In vivo analysis of polarized lipid/GTPase function and dynamics in yeast


Supervising PI

Robert Arkowitz (website)

ESR3 Charles Puerner

Project Description

Background

Lipids, such as phosphatidylinositol phosphate, and small GTPases, such as the Rho GTPase Cdc42, have been shown to play critical roles in polarized growth in a range of organisms. However, whether clusters of lipids or activated GTPases can drive polarized growth in response to external stimuli remain unknown. Specifically, can cellular asymmetries be initiated and maintained by generation of such clusters? and by what mechanisms? Understanding the dynamics and functions of localized lipids and activated GTPases will also require mathematical modeling. In this project we will take advantage of light dependent recruitment systems to probe the distribution, dynamics and function of lipids and activated GTPases in polarized growth in response to external stimuli.

Polarized tip growth requires a coordinated organization of cellular components, in particular components of the membrane transport pathway located in the filament tip. We are interested in the distribution and dynamics of membrane transport compartments, and additionally whether these compartments are altered during substrate invasion. We have used the dimorphic fungus Candida albicansthat switches between a budding yeast form and a filamentous form, in which growth occurs at the apex.

Objectives

  • Determine the role of site-specific lipid and GTPase clusters in fungal external signal-mediated polarized growth and force generation.
  • Determine whether artificially generated clusters of lipid and activated GTPases can drive polarized growth and/or compete with endogenous growth sites.

Summary of Results

Initially, we established a method to quantitate the number of secretory vesicles in the Spitzenkörper, a fungal specific cluster of secretory vesicles at the filament tip, using fluorescence microscopy, and this number was comparable to that determined by serial-section transmission electron microscopy [1]. Subsequent to the establishment of the Spitzenkörper, the number of secretory vesicles in this structure remains constant during growth. Using multi-channel imaging with spectrally distinct fluorescent proteins, we observed the distribution of multiple organelles in a single cell, i.e.secretory vesicles, Golgi, and endoplasmic reticulum, providing insight into the organization required to maintain polarized tip growth.

In order to study C. albicans substrate invasion, we have generated PDMS microchambers, in which cells are entrapped, and monitored filamentous growth using live-cell time-lapse microscopy. The stiffness of the microchambers can be varied during fabrication, allowing us to investigate invasion under a range of resistive forces. Initially, we observed a decrease in the percentage of invasive growth with an increase in PDMS stiffness and a stiffness threshold where filaments are no longer able to invade. This threshold is likely the growth-stalling force [2], [3], a value where resistive force should be equal to the turgor pressure in the cell. The cell filaments, which were unable to invade, bent during extension, eventually filling the microchamber. We have followed filament invasion within the PDMS substrate and observed a reduction in the rate of filament extension, compared to cells growing on the substrate surface. Additionally, we have measured an increase in cell diameter and a decrease in cell compartment length during invasive growth, resulting in a cell volume similar to that of surface growing cells.

These results suggest that invasive growth leads to a disruption of cell polarity, as has been seen upon dramatic perturbation of growth in Schizosaccharomyces pombe[4]. To address this, we are measuring the distribution of activated Cdc42 in the filament tip during invasion, using a fluorescent reporter [5].

Furthermore, we are interested in the effects of resistive force on organelle distribution. We have shown that, when a hyphal filament bursts out of the PDMS into a liquid filled chamber, i.e.a dramatic reduction of resistive force, i) there is a transient peak in secretory vesicle signal in the filament tip, ii) the endocytic collar shifts transiently back from the tip.

These results suggest that cellular organization and growth are altered by mechanical stress.

References

  1. A. Weiner et al., “On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicansCell. Microbiol.2019, 21(1): e12963.
  2. N. Minc, Microfabricated Chambers as Force Sensors for Probing Forces of Fungal Growth, Methods in Cell Biology,1st Ed.,120. Elsevier Inc., 2014.
  3. N. Minc et al, “Mechanical Forces of Fission Yeast Growth” Curr. Biol. 2009, 19(13): 1096–1101.
  4. A. Haupt, et al., “A Positive Feedback between Growth and Polarity Provides Directional Persistency and Flexibility to the Process of Tip Growth” Curr. Biol 2018,28(20):3342-3351.
  5. V. Corvest,et al., “Spatio temporal regulation of Rho1 and Cdc42 activity during Candida albicansfilamentous growth”Mol. Microbiol.2013, 89(4):626-48.