Helical growth during the phototropic response, avoidance response, and in stiff mutants of Phycomyces blakesleeanus

The sporangiophores of Phycomyces blakesleeanus have been used as a model system to study sensory transduction, helical growth, and to establish global biophysical equations for expansive growth of walled cells. More recently, local statistical biophysical models of the cell wall are being constructed to better understand the molecular underpinnings of helical growth and its behavior during the many growth responses of the sporangiophores to sensory stimuli. Previous experimental and theoretical findings guide the development of these local models. Future development requires an investigation of explicit and implicit assumptions made in the prior research. Here, experiments are conducted to test three assumptions made in prior research, that (a) elongation rate, (b) rotation rate, and (c) helical growth steepness, R, of the sporangiophore remain constant during the phototropic response (bending toward unilateral light) and the avoidance response (bending away from solid barriers). The experimental results reveal that all three assumptions are incorrect for the phototropic response and probably incorrect for the avoidance response but the results are less conclusive. Generally, the experimental results indicate that the elongation and rotation rates increase during these responses, as does R, indicating that the helical growth steepness become flatter. The implications of these findings on prior research, the “fibril reorientation and slippage” hypothesis, global biophysical equations, and local statistical biophysical models are discussed.

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

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.

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

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.

Dimensionless Numbers to Analyze Expansive Growth Processes

Cells of algae, fungi, and plants have walls and exhibit expansive growth which can increase their volume by as much as 10,000 times. Expansive growth is central to their morphogenesis, development, and sensory responses to environmental stimuli. Equations describing the biophysical processes of the water uptake rate and the wall deformation rate have been derived, validated, and established. A significant amount of research provides insight into the molecular underpinnings of these processes. What is less well known are the relative magnitudes of these processes and how they compare during expansive growth and with walled cells from other species. Here, dimensionless numbers (Π parameters) are used to determine the magnitudes of the biophysical processes involved in the expansive growth rate of cells from algae (Chara corallina), fungi (Phycomyces blakesleeanus), and plants (Pisum satinis L.). It is found for all three species that the cell’s capability for the water uptake rate is larger than the wall plastic deformation rate and much larger than the wall elastic deformation rate. Also, the wall plastic deformation rates of all three species are of similar magnitude as their expansive growth rate even though the stress relaxation rates of their walls are very different. It is envisioned that dimensionless numbers can assist in determining how these biophysical processes change during development, morphogenesis, sensory responses, environmental stress, climate change, and after genetic modification.