Fungal Cell BiologyCurrent Projects Completed Projects
Filamentous fungi such as moulds and mildews, form tubular cells called hyphae. Some fungi colonize a diversity of non-living organic substrates, and others interact with living hosts through symbioses or by causing disease. Advantages to studying basic processes in fungal model systems include compact size, fast growth rate, morphological simplicity, and a wealth of experimental and genetic tools. Fungal genomes are smaller and less complex than those of animals (many fungal genomes are completely sequenced), and yet some animal and fungal genes are functionally homologous.
Fungal cells are surrounded and protected by walls that mediate their environmental interactions. My research uses chemical and cell molecular genetic methods and microscopy to explore aspects of fungal growth and interaction.
Our main research system is the model organism Aspergillus nidulans, particularly genes that affect hyphal morphogenesis and/or cell wall structure. These include galactofuranose biosynthesis and cell secretion genes. Along with well-established types of microscopy (confocal fluorescence, TEM, SEM), we are using high spatial resolution chemical analytical methods (including AFM, FTIR, and SERS) for characterizing fungal hyphae and their wall composition and structure.
Aspergillus Galactofuranose biosynthesis:
Kausar Alam Sean Xiaoxiao He Jill Shengnan Li
Fatemeh Farazkhorasani Martin Prusinkiewicz
Our paper on antifungal drug sensitivity (Alam, El-Ganiny, Afroz et al 2012 Fungal Genetics and Biology 49: 1033-1043) was chosen for the cover image of that issue.
I am using site-directed mutagenesis to assess the role of certain amino acid residues in function of two galactofuranose (Galf) pathway genes, UgmA (whose structure was recently solved by our collaborators; see van Straaten et al 2012 J Biol Chem 287:10780-90) and the UgtA (see Afroz et al 2011, FGB 48:896-903).
The genus Aspergillus includes species used in biotechnology, and others that are increasingly important human and plant pathogens. The fungal cell wall is essential for its survival, and is an excellent target for antifungal drug development, since wall components are not found in humans. However, fungi are rapidly evolving resistance to most drugs. I am using strains deleted for UgmA and related enzymes to explore resistance mechanisms against wall-targeting compounds.
I have been characterizing genes that confer tolerance to anti-fungal drugs. We are using Calcofluor White (CFW) to test generation and inheritance of tolerance mutations.
Fig 1 shows a CFW mutant (AXE5, compared to wildtype strain (P2)
Fig 2 shows meiotic progeny from a mating between AXE5 (CFW-resistant, yellow square) and AXM5 (CFW-sensitive, green oval) wild type. For 166 analyzed, CFW resistance:sensitivity was 81:85, for all three spore colours (white, chartreuse, and green).)
I am working with Sean He. Identification of the genetic lesion responsible for mutation in a wild type strain is the most challenging stage in gene characterization and analysis. I used whole-genome sequencing to assess single nucleotide polymorphisms (snp’s) in CFW-resistant mutants. The diagram shows that the AXE5 snp’s were evenly distributed across the 17 A. nidulans supercontigs (available at the Broad Institute). The AXE5 lesion caused a premature stop in ANID 10647, nearby but distinct from the mutation that caused the abnormal hyphal branching mutant ahbB. I have also identified the site of the AXE8 lesion, a premature stop in an uncharacterized gene..
Gough Lab, Dept Chemistry, U Manitoba
Nanoparticles (NPs) are natural and engineered aggregates whose dimension is on the order of nanometers, which are millionths of millimeters. Fungi grown in metal-containing environments become associated with metallic NPs that can be used for analytical imaging using surface-enhanced Raman spectroscopy (SERS). NPs may provide advantages over SERS analysis using nanopatterned substrates (see Szeghalmi et al 2007, Publications page) which might report only on cell surfaces.
Our paper on gold nanoparticles (Prusinkiewicz, Farazkhorasani et al 2012 Analyst 137: 4934-42) was chosen for the cover image of that issue.
I will be using metallic nanoparticles (NPs) as an probe for surface enhanced Raman spectroscopy (SERS), a high sensitivity and high spatial resolution chemical analysis method. We expect that SERS-NP analysis will provide results that are complementary to Fourier transform infrared spectroscopy.
The chemical and biological activity of NPs depends on their composition, size, and location. When I was with the Kaminskyj lab, we were developing methods for growing NPs in fungal cells. We were combining light and electron microscopy to assess where the NPs form under different biosynthesis conditions, and whether they affect cell physiology.
Sharmin Afroz Robyn Pollock Geoff Bray Michelle Hubbard Miryha Retzlaff
I am studying two enzymes in the UDP-galactofuranose biosynthesis pathway: UPD-glucose-4-epimerase (UgeA) and UDP-galactopyranose mutase (UgmA). Galactofuranose is found in the cell walls of many microorganisms including fungi. If inhibitors can be developed against these enzymes, they could be effective antifungal drugs.
I am studying the UDP-galactofuranose transporter, which is expected to function downstream of UgmA. UDP-galactofuranose is synthesized in the cytoplasm, and must be transported into a membrane-bound compartment, likely the Golgi equivalent, prior to galactofuranose incorporation into the cell wall.
Robyn Pollock : Effects of disease and environmental factors on alarm substance cell investment in fathead minnows (Pimephales promelas)
Fish in the order Ostariophyi have specialized epidermal cells known as alarm substance cells (ASCs). Alarm substance is released when ASCs are ruptured. Nearby conspecific fish use alarm substance as an indication of predation risk, leading to antipredator behaviour (e.g. dashing, freezing, schooling). Recently, however, we discovered that ASCs may also have an antipathogen function.
My research examines ASC investment in fathead minnows (Pimephales promelas), a model Ostariophysan, throughout the year and whether exposure to a pathogenic water mold (Saprolegnia ferax) influences ASC investment. I will also examine how intra-population differences, virulence of the pathogen, and pathogen density may affect ASC investment in fathead minnows. My work is co-supervised with Dr. Doug Chivers (Department of Biology).
Aspergillus nidulans hypB5 has a temperature sensitive morphogenetic defect. Its hyphae grow like wildtype strains at 28°C but not at 42°C. The hypB5 defect can be partially complemented by a Sec7 domain protein, cloned by Yi Yang during her M. Sc. research. I am working to resolve some questions remaining about the relationship between AnSec7 and hypB.
Fungal tip growth uses targeted secretion of wall forming materials at the hyphal tip to produce tubular cells called hyphae. These materials are synthesized and transported within the hypha by components of the endomembrane system: the endoplasmic reticulum (ER), Golgi body equivalents (GEs), vacuoles and vesicles. Organization and motility of endomembrane cargo requires F-actin and microtubules, components of the cytoskeleton.
Laser scanning confocal epifluorescence microscopy is a superb tool for studying growth dynamics. I am using fluorescent-protein tagged gene products and fluorescent chemical probes to study endomembranes and cytoplasmic microtubules in growing Aspergillus hyphae.
The images to the right show relatively stationary FM 4-64 stained endomembranes (red) and relatively motile granules GFP-tagged sodVIC (green, in a strain generated by Andy Breakspeare during his PhD research in Susan Assinder's lab, Dept Biological Sciences, Univ Wales-Bangor) that localize to fungal Golgi equivalents.
My research is continuing work begun by Michelle Hubbard, studying the relationship between endomembrane arrays and hyphal morphology in Aspergillus nidulans. This image is a confocal micrograph of a living A. nidulans morphogenesis mutant colony after 20 h growth, stained with the endomembrane selective dye FM4-64.
I am studying the relationship between endomembrane array dynamics and hyphal morphology in Aspergillus nidulans. This image is a confocal micrograph of actively growing A. nidulans hyphal tips stained with the endomembrane selective dye FM4-64.