Ras hyperactivation versus overexpression: Lessons from Ras dynamics in Candida albicans

Abstract

Ras signaling in response to environmental cues is critical for cellular morphogenesis in eukaryotes. This signaling is tightly regulated and its activation involves multiple players. Sometimes Ras signaling may be hyperactivated. In C. albicans, a human pathogenic fungus, we demonstrate that dynamics of hyperactivated Ras1 (Ras1G13V or Ras1 in Hsp90 deficient strains) can be reliably differentiated from that of normal Ras1 at (near) single molecule level using fluorescence correlation spectroscopy (FCS). Ras1 hyperactivation results in significantly slower dynamics due to actin polymerization. Activating actin polymerization by jasplakinolide can produce hyperactivated Ras1 dynamics. In a sterol-deficient hyperfilamentous GPI mutant of C. albicans too, Ras1 hyperactivation results from Hsp90 downregulation and causes actin polymerization. Hyperactivated Ras1 co-localizes with G-actin at the plasma membrane rather than with F-actin. Depolymerizing actin with cytochalasin D results in faster Ras1 dynamics in these and other strains that show Ras1 hyperactivation. Further, ergosterol does not influence Ras1 dynamics.

To each its own: Mechanisms of cross-talk between GPI biosynthesis and cAMP-PKA signaling in Candida albicans versus Saccharomyces cerevisiae

“…6 B ) and enable increased cAMP production. As expected, the wild type strain overexpressing CaGPI2 is hyperfilamentous in Spider medium or YEPD at 30 °C, has high levels of F-actin even in yeast cells, and possesses ‘hyperactivated’ CaRas1 as tracked by FCS ( 22 , 162 ).…”

“…Some clues are available, however, from studies on the diffusion dynamics of N-terminally mRFP-tagged CaRas1 in wild type cells versus cells that overexpress either CaRas1 ( CaRAS1oe strain) or a constitutively activated variant, CaRas1 G13V ( CaRAS1 G13V oe strain) which cannot interact with Ira1 to return to its inactive state ( 162 ). Hyperfilamentation in Spider medium is exhibited by both the overexpression strains.…”

Ras signaling activates glycosylphosphatidylinositol (GPI) anchor biosynthesis via the GPI–N-acetylglucosaminyltransferase (GPI–GnT) in Candida albicans

“…10). We recently demonstrated this to be the case in the CaGPI19 conditional null strain, where CaGpi2 is also overexpressed (33).…”

The ER-resident Ras Inhibitor 1 (Eri1) ofCandida albicansinhibits hyphal morphogenesis via the Ras-independent cAMP-PKA pathway

“…For hyphal morphogenesis, as shown in Figure 4A, Cyr1 binds to the CAP1-G-actin complex via its CAP1-binding domain and triggers actin polymerization (23). Conversely, polymerization of actin can hyperactivate the cAMP-PKA pathway and produce hyperfilamentation (24). In mutants of CaERI1 we see increased F-actin levels in yeast cells, indicating that they are already primed for filamentation.…”