Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating

Fus2p is a pheromone-induced protein associated with the amphiphysin homologue Rvs161p, which is required for cell fusion during mating in Saccharomyces cerevisiae. We constructed a functional Fus2p–green fluorescent protein (GFP), which exhibits highly dynamic localization patterns in pheromone-responding cells (shmoos): diffuse nuclear, mobile cytoplasmic dots and stable cortical patches concentrated at the shmoo tip. In mitotic cells, Fus2p-GFP is nuclear but becomes cytoplasmic as cells form shmoos, dependent on the Fus3p protein kinase and high levels of pheromone signaling. The rapid cytoplasmic movement of Fus2p-GFP dots requires Rvs161p and polymerized actin and is aberrant in mutants with compromised actin organization, which suggests that the Fus2p dots are transported along actin cables, possibly in association with vesicles. Maintenance of Fus2p-GFP patches at the shmoo tip cortex is jointly dependent on actin and a membrane protein, Fus1p, which suggests that Fus1p is an anchor for Fus2p. In zygotes, Fus2p-GFP forms a dilating ring at the cell junction, returning to the nucleus at the completion of cell fusion

GMF Severs Actin-Arp2/3 Complex Branch Junctions by a Cofilin-like Mechanism

SummaryBackgroundBranched actin filament networks driving cell motility, endocytosis, and intracellular transport are assembled in seconds by the Arp2/3 complex and must be equally rapidly debranched and turned over. One of the only factors known to promote debranching of actin networks is the yeast homolog of glia maturation factor (GMF), which is structurally related to the actin filament-severing protein cofilin. However, the identity of the molecular mechanism underlying debranching and whether this activity extends to mammalian GMF have remained open questions.ResultsUsing scanning mutagenesis and total internal reflection fluorescence microscopy, we show that GMF depends on two separate surfaces for debranching. One is analogous to the G-actin and F-actin binding site on cofilin, but we show using fluorescence anisotropy and chemical crosslinking that it instead interacts with actin-related proteins in the Arp2/3 complex. The other is analogous to a second F-actin binding site on cofilin, which in GMF appears to contact the first actin subunit in the daughter filament. We further show that GMF binds to the Arp2/3 complex with low nanomolar affinity and promotes the open conformation. Finally, we show that this debranching activity and mechanism are conserved for mammalian GMF.ConclusionsGMF debranches filaments by a mechanism related to cofilin-mediated severing, but in which GMF has evolved to target molecular junctions between actin-related proteins in the Arp2/3 complex and actin subunits in the daughter filament of the branch. This activity and mechanism are conserved in GMF homologs from evolutionarily distant species

Antagonistic regulation of Fus2p nuclear localization by pheromone signaling and the cell cycle

When yeast cells sense mating pheromone, they undergo a characteristic response involving changes in transcription, cell cycle arrest in early G1, and polarization along the pheromone gradient. Cells in G2/M respond to pheromone at the transcriptional level but do not polarize or mate until G1. Fus2p, a key regulator of cell fusion, localizes to the tip of the mating projection during pheromone-induced G1 arrest. Although Fus2p was expressed in G2/M cells after pheromone induction, it accumulated in the nucleus until after cell division. As cells arrested in G1, Fus2p was exported from the nucleus and localized to the nascent tip. Phosphorylation of Fus2p by Fus3p was required for Fus2p export; cyclin/Cdc28p-dependent inhibition of Fus3p during late G1 through S phase was sufficient to block exit. However, during G2/M, when Fus3p was activated by pheromone signaling, Cdc28p activity again blocked Fus2p export. Our results indicate a novel mechanism by which pheromone-induced proteins are regulated during the transition from mitosis to conjugation

MY9184 expressing Fus2-GFP expressed from its own promoter was treated with α-factor for 1

5 h and either mock treated (A and C) or treated with nocodazole (B and D) for 10 min. Live cell image stacks were acquired (A and B). Arrows indicate directed movement of Fus2p-GFP puncta. Alternatively, cells were fixed and stained with anti-tubulin antibody (C and D) to demonstrate microtubule depolymerization. Bars, 1 μm.<p><b>Copyright information:</b></p><p>Taken from "Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating"</p><p></p><p>The Journal of Cell Biology 2008;181(4):697-709.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386108.</p><p></p

(A) Mating of wild type (MY9184) expressing Fus2p-GFP to wild type (MY8093) for 1 h

Flattened projections of deconvolved images are shown (top). To reveal the ring, deconvolved images were rotated ∼150° around the x axis. Fus2p-GFP localizes first to a restricted patch at the ZCF (left zygote, 0 min), dilates into a ring (10–40 min), and then accumulates in the nucleus (50 min). (B) Mating of wild type (MY9184) expressing Fus2p-GFP to a mutant (JY429). Deconvolved images were rotated slightly around the y axis to show the ring. (C) Indicated strains expressing Fus2p-GFP (MY9184, MY9213, MY9216, MY9208, MY9217, and 9211) were mated to a mutant (JY429). Bars, 1 μm.<p><b>Copyright information:</b></p><p>Taken from "Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating"</p><p></p><p>The Journal of Cell Biology 2008;181(4):697-709.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386108.</p><p></p

(A) Mitotically expressed Fus2p-GFP localizes to the nucleus at all stages of mitotic growth

MY9201 containing was grown in media containing galactose (top, DIC; bottom, GFP). (B) α-factor treatment causes Fus2p-GFP to exit the nucleus. MY9201 was grown in galactose-containing media and induced with α-factor. As the cell completed mitosis, nuclear Fus2p exited the nucleus and localized to the shmoo tip in pheromone-responding cells (left, DIC at 0 min; remaining panels, GFP). (C) Preexisting nuclear Fus2p-GFP exits the nucleus. MY9201 grown in galactose was transferred to media containing dextrose for the indicated times (10, 20, or 30 min) to shut off expression before treatment with pheromone for 1.5 h. Fus2p-GFP was nuclear in mitotic cells but localized to the shmoo tip in shmoos (top, DIC; bottom, GFP). (D) Fus2p-GFP nuclear exit is dependent on high levels of pheromone. MY9184 was treated with 6 μM α-factor (top) or 0.6 μM α-factor (bottom) and imaged at various times (left, transmitted or DIC at 0 min; right, GFP). (E) Nuclear exit requires the Fus3p MAP-kinase. MY9211 was treated with α-factor for 1.5 h. Transmitted light (first panel), DAPI of the nucleus (second panel), and GFP (remaining panels) are shown. Bars, 1 μm.<p><b>Copyright information:</b></p><p>Taken from "Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating"</p><p></p><p>The Journal of Cell Biology 2008;181(4):697-709.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386108.</p><p></p

Cdc42p and Fus2p act together late in yeast cell fusion

Cdc42p is the master regulator of morphogenesis in eukaryotic cells. It has an additional role in cell fusion, acting later in the pathway, after cells have undergone the changes in polarization and growth required for fusion. Cdc42p acts in concert with Fus2p to allow cell fusion