Fungal cell structure and organization

Human pathogenic fungi produce three basic ‘cell’ types: hyphae, yeast cells, and spores. The organization and subcellular structure of these different cell types and their modes of growth and formation are reviewed. Growth and form is the consequence of how new cell surface is formed. This is generated by the delivery of vesicles to the surface which provides new membrane and the enzymes for cell wall synthesis. To generate these various cell types, the pathway of vesicle secretion to the surface has to be carefully regulated. These vesicles have to be transported through the cell by the cytoskeleton, and in filamentous cells these vesicles accumulate at a supply centre called the Spitzenkörper before docking and fusion with the hyphal apex. Ultimately, membrane is also endocytosed and recycled behind actively expanding regions of the fungal surface. These various processes are described and particular emphasis is given to the structural and organizational features of fungal cells that play roles in their pathogenesis and virulence.

A Pericentrin-Related Protein Homolog in Aspergillus nidulans Plays Important Roles in Nucleus Positioning and Cell Polarity by Affecting Microtubule Organization

ABSTRACTPericentrin is a large coiled-coil protein in mammalian centrosomes that serves as a multifunctional scaffold for anchoring numerous proteins. Recent studies have linked numerous human disorders with mutated or elevated levels of pericentrin, suggesting unrecognized contributions of pericentrin-related proteins to the development of these disorders. In this study, we characterized AnPcpA, a putative homolog of pericentrin-related protein in the model filamentous fungusAspergillus nidulans, and found that it is essential for conidial germination and hyphal development. Compared to the hyphal apex localization pattern of calmodulin (CaM), which has been identified as an interactive partner of the pericentrin homolog, GFP-AnPcpA fluorescence dots are associated mainly with nuclei, while the accumulation of CaM at the hyphal apex depends on the function of AnPcpA. In addition, the depletion of AnPcpA by an induciblealcApromoter repression results in severe growth defects and abnormal nuclear segregation. Most interestingly, in mature hyphal cells, knockdown of pericentrin was able to significantly induce changes in cell shape and cytoskeletal remodeling; it resulted in some enlarged compartments with condensed nuclei and anucleate small compartments as well. Moreover, defects in AnPcpA significantly disrupted the microtubule organization and nucleation, suggesting that AnPcpA may affect nucleus positioning by influencing microtubule organization.

Apical Sterol-rich Membranes Are Essential for Localizing Cell End Markers That Determine Growth Directionality in the Filamentous FungusAspergillus nidulans

In filamentous fungi, hyphal extension depends on the continuous delivery of vesicles to the growing tip. Here, we describe the identification of two cell end marker proteins, TeaA and TeaR, in Aspergillus nidulans, corresponding to Tea1 and Mod5 in Schizosaccharomyces pombe. Deletion of teaA or teaR caused zig-zag-growing and meandering hyphae, respectively. The Kelch-repeat protein TeaA, the putatively prenylated TeaR protein, and the formin SepA were highly concentrated in the Spitzenkörper, a vesicle transit station at the tip, and localized along the tip membrane. TeaA localization at tips depended on microtubules, and TeaA was required for microtuble convergence in the hyphal apex. The CENP-E family kinesin KipA was necessary for proper localization of TeaA and TeaR, but not for their transportation. TeaA and TeaR localization were interdependent. TeaA interacted in vivo with TeaR, and TeaA colocalized with SepA. Sterol-rich membrane domains localized at the tip in teaA and teaR mutants like in wild type, and filipin treatment caused mislocalization of both proteins. This suggests that sterol-rich membrane domains determine cell end factor destinations and thereby polarized growth.

Basic-Zipper-Type Transcription Factor FlbB Controls Asexual Development in Aspergillus nidulans

ABSTRACT The fungal colony is a complex multicellular unit consisting of various cell types and functions. Asexual spore formation (conidiation) is integrated through sensory and regulatory elements into the general morphogenetic plan, in which the activation of the transcription factor BrlA is the first determining step. A number of early regulatory elements acting upstream of BrlA (fluG and flbA-E) have been identified, but their functional relations remain to be further investigated. In this report we describe FlbB as a putative basic-zipper-type transcription factor restricted to filamentous fungi. FlbB accumulates at the hyphal apex during early vegetative growth but is later found in apical nuclei, suggesting that an activating modification triggers nuclear import. Moreover, proper temporal and quantitative expression of FlbB is a prerequisite for brlA transcription, and misscheduled overexpression inhibits conidiation. We also present evidence that FlbB activation results in the production of a second diffusible signal, acting downstream from the FluG factor, to induce conidiation.

Polarized Gene Expression Determines Woronin Body Formation at the Leading Edge of the Fungal Colony

The Woronin body (WB) is a peroxisome-related organelle that is centered on a crystalline core of the HEX-1 protein, which functions to seal septal pores of filamentous ascomycetes in response to cellular damage. Here, we investigate the cellular and genetic control of WB-formation and show that polarized hex-1 gene expression determines WB-biogenesis at the growing hyphal apex. We find that intron splicing is coupled to efficient hex-1 gene expression and strikingly, when the yellow fluorescent protein was expressed from hex-1 regulatory sequences, we observed a fluorescent gradient that was maximal in apical cells. Moreover, endogenous hex-1 transcripts were specifically enriched at the leading edge of the fungal colony, whereas other transcripts accumulated in basal regions. Time-lapse confocal microscopy showed that HEX-1 crystals normally formed in the vicinity of the hyphal apex in large peroxisomes, which matured and were immobilized at the cell periphery as cells underwent septation. When the hex-1 structural gene was expressed from regulatory sequences of an abundant, basally localized transcript, WB-core formation was redetermined to basal regions of the colony, and these strains displayed loss-of-function phenotypes specifically in apical hyphal compartments. These results show that apically localized gene expression is a key determinant of spatially restricted WB-assembly. We suggest that this type of regulation may be widely used to determine cellular activity in apical regions of the fungal hypha.

Isolation and regeneration of protoplasts fromPenicillium digitatum

The effects of some factors on the isolation of protoplasts fromPenicillium digitatumwere studied, including the appropriate material (young mycelia and generating spores), the concentrations of enzyme and osmotic pressure stabilizers, reaction time and reaction temperature. Results demonstrated that germinating spores were an ideal material resource for the isolation ofP. digitatumprotoplasts. Highest yield and quality ofP. digitatumprotoplasts were obtained by shaking germinating spores suspended in a solution of 10 mg/ml Lywallzyme™ dissolved in 0.7 M NaCl as osmotic pressure stabilizer at 80 rev/min and 30°C for 3.0–3.5 h. The regeneration rate of the isolated protoplasts was as high as 24.9% on double-layer Czapek medium containing 0.7 M NaCl. Additionally, observation of the protoplast release pattern showed that the protoplasts ofP. digitatumwere released primarily from the hyphal apex and occasionally from the subapical or original sites of germinating tubes. The protoplasts ofP. digitatumwere regenerated in a direct manner of either yeast-like cell development or mycelium formation.

Laser Microsurgery Permits Fungal Plasma Membrane Single-Ion-Channel Resolution at the Hyphal Tip

ABSTRACT A method for formation of high-electrical-resistance seals on theNeurospora crassa plasma membrane, allowing resolution of single-ion-channel activity by patch clamp electrophysiology, is reported. Laser microsurgery permits access to the hyphal apex without enzymatic cell wall digestion and loss of morphological polarity. Cell wall reformation is delayed by brefeldin. This method can allow full characterization of apical plasma membrane channels, which are implicated in tip growth.