Fusion between MAK-2-GFP-expressing cell fusion mutants and RFP-expressing wild type cells.

<p>Conidia samples containing equal number of RFP-expressing wild type conidia and MAK-2-GFP-expressing wild type or mutant conidia were grown under CAT induction conditions for 4 hours. DIC images, GFP fluorescent images, RFP fluorescent images, and merged images for each combination of conidia types are shown in the columns from left to right respectively. Each row shows the images for RFP-expressing wild type conidia mixed with MAK-2-GFP-expressing wild type (WT) (row 1), <i>Δham-6</i> (row 2), <i>Δham-7</i> (row 3), <i>Δham-8</i> (row 4), <i>Δham-9</i> (row 5), <i>Δham-10</i> (row 6), <i>Δamph-1</i> (row 7), and <i>Δwhi-2</i> (row 8) conidia. Wild type conidia frequently engaged in cell fusion, while <i>Δamph-1</i> and <i>Δwhi-2</i> conidia engaged in cell fusion with w conidia at a low frequency.</p

Characterization of the <i>Neurospora crassa</i> Cell Fusion Proteins, HAM-6, HAM-7, HAM-8, HAM-9, HAM-10, AMPH-1 and WHI-2

<div><p>Intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. Cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. Approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the model filamentous fungus <i>Neurospora crassa</i>. Genetic, biochemical, and microscopic techniques were used to characterize the functions of seven previously poorly characterized cell fusion proteins. HAM-6, HAM-7 and HAM-8 share functional characteristics and are proposed to function in the same signaling network. Our data suggest that these proteins may form a sensor complex at the cell wall/plasma membrane for the MAK-1 cell wall integrity mitogen-activated protein kinase (MAPK) pathway. We also demonstrate that HAM-9, HAM-10, AMPH-1 and WHI-2 have more general functions and are required for normal growth and development. The activation status of the MAK-1 and MAK-2 MAPK pathways are altered in mutants lacking these proteins. We propose that these proteins may function to coordinate the activities of the two MAPK modules with other signaling pathways during cell fusion.</p></div

Immunofluorescent localization images for HA-tagged proteins.

<p>Anti-HA primary antibody and Alexa Fluor 488-conjugated secondary antibody were used to label HA-tagged protein in fixed germ tubes/CATs. Typical DIC images (left), fluorescent images (middle), and merged images (right) are shown. Images are shown for Wild type (WT) control (top row), <i>HA-ham-7</i> transformant germ tubes (row 2), and CATs (row 3), <i>HA-ham-8</i> transformant germ tube (row 4) and CATs (row 5), <i>HA-amph-1</i> transformant germ tube (row 6) and CATs (row 7), <i>HA-whi-2</i> transformant germ tube (row 8) and CATs (bottom row).</p

Schematic model for the regulatory network involved in CAT fusion.

<p>PP-1, ADV-1, SNF-5, and RCO-1/RCM-1 are transcription factors required for CAT fusion. MIK-1/MEK-1/MAK-1 and NRC-1/MEK-2/MAK-2 are two MAP kinase pathways required for CAT fusion. HAM-2/HAM-3/HAM-4/MOB-3/PP2A/PPG-1 form the STRIPAK complex that regulates MAK-1 nuclear accumulation. HAM-1/SO and MAK-2 engage in Ping-Pong signaling behavior during CAT fusion. HAM-6/HAM-7/HAM-8 are required at the plasma membrane/cell wall for MAK-1 pathway activation. HAM-10 may regulate vesicular trafficking and could potentially respond to calcium signaling during cell fusion. AMPH-1 regulates vesicular trafficking and endocytosis during cell fusion. WHI-2 may regulate the MAP kinase pathways through a general stress response pathway. The role of HAM-9 during CAT fusion remains to be determined.</p

Peroxidase activation of MAK-1 and MAK-2 pathways in cell fusion mutant vegetative hyphal cells.

<p>Western blot analyses using Phospho-p44/42 MAPK antibody were performed to evaluate MAK-1 and MAK-2 activation in wild type (WT) and mutant vegetative hyphal cells in response to peroxidase treatments. Quantitative analyses of the Western blots were performed to determine the levels of phosphorylated MAK-1 and MAK-2 in non-stressed and oxidative-stressed samples (wild type, <i>Δham-6</i>, <i>Δham-7</i>, <i>Δham-8</i>, <i>Δham-9</i>, <i>Δham-10</i>, <i>Δamph-1</i>, and <i>Δwhi-2</i>). A) MAK-1 activation in wild type and mutants in response to peroxidase treatment. B) MAK-2 activation in wild type and mutants in response to peroxidase treatment. The levels of MAK-1 and MAK-2 in the non-stressed wild type sample were set as 100% for the quantitative analysis.</p

Strains used in this study.

<p>Strains used in this study.</p

MAK-2-GFP localization in wild type and mutant germ tubes/CATs.

<p>MAK-2-GFP expressing wild type (WT) and mutant conidia were grown under CAT induction conditions for 4 hours. DIC images (left column), GFP fluorescent images (middle column), and merged images (right column) are shown. The images show germ tubes/CATs for wild type (WT) (row 1), <i>Δham-6</i> (row 2), <i>Δham-7</i> (row 3), <i>Δham-8</i> (row 4), <i>Δham-9</i> (row 5), <i>Δham-10</i> (row 6), <i>Δamph-1</i> (row 7), and <i>Δwhi-2</i> (row 8). The arrows in the WT GFP fluorescent image point to the localization of MAK-2-GFP at the sites of cell fusion.</p

Complementation of CAT fusion activities by different HA, GFP and RFP tagged proteins.

<p>A) The levels of CAT fusion activity for the gene deletion mutants and for transformants expressing a tagged version of the deleted gene are shown as a percentile of the cell fusion activity for wild type CATs. B) Photograph of CAT fusion activities in wild type (WT), <i>Δham-8</i>, and <i>Δham-8</i> transformed with <i>HA-ham-8</i>. Arrows point to examples of CAT fusion in the wild-type and <i>Δham-8</i> transformed with <i>HA-ham-8</i> panels.</p

Localization of RFP-HAM-10 and RFP-AMPH-1 in germ tubes/CATs.

<p>Confocal microscopic images were taken for CATs expressing RFP-HAM-10 (top row of panels) and RFP-AMPH-1 (bottom row of panels). Images shown from left to right are DIC images, RFP fluorescent images, and merged images.</p

MAK-1 and MAK-2 phosphorylation status in germ tubes/CATs.

<p>Western blot analysis using Phospho-p44/42 MAPK antibody was performed to determine MAK-1 and MAK-2 phosphorylation status in wild type (WT) and mutant (<i>Δham-6</i>, <i>Δham-7</i>, <i>Δham-8</i>, <i>Δham-9</i>, <i>Δham-10</i>, <i>Δamph-1</i>, <i>Δwhi-2</i>, <i>Δmik-1</i>, and <i>Δnrc-1</i>) germ tubes/CATS. The positions of the phosphorylated MAK-1 (p-MAK-1) and phosphorylated MAK-2 (p-MAK-2) in the Western blot are noted in the left margin of the figure. B) The relative MAK-1 phosphorylation status in mutant germ tubes/CATs relative to the MAK-1 phosphorylation status in wild type germ tubes/CATs (WT value is set at 100%). C) The relative MAK-2 phosphorylation status in mutant germ tubes/CATs compared to the MAK-2 phosphorylation status in wild type germ tubes/CATs (WT value is set at 100%).</p