Regulation of a Differentiation MAPK Pathway by a Novel Integrated Signaling Network and Multiple Sensors

Filamentous growth is a cell differentiation program utilized by Saccharomyces cerevisiae to respond to nutrient limitation in the environment. This process is principally controlled by a mitogen-activated protein kinase (MAPK) pathway but is also impacted by a number of other pathways including Ras2p-cAMP-PKA, Target of Rapamycin, Rim101, and mitochondrial retrograde. Using a high-throughput genetic screening approach in conjunction with directed gene-deletion analysis, I have identified 97 new regulators of the filamentous growth MAPK pathway. These new regulators created new connections to the filamentous growth MAPK pathway as well as extended previously known connections. I have linked several of the pathways governing filamentous growth together as part of an integrated signaling network by showing that these pathways regulate each other’s transcriptional targets. This network indicates an intricate level of communication and coordination among these pathways that has not been previously appreciated. I show that proper coordination of the filamentous growth MAPK pathway is essential for proper morphogenesis and this is a potential reason for the many inputs used to control this response. The filamentous growth MAPK pathway is also regulated by three transmembrane proteins – Msb2p, Sho1p, and Opy2p. Here these three proteins are compared to determine that they have specific functions in regulating filamentous growth. The three proteins exhibit different localization patterns and rates of turnover from the plasma membrane. I show that the Rim101 pathway affects the filamentous growth MAPK pathway independently of the ESCRT pathway which shares components with the Rim101 pathway. Additionally, I have shown that overexpression of the arrestin protein Aly1p results in mislocalized Msb2p and diminished pathway activity

<i>Neurospora crassa</i> Female Development Requires the PACC and Other Signal Transduction Pathways, Transcription Factors, Chromatin Remodeling, Cell-To-Cell Fusion, and Autophagy

<div><p>Using a screening protocol we have identified 68 genes that are required for female development in the filamentous fungus <i>Neurospora crassa</i>. We find that we can divide these genes into five general groups: 1) Genes encoding components of the PACC signal transduction pathway, 2) Other signal transduction pathway genes, including genes from the three <i>N. crassa</i> MAP kinase pathways, 3) Transcriptional factor genes, 4) Autophagy genes, and 5) Other miscellaneous genes. Complementation and RIP studies verified that these genes are needed for the formation of the female mating structure, the protoperithecium, and for the maturation of a fertilized protoperithecium into a perithecium. Perithecia grafting experiments demonstrate that the autophagy genes and the cell-to-cell fusion genes (the MAK-1 and MAK-2 pathway genes) are needed for the mobilization and movement of nutrients from an established vegetative hyphal network into the developing protoperithecium. Deletion mutants for the PACC pathway genes <i>palA</i>, <i>palB</i>, <i>palC</i>, <i>palF</i>, <i>palH</i>, and <i>pacC</i> were found to be defective in two aspects of female development. First, they were unable to initiate female development on synthetic crossing medium. However, they could form protoperithecia when grown on cellophane, on corn meal agar, or in response to the presence of nearby perithecia. Second, fertilized perithecia from PACC pathway mutants were unable to produce asci and complete female development. Protein localization experiments with a GFP-tagged PALA construct showed that PALA was localized in a peripheral punctate pattern, consistent with a signaling center associated with the ESCRT complex. The <i>N. crassa</i> PACC signal transduction pathway appears to be similar to the PacC/Rim101 pathway previously characterized in <i>Aspergillus nidulans</i> and <i>Saccharomyces cerevisiae</i>. In <i>N. crassa</i> the pathway plays a key role in regulating female development.</p></div

Genes from the PacC pathway are required for female development.

<p>A notation of “This report” in the reference information column indicates that the gene was identified as being needed for <i>N. crassa</i> development by our experiments.</p><p>Genes from the PacC pathway are required for female development.</p

The PACC pathway is required for the development of asci.

<p>Fertilized perithecia from wild type (left panel/A) and Δ<i>palA</i> (right panel/B) were allowed to develop for 7 days. The perithecia were squashed between a glass slide and a glass coverslip and examined with a transmitted light microscope. The wild type perithecia has generated ascospores (arrow) while the Δ<i>palA</i> peirthecia is defective in ascospore formation. The arrow in the left panel points to an ascospore.</p

Constitutively active PACC activates female development.

<p>Cells were inoculated onto agar slants and allowed to grow for 10 days at room temperature. The left panel (A) shows a Δ<i>palC</i> isolate on Vogel’s sucrose medium. The middle panel (B) shows a Δ<i>palC</i> isolate that has been transformed with the constitutively activated PACC construct growing on synthetic crossing medium. The right panel (C) shows a Δ<i>palC</i> isolate that has been transformed with the constitutively activated PACC construct growing on Vogel’s sucrose medium, a medium that represses female development. Note that the constitutively activated PACC caused protoperithecia production in the absence of PALC on both media. The arrows in the middle and right panels point to protoperithecia.</p

Signal transduction pathway genes are required for female development.

<p>PP – <u>P</u>reviously<u> p</u>ublished data demonstrated that the gene was needed for female development.</p><p>NA – a deletion strain is <u>n</u>ot <u>a</u>vailable in the single gene deletion library.</p><p>NA(het) – the deletion strain in the single gene deletion library is a <u>het</u>erokaryon and a homokaryon isolate was <u>n</u>ot <u>a</u>vailable during the screening experiments.</p><p>RIP – a RIP experiment was used to verify that the gene is required for female development.</p><p>An * by the NCU number indicates that the gene is needed for CAT (conidia anastomosis tube) formation (a cell fusion phenotype) and is likely to be a component of either the MAK-1 or MAK-2 signal pathway.</p><p>A notation of “This report” in the reference information column indicates that the gene was either newly identified or verified by co-segregation and complementation analysis as being needed for <i>N. crassa</i> development by our experiments.</p><p>Signal transduction pathway genes are required for female development.</p

Transcription factors needed for female development.

<p>PP – <u>P</u>reviously<u> p</u>ublished data demonstrated that the gene was needed for female development.</p><p>NA – a deletion strain is <u>n</u>ot <u>a</u>vailable in the single gene deletion library.</p><p>NA (het) – the deletion strain in the single gene deletion library is a <u>het</u>erokaryon and a homokaryon isolate was <u>n</u>ot <u>a</u>vailable during the screening experiments.</p><p>Transcription factors needed for female development.</p

Perithecia grafting experiments.

<p>Small pieces of cellophane containing “graft” fertilized wild type protoperithecia were placed on mutant “host” vegetative hyphal networks grown on synthetic crossing medium. The “host” shown are: 1) Δ<i>fmf-1</i> (left panel/A) which shows a host supporting the development of the graft perithecia. 2) Δ<i>ada-1</i> (middle panel/B) which shows a host not supporting the development of the graft perithecia. 3) Δ<i>palA</i> (right panel/C) which shows the graft inducing protoperithecia in the Δ<i>palA</i> host. Arrows point to examples of perithecia on the cellophane (left panel/A) and protoperithecia induced in the host vegetative hyphal network (right panel/C).</p

Schematic representation of the <i>N. crassa</i> PACC pathway.

<p>The PACC signal transduction pathway elements found in <i>N. crassa</i>, and the model for how the pathway might function are depicted. The PALH and PALF proteins are thought to be found at the plasma membrane. PALH is a seven transmembrane receptor which is sensitive to environmental cues. PALF is an arrestin type protein that associates with PALH. PALF is phosphorylated and ubiquitinated in response to the environmental cues. These events lead to the endocytosis of the PALH/PALF complex. Following endocytosis, the PALH is directed into an ESCRT compartment, where it enters into a signaling complex containing PALA, PALB, PALC, and PACC. Within the signaling complex, PALB functions as a protease which cleaves PACC. This cleavage event removes a C-terminal inhibitory domain from the PACC transcription factor, and the processed PACC is released from the signaling complex. The activated PACC then enters the nucleus and directs transcriptional activity leading to the formation of the protoperithecium.</p