). Some fungal species display fenestrated ER sheets (11, 26), a type of ER sheets described also in mammalian cells (42). It remains unclear what roles these ER sheet variants play.

Fig 3.

Endoplasmic reticulum sheets in filamentous fungi. (A) Model of an ER sheet showing a general 3D view, where multipass transmembrane proteins stabilize the membrane curvature at the edges of the sheets devoid of ribosomes. (B) Mean values of the luminal width (n = 5) observed in cryofixed hyphae of N. crassa (15), Aspergillus nidulans (28), Fusarium acuminatum (27), Athelia rolfsii (12), Pisolithus tinctorius (37), and Monoblepharis macrandra (33). Error bars represent standard deviation. (C) rER sheets in Trichosporon sporotrichoides (reproduced from reference 50 with permission from John Wiley and Sons), showing two continuous apposed elongated membranes with high curvature edges (white arrow). SP, septa. Scale bar in C: 250 nm.

The mechanisms regulating ER sheet formation can be quite complex. In mammalian cells, proteins with coiled-coil domains, such as Climp63, generate and stabilize the ER sheets (51). The multimerization of Climp63 maintains the intraluminal space of ER sheets. However, no orthologs of Climp63 appear to be present in yeast or filamentous fungi. No other proteins that could presumably participate in the biogenesis of the ER sheets in filamentous fungi have been yet identified.

ER tubules and polygonal networks

The ER tubules consist of elongated cylinders with an average diameter ranging from 30 to 50 nm in mammalian cells (2) or even narrower ER tubules in neuronal axons (~20 nm) (52) (Fig. 4A). In S. cerevisiae, ER tubule diameters range from 20 to 50 nm (47). From published transmission electron micrographs of the corn smut fungus Ustilago maydis (Basidiomycota) hyphae (31), we have estimated that ER tubule diameters range from 15 to 20 nm. Ultrastructural 2D data of fungal hyphae have revealed ER in unduloid tubular arrangements (Fig. 4B) (11, 12, 24, 31, 48, 53). In germ tubes of Uromyces phaseoli var. typica (Basidiomycota), the ER can be found as irregularly shaped single tubules (11). In intracellular pathogenic hyphae of U. maydis, small arrays of tubular sER were revealed to be associated with rER (Fig. 4C) (31).

Fig 4.

ER tubules in fungal cells. (A) Model of an ER tubule showing a general 3D view of the ER structure, where multipass transmembrane proteins from the reticulon and YOP families induce and stabilize the high membrane curvature. (B and C) TEM images of Ustilago maydis (courtesy of K. Snetselaar). In a 2D view, the ER tubules may look irregular, and in some circumstances, arrays of ER tubules appear during the early stage of intracellular-host sporulation. 3D reconstruction of a whole cell of (D) Saccharomyces cerevisiae (courtesy of K. Czymmek) and germ tube of (E) Candida albicans (courtesy of A. Weiner and R. Arkowitz) by FIB-SEM. Scale bars in B and C: 200 nm.

The best characterization of ER tubules in fungi has been achieved with 3D TEM reconstruction (47, 54). In S. cerevisiae, well-preserved ER tubules appear as a lace-like reticulum without bound ribosomes (Fig. 4D) (47). In germ tubes of C. albicans, the ER tubules are in continuity with ER sheets (Fig. 4E) (48). In hyphae of Ashbya gossypii (Ascomycota) examined by electron tomography, all organelles were identified clearly, with the exception of the ER, which was only partially identified close to nuclei, in the middle sections, and within subcortical regions (55).

In mammalian cells, ER tubules can be arranged in a higher-order structure as a polygonal interconnected network by three-way junctions (56). In S. cerevisiae and U. phaseoli var. typica, polygonal ER networks have also been recognized exclusively near the plasma membrane (11, 57). Using fluorescence microscopy and employing various ER markers, there is evidence for a polygonal ER network in U. maydis yeast-like cells (14) and Zymoseptoria tritici (Basidiomycota) hyphal tips (58). However, the polygonal ER network is not visible in the hyphal stage of U. maydis (14), A. oryzae (17, 59), A. nidulans (19), Podospora anserina (18, 60), and N. crassa (15).

Two evolutionary conserved families of proteins, the reticulons (RTN) and Yop1/DP1 [Receptor Expression Enhancing Proteins (REEPs)], are structural proteins that shape and stabilize the ER tubules (57). The reticulons and Yop1/DP1 proteins contain pairs of closely spaced transmembrane segments (61, 62). In S. cerevisiae, the absence of Rtn1 and Yop1 disrupts the stability of the polygonal ER network and results in the conversion of tubules to sheets. In contrast, overexpression of Rtn1 leads to long tubule formation (57). Studies in P. anserina support the importance of RTN1 and YOP1 to maintain the ER organization (18). In this fungus, deletion of RTN1 and YOPs causes ER rearrangements (18).

Atlastins (ATLs) in metazoans and their yeast homolog Sey1 are membrane-anchored dynamin-like GTPases mediating homotypic ER membrane fusion and forming new three-way junctions within polygonal ER networks (56, 63). Sey1 has a cytoplasmic GTPase domain followed by a helical bundle, intramembrane hairpin loops, a transmembrane-embedded region, and an amphipathic helix (62, 64). The lack of Sey1 and Rtn1 or Yop1 results in ER tubule defects in S. cerevisiae (63). In F. graminearum, Sey1 plays a role in vegetative growth, asexual development, lipid droplet formation, mycotoxin production, and pathogenicity (65).

HYPHAL DISTRIBUTION OF THE ER

Few accounts of 2D or 3D reconstructions cover hyphal regions I to III (7, 12, 15, 48, 55). Ultrastructural studies using cryopreparation protocols have shown a greater abundance of rER sheets within hyphal subapical regions II and III (11, 12, 15, 25, 28, 30–33) (Fig. 5A). Fewer cryomethod studies have revealed sER tubules in hyphal apical region I (11, 12) (Fig. 5B through D). Fluorescence dyes and selective tagging of both luminal and membrane-bound ER proteins using fluorescent markers have unveiled distinct ER distribution patterns along mature hyphae in various fungal species. These species encompass Claviceps purpurea (Ascomycota), U. maydis, A. nidulans, A. oryzae, Z. tritici, P. anserina, Aspergillus niger, Botrytis cinerea (Ascomycota), and N. crassa, as summarized in Table S3 (14, 15, 17, 18, 58, 66, 67). Within these fungal species, the majority of ER markers (77.14%) have been identified in subapical regions surrounding the nuclei at the NE and at the peripheral ER (pER) membranes (Fig. S1). There are very few markers exclusively located at the NE (2.85%) or at the pER (5.71%). The expression intensity of each marker may exhibit variations across different domains, offering insights into the relative abundance of each protein within a specific ER structure. Additionally, there is evidence of ER markers being localized at the hyphal apices, though this aspect has received comparatively less attention in the current body of research.

Fig 5.

Subcellular distribution of the ER. TEM micrographs of cryofixed and freeze-substituted hyphae of (A–C) Athelia rolfsii (12) (panels A and B reproduced from reference 12 with permission from Springer Nature; photograph in panel C by R. W. Roberson), (D) Uromyces phaseoli var. typical (reproduced from reference 11 with permission from Elsevier), (E) Neurospora crassa (reproduced from reference 15 with permission from Elsevier), and conidial cells of (F) Magnaporthe grisea (reproduced from reference 25 with permission from Springer Nature), showing the different ER distribution patterns. (A) At low magnification, peripheral ER (white arrowheads) along the hypha can be observed. (B–D) The ER can be observed at the apical domain close to the SPK and the PM (cortical ER). (E and F) The peripheral ER is observed as dispersed strands in the cytoplasm (white arrowheads). Note the outer nuclear membrane decorated with ribosomes (white arrows, E.1, F.1). Occasionally, the ER is arranged as a (F) stacked-ER close to the NE in conidia. (G) Distribution of the ER in 2D ultrastructure studies (n = 15), reported from 1963 to 2018, at hyphal structures (germ tubes/mature hyphae) of six Ascomycota: Fusarium acuminatum (27), Trichoderma reesei (26), Athelia rolfsii (12), Aspergillus nidulans (25, 28), Fusarium graminearum (24), and Neurospora crassa (15, 23); five Basidiomycota: Rhizoctonia solani (29), Polystictus versicolor (68), Uromyces phaseoli var. typica (11), Ustilago maydis (31), and Pisolithus tinctorius (37), as well as Gigaspora (32) and Monoblepharis macrandra (33), members of the Glomeromycota and the Monoblepharomycota, respectively. (H) Distribution of the ER in 2D ultrastructure studies (n = 8), reported from 1966 to 2007, in spores of two Ascomycota: Magnaporthe grisea (25) and Botrytis cinerea (69); two Basidiomycota: Gymnosporangium juniper-virginianae (70) and Sporisorium sorghi (71); three Mucoromycota: Cunninghamella vesiculosa (72), Gilbertella persicaria (73), and Rhizopus sexualis (74); and the Glomeromycota Gigaspora margarita (75). Scale bars in A: 2.8 µm; B: 280 nm; E: 1 µm; and F: 500 nm.

Nuclear envelope

In both mammalian and yeast cells, the ER forms a single interconnected and highly dynamic system that extends from the NE outer membrane through the cytoplasm (76, 77). Different ultrastructural studies of filamentous fungi that used chemical and cryofixation of hyphal cells (n = 15) and spore cells (n = 8) revealed that the NE was clearly visible in 60% of the hyphae and 62.5% of the spores (Fig. 5G and H) (Tables S1 and S2) (11, 12, 15, 25, 28, 30–33). In the hyphae of N. crassa and conidia of M. grisea, the cytosolic surface of the NE appeared decorated with ribosomes (Fig. 5E.1 and F.1) (15, 25). In addition, sheets of rER close to the NE were observed in 25% and 50% of the hyphae and spores analyzed, respectively (Fig. 5G and H). Thus far, there is no evidence by TEM indicating a direct connection between these rER sheets and the NE (Fig. 5E.1).

Genetically encoded fluorescent tags have been used to image the NE. The use of fluorescent proteins containing a KDEL or HDEL ER retention signal has proven useful in illuminating the NE and some perinuclear structures in several fungi, including U. maydis, A. oryzae, and P. anserina (Fig. 6A through C). In addition, endogenous tagging of Sec63, a component of the protein-conducting channel in the ER membrane, in A. nidulans (Fig. 6D), and of the SERCA-type Ca²⁺ ATPase NCA-1 (Fig. 6E) and the cargo adaptors ERV-25 and ERV-3 in N. crassa (16, 44) have revealed these proteins at the NE. In A. fumigatus, the sterol regulatory element-binding protein SrbA required for fungal virulence and the lipid remodeling protein PerA have both been localized in the NE (78, 79). In A. oryzae, SNARE proteins AoUfe1, AoSec20, AoUse1p, and AoSec22 illuminated the NE connected with some ER membranes (80).

Fig 6.

Endoplasmic reticulum markers in hyphal cells. (A) Expression of eGFP fused to the signal peptide of calreticulin and containing an HDEL ER retention signal in U. maydis (reproduced from reference 14). Fluorescence is excluded from a region at the subapex (arrow), and a very bright fluorescent region is observed at the hyphal tip (arrowhead). Scale bars in A: left and center: 2 µm and right: 1 µm. (B) Expression of ER proteins in Aspergillus oryzae. (Left photo [BipA-DsRed2/H2B-EGFP] reproduced from reference 17 with permission of Elsevier; center [ClxA-EGFP] and right [AoSec13-EGFP] photos courtesy of J. Maruyama.) At the tip, AoSec13-GFP is localized as puncta (arrows). Scale bars in B: 2.5 µm, center: 4 µm and right: 4.5 µm. (C) RTN1 and ER-targeted GFP in Podospora anserina. (Left photo reproduced from reference 18; center and right photos courtesy of L. Peraza-Reyes.) Scale bars in C: left: 2.5 µm; center: 5 µm; and right: 2 µm (D) Sec63 in Aspergillus nidulans reproduced from reference 19). Scale bars in B: left and center: 1.2 µm and right: 1 µm. (E) NCA-1 (subapical) (courtesy of B. J. Bowman), SEC-63 (apex and subapex) (reproduced from reference 15 with permission from Elsevier), and ERP-3-mCherry (apex) (reproduced from reference 16 with permission from John Wiley and Sons). Arrows indicate regions of condensed fluorescence. Scale bars E: left and center: 5 µm and right: 3.5 µm.

Selective ER stains have also allowed to image the NE in growing fungal cells. In N. crassa, ER-tracker blue-white DPX stains intensively the NE (Fig. 7A). The use of this dye has revealed details of the NE and tubular structures that appear as part of the ER polygonal network in conidia of M. oryzae, in mature hyphae of F. graminearum (Fig. 7B) and A. oryzae, and in germ tubes of A. niger and B. cinerea (17, 24, 67, 80–83). In Sordaria macrospora (Ascomycota), using structured illumination microscopy, the complex striatin-interacting phosphatase and kinase (STRIPAK) subunit PRO45 was localized at the cytosolic face of the NE, which was co-stained with ER-tracker blue-white DPX (Fig. 7C) (84). This study reinforces the importance of performing super-resolution microscopy to resolve better spatial distributions of candidate ER proteins at the NE.

Fig 7.

The ER stained with ER-tracker blue-white DPX in filamentous fungi. (A) In Neurospora crassa, ER-tracker shows the pER (arrow) and the NE (arrowhead) (image by J. M. Martinez-Andrade). Scale bar: 8.5 µm. (B and C) Super-resolution with structured illumination microscopy (SIM) revealed the detailed structure of the pER composed by a tubular network (asterisks) in Fusarium graminearum (reproduced from reference 24) and the NE in Sordaria macrospora (courtesy of I. Teichert). Scale bars in B: 2 µm and C: 5 µm.

Peripheral ER

Most ER morphology and distribution depictions are based on data from unicellular mammalian cells (77, 85). The ER is illustrated in 3D as one extensive organelle composed of a stack of ER sheets and a tubular network connected to the NE (85). The peripheral ER (pER) refers to the ER sheets and tubules that extend into the cytoplasm from the NE. In mammalian cells, the pER comprises mainly of ER tubules (77, 85). In yeast uninucleate cells, most of the pER is observed as sheets close to the plasma membrane, and it is referred to as cortical ER (cER), which is connected with the NE by a small portion of ER tubules (Fig. 8) (47). In the multinucleated cells of filamentous fungi, the pER consists primarily of a collection of ER sheets that appear interconnected and move in unison with the nuclei (Fig. 8).

Fig 8.

Simplified model of the ER distribution in uninucleated and multinucleated fungal cells. The pER presumably derives from each nucleus in yeast and filamentous fungi. In yeast, abundant pER is found close to the cell surface (cortical ER, cER), and some of it is connected to the NE via ER tubules. In hyphae, some cER membranes are located along the cell’s longitudinal axis and are more predominant at the endocytic subapical region.

The amphiphilic styryl dye FM4-64 stains the plasma membrane, and it is subsequently internalized by endocytosis, resulting in the membrane staining of organelles within the endomembrane system, e.g., endosomes, vacuoles, ER, and secretory vesicles associated with the SPK (86). In N. crassa and Trichoderma reesei (Ascomycota), FM4-64 has elucidated pER membranes (87, 88). Similarly, the DiOC₆ dye can stain the pER in T. reesei (89). Other strategies that allowed labeling the pER include the use of fluorescent proteins containing a KDEL or HDEL ER retaining signal sequence (Fig. 6). Dikaryotic hyphae of U. maydis expressing eGFP containing the signal peptide of calreticulin and the ER retention signal HDEL showed fluorescence at pER tubules that extended from the NE toward the tip (Fig. 6A) (14). In addition, when tagged with fluorescent proteins, proteins such as the HSP70 molecular chaperone BiP of the ER lumen and the ER membrane protein Sec63 allowed imaging of the pER in some fungal species (15, 19, 34, 44, 88). In A. oryzae and P. anserina, BipA tagged with GFP and GFP containing ER-targeting and retention signals of BiP, respectively, revealed pER with arrangements of condensed ER patches-like especially in the hyphal subapex (Fig. 6B and C) (17, 60). In A. oryzae, some pER membranes localized near the plasma membrane and appeared tightly connected with the NE (17, 80). In A. nidulans, Sec63-GFP, in addition to localizing to the NE, was found at strands of pER, including strands of pER associated with the PM (cortical pER), and at interconnecting tubules between cortical pER and the NE (Fig. 6D) (19). During autophagy conditions induced by nitrogen starvation in A. nidulans hyphae, the general distribution of the pER network remained unchanged. However, it was observed that certain ER cup-shaped structures, reminiscent of omegasomes found in mammalian cells (90), began to appear in close proximity to early autophagosomes (91). Co-expression of Sec63-GFP with the actin-binding protein Abp tagged with mRFP suggested that the subapical endocytic collar acts as a barrier to constrict the pER at the tip (Fig. 6D) (19). In N. crassa, CSE-7, a cargo receptor required for the exit of chitin synthase 4 from the ER, and SEC-63 were associated with the pER (Fig. 6E) (15). In N. crassa, SEC-63 was not closely associated with the PM nor found at the NE, as seen in A. nidulans (19). Finally, the localization of Sec13 (a component of the COPII protein complex), the ER cargo adaptor ERP-3, or the ER shaping proteins YOP2 and RTN1 have added to our understanding of pER domains at the apical regions in A. oryzae, N. crassa, and P. anserina, respectively (Fig. 6B and E) (16–18).

During mitosis in filamentous fungi, such as in A. nidulans, apical hyphal growth and secretory processes are not interrupted (92). In contrast to mammalian cells, the NE remains intact throughout mitosis (19, 93). Furthermore, observations of Sec63-GFP in A. nidulans revealed that pER strands also remain intact during mitosis (19). In A. nidulans hyphae exposed to dithiothreitol-induced reductive stress, it was observed that cortical strands of pER tend to aggregate, while the integrity of the NE remains unaffected. This finding suggests that the cortical pER is a critical subdomain of the ER responsible for secretion processes (19).

Apical ER

While some early studies revealed the presence of sER at apical hyphal regions, little attention was given to its functional implication in tip growth. A detailed review of the published data allowed us to identify sER tubules at region I in classical ultrastructural works of chemically fixed hyphal cells (7, 94). Indeed, sER can be observed as “minute membranous tubules” at the core of the SPK in the hyphae of F. oxysporum and Ascodesmis nigricans (Ascomycota) (8). More recently, the growing number of published works using fluorescence microscopy has revealed the presence of ER markers at hyphal apical regions.

In dikaryotic hyphae of U. maydis, the ER forms a cap at the very tip, excluding the apical dome during growth (Fig. 6A) (14). A similar organization was observed in Z. tritici, where the ER was very close to the plasma membrane at the tip (58). In A. oryzae, AoBipA-GFP fluorescence revealed a differential distribution of ER along the hyphae, with a brighter signal in the apical region than in the basal regions (Fig. 6B) (17). The fluorescent ER-selective stain ER-tracker blue-white DPX has revealed pleomorphic ER structures at the hyphal tips in P. tinctorius (30), A. oryzae (80), B. cinerea (81), and A. niger (67). In P. anserina, some ER was localized at the subapex, coinciding with the region identified as the endocytic subapical collar in other fungi (Fig. 6C) (18). Interestingly, using RTN-1 as a marker for ER tubules, it was shown that fluorescence extended into the core of the SPK (Fig. 6C) (18). In M. oryzae and A. nidulans, the ER is generally excluded from the apical dome (Fig. 6D) (19, 95, 96). However, in an A. nidulans strain expressing GFP fused to a chitinase export signal and having an ER retention signal, fluorescence was observed in a reticulate network throughout the cytoplasm and at the NE, and it was brighter toward the growing apex (97). Neurospora crassa hyphae exposed to brefeldin A (BFA) conjugated to BODIPY 558/568 showed fluorescently labeled ER membranes near the tip, just below the SPK, where chitosomes containing chitin synthases accumulate (98). Localization of SEC-63, a subunit of the SEC-61 ER translocon, has shown that the ER extends to the subapical region, excluding the apical dome in N. crassa (Fig. 6E). Expression of the green fluorescent protein containing an N-terminal peptide signal and an ER-retention signal from the ATP-dependent BiP chaperone (a member of the heat shock Hsp70 protein family) of P. anserina revealed the ER at the tip in N. crassa (99). Other ER-resident proteins, such as the ER cargo adaptors ERP-1, ERV-25, or ERP-3 for cellulase CBH-1, and ERP-29 for cellulase CBH-2, have also been localized at the hyphal tips of N. crassa (Fig. 6E) (16). The role of the apical sER remains unclear.

SUBDOMAINS OF THE ER

ER exit sites

The ER exit sites (ERES), also known as the transitional ER, are found in ER regions, where the deformation of the ER membrane forms coat protein complex II (COPII) vesicles. In general, ERES seem to be restricted to the sER tubules and the high curved edges of the rER sheets with no associated ribosomes (100, 101). ERES are enriched near Golgi cisternae in mammalian cells (102). Similar discrete ERES can be observed in Pichia pastoris (103). However, in S. cerevisiae, ERES are distributed and scattered throughout the ER network (102, 103). In S. cerevisiae, triple knockout mutants of RTN1, RTN2, and YOP1 and the double mutant of SEY1 and YOP1 displayed altered localization of ERES clusters, which were found at the ER cisternae edges instead of ER tubules due to the loss of high curvature of the tubule membrane (104). Thus, it is thought that inducer and stabilizer proteins of the ER that confer high curvature are essential for ERES organization. In filamentous fungi, information on ERES is relatively scarce. Sec12 is a type II single transmembrane domain protein that catalyzes the exchange of GDP for GTP on Sar1, a small GTPase that regulates the formation of COPII vesicles at ERES (105). However, Sec12 is not considered a good marker for ERES in A. nidulans, where it localizes uniformly at the NE and a network of pER strands (105). In contrast, Sec13, the core component of the COPII coat, was localized to punctate structures along pER membranes in a polarized distribution and proved to be a good ERES marker in A. nidulans (105). In A. oryzae, a similar localization was observed for AoSec13 with a higher concentration of punctae at the apices and decreasing toward subapical regions (59). This polarized distribution parallels the distribution of abundant and dispersed Golgi cisternae in filamentous fungi and suggests an active role of the ER in secretory processes that support tip growth.

In yeast, a mechanism referred to as “hug-and-kiss” has been proposed for efficient vesicle targeting and cargo transport from ERES to cis-Golgi (106). In this process, the cis-Golgi approaches and contacts ERES, resulting in the collapse of the COPII coat and the capture of the cargo from the cis-Golgi. However, in A. nidulans, the SNARE-templating-and-membrane-tethering protein AnUso1, tagged with GFP, was observed as puncta corresponding to early (=cis) Golgi cisternae. Surprisingly, these puncta did not co-localize with AnSec13, tagged with mCherry, at ERES (107). This suggests that the “hug-and-kiss” mechanism reported in yeast may not be applicable to A. nidulans.

Membrane contact sites

ER membrane contact sites (MCSs) are regions of close apposition between the ER membrane and the membranes of other organelles, including the PM. The functions of MCSs are to facilitate intracellular communication, promote functional integration of compartmentalized processes, and regulate intracellular lipid flow and distribution, as well as Ca²⁺ homeostasis (108, 109). Both membranes are tethered but not fused and are separated by a space of 10–30 nm. Ribosomes are excluded from the surface of the contact sites. In filamentous fungi, MCSs can be observed between the ER and the PM, membranes of organelles, e.g., mitochondria (11, 70), and membranes of septal regions (29, 110–112).

ER-PM membrane contact sites

In yeast, 3D reconstructions revealed clearly how the ER is adjacent to the PM (47, 54). The ER-PM MCSs constitute the most prominent ER subdomain, and it is estimated that 20%–45% of the ER is associated with the PM (47), while in mammalian cells, only 0.23%–5% of the ER is closely associated with the PM (113). In filamentous fungi, the exact surface area and volume that ER-PM MCSs occupy in cells are unknown. Use of TEM has shown that the pER is prominent near the PM and septa and in a few cases within the apical dome (Fig. 5B through D), depending on the fungal species (11, 29, 111). However, most 2D TEM studies are limited to medial sections, revealing only a partial visualization of the MCSs.

The ER-PM MCSs are highly enriched in phosphatidylserine and phosphatidylinositol and are suggested to have a role in lipid supply to the PM (114). By using fluorescently labeled reporters of the ER at the optical level, it has been shown that the ER polygonal network may play a key role in Ca²⁺ sequestration in the cER of yeast (57). A similar distribution has been observed in U. maydis and Z. tritici, where the ER was imaged using eGFP containing the signal peptide of calreticulin from rabbits and the ER-retention signal HDEL (14, 58). Calreticulin and calnexin are ER chaperones that, in addition to monitoring protein folding and glycosylation, bind Ca²⁺, participating in ER Ca²⁺ homeostasis (115). Furthermore, calnexin has been found at the cell surface of fungi, where it triggers host immune responses (116). However, it is not understood how it reaches the cell surface. It is currently unclear whether ER-PM MCSs mediate Ca²⁺ regulation in filamentous fungi. In mammals, store-operated-Ca²⁺ entry is orchestrated by the ER membrane protein STIM1, and a depletion of intracellular Ca²⁺ increases ER-PM MCSs (113). Recent quantitative proteomic analysis of ER tubules of S. cerevisiae (117) revealed the presence of tethering proteins that physically interact with the PM, including tricalbin proteins Tcb1, Tcb2, and Tcb3, homologs of synaptotagmins conserved in mammalian cells, and Scs2, a homolog of the VAMP-associated protein VAP. Tricalbins are implicated in the Ca²⁺-dependent non-vesicular transfer of glycophospholipids (109). VAPs form multi-subunit bridges that connect ER to the PM as alternative mechanisms for membrane tethering. The elimination of Scs2 and its paralog Scs22 severely affects the ER-PM MCSs in yeast (118–120). Scs2 and Scs22 bind phosphoinositides (i.e., PI₄P) in their N-terminal domain (121). In addition, deletion of Ist2, which belongs to the anoctamin/TMEM16 protein family of Ca²⁺-gated channels, causes a significant reduction in ER-PM MCSs in yeast (122). In A. fumigatus, afTMEM16 has been characterized in vitro as a Ca²⁺-gated ion channel and a Ca²⁺-dependent scramblase (123). Labeling some ER-PM proteins with fluorescent proteins may give information about ER-PM MCSs in filamentous fungi.

ER-septum membrane contact sites

While most MCSs occur between the membrane of the ER and the membrane of another organelle or the PM, MCSs have also been observed at dolipore-septal pore caps (SPC) or parenthesomes in Basidiomycota) (Fig. 9A) and at simple septa in Ascomycota (Fig. 9B). For instance, an ultrastructural study of the septal pore apparatus of Serendipita “williamsii” (Basidiomycota) revealed ER membranes adjacent or attached to the SPC (112). Another ultrastructural study in Pisolithus tinctorius (Basidiomycota) revealed rER membranes in contact with the septa with ribosomes only on the cytosolic side (110). Additionally, scanning electron microscopy in Ceratobasidium cornigerum showed in 3-D tubules of ER surrounding the dome-shaped SPC (49). The association between the ER and the SPC was confirmed by zinc iodide-osmium tetroxide staining and freeze-fracture technique by SEM (124), the use of the selective fluorochromes ER-tracker blue-white DPX and BODIPY-BFA (125), and the analysis of isolated SPC fractions and SPC18 associated to the ER (125, 126). In addition, microfilaments, which may be part of the actin cytoskeleton, have also been observed in the septal region in Trametes versicolor (Basidiomycota) (127) and at the SPC in Rhizoctonia solani (Basidiomycota) (128). In N. crassa, the ER has been observed near the PM in the lateral walls of septa at the ultrastructural level (111), suggesting it might be involved in septum biogenesis.

Fig 9.

ER membrane contact sites in filamentous fungi. The models based on (A) Rhizoctonia solani (29) and (B) Neurospora crassa (111) are a general representation of the subcellular organization of septa at the ultrastructural level. The ER is flanking the lateral cell wall and septa, where there are MCSs with the PM.

CONCLUDING REMARKS

The ER is a complex, extensive, dynamic organelle that performs a wide array of functions in eukaryotic cells. In filamentous fungi, the ER consists of two distinct domains: the NE and the pER. The pER exhibits remarkable morphological diversity, with the rER forming extensive flattened sheets and the sER manifesting as tubular structures. These morphologies can undergo various arrangements, including stacked sheets, polygonal networks, or OSERs, depending on metabolic conditions. Unlike yeast cells, filamentous fungal cells are multinucleated, and this unique characteristic, along with their highly polarized growth mode, likely contributes to the diverse ER arrangements.

Despite its complexity, evidence of structural continuity within the ER is often lacking in 2D TEM studies, which primarily rely on thin sections (~70 nm) and are insufficient to demonstrate potential membrane continuity. Fluorescence microscopy studies in fungi typically provide a 2D perspective using ER markers, with a few including dynamic analysis, albeit at low resolution, and showing some degree of continuity between membranes (14, 19, 24, 44, 80). However, due to limitations in temporal and spatial resolution, it remains challenging to establish conclusive evidence of membrane continuity between the NE and the pER. This question of whether the ER membranes form an interconnected network spanning the fungal cytoplasm remains unanswered and calls for further investigation through comprehensive 3D analysis.

The fungal Kingdom boasts a remarkable diversity of cellular morphologies, with species like N. crassa exhibiting as many as 28 morphologically distinct cell types (129). This diversity likely contributes to functional and morphological differences of the ER. Plasticity of the ER may exist at different stages of morphological development, such as during the maturation of germ tubes into mature hyphae of N. crassa. In germ tubes of N. crassa, organelles are evenly distributed, and SPK is absent (130). Upon reaching a length of ~150 µm, organelles adopt a polarized distribution, SPK are present, and cell extension rates increase, thus establishing mature hyphae (130). Furthermore, the ER distribution undergoes changes during sexual development in Ascomycota species (131, 132). Most published studies on the ER in fungi do not provide direct comparisons of ER membrane distribution or morphology across different cell types or developmental stages. Furthermore, various research laboratories utilize diverse microscopy techniques, sample preparation methods, growth media, and other variables. Consequently, drawing conclusions about ER distribution patterns based on specific taxa, cell types, or developmental stages comes with significant risks. Additionally, it has been observed that pER distribution may adapt to cellular needs and environmental conditions. Accordingly, the pER organization must be studied under both optimal and stressful growth conditions to understand the function-structure correlation fully.

To unravel the ER’s morphological and functional diversity, behavior, dynamics, and 3D organization, it is essential to employ a combination of both basic and advanced bioimaging technologies (e.g., super-resolution fluorescence microscopy and electron tomography) alongside biochemical and molecular investigations. Furthermore, correlative light and electron microscopy approaches that compare information from different imaging modalities should be pursued. However, studying fungal hyphae using bioimaging is challenging, particularly when it comes to maintaining optimal growth conditions during live-cell fluorescence microscopy imaging and prior to fixation. Coordinated efforts are crucial, aiming to analyze various cell types under standardized conditions, ensuring consistent sample preparation, imaging methods, and markers. Hyphal tip cells, in particular, are highly susceptible to environmental perturbations, which can halt growth and disrupt cytoplasmic organization, highlighting the need for meticulous cell preparation. Observing the presence of a SPK at the hyphal tip and assessing the overall shape of the hyphal tip serve as reliable indicators of cellular health. In light of these considerations, it is imperative to further investigate whether the apical ER plays a role in apical growth and/or in calcium regulation.

Certain complex ER arrangements seen in mammalian or yeast cells, such as polygonal ER networks, are rarely observed in fungal hyphae. This limitation is attributed to the technical challenges of preparing high-quality images from the cortical regions of hyphae, which are essential for comprehensive 3D reconstructions. The polygonal ER network may be susceptible to changes due to prefixation handling and may not consistently appear in cells preserved via cryopreparation protocols. Furthermore, the contrast of ER membranes can vary depending on the preparation protocols employed.

Most published research has concentrated on understanding the enzymatic functions and protein complexes localized within the ER, with a focus on well-studied targets like ergosterol biosynthesis enzymes. However, some studies have suggested potential drug targets in animal or plant fungal pathogens, such as oxysterol-binding proteins, GPI-phospholipases, and ER-shaping proteins like Sey1 (65, 78, 79, 95, 133).

Despite being conserved, some commonly studied ER proteins exhibit notable differences in subcellular ER distribution between mammalian, yeast, and fungal cells, reflecting the differences in their modes of growth and division. Notably, it has been proposed that the distribution gradient of the ER observed in hyphae might facilitate polarized growth and apical exocytosis (17). What is particularly intriguing is the presence of numerous hypothetical proteins within the growing collection of annotated fungal genomes, many of which remain uncharacterized. Some of these proteins, believed to be synthesized in the ER and incorporated into the plasma membrane or cell wall, have unique functions in fungi. Investigating the sorting processes of these distinctive proteins could unveil novel insights into the ER organization and its role in fungal biology.

In conclusion, comprehending the structural and functional intricacies of the ER within fungal hyphae necessitates the combined application of advanced light and electron microscopy techniques, as well as standardized preparation, imaging, and analytical methods.

Biographies

Juan Manuel Martínez Andrade obtained his bachelor’s degree in dentistry at the Universidad Nacional Autónoma de Mexico (UNAM). He completed a M.Sc. in Life Sciences at the Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), in Baja California, Mexico. He has gained valuable experience as Electron Microscopy technician at the Instituto Potosino de Investigación Científica y Tecnológica (IPICYT) in San Luis Potosí, Mexico, and at the Laboratorio Nacional de Microscopía Avanzada (LNMA-CICESE) at CICESE. Currently, he is undertaking his Ph.D. at CICESE, focusing on the the structure and function of the endoplasmic reticulum in the model filamentous fungus Neurospora crassa. His research contributes to a deeper understanding of celular processes within this fungal model.

Robby Roberson received his M.Sc. at Stephen F. Austin State University under the direction of Charles Mims and his Ph.D. in Plant Biology at the University of Georgia working under Melvin Fuller. After obtaining his Ph.D. in 1989 he accepted a position at Arizona State University as an Assistant Professor. His primary research goal is the better understanding of cytoplasmic order and behavior in hyphal cells of various filamentous fungi including Allomyces macrogynus, Neurospora crassa, and members of the zygomycetous fungi.

Meritxell Riquelme obtained her Biology degree at the University of Barcelona, Spain. After completion of a M.Sc. in Plant Pathology and a Ph.D. in Microbiology at the University of California, Riverside, United States, she was a postdoctoral fellow at the University of Oxford, United Kingdom, where she investigated the mating type genes in the mushroom Coprinopsis cinerea. In 2004, she joined the Department of Microbiology, at the Centro de Investigación Científica y de Educación Superior de Ensenada, CICESE, Baja California, Mexico, where she is a Research Professor. From 2012-2013 she was visiting Professor at the Department of Cellular and Molecular Medicine at the University of California, San Diego. Her research interests include the secretory pathways involved in hyphal polarity and morphogenesis in Neurospora crassa. She has revealed that the differently sized secretory vesicles that accumulate in the Spitzenkörper of N. crassa contain distinct polysaccharide synthases. In addition, she studies the distribution of the human pathogen Coccidioides spp. in semi-arid regions of Baja California and fungi of the deep-sea sediments of the Gulf of Mexico

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/mmbr.00027-23.

Legend of Fig. S1. mmbr.00027-23-s0001.tif.

Supplemental figure legend.

Figure S1. mmbr.00027-23-s0002.docx.

Venn diagram of proteins found in the ER in filamentous fungi.

Tables S1 to S3. mmbr.00027-23-s0003.docx.

Summary of the endoplasmic reticulum distribution at the ultrastructure level in hyphal cells and spores and by fluorescence microscopy.

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