THE DAMPENING OF LIPID DROPLET OSCILLATORY MOVEMENT IN NITROGEN STARVED FILAMENTOUS FUNGI BY A LOW DOSE OF MITOCHONDRIAL RESPIRATION INHIBITOR

2021 ◽  
Author(s):  
Tanja Pajic ◽  
◽  
Miroslav Zivic ◽  
Mihailo Rabasovic ◽  
Aleksandar Krmpot ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Filamentous Fungus ◽  
Sodium Azide ◽  
Mitochondrial Respiration ◽  
Lipid Droplets ◽  
Low Dose ◽  
Eukaryotic Cells ◽  
Confined Space ◽  
Phycomyces Blakesleeanus ◽  
Oscillatory Movement

Lipid droplets (LDs) are small mobile organelles conserved in all eukaryotic cells. We wanted to test if the LD movement can be muffled by an incomplete inhibition of mitochondrial respiration, induced by treating hyphae of filamentous fungus Phycomyces blakesleeanus with 0.5 mM sodium azide. Nitrogen starved hyphae were used, in order to obtain LDs in larger sizes and numbers. The data obtained unequivocally showed: 1. Sodium azide treatment dramatically reduces the LD velocity and the distances LDs travel; 2. LDs in both controls and in azide-treated hyphae oscillate in a small confined space instead of travelling through the cell; 3. Azide-treated LDs oscillate less frequently and in smaller confinement than controls.

scholarly journals Lipid droplets and the host–pathogen dynamic: FATal attraction?

2021 ◽  
Vol 220 (8) ◽  
Author(s):  
Marta Bosch ◽  
Matthew J. Sweet ◽  
Robert G. Parton ◽  
Albert Pol
Keyword(s):  
Innate Immunity ◽  
Lipid Droplets ◽  
Innate Immune ◽  
Metabolic Energy ◽  
Eukaryotic Cells ◽  
Bioactive Lipids ◽  
Front Line ◽  
Antimicrobial Proteins ◽  
Current State ◽  
Fatal Attraction

In the ongoing conflict between eukaryotic cells and pathogens, lipid droplets (LDs) emerge as a choke point in the battle for nutrients. While many pathogens seek the lipids stored in LDs to fuel an expensive lifestyle, innate immunity rewires lipid metabolism and weaponizes LDs to defend cells and animals. Viruses, bacteria, and parasites directly and remotely manipulate LDs to obtain substrates for metabolic energy, replication compartments, assembly platforms, membrane blocks, and tools for host colonization and/or evasion such as anti-inflammatory mediators, lipoviroparticles, and even exosomes. Host LDs counterattack such advances by synthesizing bioactive lipids and toxic nucleotides, organizing immune signaling platforms, and recruiting a plethora of antimicrobial proteins to provide a front-line defense against the invader. Here, we review the current state of this conflict. We will discuss why, when, and how LDs efficiently coordinate and precisely execute a plethora of immune defenses. In the age of antimicrobial resistance and viral pandemics, understanding innate immune strategies developed by eukaryotic cells to fight and defeat dangerous microorganisms may inform future anti-infective strategies.

THE EFFECTS OF SELENITE ON FILAMENTOUS FUNGI LIPID DROPLETS MONITORED „IN VIVO“ LABEL FREE USING ADVANCED NONLINEAR MICROSCOPY TECHNIQUE 2021ICCBIKG (2021)

2021 ◽  
Author(s):  
Tanja Pajic ◽  
◽  
Natasa Todorovic ◽  
Dunja Stefanovic ◽  
Mihailo Rabasovic ◽  
...  
Keyword(s):  
Size Distribution ◽  
Filamentous Fungi ◽  
Lipid Droplet ◽  
Laser Scanning ◽  
Biological Effects ◽  
Laser Scanning Microscopy ◽  
Cellular Stress Response ◽  
Phycomyces Blakesleeanus ◽  
Phase Duration

Third Harmonic Generation (THG) microscopy was employed as a method of choice for lipid droplet (LD) measurements and quantification of the effect of selenite on LDs. Nonlinear laser scanning microscopy (NLSM) employs ultra-short laser pulses for imaging. THG microscopy is the modality of NLSM. Strong THG signals can only be observed from regions with non- uniformities with respect to their refractive index. Such regions in biological samples are lipid-water interfaces, and by far the brightest features in cells are LDs. For that reason, THG microscopy is the appropriate method for imaging of LDs from live unfixed cells, without the need for additional labeling. The biological effects of spore- to- end- of- exponential- phase duration (27 – 30 h) of exposure to 1 mM selenite were monitored in vivo on the cells of filamentous fungi in liquid culture. We measured the lipid droplet density and size distribution in a model fungi Phycomyces blakesleeanus. The in-house built microscope frame complemented with Yb KGW laser (1040 nm, 200 fs pulses) was used, while detection was enabled in the transmission arm by PMT through the Hoya glass UV filter (peak at 340 nm). From THG images of control and Se+4–treated hyphae, LD size and number were measured, showing that LD density was increased by more than 60% in Se+4–treated hyphae, compared to control. The average LD size distribution seemed slightly changed by Se+4 -treatment. The obtained results suggest that 1 mM selenite treatment probably induces cellular stress response in filamentous fungi.

scholarly journals Defining individual size in the model filamentous fungus Neurospora crassa

2016 ◽  
Vol 283 (1826) ◽  
pp. 20152470 ◽  
Author(s):  
Linda Ma ◽  
Boya Song ◽  
Thomas Curran ◽  
Nhu Phong ◽  
Emilie Dressaire ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Filamentous Fungi ◽  
Filamentous Fungus ◽  
Reproductive Potential ◽  
Fungal Mycelium ◽  
Dependent Property ◽  
Mycelial Network ◽  
And Function ◽  
Individual Size ◽  
Over Time

It is challenging to apply the tenets of individuality to filamentous fungi: a fungal mycelium can contain millions of genetically diverse but totipotent nuclei, each capable of founding new mycelia. Moreover, a single mycelium can potentially stretch over kilometres, and it is unlikely that its distant parts share resources or have the same fitness. Here, we directly measure how a single mycelium of the model ascomycete Neurospora crassa is patterned into reproductive units (RUs), meaning subpopulations of nuclei that propagate together as spores, and function as reproductive individuals. The density of RUs is sensitive to the geometry of growth; we detected 50-fold smaller RUs when mycelia had expanding frontiers than when they were constrained to grow in one direction only. RUs fragmented further when the mycelial network was perturbed. In mycelia with expanding frontiers, RU composition was strongly influenced by the distribution of genotypes early in development. Our results provide a concept of fungal individuality that is directly connected to reproductive potential, and therefore to theories of how fungal individuals adapt and evolve over time. Our data show that the size of reproductive individuals is a dynamic and environment-dependent property, even within apparently totally connected fungal mycelia.

scholarly journals Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei

2017 ◽  
Vol 83 (15) ◽  
Author(s):  
Pak Yang Chum ◽  
Georg Schmidt ◽  
Markku Saloheimo ◽  
Christopher P. Landowski
Keyword(s):  
Dna Repair ◽  
Trichoderma Reesei ◽  
Filamentous Fungi ◽  
Filamentous Fungus ◽  
Unintended Consequences ◽  
Dna Repair Genes ◽  
Content Type ◽  
Targeted Integration ◽  
Gene Integration ◽  
Repair Genes

ABSTRACT Trichoderma reesei is a filamentous fungus that is used worldwide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement using mutagenesis and screening approaches. It is also desirable to specifically manipulate the genes of the organism to further improve and to modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation, we have developed a simple transient method for silencing genes in T. reesei. Using gene-specific small interfering RNAs (siRNAs) targeted to mus53, we could achieve up to 90% knockdown of mus53 mRNA. As a practical example, we demonstrated that transient silencing of DNA repair genes significantly improved homologous integration of DNA at a specific locus in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59% marker gene integration. IMPORTANCE The previous solution for improving targeted integration efficiency has been deleting nonhomologous end joining (NHEJ) DNA repair genes. However, deleting these important repair genes may lead to unintended consequences for genomic stability and could lead to the accumulation of spontaneous mutations. Our method of transiently silencing NHEJ repair pathway genes allows recovery of their important repair functions. Here we report a silencing approach for improving targeted DNA integration in filamentous fungi. Furthermore, our transient silencing method is a truly flexible approach that is capable of knocking down the expression of a target gene in growing mycelial cultures, which could facilitate the broad study of gene functions in T. reesei.

scholarly journals FERRITIN IN THE FUNGUS PHYCOMYCES

1971 ◽  
Vol 48 (1) ◽  
pp. 15-28 ◽  
Author(s):  
Charles N. David ◽  
Kenneth Easterbrook
Keyword(s):  
Lipid Droplets ◽  
Developmental Stages ◽  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Fold Increase ◽  
Iron Level ◽  
Phycomyces Blakesleeanus ◽  
Thin Sections ◽  
Iron Protein ◽  
Ferritin Iron

The iron-protein ferritin has been purified from mycelium, sporangiophores, and spores of the fungus Phycomyces blakesleeanus. It has a protein-to-iron ratio of 5, a sedimentation coefficient of 55S, a buoyant density in CsCl of 1.82 g/cm3, and the characteristic morphology of ferritin in the electron microscope. Apoferritin prepared from Phycomyces ferritin has a sedimentation coefficient of 18S and consists of subunits of molecular weight 25,000. In the cytoplasm of Phycomyces, ferritin is located on the surface of lipid droplets (0.5–2.0 µ in diameter) where it forms crystalline monolayers which are conspicuous in electron micrographs of sporangiophore thin-sections. Ferritin is found in all developmental stages of Phycomyces but is concentrated in spores. The level of ferritin iron is regulated by the iron level in the growth medium, a 50-fold increase occurring on iron-supplemented medium.

scholarly journals The autophagy protein ATG9A enables lipid mobilization from lipid droplets

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Elodie Mailler ◽  
Carlos M. Guardia ◽  
Xiaofei Bai ◽  
Michal Jarnik ◽  
Chad D. Williamson ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Membrane Protein ◽  
Human Cell ◽  
Mitochondrial Respiration ◽  
Lipid Droplets ◽  
Critical Role ◽  
Human Cell Lines ◽  
Lipid Mobilization ◽  
C Elegans ◽  
Close Proximity

AbstractThe multispanning membrane protein ATG9A is a scramblase that flips phospholipids between the two membrane leaflets, thus contributing to the expansion of the phagophore membrane in the early stages of autophagy. Herein, we show that depletion of ATG9A does not only inhibit autophagy but also increases the size and/or number of lipid droplets in human cell lines and C. elegans. Moreover, ATG9A depletion blocks transfer of fatty acids from lipid droplets to mitochondria and, consequently, utilization of fatty acids in mitochondrial respiration. ATG9A localizes to vesicular-tubular clusters (VTCs) that are tightly associated with an ER subdomain enriched in another multispanning membrane scramblase, TMEM41B, and also in close proximity to phagophores, lipid droplets and mitochondria. These findings indicate that ATG9A plays a critical role in lipid mobilization from lipid droplets to autophagosomes and mitochondria, highlighting the importance of ATG9A in both autophagic and non-autophagic processes.

scholarly journals Lis1 is an initiation factor for dynein-driven organelle transport

2012 ◽  
Vol 197 (7) ◽  
pp. 971-982 ◽  
Author(s):  
Martin J. Egan ◽  
Kaeling Tan ◽  
Samara L. Reck-Peterson
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungus ◽  
Molecular Motor ◽  
Neurological Diseases ◽  
Cytoplasmic Dynein ◽  
Initiation Factor ◽  
Eukaryotic Cells ◽  
Organelle Transport ◽  
General Role ◽  
Reduced Frequency

The molecular motor cytoplasmic dynein is responsible for most minus-end–directed, microtubule-based transport in eukaryotic cells. It is especially important in neurons, where defects in microtubule-based motility have been linked to neurological diseases. For example, lissencephaly is caused by mutations in the dynein-associated protein Lis1. In this paper, using the long, highly polarized hyphae of the filamentous fungus Aspergillus nidulans, we show that three morphologically and functionally distinct dynein cargos showed transport defects in the genetic absence of Lis1/nudF, raising the possibility that Lis1 is ubiquitously used for dynein-based transport. Surprisingly, both dynein and its cargo moved at normal speeds in the absence of Lis1 but with reduced frequency. Moreover, Lis1, unlike dynein and dynactin, was absent from moving dynein cargos, further suggesting that Lis1 is not required for dynein-based cargo motility once it has commenced. Based on these observations, we propose that Lis1 has a general role in initiating dynein-driven motility.

scholarly journals Poly-Saturated Dolichols from Filamentous Fungi Modulate Activity of Dolichol-Dependent Glycosyltransferase and Physical Properties of Membranes

2019 ◽  
Vol 20 (12) ◽  
pp. 3043 ◽  
Author(s):  
Gryz ◽  
Perlińska-Lenart ◽  
Gawarecka ◽  
Jozwiak ◽  
Piłsyk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Differential Scanning Calorimetry ◽  
Filamentous Fungi ◽  
Fluorescence Anisotropy ◽  
Eukaryotic Cells ◽  
Fatty Acids Composition ◽  
Scanning Calorimetry ◽  
Yeast Saccharomyces Cerevisiae ◽  
Almost All ◽  
Pe Ratio

 Mono-saturated polyprenols (dolichols) have been found in almost all Eukaryotic cells, however, dolichols containing additional saturated bonds at the ω-end, have been identified in A. fumigatus and A. niger. Here we confirm using an LC-ESI-QTOF-MS analysis, that poly-saturated dolichols are abundant in other filamentous fungi, Trichoderma reesei, A. nidulans and Neurospora crassa, while the yeast Saccharomyces cerevisiae only contains the typical mono-saturated dolichols. We also show, using differential scanning calorimetry (DSC) and fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) that the structure of dolichols modulates the properties of membranes and affects the functioning of dolichyl diphosphate mannose synthase (DPMS). The activity of this enzyme from T. reesei and S. cerevisiae was strongly affected by the structure of dolichols. Additionally, the structure of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) model membranes was more strongly disturbed by the poly-saturated dolichols from Trichoderma than by the mono-saturated dolichols from yeast. By comparing the lipidome of filamentous fungi with that from S. cerevisiae, we revealed significant differences in the PC/PE ratio and fatty acids composition. Filamentous fungi differ from S. cerevisiae in the lipid composition of their membranes and the structure of dolichols. The structure of dolichols profoundly affects the functioning of dolichol-dependent enzyme, DPMS.

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