Mechanisms of Cdc42 Polarization in Yeast

Polarization is important for the function and morphology of many different cell types. The keys regulators of polarity in eukaryotes are the Rho-family GTPases. In the budding yeast Saccharomyces cerevisiae, which must polarize in order to bud and to mate, the master regulator is the highly conserved Rho GTPase, Cdc42. During polarity establishment, active Cdc42 accumulates at a site on the plasma membrane characterizing the “front” of the cell where the bud will emerge. The orientation of polarization is guided by upstream cues that dictate the site of Cdc42 clustering. However, in the absence of upstream cues, yeast can still polarize in a random direction during symmetry breaking. Symmetry breaking suggests cells possess an autocatalytic polarization mechanism that can amplify stochastic fluctuations of polarity proteins through a positive feedback mechanism.

Two different positive feedback mechanisms have been proposed to polarize Cdc42 in budding yeast. One model posits that Cdc42 activation must be localized to a site at the plasma membrane. Another model posits that Cdc42 delivery must be localized to a particular site at the plasma membrane. Although both mechanisms could work in parallel to polarize Cdc42, it is unclear which mechanism is critical to polarity establishment. We directly tested the predictions of the two positive feedback models using genetics and live microscopy. We found that localized Cdc42 activation is necessary for polarity establishment.

While this explains how active Cdc42 localizes to a particular site at the plasma membrane, it does not address how Cdc42 concentrates at that site. Several different mechanisms have been proposed to concentrate Cdc42. The GDI can extract Cdc42 from membranes and selective mobilize GDP-Cdc42 in the cytoplasm. It was proposed that selectively mobilizing GDP-Cdc42 in combination with local activation could locally concentrate total Cdc42 at the polarity site. Although the GDI is important for rapid Cdc42 accumulation at the polarity site, it is not essential to Cdc42 concentration. It was proposed that delivery of Cdc42 by actin-mediated vesicle can act as a backup pathway to concentrate Cdc42. However, we found no evidence for an actin-dependent concentrating pathway. Live microscopy experiments reveal that prenylated proteins are not restricted to membranes, and can enter the cytoplasm. We found that the GDI-independent concentrating pathway still requires Cdc42 to exchange between the plasma membrane and the cytoplasm, which is supported by computational modeling. In the absence of the GDI, we found that Cdc42 GAP became essential for polarization. We propose that the GAP limits GTP-Cdc42 leak into the cytoplasm, which would be prohibitive to Cdc42 polarization.

Epithelial-mesenchymal transitions and hepatocarcinogenesis.

Epithelial-mesenchymal transitions (EMTs) are believed to play a role in invasion and metastasis of many types of tumors. In this issue of the JCI, Chen et al. show that a gene that has been associated with aggressive biology in hepatocellular carcinomas initiates a molecular cascade that results in EMT.

BDNF-TrkB Signaling in Single-Spine Structural Plasticity

Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.

From Polarity to Morphogenesis PAK Behaviors and Mechanism for Bud Sensing in Morphogenesis Checkpoint

Bud formation by Saccharomyces cerevisiae is a fundamental process for yeast proliferation. Bud emergence is initiated by the polarization of the cytoskeleton, leading to local secretory vesicle delivery and gulcan synthase activity. The master regulator of polarity establishment is a small Rho-family GTPase – Cdc42. Cdc42 forms a clustered patch at the incipient budding site in late G1 and mediates downstream events which lead to bud emergence. Cdc42 promotes morphogenesis via its various effectors. PAKs (p21-activated kinases) are important Cdc42 effectors which mediate actin cytoskeleton polarization and septin filament assembly. The PAKs Cla4 and Ste20 share common binding domains for GTP-Cdc42 and they are partially redundant in function. However, we found that Cla4 and Ste20 behaved differently during the polarization and this depended on their different membrane interaction domains. Also, Cla4 and Ste20 compete for a limited number of binding sites at the polarity patch during bud emergence. These results suggest that PAKs may be differentially regulated during polarity establishment.

Morphogenesis of yeast must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site prior to bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a “bud sensor”. Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed, and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence.