Trade-off between Plasticity and Velocity in Mycelial Growth

ABSTRACT Tip-growing fungal cells maintain cell polarity at the apical regions and elongate by de novo synthesis of the cell wall. Cell polarity and tip growth rate affect mycelial morphology. However, it remains unclear how both features act cooperatively to determine cell shape. Here, we investigated this relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 μm-width channels of microfluidic devices. Since the channels are much narrower than the diameter of hyphae, any hypha growing through the channel must adapt its morphology. Live-cell imaging analyses revealed that hyphae of some species continued growing through the channels, whereas hyphae of other species often ceased growing when passing through the channels, or had lost apical polarity after emerging from the other end of the channel. Fluorescence live-cell imaging analyses of the Spitzenkörper, a collection of secretory vesicles and polarity-related proteins at the hyphal tip, in Neurospora crassa indicates that hyphal tip growth requires a very delicate balance of ordered exocytosis to maintain polarity in spatially confined environments. We analyzed the mycelial growth of seven fungal species from different lineages, including phytopathogenic fungi. This comparative approach revealed that the growth defects induced by the channels were not correlated with their taxonomic classification or with the width of hyphae, but, rather, correlated with the hyphal elongation rate. This report indicates a trade-off between morphological plasticity and velocity in mycelial growth and serves to help understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology, and pathogenicity. IMPORTANCE Cell morphology, which is controlled by polarity and growth, is fundamental for all cellular functions. However how polarity and growth act cooperatively to control cell shape remains unclear. Here we investigated their relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 μm-width channels of microfluidic devices. We found that most fast growing hyphae often lost the cell polarity after emerging from the channels, whereas slow growing hyphae retained polarity and continued growing, indicating a trade-off between plasticity and velocity in mycelial growth. These results serve to understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology and pathogenicity.

Trade-off between plasticity and velocity in mycelial growth

Tip-growing fungal cells maintain the cell polarity at the apical regions and elongate by de novo synthesis of cell wall. Cell polarity and growth rate affect the mycelial morphogenesis, however, it remains unclear how they act cooperatively to determine cell shape. Here we investigated their relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 μm-width channels of microfluidic devices. Since the channels are much narrower than the diameter of hyphae, the hyphae must change its morphology when they grow through the channels. Live imaging analysis revealed that hyphae of some species continued growing through the channels, whereas hyphae of other species often ceased growing when passing through the channels or lost the cell polarity after emerging from the channels. Fluorescence live imaging analysis of the Spitzenkörper, a collection of secretory vesicles and polarity-related proteins at hyphal tips, in Neurospora crassa hyphae indicates that hyphal tip growth requires a very delicate balance of ordered exocytosis to maintain polarity in spatially confined environments. We analyzed the mycelial growth of seven fungal species from different lineages, which also include phytopathogenic fungi. This comparative cell biology showed that the growth defects in the channels were not correlated with their taxonomic classification nor with the width of hyphae, but, correlated with the hyphal elongation rate. This is the first report indicating a trade-off between plasticity and velocity in mycelial growth, and serves to understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology and pathogenicity.

Potent Chitin Synthase Inhibitors from Plants

Background: Chitin is the main component of fungal, protozoan and helminth cell wall. They help to maintain the structural and functional characteristics of these organisms. The chitin wall is dynamic and is repaired, rearranged and synthesized as the cells develop. Active synthesis can be noticed during cytokinesis, laying of primary septum, maintenance of lateral cell wall integrity and hyphal tip growth. Chitin synthesis involves coordinated action of two enzymes namely, chitin synthase (that lays new cell wall) and chitinase (that removes the older ones). Since chitin synthase is conserved in different eukaryotic microorganisms that can be a ‘soft target’ for inhibition with small molecules. When chitin synthase is inhibited, it leads to the loss of viability of cells owing to the self- disruption of the cell wall by existing chitinase. Methods: In the described study, small molecules from plant sources were screened for their ability to interfere with hyphal tip growth, by employing Hyphal Tip Burst assay (HTB). Aspergillus niger was used as the model organism. The specific role of these small molecules in interfering with chitin synthesis was established with an in-vitro method. The enzyme required was isolated from Aspergillus niger and its activity was deduced through a novel method involving non-radioactively labelled substrate. The activity of the potential lead molecules were also checked against Candida albicans and Caenorhabditis elegans. The latter was adopted as a surrogate for the pathogenic helminths as it shares similarity with regard to cell wall structure and biochemistry. Moreover, it is widely studied and the methodologies are well established. Results: Out of the 11 compounds and extracts screened, 8 were found to be prospective. They were also found to be effective against Candida albicans and Caenorhabditis elegans. Conclusion: Purified Methyl Ethyl Ketone (MEK) Fraction1 (F1) of Coconut (Cocos nucifera) Shell Extract (COSE) was found to be more effective against Candida albicans with an IC50 value of 3.04 μg/mL and on L4 stage of Caenorhabditis elegans with an IC50 of 77.8 μg/mL.

Hyphal tip growth and cytoplasmic characters of Conidiobolus coronatus (Zoopagomycota, Entomophthoromycotina)

Characteristics of hyphal structure and growth can provide insights into the mechanisms of polarized growth and support investigations of fungal phylogeny. To assist with the resolution of evolutionary relationships of the zygomycetes, the authors used comparative bioimaging methods (light [LM] and transmission electron [TEM] microscopy) to describe selected subcellular characters of hyphal tips of Conidiobolus coronatus. Growing hyphae of C. coronatus contain Spitzenkörper (Spk). Spk are most commonly present in hyphae of Dikarya (Ascomycota and Basidiomycota) and are rarely reported in zygomycete hyphae, which possess an apical vesicle crescent (AVC). Such findings raise questions regarding the evolution of the Spk and its relationship with the AVC. Descriptions of additional subcellular characters (e.g., mitotic-phase spindle pole bodies, cytoplasmic behavior, organelle structure) are also presented.

A Cellulose Synthase-Like Protein Involved in Hyphal Tip Growth and Morphological Differentiation in Streptomyces

ABSTRACT Cellulose synthase and cellulose synthase-like proteins, responsible for synthesizing β-glucan-containing polysaccharides, play a fundamental role in cellular architectures, such as plant cell and tissue morphogenesis, bacterial biofilm formation, and fruiting-body development. However, the roles of the proteins involved in the developmental process are not well understood. Here, we report that a cellulose synthase-like protein (CslASc) in Streptomyces has a function in hyphal tip growth and morphological differentiation. The cslASc replacement mutant showed pleiotropic defects, including the severe delay of aerial-hyphal formation and altered cell wall morphology. Calcofluor white fluorescence analysis demonstrated that polysaccharide synthesis at hyphal tips was dependent on CslASc. cslASc was constitutively transcribed, and an enhanced green fluorescent protein-CslASc fusion protein was mostly located at the hyphal tips. An extract enriched in morphogenetic chaplin proteins promoted formation of aerial hyphae by the mutant. Furthermore, a two-hybrid experiment indicated that the glycosyltransferase domain of CslASc interacted with the tropomyosin-like polarity-determining DivIVA protein, suggesting that the tip-located DivIVA governed tip recruitment of the CslASc membrane protein. These results imply that the cellulose synthase-like protein couples extracellular and cytoskeletal components functioning in tip growth and cell development.